CA2230967C - Improved nucleic acid assays - Google Patents

Abstract

The present invention relates to the detection of specific nucleic acid sequences, either by a process of amplification of specific nucleic acid sequences or not. More particularly the invention provides for improved compositions and methods for reducing the chance for contamination from manipulation of reagents, internal controls for amplification, and the use of automated apparatus for the automated detection of one, or more than one amplified nucleic acid sequences.

Description

Improved Nucleic Acid Assays Field of the Invention The present invention relates to the detection of specific nucleic acid sequences in a target test sample.

In particular, the present inventioni relates to the automated detection of specific nucleic acid sequences v/hich are either unamplified or amplified nucleic acid sequences (amplicons).
In addition, the present invention 1-elates to the use of automated amplification, methods and compositions for monitoring successful amplification, improved methods for reducing the lo chance for contamination, and the use of ianified reaction buffers and unit dose aliquots of reaction components for amplification.
Finally, the present invention also relates to unique constructs and methods for the conventional or automated detection of one, or more than one different nucleic acid sequences in a single assay.

The Backg~rounci of the Invention The development of techniques for the nianipulation of nucleic acids, the amplification of such nucleic acids when necessary, and t?he subsequent detection of specific sequences of nucleic acids or amplicons has generated extremely sensitive and nucleic acid sequence specific assays fo r the diagnosis of disease and/or identification of pathogenic organisms in a test sample.
Amplification of nucleic acids When necessary, enzymatic amplification of nucleic acid sequences will enhance the ability to detect such nucleic acid sequer.ices. Generally, the currently known amplification schemes can be broadly grouped into two classes based on whether, the enzymatic amplification reactions are driven by continuous cycling of the temperature between the denaturation temperature, the primer annealing temperature, and the amplicon (product of enzymatic arnplification of nucleic acid) synthesis temperature, or whether the temperature is kept constant throughout the enzymatic amplification process (isothermal amplification).
Typical cycling nucleic acid amplification technologies P,thermocycling) are polymerase chain reaction (PCR), McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Cliicago, Illinois 60606 (312)913-0001 2 and iizase chain reaction (LCR). Specific protocols for such reactions are discussed in. for example. Short Protocols in Molecular Biology, 22"` Edition, A Compendium of Methods from Current Protocols in iviolecular Biolow, (Eds. Ausubel et al., Tohn Wiiev &
Sons, New York.
1992) chapter 15. Reactions which are isothemial include: transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), and strand displacement amplification (SDA).
ti.S. Patent documents which discuss nucleic acid amplification include 4,683,195;
4,683,202; 5,130,238; 4,876,187; 5,030,557; 5,399,491; 5,409,818; 5,485,184;
5,409,818;
5,554,517; 5,437,990 and 5,554,516. It is well known that methods such as those described in these i c~ patents permit the amplification and detection of nucleic acids without requiring cloning, and are responsible for the most sensitive assays for nucleic acid sequences. However, it is equally well recognized that along with the sensitivity of detection possible with nucleic acid amplification, the ease of contamination by minute amounts of unwanted exoaenous nucleic acid sequences is extremely great. Contamination by unwanted exogenous DNA or RNA nucleic acids is equally likely. The 15 utility of amplification reactions will be enhanced by methods to control the introduction of unwanted exogenous nucleic acids and other contaminants.
Prior to the discovery of thermostable enzymes, methods that used therrnocycling were made extremely difficult by the requirement for the addition of fresh enzyme after each denaturation step, since initially the elevated temperatures required for denaturation also 20 inactivated the polymerases. Once thermostable enzymes were discovered, cycling nucleic acid amplification became a far more simplified procedure where the addition of enzyme was only needed at the beginning of the reaction. Thus reaction tubes did not need to be opened and new enzyme did not need to be added during the reaction, allowed for an improvement in efficiency and accuracy as the risk of contamination was reduced, and the cost of enzymes was also reduced.
25 An example of a thermostable enzyme is the polymerase isolated from the organism Thermopliilus aquaticus.
In aeneral, %sothermal amplification can require the combined activity of multiple enz'VnIe activities for which no optimal thermostable variants have been described. The initial step of an amplification reaction will usually require denaturation of the nucleic acid target, for example in the TMA reaction, the initial denaturation step is usually _ 65 C, but can be typically _ 95 C, and is used when required to remove the secoridary structure of the target nucleic acid.
The reaction mixture is then cooled to a lower temperature which allows for primer annealing, and is the optimal reaction temperature for the combined activities of the amplification enzymes. For example, in TMA the enzymes are generally a T7 RNA polymerase and a reverse trariscriptase (which includes endogenous RNase H activity). The temperature of the reaction is kept constant through out the subsequent -isothermal amplification cycle.
Because of the lack of suitable thermostable enzymes, some isothermal amplifications will generally require the addition of enzymes to the reaction mixture after denaturation at high temperature, and cool-down to a lower teinperature. This requirement is inconvenient, and requires the opening of the amplification reaction tube, which introduces a major opportunity for coritamination.
Thus, it would be most useful if such reactions could be more easily performed with a reduced risk of contamination by methods which would allow for integrated denaturation and amplification wiithout the need for manual enzyme transfer.

Amplification Buffer and Single Ts:eaction Aliquot of Reagents Typical reaction protocols require the use of several different buffers, tailored to optimize the activity of the particular enzyme being used at certain steps in the reaction, or for optimal 2o resuspension of'reaction components. For example, while a typical PCR 10x amplification buffer will contain 500inM KCI and 100mM Tris HC1, pH 8.4, the concentration of MgC12 will depend upon the nucleic acid target sequence and primer set of interest. Reverse transcription buffer (5x) typically contains 400mM Tris-Cl, pH 8.2; 400mM KCl and 300 mM MgC1Z1 whereas Murine Maloney Leukemia Virus reverse transcriptase buffer (5x) typically contains 250mM Tris-Cl, pH
8.3; 375mM KC1; 50mM DTT (Dithiothreitol) and 15mM MgC1z.
While such reaction buffers can be prepared in bulk from stock chemicals, most commercially available amplification products provide bulk packaged reagents and specific buffers for use with the amplification protocol. For example, commercially available manual arnplification assays for detection of clir-ically significant pathogens (for example Gen-Probe Inc.
Chlamydia, an(i Mycobacterium tuberculosis detection assays) requires several manual McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 4 manipulations to perforrn the assay, including dilution of the test sample in a sample dilution buffer ( SDB). combination of the diiuted sample with amplification reaction reagents such as oliaonucleotides and specific oliQonucleotide promoteriprimers which have been reconstituted in an amplification reconstitution buffer (ARB). and finally, the addition to this reaction mixture of enzymes reconstituted in an enzvme dilution buffer (EDB).
The preparation and use of multiple buffers which requires multiple manual additions to the reaction mixture introduces a greater chance for contamination. It would be most useful to have a sinale unified buffer which could be used in all phases of an amplification protocol. In particular, with the commercially available TMA assays described above, the requirement for 1 o three buffers greatly complicates automation of such a protocol.
Bulk packaging of the enzyme or other reaction components by manufacturers, may require reconstitution of the components in large quantities, and the use of stock amounts of multiple reagents, can be wasteful when less than the maximal number of reactions are to be carried out, as some of these components may be stable for only a short time.
This process of 15 reconstitution also requires multiple manipulations by the user of the stock reagents, and aliquoting of individual reaction amounts of reagents from stocks which creates a major opportunity for contamination.
Methods and compositions for the preparation of bulk quantities of preserved proteins are known, see for example, U.S. Patent 5,098,893; 4,762,857; 4,457,916;
4,891,319; 5,026,566 and 20 interr.ational Patent Publications WO 89/06542; WO 93/00806; WO 95/33488 and WO

89/00012. However, the use of pre-aliquoted and preserved reagent components ui single reaction quantities/dose is both very useful and economical. Single aliquots of enzyme reagent avoids multiple use of bulk reagents, reducing waste, and greatly reducing the chance of contamination. Further, such single reaction aliquots are nlost suitable for the automation of the reaction process.
The requirement for m,any changes of buffer and the multiple addition of reagents compiicates the automation of such reactions. A single dose unit of reaction buffer mixture. and a unified combination buffer will both simplifi- automation of the process and reduce the chance of contamination.

.qistomation of.N-ucleic Acid Detection witi7 or l~itliout.qnzplification ~ucleic acid probe assays. and combination arnplification probe assays can be rapid.
sensitive. hi zhlv specific, and usually require precise handlin2 in order to mini-nize contamination with non-specific nucleic acids, and are thus prime candidates for automation. As -with conventional nucleic acid detection protocols. it is Qenerally required to utilize a detection probe oliQonucleotide sequence which is linked by some means to a detectable sivnal ocneratinv component. One possible probe detection system is described in U.S. Patent 4,581,33 In addition, automation of a nucleic acid detection system targeting unamplified or 1o amplified nucleic acid, or a combined automated amplification/detection system will generally be adaptable to the use of nucleic acid capture oligonucleotides that are attached to some form of solid support system. Examples of such attachment and methods for attachment of nucleic acid to solid support are found in U.S. Patent 5,489,653 and 5,510,084.

15 Automation of amplification, detection, and a combination of amplification and detection is desirable to reduce the requirement of multiple user interactions with the assay. Apparatus and methods for optically analyzing test materials are described for example in U.S. Patent 6,122,28=.
Automation is generally believed to be more economical, efficient, reproducible and accurate for the processing of clinical assays. Thus with the superior sensitivity and specificity of nucleic acid detection assays, the use of amplification of nucleic acid sequences, and automation at one or more phases of an assay protocol can enhance the utility of the assay protocol and its utility in a clinical setting.

Advantage of Internal Control Sequences 25 Nucleic acid amplification is highly sensitive to reaction conditions, and the failure to amplify and/or detect any specific nucleic acid sequences in a sample may be due to error in the amplification process as much as being due to absence of desired target sequence. Amplification reactions are notoriously sensitive to reaction conditions and have generally required includina control reactions with known nucleic acid target and primers in separate reaction vessels treated 3o at the same time. However, internal control sequences added into the test reaction mixture would truly control for the success of the amplification process in the subject test reaction miature and would be most useful. U.S. Patent 5,457,027 teaches certain internal control sequences which are useful as an internal oligonucleotide standard in isothermal aanplification reactions for Mycobacteriunz tuberculosis.
i - Howevcr it wouid b: extremely useful to have a general method of yeneratinL, internal control sequences, that would be useful as internal controls of the various amplification procedures, which are specincally tailored to be unaffected by the nucleic acid sequences present in the target organism, the host orzanism, or nucleic acids present in the normal flora or in the environment. Generally, such internal control sequences should not be substantially similar to anv nucleic acid sequences present in a clinical setting, including human, pathogenic organism, normal flora orEanisms, or environmental orcranisms which could interfere with the amplification and detection of the internal control sequences.

Detection of More than one Nucleic Acid Sequence in a Single Assay In general, a sinale assay reaction for the detection of nucleic acid sequences is limited to the detection of a single target nucleic acid sequence. This single target limitation increases costs and time required to perform clinical diagnostic assays and verification control reactions. The detection of more than one nucleic acid sequence in a sample using a single assav would greatly enhance the efficiency of sample analysis and would be of a gxeat economic benefit by reducing costs, for example helping to reduce the need for multiple clinical assays.
Multiple analyte detection in a single assay has been applied to antibody detection of analyte as in for example Intemational Patent Publication number WO 89/00290 and WO
93/21346.

In addition to reducing cost. time required, the detection of more than one nucleic acid taraet sequence in a single assay would reduce the chance of erroneous results. In particular multiple detection would greatly enhance the utility and benefit using internal control sequences and allow for the rapid validation of negative results.

Summary of the Invention The present invention comprises methods for the automated isothermal amplification and detection of a specific nucleic acid in a test sample to be tested comprising:
a) combining a test sample to be tested with a buffer, a mixture of free nucleotides, specific oligonucleotide primers, and optionally thermostable nucleic acid polymerization enzyme, in a first reaction vessel and placing the reaction vessel in an automated apparatus such that;
b) the automated apparatus heats the first reaction vessel to a temperature, and for a time sufficient to denature, if necessary, the nucleic acid in the sample to be tested;
c) the automated apparatus cools the first reaction vessel to a temperature such that oligonucleotide primers can specifically anneal to the target nucleic acid;
d) the automated apparatus transfers the reaction mixture from the first reaction vessel to a second reaction vessel, and brings the reaction mixture in contact with thermolabile nucleic acid aniplification erzzyme;
e) the automated apparatus maintains the temperature of the second reaction vessel at a temperature which allows primer mediated amplification of the nucleic acid;
f) the automated apparatus contacts the amplified nucleic acid in the second reaction vessel with a capture nucleic acid specific for the nucleic acid am licon to be tested such that they form a specifically-bound nucleic acid-capture probe complex;
g) the automated apparatus optionally washes the specifically captured amplified nucleic acid such that non-specifically bound nucleic acid is washed away from the specifically-bound nucleic acid-capture probe complex;
h) the automated apparatus contacts the specifically-bound nucleic acid-capture probe complex with a labeled nucleic acid probe specific for the amplified nucleic acid such that a complex is fornned between the specifically amplified nucleic acid and the labeled nucleic acid probe;
i) the automated apparatus washes the specifically-bound nucleic acid-capture probe-labeled probe complex such that non-specifically bound labeled probe nucleic acid is washed away from the specifically bound complex;

McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 8 j) the automated apparatus contacts the specifically bound complex with a solution wherein an detection reaction between the labeled nucleic acid probe is effected betu-een the solution and the label attached to the nucleic acid such that a detectable si--nal is aenerated from the sample in proportion the amount of specifically-bound amplified nucleic acid in the sample;
wherein the steps h, i., and j may occur sequentially or simultaneously;
k) the automated apparatus detects the signal and optionally displays a value for the signal, or optionally records a value for the signal, As used herein, the terrn test sample includes samples taken from living patients, from non-living patients, from surfaces, gas, vacuum or liquids, from tissues, bodily fluids, swabs from 1o body surfaces or cavities, and any similar source. The term buffer as used here encompasses suitable formulations of buffer which can support the effective activity of a label, for example an enzyme placed into such buffer when treated at the appropriate temperature for activity and given the proper enzymatic substrate and templates as needed. The term specific oligonucleotide nucleic acid primers means an oligonucleotide having a nucleic acid sequence which is 15 substantially complementary to and will specifically hvbridize/anneal to a target nucleic acid of interest and may optionally contain a promoter sequence recognized by RNA
polymerase. The term reaction vessel means a container i.n which a chemical reaction can be performed and preferably capable of withstanding temperatures of anywhere from about -80 C
to 100 C.

The present invention further provides a method for the detection of the presence or absence of a single stranded or double stranded first nucleic acid in a sample, by automated isothermal amplification of said first nucleic acid in a dual chamber reaction vessel, wherein said dual chamber reaction vessel comprises two reaction chambers, a first and a second, which can be placed in fluid communication with each other, whereby said fluid communication can be controllably interrupted, said method comprising: a) combining in said first reaction chamber: a sample, said sample potentially containing said first nucleic acid, reaction buffer, a mixture of free nucleotides, a first and second specific oligonucleotide primer, and placing said reaction vessel in an automated apparatus such that; b) the automated apparatus heats the first reaction chamber to a sufficient temperature, and for a sufficient time to render any double stranded first nucleic acid in the sample to be tested into sufficient single stranded nucleic acid such that a hybridization product can form, said hybridization product comprising said first nucleic acid and at least one of said first and second oligonucleotide primer; c) the automated apparatus then cools the first reaction chamber to a sufficient temperature such that said hybridization product forms, if said first nucleic acid is present; d) the automated apparatus then transfers the reaction mixture from the first reaction chamber to said second reaction chamber via said controllable fluid communication, such that the reaction mixture is brought into contact with nucleic acid polymerization enzyme; e) the automated apparatus maintains the temperature of the second reaction chamber at a sufficient temperature which allows for the specific oligonucleotide primer mediated amplification of said first nucleic acid, if present; f) the automated apparatus then 9a contacts any amplicon product from said first nucleic acid in the second reaction chamber with a capture nucleic acid specific for said amplicon product from said first nucleic acid such that a specifically-bound nucleic acid-capture probe hybridization complex can form; g) the automated apparatus optionally washes the hybidization complex mixture such that non-specifically bound nucleic acid is washed away from the specifically-bound nucleic acid-capture probe complex; h) the automated apparatus contacts the specifically-bound nucleic acid-capture probe complex with a labeled nucleic acid probe specific for said amplicon product produced from said first nucleic acid such that a specifically-bound nucleic acid-capture probe-labeled probe complex can form; i) the automated apparatus optionally washes the specifically-bound nucleic acid-capture probe-labeled probe complex such that non-specifically bound labeled probe nucleic acid is washed away from the specifically-bound nucleic acid-capture probe-labeled probe complex; j) and the automated apparatus detects the presence or absence of said generated signal and optionally displays a value for the signal, and optionally records a value for the signal, wherein the automated apparatus contacts the specifically-bound nucleic acid-capture probe-labeled probe complex with a solution wherein a detectable signal is generated if said amplicon product and first nucleic acid is present, wherein the signal generated from the sample is proportional to the amount of said first nucleic acid in the sample; wherein each of steps h, i and j can be performed sequentially or concurrently.

The present invention further provides a device for the automated detection of a first target nucleic acid and a second target nucleic acid, said apparatus comprising a solid phase receptacle, wherein said receptacle comprises 9b a pipet-like device having a pipet-like tip and is coated with a first capture nucleic acid which can form a specific hybridization complex with said first nucleic acid, and a second capture nucleic acid which can form a specific hybridization complex with said second nucleic acid.

The present invention further provides a method for the automated detection of the presence or absence of a first target nucleic acid and a second target nucleic acid in a sample, said method comprising: a) contacting said sample with a solid phase receptacle, wherein said receptacle comprises a pipet-like device having a pipet-like tip and is coated with a first capture nucleic acid which can form a specific hybridization complex with said first nucleic acid, and a second capture nucleic acid which can form a specific hybridization complex with said second nucleic acid; b) allowing specific hybridization complex to form if said nucleic acid is present; c) contacting said solid phase receptacle hybridization complex with a first detection nucleic acid, wherein said first detection nucleic acid can form a specific hybridization detection complex with said first nucleic acid, and is conjugated to a means for generating a detectable signal selected from the group consisting of enzyme, chromophore, chemiluminescent compound, radioisotope, and fluorophore; d) allowing specific detection complex to form, then generating said detectable signal; e) detecting said signal if said first nucleic acid is in said sample; f) contacting said solid phase receptacle hybridization complex with a second detection nucleic acid, wherein said second detection nucleic acid can form a specific hybridization detection complex with said second nucleic acid, and is conjugated to a means for generating a detectable signal selected from the group consisting of enzyme, chromophore, chemiluminescent 9c compound, radioisotope, and fluorophore; g) allowing specific detection complex to form, then generating said detectable signal; h) detecting said signal if said second nucleic acid is in said sample; i) and wherein optionally, between steps, said hybridization complex can be washed to remove excess non-specifically bound nucleic acid; j) wherein the absence of a detectable signal correlates with the absence of said nucleic acid in said sample.

The instant invention further provides for the method described above, wherein the reaction buffer is a unified buffer and as such is suitable for denaturation nucleic acids and annealing of nucleic acids, and is further capable of sustaining the enzymatic activity of nucleic acid polymerization and amplification enzyme. Further encompassed by the invention is the method wherein the nucleic acid amplification enzyme is administered in the second reaction chamber as a single assay dose amount in a lyophilized pellet, and the reaction chamber is sealed prior to the amplification step.

The invention teaches an apparatus for the automated detection of more than one nucleic acid target sequences or amplicons comprising a solid phase receptacle (SPR pipet-like device) coated with at least two distinct zones of a capture nucleic acid oligonucleotide.

The invention teaches a method for the automated detection of more than one nucleic acid target sequence comprising contacting a solid phase receptacle (SPR
pipet-like device) coated 9d with at least two distinct capture nucleic acid oligonucleotides in a single or multiple zones to a sample to be tested and detecting a signal(s) from specifically bound probe.
In one embodiment of the invention, the SPR is coated with two distinct zones of capture nucleic acid oligonucleotides which are specific for different nucleic acid sequence targets. In another :5 embodiment of the invention, the SPR is coated with at least one capture probe for a target nucleic acid seqluence, and one capture probe for an amplification control nucleic acid sequence which when detected confirms that amplification did take place.
The present invention also compriises an internal amplification randomly generated positive control nucleic acid including the nucleic acid sequence of RICI and a second internal lo amplification positive control nucleic acid having the nucleic acid sequence of RIC2.
The present invention also comprises internal amplification positive control nucleic acids having the nucleic acid sequence of CRIC-2, GRIC, MRIC, and HRIC.
The present invention further coniprises a method for generating an internal amplification positive control nucleic acid consisting of:
l5 generating random nucleic acid sequences of at least 10 nucleotides in length, screening said random nucleic acid sequence and selecting for specific functionality, combining in tandem a number of such functionally selected nucleic acid sequences, and screening the combined nucleic acid sequence and optionally selecting against formation of intra-strand nucleic acid dimers, or the formation of hairpin structures.

Brief Description of the Drawinas Present:ly preferred embodiments of the invention will be described in conjunction with the appended drawings, wherein like reference numerals refer to like elements in the various views, and in which:
Figure 1 is a graph illustrating single dose reagent pellet temperature stability;
Figure :2 illustrates the general TMA protocol;
Figure 3A is a schematic representation of a disposable dual chamber reaction vessel and the heating steps associated therewith ta perform a TMA reaction in accordance with one possible embodiment of the invention;

McDonnell Boehnen Hulbert & Berghotf 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 10 Figure 3B is a schematic represeritation of altelnative form of the invention in which two separate reaction chambers are combined to form a dual chamber reaction vessel;
Figure 3C is a schematic representation of two alternative embodiments of a dual chamber reaction vessel that are snapped into place in a test strip for processing with a solid phase receptacle and optical equipment in accordance with a preferred embodiment of the invention;
Figure 4 is a schematic representation of an alternative embodiment of a dual chamber reaction vessel formed from two separal:e chambers that are combined in a manner to permit a fluid sample in one chamber to be transferred to the other chamber, with the combined dual chamber vessel placed into a test strip such as illustrated in Figure 3C;
Figure 5 is a perspective view of a stand-alone amplification processing station for the test strips having the dual chamber reaction vessels in accordance with a presently preferred form of the invention;
Figure 6 is a perspective view of one of the amplification modules of Figure 4, as seen from the rear of'the module;
Figure 7 is a perspective view of the front of the module of Figure 5;
Figure S. is another perspective view of the module of Figure 7;
Figure 9 is a detailed perspective view of a portion of the test strip holder and 95 C
Peltier heating subsystems of the module of Figures 6-8;
Figure 1.0 is an isolated perspective view of the test strip holder of Figure 9, showing two test strips installed in the test strip holder;
Figure 1.1 is a detailed perspective view of the test strip holder or tray of Figure 7;
Figure 12 is a block diagram of the electronics of the amplification processing station of Figure 7;
Figure 13 is a diagram of the vacuum subsystem for the amplification processing station of Figure 6; anci Figure 14 is a graph of the thermal cycle of the station of Figure 6.
Figure 15 illustrates a schematic of the operation of the multiplex VIDAS
detection.
Figure 16 illustrates the production of SPR with two distinct capture zones;
Figure 17 illustrates the VIDAS apparatus strip configuration for multiplex detection;
McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 1 1 Figure 18 illustrates and graphs the results of verification of the VIDAS
multiplex protocol detectirig only Neisseria gonorrhoeae (NG) target;

Figure 19A is a graph showing the results when 1x1012 CT targets were mixed with 0, 1 x 109, 1 x 10' , 1 x 10", or 1 x 10' z, NG targets, and detected with the VIDAS instrument using the multiplex protocol and SPRs coated with Chlamydia trachomatis (CT) capture probes on the bottom zone of the SPR, and NG capture probes on the top zone of the SPR.
Figure 19B illustrates the results when 1 x 1012 NG targets was mixed with 0, 1 x 109, 1 x 1010, l x 10", or l x 10'2, NG targets, and. detected with the VIDAS
instrument using the multiplex protocol and SPR coated with CT capture probes on the bottom zone of the SPR, and 1o NG capture probes on the top zone of the SPR.
Figure 2OA is a diagram showing detection of M.tb nucleic acid by VIDAS
apparatus after amplification.
Figure 20B is a diagram showing detection of M.tb nucleic acid by VIDAS
apparatus.
Figure 21 is a diagram showing detection of M.tb nucleic acid by VIDAS
apparatus after amplification.
Figure 22 is a diagram showing detection of M.tb nucleic acid by VIDAS
apparatus after amplification using the binary/dual chamber protocol.
Figure 23 illustrates the results generated by the method described showing a collection of strings of nucleic acid sequences and screening for specific functional parameters.
Figure 24 shows the nucleic acid sequence of Random Internal Control 1(RIC 1) with the possible oligonucleotide primers/probes for amplification and detection of the control sequence.
Figure 25 shows an analysis of the possible secondary structural components of the RIC1 sequence.
Figure 26 shows the nucleic acid sequence of Random Internal Control 2 (RIC2) with the possible oligonucleotide primers/probes for amplification and detection of the control sequence.
Figure 27 shows an analysis of the possible secondary structural components of the RIC2 sequence.
Figure 28 illustrates results from detection of RIC1 DNA, where the ran2l was the capture probe and ran33 was an enzyme-linked detector-probe, and shows that amplification and detection occurs under standard assay conditions.

McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (3 l2)913-0001 12 5062.1-4 Figure 29 shows that RICI RN'A, amplified by T1V1A and the chemically activated siQnal detected on a VIDAS instrument (bioMerieux Vitek, Inc.) using the enzyme-linked detection systern, has a limit of sensitivity of about 1000 molecules of RIC1 RNA
(,Aithout optimization of conditions).

Fi.2ure 30 shows the nucleic acid sequence for internal control oligonucleotides designed for assavs for detecting: Chlamydia trachomatis (CT) identified as CRIC-2; for Neissei-ia conorrhoeae (NG) identified as GRIC; for Mvco%)acteriurn tuberculosis (MT) identified as MRIC; and internal control for HIV identified as HRIC.

1o Description of the Invention The following examples are provided to better illustrate certain embodiments of the present invention without intending to limit the scope of the invention.

Exampie 1 Single Dose Reagents and Unified Buffer The implementation of a TMA reaction (see U.S. Patent 5,437,990) on-line in a VIDAS or off-line in a separate instrument (with detection occurring on a VIDAS instrument) requires modification of the chemistry used to perform the reaction manually.
First, bulk packaged reagents have been modified into single aliquot doses, and second, the buffer components of the reaction have been altered to form a single comprehensive multifunctional unified buffer solution.
Under the current manual technology, the reagents are prepared as lvophilized "cakes" of multiple-assay quantities. The amplification and enzyme reagents thus must be reconstituted in bulk and aliquoted for individual assays.
Thus the automated form of TMA on the VIDAS system improves on the above manual method by utilizing single dose pellets of lyophilized reaction components that can be resuspended in a single unified buffer which will support sample dilution, denaturation of nucleic acids, annealing of nucleic acids, and desired enzymatic activity.

A) Unified Buffer and Sin lge Dose Reagents To test the feasibility of single dose amplification reagents, standard Chlamydia TMA
Amplification artd Enzyme reagents (Gen-Probe Inc.), the bulk reagents were reconstituted in 0.75 ml of water. 12.5 1 of either the water reconstituted amplification or enzyme reagent (i.e. a single dose aliquot) were aliquoted into microcentrifuge tubes. These tubes were placed in a vacuum centrifuge with low heat to remove water. The end result of this procedure was microcentrifuge tube containing a small, dry cake of either enzyme or amplification reagent at the bottom of the tube.

The combined Unified Buffer used in this example, consists of a combination of standard 1o commercially available Gen-Probe Inc. Sample Dilution Buffer (SDB), Amplification Reconstitution Buffer (ARB), and Enzyrrie Dilution Buffer (EDB) in a 2:1:1 ratio. To each dried amplification reagent microfuge tube was added 100 1 of the combined Unified Buffer, and positive control nucleic acid (+), and overlaid with 100 1 of silicone oil.
The tube was then heated to 95 C for 10 minutes and then cooled to 42 C for 5 minutes. The 200 1 total volume 15 was then transferred to a tube containing the dried enzyme reagent. This was then gently mixed to resuspend the el--zyme reagent, and the solution was heated for one hour at 42 C.
Control reactions were prepared using Gen-Probe Control reagents which were reconstituted in the normal 1.5m1 of AR13 or EDB according to instructions provided in the Gen-Probe kit. In each control reaction 25 1 of the reconstituted amplification reagent was combined 2o with 50 1 of the SDB with the positive control nucleic acid (+). The mixture was also heated to 95 C for 10 minutes and then cooled to 42 C for 5 minutes. To this was added 25 l of the reconstituted enzyme reagent and incubated at 42 C for one hour. Negative control had no nucleic acid.
Both the test Unified Buffer (Unified) reactions and the standard Control (Control) 25 reactions were then subjected to the Gen-Probe Inc. standard Hybridization Protection Assay (HPA) protocol. Briefly, 100 1 of a Chlamydia trachomatis specific nucleic acid probe was added to each tube and allowed to hybridize for 15 minutes at 60 C. Then 300 1 of Selection Reagent was acided to each tube and the differential hydrolysis of hybridized and unhybridized probe was allowed to occur for 10 minutes. The tubes were then read in a Gen-Probe Inc. Leader McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 14 50 luminometer and the resultant data recorded as Relative Light Units (RLU) detected from the label, as shown i.n Table 1 below. Data re.ported as RLU, standard C.
Trachomatis TMA/HPA
reaction.

'rABLE 1 Unified single dose aliquot of amplification and enzyme reagents Control (+) Unified (+) Control (-) LJnified (-) 2,264,426 2,245,495 6,734 3,993 2,156,498 2,062,483 3,484 3,765 1,958,742 2,418,531 5,439 5,836 2,451,872 2,286,773 2,346,131 1,834,198 The data. in Table 1 demonstrates that comparable results are obtained when using the single dose aliquots of dried amplification and enzyme reagent. In addition, the data shows that the results were comparable using three separate buffers (ARB, EDB and SDB) and one unified combined buffer (SDB, ARB and EDB combined at a ratio of 2:1:1) to resuspend the reagents and run the reactions.

B) Pellil:ization of Single Dose R-agents In order to simplify the single dose aliquoting of reagents, methods which will allow for pelletization of these reagents in single dose aliquots were used. Briefly, reagent pellets (or beads) can be niade by aliquoting an aqueous solution of the reagent of choice (that has been combined with an appropriate excipient, such as D(+) Trehalose (a-D-Glucopyranosyl-(X-D-glucopyranoside, purchased from Pfanstiehl Laboratories, Inc., Waukegan, IL) into a cryogenic fluid, and then using sublimation to remove the water from the pellet. Once the reagent/trehalose m mixture is aliquoted (drops) into the cryogenic fluid, it forms a spherical frozen pellet. These pellets are then placed in a lyophilizer where the frozen water molecules sublimate during the vacuum cycle. The result of this procedure is small, stable, non-flaking reagent pellets which can be dispensed ir.ito the appropriate packaging. Single dose aliquot pellets of reagents which contained RT, T7 and sugar were subjected to a wide range of temperatures to examine pellet stability. After being subject to a test ternperature for 10 minutes, the pellets were then used for McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Cliicago, Illinois 60606 (312)913-0001 15 CT amplification. The results are graphed in Figure 1. The results show that the single dose reagent pellet remains stable even after to exposure, to high temperatures for 10 minutes.
The extraordinary stability of enzymes dried in trehalose has been previously reported (Colaco et al., 1992, Bio/Technology, 10, 1007) which has renewed interest in research on long-term stabilization of proteins has become a topic of interest (Franks, 1994, Bio/Technology, 12, 253). The resultiing pellets of the amplification reagent and enzyme reagents were tested by use in C. Trachomatis TMA/HPA reactions.

The prepared amplification pellets were placed in a tube to which was added 75 l of a mixture of ARB and SDB (mixed in a 1:2 ratio) with positive control nucleic acid. This sample was then heated to 95 C for 10 minutes and then cooled to 42 C for 5 minutes.
To this was added 25 1 of enzyme reagent, which had been reconstituted using standard Gen-Probe Inc. procedure.
This mixture was allowed to incubate for one hour at 42 C. The reactions were then analyzed by the HPA procedure, as described above. 'The results of this test are reported as RLU in Table 2, and labeled AMP Pellets(+). As above, negative control reactions were run without nucleic acid (-).

The prepared enzyme pellets were tested by heating 100 1 of a combination of SDB with positive control nucleic acid, EDB, and the standard reconstituted amplification reagent (in a 2:1:1 ratio) at 95 C for 10 minutes and then cooled to 42 C for 5 minutes. The total volume of the reaction mix was added to the prepar=ed enzyme pellet. After the pellet was dissolved, the :2o reaction was heated to 42 C for one hour and then subjected to HPA
analysis as above. The results of this test are reported as RLU in Table 2 below, labeled Enzyme Pellet (+). Control reactions were prepared using standard (3en-Probe Inc. reagents following standard procedure.
Data reported as RLU, standard C. Trachomatis TMA/HPA reaction.

TABLE 2 Single dose aliquot of pelleted amplification and enzyme reagents Control (+) Amp Pellets Amp Pellets :Enzyme Enzyme + Pellets + Pellets 2,363,342 2,451,387 2,619 2,240,989 3,418 2,350,028 2.215,235 2,358 3,383,195 1,865 2,168,393 2.,136,645 3,421 2,596,041 2,649 2,412,876 2,375,541 2,247 2,342,288 1,653 McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago. Illinois 60606 (312)913-0001 16 The data in Table 2 demonstrates that there was no significant difference when using the standard Gen-Probe Inc. reagents, or the (iried, prepared, single dose amplification reagent pellet, or the enzyme reagent pellet. Thus the sinlgle dose aliquots of reagents are suitable for use with a :5 single unified buffer for application to automation using a VIDAS system.

Example 2 Automated Isothermal Amplification Using Thermolabile Enzymes In order to automate the isothermal amplification assay reaction for use with clinical assay 1o apparatus, such as a VIDAS instrument (bioMerieux Vitek, Inc.), a novel dual-chamber reaction vessel has been designed to implement thie use of the unified buffer and single reaction aliquot reagent pellets clescribed above in isothelmal amplification assay of test samples which can be further used in combination with a stand alone processing station.

15 A) Dual reaction chambers The use of two chambers will facilitate keeping separate the heat stable sample/amplification reagent (containing the specific primers and nucleotides) from the heat labile enzymatic components (i.e. RNA 1-everse transcriptase, RNA polymerase RNase H).
Figure 3A is a schematic represe;ntation of a disposable dual chamber reaction vessel 10 ?0 and the heating steps associated therewith to perform a TMA reaction in accordance with one possible embodiment of the invention, Chamber A contains the amplification mix, namely nucleotides, primers, MgC12 and other salts and buffer components. Chamber B
contains the amplification enzyme that catalyzes the amplification reaction, e.g., T7 and/or RT. After addition of the targets (or patient sample) into chamber A, heat is applied to chamber A to denature the 25 DNA nucleic acid targets and/or remove RNA secondary structure. The temperature of chamber A is then cooled down to allow primer annealing. Subsequently, the solution of chamber A is brought into contact with chamber B. Chambers A and B, now in fluid communication with each other, are then maintained at the optimum temperature for the amplification reaction, e.g., 42 degrees C. By spatially separating chamber A from chamber B, and applying the heat for McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 17 denaturation to chamber A only, the thermolabile enzymes in chamber B are protected from inactivation during the denaturation step.

Figure 313 is a schematic represer.itation of an alternative form of the invention in which two separate reaction chambers 12 and 14 are combined to form a dual chamber reaction vessel 10. Like the ernbodiment of Figure 3A, Chamber A is pre-loaded during a manufacturing step with an amplification mix, namely nucleotides, primers, MgC12 and other salts and buffer components. Chamber B is pre-loaded during manufacturing with the amplification enzyme that catalyzes the arriplification reaction, e.g... T7 and/or RT. Fluid sample is then introduced into chamber A. The targets are heated for denaturation to 95 C in chamber A. After cooling lo chamber A to 42 C, the solution in chamber A is brought into contact with the enzymes in chamber B to trigger the isothermal amplification reaction.
If the reaction vessel is designed such that, after having brought the contents of chambers A and B into contact, the amplification chamber does not allow any exchange of materials with the environmerit, a closed system arnplification is realized that minimizes the risk of 1:5 contaminating the amplification reaction with heterologous targets or amplification products from previous reactions.
Figure 3C is a schematic representation of two alternative dual chamber reaction vessels and 10' thalt are snapped into place; in a test strip 19 for processing with a solid phase receptacle and optical equipment in acco;rdance with a preferred embodiment of the invention. In the embodiments of Figure 3, a unidirectional flow system is provided. The sample is first introduced into chamber A for heating to the denaturation temperature. Chamber A contains the dried amplification reagent mix 16. After cooling, the fluid is transferred to chamber B containing the dried enzynie 18 in the form of a pellet. Chamber B is maintained at 42 C
after the fluid sample is introduced into Chamber B. The amplification reaction takes place in Chamber B at the optimum reaction temperature (e.g., 42 C). After the reaction is completed, the test strip 19 is then processed in a machine such as the VIDAS instrument available from bioM6rieux Vitek, Inc., the assignee of the present invention. Persons of skill in the art are familiar with the VIDAS
instrument.
The steps of heating and cooling; of chamber A could be performed prior to the insertion _30 of the dual chamber disposable reaction. vessel 10 or 10 ' into the test strip 19, or, alternatively, McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 18 suitable heating elements could be placed adjacent to the left hand end 24 of the test strip 19 in order to provide the proper temperature control of the reaction chamber A. The stand alone amplification processing station of Figures 4-14, described below, incorporates suitable heating elements and coritrol systems to provide the proper temperature control for the reaction vessel 10.
Figure 4 is a schematic representation of an alternative embodiment of a dual chamber reaction vessel 10 " formed from two separate interlocking vessels 10A and lOB
that are combined in a manner to permit a fluid sample in one chamber to flow to the other, with the combined dual chamber vessel 10 " placed into a test strip 19 such as described above in Figure 3C. The fluid sample is introduced irito chamber A, which contains the dried amplification reagent mix 16. Vessel A is then heated off-line to 95 degrees C, then cooled to 42 degrees C.
The two vessels A and B are brought together by means of a conventional snap fit between complementary locking surfaces on the tube projection 26 on chamber B and the recessed conduit 28 on chamber A. The mixing of the sample solution from chamber A with the enzyme from chamber B occurs since the two chambers are in fluid communication with each other, as indicated by the arrow 30. The sample can then be amplified in the combined dual chamber disposable reaction vessel 10 " off-line, or on-line by snapping the combined disposable vessel 10 " into a modified VIDAS strip. The VIDAS instrument could perform the detection of the amplification reaction in known fashion.

B) Amn.lification Station Figure 5 is a perspective view of a stand-alone amplification processing system 200 for the test strips 19 having the dual chamber reaction vessels in accordance with a presently preferred form of the invention. The system 200 consists of two identical amplification stations 202 and 204, a power supply module 206, a control circuitry module 208, a vacuum tank 210 and connectors 212 for the power supply module 206. The tank 210 has hoses 320 and 324 for providing vacuum to amplification stations 202 and 204 and ultimately to a plurality of vacuum probes (one per strip) in the manner described above for facilitating transfer of fluid from the first chamber to the second chamber. The vacuum subsystem is described below in conjunction with Figure 14.

McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 19 The amplification stations 202 and 204 each have a tray for receiving at least one of the strips and associated temperature control, vacuum and valve activation subsystems for heating the reaction wells of the strip to the proper temperatures, transferring fluid from the first chamber i.n the dual chamber reaction wells to the second chamber, and activating a valve, such as a thimble valve or preferably a ball valve, to open the fluid channel to allow the fluid to flow between the two chambers.

The stations 202 and 204 are designed as stand alone - amplification stations for performing the amplification reaction in an automated manner after the patient or clinical sample has been added to the first chamber of t:he dual chamber reaction vessel described above. The processing of the strips after the reaction is completed with an SPR takes place in a separate machine, such as the VIDAS instrument. Specifically, after the strips have been placed in the stations 202 anct 204 and the reaction rull in the stations, the strips are removed from the stations 202 and 204 and placed into a VIDAS instrument for subsequent processing and analysis in known fashion.
The entire system 200 is under microprocessor control by an amplification system interface board (not shown in Figure 5). The control system is shown in block diagram form in Figure 12 and will be described later.
Referrin.g now to Figure 6, one of the amplification stations 202 is shown in a perspective view. The other amplification station is of identical design and construction.
Figure 7 is a perspective view of the front of the module of Figure 6.
n Referrir.Lg to these figures, the station includes a vacuum probe slide motor 222 and vacuum probes slide cam wheel 246 that operate to slide a set of vacuum probes 244 (shown in Figure 7) for the thimble valves up and down relative to a vacuum probes slide 246 to open the thimble valves and apply vacuum so as to draw the fluid from the first chamber of the reaction vessel 10 to thie second chamber. The vacuum probes 244 reciprocate within annular recesses provided in the vacuum probes slide 246. Obviously, proper registry of the pin structure and vacuum probe 244 with corresponding structure in the test strip as installed on the tray needs to be observed.

McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 20 The station includes side walls 228 and 230 that provide a frame for the station 202.
Tray controller board 229 is mounted between the side walls 228 and 230. The electronics module for the station 202 is installed ol;l the tray controller board 229.
A set of tray thermal insulation covers 220 are part of a thermal subsystem and are provided to envelop a tray 240 (Figure 7) that receives one or more of the test strips. The insulation covers 220 help maintain the temperature of the tray 240 at the proper temperatures.
The thermal subsystem also includes a 42 C Peltier heat sink 242, a portion of which is positioned adjacent to the second chamber in the dual chamber reaction vessel in the test strip to maintain that chamber at the proper temperature for the enzymatic amplification reaction. A

zo 95 C heat sink 250 is provided for the front of the tray 240 for maintaining the first chamber of the reaction well in the test strip at the denaturation temperature.

Figure 8 is another perspective view of the module of Figure 7, showing the 95 C heat sink 250 and a set of fins 252. Note that the 95 C heat sink 250 is positioned to the front of and slightly below the tray 240. The 42 C lieat sink 242 is positioned behind the heat sink 250.

Figure 9 is a detailed perspective view of a portion of the tray 240 that holds the test strips (not shown) as seen from above. The tray 240 includes a front portion having a base 254, a plurality of discontinuous raised paralllel ridge structures 256 with recessed slots 258 for receiving the test strips. The base of the: front 254 of the tray 240 is in contact with the 95 C heat sink 250. The side walls of the parallel raised ridges 256 at positions 256A
and 256B are placed as close as possible to the first and second chambers of the reaction vessel 10 of Figure 3A so as to reduce thermal resistance. The base of the rear of the tray 240 is in contact with a 42 C Peltier heat sink, as best seen in Figure 8. The portion 256B of the raised ridge for the rear of the tray is physically isolated from portion 256A for the front of the tray, and portion 256B is in contact with the 42 C heat sink so as to keep the second chamber of the reaction vessel in the test strip at the proper temperature.
Still referring to Figure 9, the vacuum probes 244 include a rubber gasket 260. When the vacuum probes 244 are lowered by the vacuum probe motor 222 (Figure 6) the gaskets 260 are positioned on the upper surface of the test strip surrounding the vacuum port in the dual chamber reaction vessel so as to make a tight seal and permit vacuum to be drawn on the second chamber.
McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 606(6 (312)913-0001 21 Figure 10 is an isolated perspective view of the test strip holder or tray 240 of Figure 9, showing two test strips installed in the tray 240. The tray 240 has a plurality of lanes or slots 241 receiving up to six test strips 19 for silnultaneous processing. Figure 10 shows the heat sinks :242 and 250 for maintaining the respective portions of the tray 240 and ridges 256 at the proper temperature.
Figure 11 is a detailed perspective view of the test strip holder or tray 240 as seen from below. The 95 C Peltier heat sink whiclh would be below front portion 254 has been removed in order to better illustrate the rear heat sink: 242 beneath the rear portion of the tray 240.
Figure 12 is a block diagram of the electronics and control system of the amplification 1o processing system of Figure 5. The control system is divided into two boards 310 and 311, section A 310 at the top of the diagram devoted to amplification module or station 202 and the other board 311 (section B) devoted to lthe other module 204. The two boards 310 and 311 are identical and only the top section 310 will be discussed. The two boards 310 and 311 are connected to an amplification station interface board 300.
The interface board 300 communicates with a stand alone personal computer 304 via a high speed data bus 302. The personal computer 304 is a conventional IBM
compatible computer with hard disk drive, video monitor, etc. In a preferred embodiment, the stations 202 and 204 are uncler control by the interface board 300.
The board 310 for station 202 controls the front tray 240 which is maintained at a 2o temperature of 95 C by two Peltier heat sink modules, a pair of fans and a temperature sensor incorporated into the front portion 254 of the tray 240. The back of the tray is maintained at a temperature of 42 C by two Peltier madules and a temperature sensor. The movement of the vacuum probes. 244 is controlled by the probes motor 222. Position sensors are provided to provide input signals to the tray controller board as to the position of the vacuum probes 244.
The tray controller board 310 includes a set of drivers 312 for the active and passive components of the system vvhich receive data from t:he temperature and position sensors and issue commands to the active components, i.e., motors, fans, Peltier modules, etc. The drivers are responsive to commands from the amplification interface board 300. The interface board also issues commands to the vacuum pump for the vacuum subsystem, as shown.

McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 22 Figure 13 is a diagram of the vacuum subsystem 320 for the amplification processing stations 202 and 204 of Figure 5. The subsystem includes a 1 liter plastic vacuum tank 210 which is connected via an inlet line 322 to a vacuum pump 323 for generating a vacuum in the tank 210. A vacuum supply line 324 is provided for providing vacuum to a pair of pinch solenoid valves 224 (see Figure 6) via supply lines 324A and 324B. These vacuum supply lines 324A and 324B supply vacuum to a manifold 22ti distributing the vacuum to the vacuum probes 244.
Note the pointed tips 245 of the vacuum probes 244 for piercing the film or membrane 64 covering the strip 19. The vacuum system 320 also includes a differential pressure transducer 321 for monitoring the presence of vacuum in the tank 210. The transducer 321 supplies pressure signals to the interface board 300 of Figw=e 12.
Figure 14 is a representative graph of the thermal cycle profile of the station of Figure 5.
As indicated in line 400, after an initial ramp up 402 in the temperature lasting less than a ininute, a first temperature T1 is reached (e.g., a denaturation temperature) which is maintained for a predetermined time period, such as 5-10 minutes, at which time a reaction occurs in the first chamber of the reaction vessel. Thereafter, a ramp down of temperature as indicated at 404 occurs and the temperature of the reaction solution in the first chamber of the reaction vessel 10 cools to temperature T2. After a designated amount of time after cooling to temperature T2, a fluid transfer occurs in which the solutioii in the first chamber is conveyed to the second chamber.
Temperature T2; is maintained for an appropriate amount of time for the reaction of interest, such 2o as one hour. At time 406, the temperature is raised rapidly to a temperature T3 of 65 C to stop the amplification reaction. For a TMA reaction, it is important that the ramp up time from time 406 to time 408 is brief, that is, less than 2 minutes and preferably less than one minute.
Preferably, all the ramp up and ramp down of temperatures occur in less than a minute.
Other elnbodiments of reaction vessels and amplification station components are also envisioned, and. such alternative embodiinents are encompassed in the present disclosure.
Example 3 Automated VIDAS Test for Non-amplified and Amplified Detection of Mycobacterium tuberculosis (M.tb) Using the VIDAS instrument (bioMerieux Vitek, Inc.), modified to 42 C, we have developed an iii-line simple rapid nucleic acid amplification and detection assay for the clinical McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 23 laboratory for the detection of M.tb in test samples which can be completed in a short time. The entire assay is designed to take place on a single test strip, minimizing the potential for target or amplicon containination. The amplification based assay is capable of detection of M.tb where the sample contains only 5 cells similar to the sensitivity achieved by the Gen-Probe commercial kit.
The amplification based assay utilizes isothermal transcription-mediated amplification (TMA) targeting unique sequences of rRNA, followed by hybridization and enzyme-linked fluorescent detection of nucleic acid probe (amplicon) in the VIDAS
instrument.
The amplification/detection assay can detect approximately lfg of M.tb rRNA, or less than one M.tb organism per test, and is s-pecific for all members of the M.tb complex. Specific w probes for the detection of M.tb can be found in C. Mabilat, 1994, J. Clin.
Microbiol. 32, 2707.
Standard smears for acid-fast bacilli are not always reliable as a diagnostic tool, and even when positive r.nay be a mycobateria other than M.tb. Currently, standard methods for diagnosis of tuberculosis requires culturing the slow-growing bacteria, and may take up to 6 weeks or longer. During this time, the patient is usually isolated. Initial results are that this automated test matches or exceeds the clinical sensitivity of the culture method, and offers a highly sensitive method to rapiclly (in less than three hours) detect M.tb in infected samples, thereby aiding rapid diagnosis, isolation and treatment.

A) Sample Preparation A 450 1 volume of specimen is added to 50 l of specimen dilution buffer in a lysing tube containing glass beads, sonicated for 15 minutes at room temperature to lyse organisms, heat inactivated for 15 minutes at 95 C. Where required, isothermal amplification was conducted as per a commercially available manual assay kit (Gen-Probe Inc.) following the kit instructions using standard kit reagents. However, similar assays can be conducted using the modified components as described in the Examples above.
B) Dete=ction In order for the automated detection assay to operate, the detection system requires hybridization of the target nucleic acid or amplicon to a specific capture nucleic acid bound to a solid support, (in the VIDAS system called a "solid phase receptacle" SPR
pipet-like devise), and McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, [llinois 60606 (312)913-0001 24 to a labeled detection probe nucleic acid i(for example where the label can be alkaline phosphatase, a chemiluminescent signal compound, or other reagent that will allow for specific detection of botmd probe).
In an automated system such as the VIDAS, after several wash steps to remove unbound probe, the SPR transfers the probe-target hybrid to an enzyme substrate, whereby the detectable signal is triggered from the bound probe and detected by the assay instrument.
In one embodiment, the detection probe is conjugated to alkaline phosphatase, and once placed in contact with substrate of methyl umbelliferyl phosphate (MUMP), the substrate is converted into 4-methyl umbel.liferone (4-MU) by the alkaline phosphatase. The 4-MU produces fluorescence which is measured and recorded by the standard VIDAS instrument as relative fluorescence units (RFU). When target nucleic acid is not present, no detection probe is bound, and no substrate is converted, thus no fluorescence is detected.

C) AnalZical sensitivity: Controls 1,5 Generally controls are prepared in a matrix of specimen dilution buffer with positive controls containing 5fg of M.tb rRNA, or the equivalent rRNA of approximately 1 M.tb cell.
Sensitivity of the automated probe assay can be determined by testing dilutions of lysed M. tb cells. The cell lysates can generally be plrepared with a 1 l loop of cells (the assumption being that there are approximately 1x109 colony forming units (CFU) per l l loop-full, based upon n previous titration and CFU experiments). Dilutions of the M.tb lysates can then be tested with the automated probe assay.

Figure 20A is a graph showing detection of M.tb amplicons according to the Gen-Probe kit. Figure 20B is a graph showing detection of M.tb amplicons from the same reactions as in 25 Figure 20A by the VIDAS instrument.
Figure 21 is a graph showing amplification and detection of M.tb nucleic acids on the modified VIDAS apparatus. Enzyme was used in liquid form and amplification was performed in-line with VIDAS assay instrument.
Figure 22 is a graph showing amplification and detection of M.tb nucleic acids on the 30 modified VIDAS apparatus using the bi:riary/dual chamber disposable reaction vessel. The McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 25 denaturation step was performed off-line of the VIDAS instrument, amplification and amplicon detection was performed in-line with VIDAS instrument.

Example 4 Automated VIDAS Test for Amplified Detection of Chlamydia trachomatis (CT) Using the VIDAS instrument (bioMerieux Vitek, Inc.), we have developed a simple, fully automated, highly specific assay for the rapid detection of Chlamydia trachomatis (CT) from test samples. The test utilizes isothermal TMA targeting unique sequences of the rRNA followed by hybridization and enzyme-linked fluorescence detection. The automated test specifically detects lo all the clinically important serovars of Chlamydia trachomatis (CT) from urogenital specimens in less than two hours. We obtained an analytical sensitivity of 0.5fg of rRNA, or the equivalent of approximately 1/10' of an elementary body of Chlamydia trachomatis (CT).
Agreement between the automated test and Gen-Probe's Amplified CT test for two-hundred seven (207) clinical endocervical svvabs and urines showed complete agreement.
Chlamydia trachomatis (CT) infection is the leading cause of sexually transmitted disease in the United Sitates and Europe. It is currently estimated that about four million new CT infection occur each yeai= in the United States.
Chlamydia trachomatis (CT) is a. small obligate intracellular parasite that causes infections in both females and males, adults and newborns. The greatest challenge to the control :20 of CT infection is that as many as 75% of infected women and 50% of infected men are asymptomatic. This results in a large reservoir of unrecognized infected individuals who can transmit the CT infection. The rapid and simple detection of CT infection would greatly assist identification infected individuals.

A) Patient Specimens and sample preparation Coded samples (n=207) were obtained from patients with symptoms consistent with CT
infection. The cervical samples were collected with a Gen-Probe sample collection kit containing Gen-Probe transport medium; the urine samples were collected into standard urine collection devices. All samples were stored at 4 C.

McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 26 Cervical. swabs were centrifuged at 425xg for 5 minutes to bring all liquid to the bottom of the tube. The swabs were then treated with 40 1 Gen-Probe Specimen Preparation Reagent and incubated at 60"C for 10 minutes. 20 l of the treated sample was then pipetted into 400 l of sample dilution buffer (SDB).

Two ml of each urine sample was warmed to 37 C for 10 minutes and microfuged at 12,000xg for 5 minutes. The supernatant was discarded and 300 1 of sample dilution buffer was added to each specimen. All 15 serovars of CT were used for inclusive samples, specimens were quantified and 20 1 of specimens containing 4x 1 02 IFU/ml (inclusion forming unit per ml) of each serovar was added to 400 1 of SDB. A panel of exclusive urogenital micororganisms was to obtained and quantified and 20 1 of 2x109/ml microorganisms were pipeted into 400 l of SDB.
Positive contro:l containing 0.5fg rRNA or the equivalent of 0.1 CT elementary body was diluted in SDB.

B) Sample am_plification and VIDAS detection Samples were amplified using the TMA protocol, and rRNA targets were hybridized to oligomer conjugated to AMVE copolymer and an oligomer conjugated to alkaline phosphatase.
See for example U.S. Patent 5,489,653 and 5,510,084. As described above, the solid phase receptacle (SPR pipet-like devise) carries the bound hybrids through successive wash steps and finally into the substrate 4-MUP. The alkaline phosphatase converts the substrate to fluorescent 4-MU, which is detected by the VIDAS assay machine and recorded as relative fluorescence units.
Table 2B below illustrates detection of CT by VIDAS automated assay following amplification as RFV (RFV = RFU - Background RFU) against concentration of CT
rRNA.
Dilutions of C. trachomatis purified rRl`1A from 0 to 200 molecules were amplified (n=3) and detected in the VIDAS automated probe assay. Detection limit is 20 molecules of purified rRNA.
McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 27 TABLE 2B: CT Detection by VIDAS

rRNA Input Molecules VIDAS RFV

C) Analytical specificity and Results Amplifications and detection were carried out in the presence of each of the following ATCC organisms with detections reported as RFV in Table 3 below.

TABLE 3 F-xclusivity panel for CT

Bacillus subtilis Branhamella Candida albicans Chlamydia Chlamydia 33 catarrhalis 26 pneumoniae psittaci Escherichia coli Klebsiella Lactobacillus Neisseria Neisseria 11 pneumoniae acidophilus elongata lactamica Neisseria Neisseria Propionibacterium Pseudomonas Staphylococcus meningitidis-D meningitidis-Y acnes aeruginosa aureus Streptococcus Streptococcus Streptococcus Yersinia Chlamydia agalactiae bovis pneunzoniae enterolitica trachomatis Negative Control 10 Analytical specificity for Chlamydia serovars data reported as RFV is shown in Table 4 below.

TABLE 4 Inclusivity Panel for CT

Serovar A Serovar B Serovar Ba Serovar C Serovar D

Serovar E Serovar F Serovar G Serovar H Serovar I
4608 9916 1008.2 7769 9733 Serovar J Serovar K Serovar L1 Serovar L2 Serovar L3 Positive Control Negative Control McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 28 Table 5 below illustrates the results of clinical cervical swab specimen testing for CT
comparing resullts from the Gen-Probe manual AMP-CT assay and the VIDAS
automated probe assay.

TABLE 5 Amplified Clinical Cervical Specimen Detection of CT
Gen-Probe rrianual AMP-CT assay VIDAS off-line + -automated probe + 35 0 assay - 0 85 Table 6 below illustrates the results of clinical urine specimen testing comparing the results of manual AMP-CT assay and the VIDAS automated probe assay.

TABLE 6 Amplified Clinical Urine Specimen detection of CT
Gen-Probe nianual AMP-CT assay VIDAS off-line + -automated probe + 25 0 assay - 0 62 Thus there was perfect agreement in assay results between the automated probe assay using the VID?.S instrument and the manual Gen-Probe AMP-CT assay.

:20 Example 5 Multiplex (Multiple Sequence) Nucleic Acid Detection The value of diagnostic tests based on nucleic acid probes can be substantially increased through the detection of multiple different nucleic acid molecules, and the use of internal positive controls. An automated method has beeri devised for use with the VIDAS
instrument (bioMerieux Vitek, Inc.) which can discretely detect at least two different nucleic acid sequences in one assay McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 29 reaction, and is termed the Multiplex protocol. Thus a nucleic acid amplification procedure, or a processed test sarnple may be screened for more than one amplified nucleic acid molecule in the same assay. This method relies on the spatial separation of discrete nucleic acid probes which can specifically capture different target nucleic acid sequences (amplicons), on the SPR pipet-like devise of the VIDAS instrument. The SPR is a disposable pipet-like tip which enables fluid movements as well as acting as the solid support for affinity capture. The multiplex capture by SPR is demonst:rated using capture probes specific for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG).
Figure 15 illustrates a schematic of the operation of the multiplex VIDAS
detection. The i.o SPR tips are coated in two distinct zones with oligonucleotide nucleic acid sequences which are used to specifically capture complementary nucleic acid sequences (amplicons) with their corresponding specific reporter probe or detector probe nucleic acids labeled with alkaline phosphatase (AKP). Following washes to remove unbound reporter probes, AKP
localized to the SPR bottom is detected with the fluorescent substrate 4-MUP. The AKP is stripped from the bottom of the SPR with NaOH or other reagents which promote denaturation of nucleic acid hybrids or inaciitvates AKP activity. The enzyme reaction well is emptied, washed, and re-filled with fresh 4-MUP. To confirm removal of AKP from the bottom of the SPR, the new substrate is exposed to the bottom of the SPR and arly residual fluorescence is measured.
Finally, AKP-reporter probe bound to the top of the SPR is detected by immersing the SPR in the 4-MUP, and :20 representing the presence of the second amplicons.
Figure 16 illustrates the production of SPR with two distinct capture zones.
The SPR is inserted tip-first into a silicon plug, which are held in a rack. Differential pressure is used to uniformly draw a solution of a specific capture probe at about 1 g/ml, conjugated to AMVE
copolymer, into all SPRs at one time. The amount of fluid drawn into each SPR, and thus the size of the zone, is controlled by regulating the amount of pressure in the system.
Attachment of the conjugate to the SPR surface is achieveci by passive adsorption for several hours at room temperature. After washing, and drying, the SPRs are capped with a small adhesive disc and inserted into new racks in a tip-down orientation. The lower portion of the SPR is then similarly coated with a second capture probe conjugate. SPRs are stable when stored dry at 4 C.

McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 30 Figure 17 illustrates a preferred embodiment of the VIDAS apparatus strip configuration for Multiplex detection. The strip can be pre-filled with 200 l of AKP-probe mix (about 1x101z molecules of each probe) in hybridization buffer in well X1, 600 1 of wash buffer in wells X3, X4, X5, 600 1 of stripping reagent in wells X6 and X7, and 400 1 of AKP
substrate in X8 and sealed with foil, A foil-sealed optical cuvette (XA) containing 300 1 of 4-MUP
is snapped into the strip, and the strips are inserted into the VIDAS instrument at 37 C. The Multiplex VIDAS
protocol is then executed using SPRs coated with two capture probes in distinct zones.
The VII)AS Multiplex protocol can involve many steps. For example the validation test protocol contained thirteen (13) basic steps as follows:

1o l. Transfer of 203 1 target from X0 to AKP-probes in X1, 2. Hybridize and capture to the entire SPR, 3. Wash SIPR (316 l) twice with PBS/Tween (X3, X4), 4. 4-MUP to SPR bottom (89.6 l) in XA for 5.3 minutes then read signal, 5. 4-MUP to SPR bottom (89.641) in XA for 14.8 minutes then read signal (optional), 1.5 6. Transfer used substrate from XA to X2 (5 x 67.1 l), 7. Strip AKP from SPR bottom (112.6 1) with NaOH (X7), 8. Wash XA with fresh NaOH (3 x 112.6 1; X6 to XA to X6), 9. Wash XA with PBS/Tween (3 x 112.6 l; X5 to XA to X5), 10. Transfer fresh 4-MUP from X8 to XA (6 x 48 1), 20 11. 4-MUP to SPR bottom (89.6 l) in XA for 10.7 minutes then read signal, 12. 4-MUP to SPR top (294 l) in XA for 5.5 minutes then read signal, 13. 4-MUP to SPR top (294 1) in XA for 15 minutes then read signal (optional).
Hybridization, substrate, wash and stripping steps can all involve multiple cycles of 25 pipeting the respective solution into the SPR, holding the solution for a defined period of time, and pipeting the solution out of the SPR,. Hold times for hybridization, substrate and washing or stripping are 3.0, 0.5 and 0.17 minutes respectively. The fluorescence signal is detected by the apparatus. Total assay time for the research protocol was about 1.75 hours but can be reduced to about 75 minutes.

McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 3 1 F'igure 18 illustrates and graphs the results of verification of the VIDAS
Multiplex protocol executed as described above, except the SPR was homogeneously coated with only a single capture probe for Neisseria gonorr=hoeae (NG). The number of NG
oligonucleotide targets in the test sample was varied from 0, 1x1010, or 1x10" molecules in the test sample. The data shown are averalges of replicate samples. The graph as illustrated is divided into two parts; the left and right halves show the results of two iluorescent measurements from the lower and the upper zones of the SPR, respectively. The measurements taken from the bottom zone after stripping the lower area of bound nucleic acid, and exposure for about 11 minutes in fresh 4-MUP substrate was approximately 46 RFU for all samples tested, and was equivalent to background fluorescence measured. This measurement is shown by the 0 time point in the center of the graph. Thus the graph illustrates two sequential sets of measurements of fluorescence from a single SPR, the first set of ineasurements being taken from the bottom half of the SPR (left half of the graph), and a second set of measurements taken from the top of the SPR (the right of the graph). This experiment validates that the multiplex protocol and zone coated SPR prcedure yield essentially 1.5 idnetical results. As indicated by the fluoresecense intensities in the left and right hand parts of the graph, from the lower and upper portions of the SPR.
Figure 19 illustrates Multiplex detection of CT and NG oligonucleotide targets at different input amounts. Figure 19A is a graph showing the results when 1x1012 CT
targets were mixed with 0, 1 x 109, 1 x 10' , 1 x 10", or 1 x 10' z, "NG targets, and detected with the VIDAS instrument using the Multiplex protocol and SPRs coated with CT capture probes on the bottom zone of the m SPR, and NG capture probes on the top zone of the SPR. Figure 19B illustrates the results when 1 x 10" 2 NG targets was mixed with 0, 1 x 109, 1 x 10' , 1 x 10", or 1 x 10' 2, CT targets, and detected with the VIDAS instrument using the Multiplex protocol and SPRs coated with CT
capture probes on the bottom zone of the SPR, and NG capture probes on the top zone of the SPR. The 25 data is graphed as above where the graph illustrates two sequential sets of measurements of fluorescence from a single SPR, the first. set of measurements being taken from the bottom half of the SPR (left half of the graph), Strippect and verified (the center of the graph) and a second set of measurements taken from the top of the SPR (the right of the graph) with verification of stripping of the SPR in t'he center of the graph. Irr.lportantly, this experiment shows that the two zones of 30 the SPR act independently in the multiplex protocol, since high fluorescence signals from one McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 32 zone do not interfere with signals produced for the second zone. This is regardless of whether these latter signals are high (lxl0`2), or low (1x10), or negative.
Table 7 below summarizes the data obtained by Multiplex VIDAS detection of CT
and NG in a sample at various target levels, reported in RFUs.

TABLE 7 Detection of CT and NG targets in saunple RFUSA noneB 1x109 1x1010 1x10" 1x101lx10"
nonec 43 /40E 43/116 46/693 62/7116 174/11817 273/12136 1x109 189/41 246/118 169/773 220/5750 422/12522 399/11401 1x1010 1736/41 2258/125 1937/734 1931/6639 2128/12390 2371/11180 1x10" 10339/48 9815/145 9858/760 9369/4571 9784/11825 10252/10312 1x10'2 12149/49 13520/148 12940/796 13593/4397 11239/11786 10158/9900 1x10" 11545/57 11713/121 10804/815 12805/5404 12305/12326 11416/10490 ^ Data is reported in RFUs, after -5 minute exposure of 4-MUP to bound AKP-probe B Columns are data for that number of NG targets in sample c Rows are the data for that number of CT targets in sample The first value reported is RFU detected from the CT assay portion E The second value reported is RFU detected fro;m the NG assay portion Thus the Multiplex VIDAS protocol is clearly operative and enables the rapid and discrete detection of more than one different nucleic acid in a sample. This protocol, and the SPR coating can be manipulated in many formats to present coating zones of different surface area with different sized gaps between two or more detection zones. The SPR can be coated with nucleic acids which are designed to capture different regions of the same nucleic acid sequence to detect, for example, tn.incated gene expression, different alleles or alternatively spliced genes. The SPR
can be coated to capture amplicons from internal control nucleic acid molecules which can be used to detect and confirm successful nucleic acid amplification reactions.
Thus the VIDAS
Multiplex protocol is a flexible method for detection of more than one nucleic acid sequence in the same saunplie, in a single assay, with or without amplification.

EXAMPLE 6 Internal Control Sequence and Method McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 33 The construction of internal control sequences composed of functional building blocks of sequences chosen by random generation of nucleic acid sequences for use as amplification reaction internal positive controls ideally requires that the control sequences be specifically designed to be used for the various nucleic acid amplification protocols including but not limited to PCR, LCR, TMA, NASBA, and SDA. The internal control nucleic acid sequence, in combination with the appropriate sequence specific oligonucleotide primers or promoter-primers will generate a positive amplification signal if the amplification reaction was successfully completed.
Ideally, the internal control nucleic acid is useful regardless of the nucleic acid sequences present in the target organism, the host organism, or nucleic acids present in the normal flora or in the environmer.it. Generally, the internal control sequences should not be substantially similar to any nucleic acid sequences present in a clinical setting, including human, pathogenic organisms, normal flora organisms, or environmental organisms which could interfere with the amplification and detection of the internal control sequences.
The internal control sequences of the instant invention are comprised of functional blocks of sequences cliosen from a list of randomly generated nucleic acid sequences.
The functional blocks are segments which provide for a. special property needed to allow for amplification, capture, and detection of the amplification product. For example, in a TMA
reaction, the internal control sequences are most useful when the functional blocks meet certain functional requirements of the amplification protocol, such as: a) a primer binding site on the anti-sense strand; b) a capture site; c) a detector probe binding site; d) a T7-promoter containing primer binding site on the sense strand. Each oi.'these functional elements has its own particular constraints, such as length, %G-C content, Tm, lack of homology to known sequences, and absence of secondary structural features (i.e. free from dimer fonmation or hairpin structures) which can be used to select the appropriate sequence. Thus randomly generated functional blocks of sequences can be screened for the desired functional properties before use in constructing internal control sequences.
In order to construct internal coiitrol sequences having the desired properties comprising a specified number of functional blocks and satisfying the desired constraints within each block, a random sequence generator was used to generate strings of numbers; each number being limited McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 34 to the range from 0.000 to 4.000. The lerigth of the strings is flexible and chosen based upon the desired lengths of the functional blocks.
Each number in the string (i.e. nl, n2, n3, n4 ... nx where x is the length of the string) was then assigned a corresponding nucleotide as follows: guanosine (G) if 0< n<_ 1; adenosine (A) if 1< n_ 2; thymidine (T) if 2< n<_ 3; anct cytosine (C) if 3< n_ 4. A large collection of such strings was produced and screened for those meeting the sequence and structural requirements of each functional block. Figure 23 illustrates the results generated by the method described showing a collection of strings of nucleir, acid sequences and screening for specific functional parameters. The internal control sequence can include DNA, RNA, modified oligonucleotides, or io any combination of nucleic acids, such that the illustrated sequences using DNA nomenclature can be readily adapted as desired to the appropriate nucleic acid.
Potential internal control (IC) sequences were then constructed by assembling the functional blocks (selected at random) in the proper order. Finally, the assembled internal control sequences were; then examined to insure that overall sequence and structural constraints were maintained. For example, in a TMA reaction, the internal control sequence should not have significant base-pairing potential betwee:n the two primer binding sites or form stable 3' dimer structures. Those internal control sequences which pass thorough these layers of screening were then physically produced using overlapping oligonucleotides and tested for performance in actual amplification/d.etection assays.
Although any one functional block may have some homology to sequences present in a clinical setting (a perfect match of a 21 nucleotide block is expected at a random frequency of 1 in 4e 21 sequences or about 4 x 101z; generated sequences were screened against the GenBank data base) it is high:ly unlikely that all functional blocks will be found to have substantial homology.
Since the internal control nucleic acid sequences are constructed of a group of functional blocks placed in tandem, the chance possibility that a natural nucleic acid sequence will have an identical string of nucleic acid sequence blocks in the same tandem organization is remote.
Two specific intern.al control sequences have been constructed using the method described above. Random Internal Control 1(RICI) is shown in Figure 24 with the possible oligonucleotide primers/probes for amplification and detection of the control sequence. Figure 25 shows an analysis of the possible secondary struciiure of the RIC1 molecule. RIC1 was constructed using McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 35 randomly generated strings ranl6, ran 19, ran21 and ran33. The functional blocks requiring primer binding were met by ran 16 and ran 19, while the capture site was satisfied by ran2l and the detector probe binding site was met by ran33. The choice of a capture probe or detection probe sequence designation can be interchanged, as long as the appropriate linker molecule is attached to the appropriate probe, wherein a reporter probe oligonucleotide is linked to a means for generating a detectable signal, and the capture probe oligonucleotide is linked to a means for adhering the capture probe to an appropriate support. The probes and oligos are described with the understanding that in the case of doulble stranded DNA, the complementary strand can be the target or as appropriate can be converted for use as the strand for detection.
Thus in the 1.0 appropriate circumstance, one of ordinary skill in the art will be able to modify the sequences as disclosed to getierate alternative probes and primers which are suitable for use in an equivalent fashion as described herein.
Random Internal Control 2 (RIC2) is shown in Figure 26 with the possible oligonucleotide primers/probes for ampl:ification and detection of the control sequence. Figure 27 shows an analysis of the possible secondary structure of the RIC2 sequence.
Similarly to RIC1, RIC2 was constructed using randomly generated strings ran27, ran32, ran39 and ran5 1. Thus, illustrating that it is also possible that the functional blocks requiring primer binding, capture probe binding, detector probe binding can be met by alternative random sequences generated by the method described above.
:20 Figure 28 illustrates results from detection of RIC1 DNA, where the ran21 was the capture probe and ran33 was an enzyme-linked detectar-probe, and shows that detection occurs under standard assay conditior.is with expected fluorescence intensities. Figure 29 shows that RIC 1 RNA, amplified by TMA and detected on a VIDAS instrument (bioMerieux Vitek, Inc.) using the enzyme-linked detection system, has a limit of sensitivity of about 1000 molecules of RIC1 RNA
(without optimization of conditions). Siinilar analysis of RIC2 sequences was performed and found to be similar to RIC 1. It is significant that the amplification and detection system of the internal control functioned effectively under the conditions optimized for the selected target.
As an alternative approach for Multiplex detection using internal controls (IC), SPRs can be homogeneously coated with a mixture of different capture nucleic acid sequences in a single, whole-SPR zone. For example, two capture nucleic acid sequences can be combined in one zone, McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 606C6 (312)913-0001 36 one specific for a target test sequence, and one specific for an internal control sequence. Target amplicons, if present, and internal contro:l amplicons are simultaneously hybridized to the SPR, amplicons. In the presence of labeled probe nucleic acid sequences specific for the target test nucleic acid sequence. Following washing, a first signal read is done to so that the presence or absence of label on the SPR is determined to ascertain the presence or absence of the test target.
.A second hybriclization is then done (sequential hybridization) to the SPR
using a labeled specific for the internal control. The SPR is washed to remove excess unbound detection probe, and the second label is measured to indicate the presence or absence of the internal control. If the first signal is negative, a positive signal from the IC second read confirms the functionality of the amplification/detection system. In this case, one can conclude that the test target nucleic acid sequence was truly absent or below detection (true negative). If the first signal is positive, this alone is enough to confirm functionality of the amplification and detection system, and the second signal is immaterial (positive result). If the first and second label are the same, an additive signal will result from the positive first read and the positive second IC
read. If both the first signal is negative and the second IC signal is also negative, then the amplification/detection functionality fa:iled, which could be due to for example, sample interference or mechanical failure. In this case the test result is reported invalid (false negative) and re-testing is recommended. If the labels used are different then neither sequential hybridization or sequential detection steps would be necessary.
There is great interest in the use of internal controls, the underlying rational being that "...
if the sample will not support the amplifiication of the internal control, it is unlikely to support the amplification of the target nucleic acid sequence." (NCCLS Document MM3-A, Molecular Diagnostic Methods for Infectious Diseases; Approved Guideline, p. 55, March 1995).
Using a sequential hybridization approach with multiple detector probes, it has been :25 possible to design protocols which allow for the discrete detection of first signal read (ie. pure CT
signal) and an additive "mixed" second signal read (ie. additive CT and IC
signals; see Table 7A
below). This protocol will not need stripping. For example, Table 7A shows the results when different mixtures of CT and IC synthetic targets were first captured with homogeneously coated SPRs and first hybridized with the CT detector probe. After the first read, hybridization was performed with the IC detector probe, followed by a second read (same substrate).
McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 37 This type of protocol can also be used for a combined GC/CT/internal control assay, if a screening approach is allowed (no discrimination between GC and/or CT
positives during the first read). GC and CT specific signals have to be resolved by running the CT
and GC specific assays on screen positive samples (5-10% of cases, depending on prevalence) SPRs would be coated homogerleously with 3 capture probes (CT/GC/internal control).
Alternatively, the IC
could share a capture probe with either CT or GC.

TABLE 7A: Homogeneous Coated SPR Detection of multiple signals Target CT 1s` Read IC 2nd Read Bkg. RFU

no target 66 89 57 Thus internal control sequences described above are useful for application with VIDAS
apparatus with coated SPR and the use of the Multiplex system to provide for combined assay detection of a nucleic acid and monitorinlg control for successful reaction.

EXAMPLE 7 Internal Control Sequence Refinenient of the randomly generated internal control sequences will allow for optimization of'such internal control sequences for specific assay systems.
Following the methods described above, internal control nucleic acid sequences have been designed and validated for use in various amplification and detection systems including an internal control for a Chlamydia trachomatis (CT) assay identified as CRIC-2; for a Neisseria gonorrhoeae (NG) assay identified as GRIC; and for Mycobacterium tuberculosis (MT) identified as MRIC. An internal control was generated for HIV assays identified as HRIC, wherein both the capture probe sequence and reporter probe sequence were derived from random sequence. The sequence of the McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 38 internal control. and the corresponding target sequence are shown in Figure 30. In each of these internal control sequences, the Random Sequence Probe #1082 can be used as the reporter probe, when suitably conjugated to a reporter molecule as described previously. In the HIV internal control, a capture oligonucleotide Random Sequence Probe #1081 has been designed for use in the capture of the control sequence, for il;nproved quantitation by elimination of competition between the target amplicons and IC amplicons for a common capture probe.

McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 39 SEQUENCE LISTING
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(vii) PRIOR APPLICATION DATA:
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(A) NAME: SMART & BIGGAR
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(C) REFERENCE/DOCKET NUMBER: 76909-79 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)-232-2486 (B) TELEFAX: (613)-232-8440 (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "random internal control 1"
(ix) FEATURE:
(A) NAME/KEY: misc feat:ure (B) LOCATION: 4..24 (D) OTHER INFORMATION: /note= "RAN16 TMA primer"
(ix) FEATURE:
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( ix ) FEA'.CURE :
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

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McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 41 (xi) SEQtirENCE DESCRIPTION: SEQ ID NO:2:

(2) INFORMATION FOR SEQ ID NO:3:
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(A) LENGTH: 56 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "RIC1 top oligo (ix) FEATURE:
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(2) INFORMATION FOR SEQ ID NO:4:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

(2) INFORMATION FOR SEQ ID NO:6:
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(ix) FEATURE:
(A) NAME/KEY: misc feature (B) LOCATION: 1..96 (D) OTHER INFORMATION: /note= "RIC2 target"
(ix) FEATURE:
(A) NAME/KEY: misc feat:ure (B) LOCATION: 1..26 (D) OTHER INFORMATION: /note= "RAN51 TMA primer"
( ix ) FEA".."URE :
(A) NAME/KEY: misc feat:ure (B; LOCATION: 28..48 (D) OTHER INFORMATION: /note= "RAN27 AMVE-probe, amino link at 5' end"
(ix) FEA'f'URE :
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(xi) SEQ'JENCE DESCRIPTION: SEQ ID NO:7:

(2) INFORMATION FOR SEQ ID NO:8:
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(ix) FEATURE:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

(2) INFORMAT'ION FOR SEQ ID NO:9:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

(2) INFORMA7.'ION FOR SEQ ID N0:10:
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(A) LENGTH: 52 base pairs (E3) TYPE: nucleic acici (C) STRANDEDNESS: single McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 44 (D) TOPOLOGY: linear (ii) MOLE;CULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "T7 promoter/RAN39 primer"
( ix ) FEA'I'URE :
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(xi) SEQIJENCE DESCRIPTION: SEQ ID NO:10:

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(xi) SEQIJENCE DESCRIPTION: SEQ ID NO:11:

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(xi) SEQUENCE DESCRIPTION: S:EQ ID NO:12:

(2) INFORMATION FOR SEQ ID NO:13:

McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 45 (i) SEQUENCE CHARACTERISTICS:
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1.0 UCGAAAGAUC AGCUAAUACC GCAUACGUCU UGAGAGGGAA AGCAGGGGAC 110 (2) INFORMATION FOR SEQ ID NO:14::
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
'25 GGCGAGUGGC GAACGGGUGA GUAACAUAAU GGGUGAGCAA GUCUUUCUGG GGAUAACUGA 60 (2) INFORMATION FOR SEQ ID NO:15:
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McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 46 (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQL''ENCE CHARACTERISTICS:
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(ix) FEATURE:
(A) NAME/KEY: misc feature (B) LOCATION: 54..74 (D) OTHER INFORMATION: /note= "Random Seq Probe #1082 (Reporter)"
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McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 47 (ix) FEATURE:
(A) NAME/KEY: misc feature (B) LOCATION: 20..40 (D) OTHER INFORMATION: /note= "Random Sequence Probe #1082 (Reporter)"
( ix ) FEA'CURE :
(A) NAME/KEY: misc feature (B) LOCATION: 41..61 (D) OTHER INFORMATION: /note= "Random Sequence Probe #1081 il0 (Capture) "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1B:

McDonnell Boehnen Hulbert & Berghoff 300 S. Wacker Drive Chicago, Illinois 60606 (312)913-0001 48

Claims (19)

1. A method for the detection of the presence or absence of a single stranded or double stranded first nucleic acid in a sample, by automated isothermal amplification of said first nucleic acid in a dual chamber reaction vessel, wherein said dual chamber reaction vessel comprises two reaction chambers, a first and a second, which can be placed in fluid communication with each other, whereby said fluid communication can be controllably interrupted, said method comprising:

a) combining in said first reaction chamber: a sample, said sample potentially containing said first nucleic acid, reaction buffer, a mixture of free nucleotides, a first and second specific oligonucleotide primer, and placing said reaction vessel in an automated apparatus such that;

b) the automated apparatus heats the first reaction chamber to a sufficient temperature, and for a sufficient time to render any double stranded first nucleic acid in the sample to be tested into sufficient single stranded nucleic acid such that a hybridization product can form, said hybridization product comprising said first nucleic acid and at least one of said first and second oligonucleotide primer;

c) the automated apparatus then cools the first reaction chamber to a sufficient temperature such that said hybridization product forms, if said first nucleic acid is present;

d) the automated apparatus then transfers the reaction mixture from the first reaction chamber to said second reaction chamber via said controllable fluid communication, such that the reaction mixture is brought into contact with nucleic acid polymerization enzyme;

e) the automated apparatus maintains the temperature of the second reaction chamber at a sufficient temperature which allows for the specific oligonucleotide primer mediated amplification of said first nucleic acid, if present;

f) the automated apparatus then contacts any amplicon product from said first nucleic acid in the second reaction chamber with a capture nucleic acid specific for said amplicon product from said first nucleic acid such that a specifically-bound nucleic acid-capture probe hybridization complex can form;

g) the automated apparatus optionally washes the hybidization complex mixture such that non-specifically bound nucleic acid is washed away from the specifically-bound nucleic acid-capture probe complex;

h) the automated apparatus contacts the specifically-bound nucleic acid-capture probe complex with a labeled nucleic acid probe specific for said amplicon product produced from said first nucleic acid such that a specifically-bound nucleic acid-capture probe-labeled probe complex can form;

i) the automated apparatus optionally washes the specifically-bound nucleic acid-capture probe-labeled probe complex such that non-specifically bound labeled probe nucleic acid is washed away from the specifically-bound nucleic acid-capture probe-labeled probe complex;

j) and the automated apparatus detects the presence or absence of said generated signal and optionally displays a value for the signal, and optionally records a value for the signal, wherein the automated apparatus contacts the specifically-bound nucleic acid-capture probe-labeled probe complex with a solution wherein a detectable signal is generated if said amplicon product and first nucleic acid is present, wherein the signal generated from the sample is proportional to the amount of said first nucleic acid in the sample; wherein each of steps h, i and j can be performed sequentially or concurrently.

2. The method as in claim 1 wherein the nucleic acid amplification enzyme is placed in said second reaction chamber as a single assay dose amount in a lyophilized pellet, and said reaction chamber is sealed prior to use.

3. The method as in claim 1 or 2 wherein the nucleic acid amplification enzyme is a thermostable enzyme.

4. The method as in claim 3 wherein said nucleic acid amplification enzyme is placed in the first reaction chamber.

5. The method as in any one of claims 1 to 4 which further incorporates internal control molecules.

6. The method as in any one of claims 1 to 5 which further includes amplification and detection of a target nucleic acid sequence.

7. The method as in any one of claims 1 to 5 which further includes detection of primer control sequences.

8. A device for the automated detection of a first target nucleic acid and a second target nucleic acid, said apparatus comprising a solid phase receptacle, wherein said receptacle comprises a pipet-like device having a pipet-like tip and is coated with a first capture nucleic acid which can form a specific hybridization complex with said first nucleic acid, and a second capture nucleic acid which can form a specific hybridization complex with said second nucleic acid.

9. The device as in claim 8 wherein said first capture nucleic acid and said second capture nucleic acid are coated on said solid phase receptacle in two distinct zones.

10. The device as in claim 8 wherein said first capture nucleic acid and said second capture nucleic acid are coated on said solid phase receptacle in a single zone.

11. The device as in any one of claims 8 to 10, modified for the detection of more than two nucleic acids, further comprising additional capture nucleic acids which can each form a specific hybridization complex with additional target nucleic acid other than said first or second nucleic acid in said sample.

12. A method for the automated detection of the presence or absence of a first target nucleic acid and a second target nucleic acid in a sample, said method comprising:

a) contacting said sample with a solid phase receptacle, wherein said receptacle comprises a pipet-like device having a pipet-like tip and is coated with a first capture nucleic acid which can form a specific hybridization complex with said first nucleic acid, and a second capture nucleic acid which can form a specific hybridization complex with said second nucleic acid;

b) allowing specific hybridization complex to form if said nucleic acid is present;

c) contacting said solid phase receptacle hybridization complex with a first detection nucleic acid, wherein said first detection nucleic acid can form a specific hybridization detection complex with said first nucleic acid, and is conjugated to a means for generating a detectable signal selected from the group consisting of enzyme, chromophore, chemiluminescent compound, radioisotope, and fluorophore;

d) allowing specific detection complex to form, then generating said detectable signal;

e) detecting said signal if said first nucleic acid is in said sample;

f) contacting said solid phase receptacle hybridization complex with a second detection nucleic acid, wherein said second detection nucleic acid can form a specific hybridization detection complex with said second nucleic acid, and is conjugated to a means for generating a detectable signal selected from the group consisting of enzyme, chromophore, chemiluminescent compound, radioisotope, and fluorophore;

g) allowing specific detection complex to form, then generating said detectable signal;

h) detecting said signal if said second nucleic acid is in said sample;

i) and wherein optionally, between steps, said hybridization complex can be washed to remove excess non-specifically bound nucleic acid;

j) wherein the absence of a detectable signal correlates with the absence of said nucleic acid in said sample.

13. The method as in claim 12 which further incorporates internal control molecules.

14. The method as in claim 12 or 13 which further includes amplification and detection of a target nucleic acid sequence.

15. The method as in claim 12 or 13 which further includes detection of primer control sequences.

16. The method as in any one of claims 12 to 15, wherein one or more of said steps c and f are performed concurrently, said steps d and g are performed concurrently, and steps e and h are performed concurrently.

17. The method as in any one of claims 12 to 16, wherein said means for generating a detectable signal is the enzyme alkaline phosphate, and generation of said detectable signal comprises contacting said means with a substrate of methyl umbelliferyl phosphate.

18. The method as in any one of claims 12 to 17, modified for the detection of more than two nucleic acids, wherein said solid phase receptacle is further coated with additional specific capture nucleic acid, wherein said additional specific capture nucleic acid can form specific hybridization complex with additional target nucleic acid other than said first and second nucleic acid in said sample, and wherein said method contacts additional hybridization capture complex with additional specific detection nucleic acid, wherein said additional detection nucleic acid can form a specific hybridization detection complex with said additional nucleic acid, and is conjugated to a means for generating a detectable signal selected from the group consisting of enzyme, chromophore, chemiluminescent compound, radioisotope, and fluorophore.

19. The method as in claim 18 wherein said first nucleic acid is specific for Chlamydia trachomatis (CT), and said second nucleic acid is specific for Neisseria gonorrhoeae (NG).