WO2009012194A1 - Methods and devices for detection of genomic material - Google Patents

Abstract

Provided are methods and devices for detection of genomic material from a virulent strain of a pathogen.

Description

METHODS AND DEVICES FOR DETECTION OF GENOMIC MA TERIAL

RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Application No: 60/949,621, filed July 13, 2007, the contents of which are incorporated by reference herein in their entirety.

BACKGROUND

[0002] Detection of the bacteria that have infected a subject, including metabolites, nucleic acids, and proteins thereof, is a fundamental component in the diagnosis and treatment of medical disorders, as well as in research. A number of methodologies are currently in use for detection. These methodologies can generally be divided into antibody-based diagnostic assays for proteins, either components of the bacteria or byproducts of the disease, and diagnostic assays for nucleic acids, such as the genetic material encoding a component of the bacteria. Improved methodologies for detecting nucleic acids using immunochromatographic test devices are needed.

SUMMARY

[0003] Provided are methods of capturing, extracting and amplifying nucleic acids from pathogens to identify the pathogens, e.g., for diagnostic or treatment purposes. The methods comprise capturing polynucleotides of a pathogen in a zone of lateral flow or other immunochromatographic test device, extracting the captured polynucleotides and amplifying the extracted polynucleotides using primers indicative of a virulent strain of the pathogen; wherein positive amplification indicates a virulent strain of the pathogen is present in the sample. [0004] The methods may be incorporated into any test format or device suitable for the practice of the methods. Also provided are kits, reagents, etc. for the practice of the methods.

[0005] Further objectives and advantages of the present invention will become apparent as the description proceeds. To gain a full appreciation of the scope of the present invention, it will be further recognized that various aspects of the present invention can be combined to make desirable embodiments of the invention. DETAILED DESCRIPTION

[0006] Unless defined otherwise above, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Where a term is provided in the singular, the inventor also contemplates the plural of that term. The nomenclature used herein and the procedures described below are those well known and commonly employed in the art.

[0007] The singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. [0008] "Amplification," refers to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art. (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.) [0009] The term "antibody" refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In exemplary embodiments, antibodies used with the methods and compositions described herein are derivatives of the IgG class.

[0010] The term "antibody fragment" refers to any derivative of an antibody which is less than full-length. In exemplary embodiments, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments. The antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, it may be recombinantly produced from a gene encoding the partial antibody sequence, or it may be wholly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multimolecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids. [0011] "Complementary" or "complementarity", refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. [0012] The terms "comprise" and "comprising" is used in the inclusive, open sense, meaning that additional elements may be included.

[0013] The term "extraction" refers to any process by which polynucleotides may be isolated from a pathogen.

[0014] "Hybridization" refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing. "Specific hybridization" of a probe to a target site of a template nucleic acid refers to hybridization of the probe predominantly to the target, such that the hybridization signal may be clearly interpreted. As further described herein, such conditions resulting in specific hybridization vary depending on the length of the region of homology, the GC content of the region, and the melting temperature "T(m)" of the hybrid. Hybridization conditions will thus vary in the salt content, acidity, and temperature of the hybridization solution and the washes.

[0015] The term "including" is used herein to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably. [0016] "Polynucleotide" or "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. ESTs, chromosomes, cDNAs, mRNAs, and rRNAs are representative examples of molecules that may be referred to as nucleic acids. [0017] The term "pathogen" refers to any organism which may cause disease in a subject, such as a bacterium, fungus, parasite, virus, etc.

[0018] "Protein" (if single-chain), "polypeptide" and "peptide" are used interchangeably herein when referring to a gene product, e.g., as may be encoded by a coding sequence. When referring to "polypeptide" herein, a person of skill in the art will recognize that a protein can be used instead, unless the context clearly indicates otherwise. A "protein" may also refer to an association of one or more polypeptides.

[0019] The term "sample" refers to any sample potentially containing a pathogen. For example, a sample may be a bodily fluid such as blood, urine, mucous or saliva, or a respiratory sample, such as a nasopharyngeal wash or aspirate, nasal swab, nasopharyngeal swab, nasal wash, throat swab, transtracheal aspirate, bronchoalveolar lavage, elution buffer used to wash a respiratory sample, etc.

[0020] Provided in one aspect is a method, comprising: [0021] (a) flowing a sample along a support comprising at least one capture zone;

[0022] (b) capturing a pathogen in the sample, if present, in the at least one capture zone;

[0023] (c) extracting the polynucleotides, if any, from the pathogen captured in the at least one capture zone; and

[0024] (d) amplifying the extracted polynucleotides, wherein positive amplification indicates a virulent strain of the pathogen is present in the sample.

[0025] The capture zone may comprise any immobilized reagent specific for the pathogens, e.g., an antibody or other protein specific for a molecule on the surface of the pathogen.

[0026] Pathogens that may be detected using the above methods include any of a bacterium, fungus, parasite, virus, etc. In certain embodiments, the pathogen is a bacterium. In certain embodiments, the bacterium is Group A Streptococcus. In certain embodiments, the pathogen is a virus. In certain embodiments, the virus is an influenza virus, for example avian influenza.

[0027] The polynucleotides may be extracted from the pathogen using any method known in the art for extracting polynucleotides. In one embodiment, the polynucleotides are obtained from a captured pathogen. It is also possible to obtain the pathogen and culture it in vitro, such as to obtain a larger population from which polynucleotides may be extracted.

Methods for establishing primary cell cultures are known in the art.

[0028] In certain embodiments, the polynucleotide is RNA. When isolating RNA from a pathogen, it may be important to prevent any further changes in gene expression in the pathogen. Changes in expression levels are known to change rapidly following perturbations, e.g., heat shock or activation with lipopolysaccharide (LPS) or other reagents. In addition, the RNA may quickly become degraded. RNA may be extracted by a variety of methods, e.g., the guanidium thiocyanate lysis followed by CsCl centrifugation (Chirgwin et al, (1979), Biochemistry 18:5294-5299). RNA from single cells may be obtained as described in methods for preparing cDNA libraries from single cells, such as those described in Dulac, C. (1998) Curr. Top. Dev. Biol 36, 245 and Jena et al. (1996) J. Immunol. Methods 190:199. Care to avoid RNA degradation must be taken, e.g., by inclusion of RNAsin.

[0029] The RNA sample may then be enriched in particular species. In one embodiment, poly(A)+ RNA is isolated from the RNA sample. In general, such purification takes advantage of the poly- A tails on mRNA. In particular and as noted above, poly-T oligonucleotides may be immobilized within on a solid support to serve as affinity ligands for mRNA. Kits for this purpose are commercially available, e.g., the MessageMaker kit (Life Technologies, Grand Island, NY).

[0030] In a preferred embodiment, the RNA population is enriched in sequences of interest, such as those of the genes involved in making the pathogen a virulent strain. Enrichment may be undertaken, e.g., by primer-specific cDNA synthesis, or multiple rounds of linear amplification based on cDNA synthesis and template-directed in vitro transcription (see, e.g., Wang et al. (1989) PNAS 86, 9717; Dulac et al., supra, and Jena et al., supra). [0031] The population of polynucleotides, enriched or not in particular species or sequences, may further be amplified. Such amplification is particularly important when using polynucleotides from a single or a few pathogens. [0032] A variety of amplification methods are suitable for use in the methods of the invention, including, e.g., PCR; ligase chain reaction (LCR) (see, e.g., Wu and Wallace, (1989) Genomics 4, 560, Landegren et al. (1988) Science 241, 1077); self-sustained sequence replication (SSR) (see, e.g., Guatelli et al., (1990) PNAS, 87, 1874); nucleic acid based sequence amplification (NASBA) and transcription amplification (see, e.g. , Kwoh et al.,(1989) PNAS 86, 1173). For PCR technology, see, e.g., PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, N.Y., N.Y., 1992); PCR Protocols: A Guide to Methods and applications (eds. Innis, et al., Academic Press, San Diego, Calif, 1990); Mattila et al., (1991) Nucleic Acids Res. 19, 4967; Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202. Methods of amplification are described, e.g., in

Ohyama et al. (2000) BioTechniques 29:530; Luo et al. (1999) Nat. Med. 5, 117; Hegde et al. (2000) BioTechniques 29:548; Kacharmina et al. (1999) Meth. Enzymol. 303:3; Livesey et al. (2000) Curr. Biol. 10:301; Spirin et al. (1999) Invest. Ophtalmol. Vis. Sci. 40:3108; and Sakai et al. (2000) Anal. Biochem. 287:32. RNA amplification and cDNA synthesis may also be conducted in cells in situ (see, e.g., Eberwine et al. (1992) PNAS 89:3010). [0033] One of skill in the art will appreciate that whatever amplification method is used, if a quantitative result is desired, care must be taken to use a method that maintains or controls for the relative frequencies of the amplified nucleic acids to achieve quantitative amplification. Methods of "quantitative" amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. A high density array may then include probes specific to the internal standard for quantification of the amplified nucleic acid.

[0034] In certain embodiments, RNA is reverse transcribed with a reverse transcriptase and a primer consisting of oligo(dT) and a sequence encoding the phage T7 promoter to provide single stranded DNA template. The second DNA strand is polymerized using a DNA polymerase. After synthesis of double-stranded cDNA, T7 RNA polymerase is added and RNA is transcribed from the cDNA template. Successive rounds of transcription from each single cDNA template results in amplified RNA. Methods of in vitro polymerization are well known to those of skill in the art (see, e.g., Sambrook, (supra) and this particular method is described in detail by Van Gelder, et al., (1990) PNAS, 87: 1663-1667 who demonstrate that in vitro amplification according to this method preserves the relative frequencies of the various RNA transcripts. Moreover, Eberwine et al. PNAS, 89: 3010- 3014 provide a protocol that uses two rounds of amplification via in vitro transcription to achieve greater than 10⁶ fold amplification of the original starting material, thereby permitting expression monitoring even where biological samples are limited. [0035] Also provided are devices, e.g., lateral flow devices, a test strip, or other support for practice of the above-described methods. For example, a device may comprise a porous test strip comprising at least one capture zone comprising an antibody or other molecule specific for a pathogen of interest.

[0036] A kit for the practice of the above methods may include a support, reagents and wash and incubation buffers. Such kits and devices can contain any number or combination of reagents or components. The kits can comprise one or more of the above components in any number of separate containers, tubes, vials and the like or such components can be combined in various combinations in such containers. Kit components may be packaged for either manual or partially or wholly automated practice of the foregoing methods. Further, instructions for the use of a device or kit may be included with the device or kit. Such kits and devices may have a variety of uses, including, for example, diagnosis, therapy, and other applications.

[0037] By way of example, generally, an immunoassay device for determining the presence or amount of an analyte of interest in a sample includes a sample application member, which is in liquid communication with a conjugate pad, which is in liquid communication with a nitrocellulose test strip having at least a capture zone. The immunoassay can also include a distal sink at the end opposite to the sample application pad to absorb any excess liquid after testing has run to completion. [0038] The sample application pad is a porous pad able to absorb the sample to be tested and transfer the absorbed sample to the conjugate pad by capillary action. The conjugate pad includes one or more dried labeled molecules or reagents, such as antibodies, capable of specifically binding to the one or more pathogens of interest forming a pathogen- labeled reagent complex. The conjugate pad may also include one or more stabilizing compounds that are able to induce thermal stability and also stability as to conditions imposed by humidity and temperature. The conjugate pad is a porous pad able to absorb the transferred sample from the sample application pad and transfer the sample to the nitrocellulose strip by capillary action. The nitrocellulose strip is able to absorb the sample from the conjugate pad and transfer the sample by capillary action downstream to the capture zone. The capture zone of the immunoassay device includes one or more immobilized molecules or reagents, such as antibodies or antibody fragments, capable of specifically binding to the one or more pathogens of interest or any portion of the pathogen-labeled reagent complex. The control zone of the immunoassay device may include one or more immobilized molecules or reagents, such as antibodies or antibody fragments, capable of specifically binding to the one or more labeled reagents.

[0039] When a liquid test sample is applied to the sample application pad of the device, the sample travels through the sample application pad, the conjugate pad, and nitrocellulose strip by capillary action. When the sample travels through the conjugate pad, the sample solublizes the dried labeled molecule or reagent, and if the pathogen of interest is present in the sample, the solubilized labeled molecule or reagent binds the pathogen of interest forming a pathogen-labeled reagent complex, otherwise, if the pathogen of interest is not present in the sample, no complex is formed. The pathogen-labeled reagent complex in the case of a positive test, or the labeled reagent alone in the case of a negative test, then travel to the nitrocellulose strip and travel through and pass the capture zone of the device. If the pathogen of interest is present in the sample, the pathogen-labeled reagent complex binds to the immobilized reagent of the test result zone forming a detectable line, and if the analyte of interest is not present in the sample, no pathogen labeled reagent complex is formed and therefore no binding occurs at the test result zone. Whether or not the pathogen of interest is present in the sample to form a complex, the labeled reagent binds to the immobilized reagent of the control zone forming a detectable line indicating that the test has run to completion. Any excess liquid sample, after the testing has run to completion, can be absorbed the distal sink of the device.

EXEMPLIFICATION

[0040] The invention, having been generally described, may be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention in any way. All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0041] Example 1: Detection of avian influenza genomic material from a lateral flow assay

[0042] Mallard /OH/421/87 H7N8 at 10⁶ EID₅₀/100 μL was used to test whether virus could be detected from the Binax FIuA NOW® device test strip. The virus was diluted in BHI from 10⁶ EID50 stock to 10³ EID50. Each virus concentration was tested in duplicate (A, B) on the Binax device in accordance with the kit instructions. The flu A positive band was scraped off with a PlOOO tip and placed in 150 μL Qiagen buffer and vortexed. 150 μL 70% ethanol was added, mixed by inversion and extracted RNA with the Qiagen Rneasy mini kit in accordance with test instructions. RNA was eluted in 50 μL of nuclease free H₂O. Standard USDA RRT-PCR was run with each sample. The RRT-PCR results were as follows:

[0043] Example 2: Testing of Lateral Flow Device for Group A Streptococcus DNA in Lightcycler Assay

[0044] Cultured Group A Streptococcus ("Strep") samples were diluted into saliva. 100 μl of spiked saliva was tested in the device per Binax protocol. The entire test was performed including the final 30 minute incubation. The device was opened within 30 min of completing the test. The strip inside the device was cut into pieces, and the pieces put into 520 μl of MagNa Pure lysis buffer plus 80 μl of MagNaPure proteinase K. It was incubated at 65⁰C for 10 minutes and at 95⁰C for 10 minutes. 100 μl of the supernatant was extracted with the MagNa Pure bacterial extraction protocol (DNA III Kit) and eluted with 100 μl of buffer.

[0045] We have detected the Group A Strep at levels of 2x10⁴ copies/ mL and have repeated detection at levels between 10⁴ and 10⁵ copies/mL (concentration of the original Group A Strep + saliva mix). We have a run where a 10³ copies per mL level was detected, but 3x10³ and 10⁴ copies/mL were not. The strips absorb a lot of lysis buffer which is difficult to recover. It appears that Group A Strep DNA is recoverable throughout the strip, but because the liquid from the device moves as the strip is being cut, it is difficult to be sure that the DNA is evenly dispersed without additional experiments. [0046] With the current protocol we may consistently detect Group A Strep in saliva from the device at input levels above 10⁴ copies/mL. Modifying the protocol to use less diluent by developing a procedure of washing the Group A Strep DNA out of the strip pads via centrifugation, would probably improve the detection at the 10⁴ and 5 x 10³ level. [0047] Example 3: PCT Test Device Preparation Protocol [0048] The devices were run as per the current "semi-wet" extraction. New-mold inner barrels were loaded with 4 porex disks containing ~23 μl of: 1) 100Ku/ml mutanolysin/5% sucrose, 2) 4M NaNO₂/5% Tween-20, 3) 10% Zwittergent 314 and 4) 20 mg/ml bromelain/5% sucrose/20mM sodium borate pH 8.0.

[0049] Loaded barrels were affixed to cassettes containing the Group A Streptococcus test strips originally prepared for the ongoing clinical trial (Rabbit FAb gold, partially FAbed Goat capture).

[0050] Positive samples were prepared by diluting IXlO⁸ stock of Group A Strep 1 : 100 to IXlO⁶ with 0.9% saline. Negative samples were prepared by diluting a lmg/ml stock of Group A Strep polysaccharide antigen 1 :40000 to 25ng/ml in 0.9% saline. 2 X 210 μl of the negative control and 3 X 210 μl of positive control were aliquotted into micro fuge tubes and 70 μl of Group A Strep reagent 2 (0.125M acetic acid/5% Tween-20) were added to each tube. [0051] 265 μl of each was applied separately to the top of the barrel of the devices assembled above. The samples were allowed to incubate for 15 minutes on the bench, the barrel was twisted initiating the flow of extracted sample onto the test strip and devices were observed for positive signal at 15 minutes. Devices were allowed to dry and transferred for PCR evaluation.

EQUIVALENTS

[0052] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification.

The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. Such equivalents are intended to be encompassed by the following claims.

REFERENCES

[0053] All publications and patents mentioned herein, including those items listed below, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. 1. D.Kozwich, K.A.Johansen, K.Landau, C.A.Roehl, S.Woronoff and P.A.Roehl.

Development of a Novel, Rapid Integrated Cryptosporidium parvum Detection Assay. Appl Environ Microbiol 66(7): 2711-2717, July 2000.

2. P.Corstjens, M.Zuiderwijk, A.Brink, S.Li, H.Feindt, R.S.Niedbala and H.Tanke. Use of Up-Converting Phosphor Reporters in Lateral-Flow Assays to Detect Specific Nucleic Acid Sequences: A Rapid, Sensitive DNA Test to Identify Human Papillomavirus Type 16 Infection. Clin Chem 47(10): 1885-1893, 2001. 3. P.L.A.M.Corstjens, M.Zuiderwijk, M.Nilsson, H.Feindt, R.S.Niedbala and HJ Tanke. Lateral-flow and up-converting phosphor reporters to detect single-stranded nucleic acids in a sandwich-hybridization assay. Analyt Biochem 312: 191-200, 2003.

4. M.Zuiderwijk, H.J.Tanke, R.S.Niedbala and P.L.A.M.Corstjens. An amplification- free hybridization-based DNA assay to detect Streptococcus pneumoniae utilizing the up- converting phosphor technology. Clin Biochem 36: 401-403, 2003.

5. K.Takada, Y.Sakaguchi, C.Oka and M.Hirasawa. New Rapid Polymerase Chain Reaction-Immunochromatographic Assay for Porphyromonas gingivalis. J Periodontol 76:508-512, 2005. 6. Y.T.Horng, P.C.Soo, B.J.Shen, Y.L.Hung, K.Y.Lo, H.P.Su, J.R.Wei, S.C.Hsieh SC, P.R.Hsueh and H.C.Lai. Development of an improved PCR-ICT hybrid assay for direct detection of Legionella and Legionella pneumophila from cooling tower water specimens. Water Research 40: 2221-2229, 2006.

7. W.K.Fong, Z.Modrusan, J.P.McNevin, J.Marostenmaki, B.Zin, and F.Bekkaoui. J Clin Microbiology, July 2000, 2525-2529.

8. J.Wang, Z.Cheh, P.L.A.M.Corstjens, M.G.Mauk and H.H.Bau. A disposable micro fluidic cassette for DNA amplification and detection. Lab on a Chip 6: 46-53, 2006.

Claims

Claimed are:

1. Provided in one aspect is a method, comprising:

(a) flowing a sample along a support comprising at least one capture zone;

(b) capturing a pathogen in the sample, if present, in the at least one capture zone;

(c) extracting the polynucleotides, if any, from the pathogen captured in the at least one capture zone; and

(d) amplifying the extracted polynucleotides, wherein positive amplification indicates a virulent strain of the pathogen is present in the sample.

2. The method of claim 1, wherein the amplifying is performed using primers indicative of a virulent strain of the pathogen.

3. The method of claim 1 , wherein the polynucleotide is a ribonucleotide.

4. The method of claim 1 , wherein the pathogen is a virus.

5. The method of claim 4, wherein the pathogen is avian influenza.

6. The method of claim 1, wherein the pathogen is a bacterium.

7. The method of claim 6, wherein the pathogen is Group A Streptococcus.

8. The method of claim 1, wherein the support is a porous strip.