CN101144775B - Bacteria real-time fluorescence quantitative polymerase chain reaction detection reagent kit - Google Patents
Bacteria real-time fluorescence quantitative polymerase chain reaction detection reagent kit Download PDFInfo
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- CN101144775B CN101144775B CN200710070629A CN200710070629A CN101144775B CN 101144775 B CN101144775 B CN 101144775B CN 200710070629 A CN200710070629 A CN 200710070629A CN 200710070629 A CN200710070629 A CN 200710070629A CN 101144775 B CN101144775 B CN 101144775B
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Abstract
The invention provides a reagent kit for testing the bacteria real time fluorescence quantitative polymerase chain reaction, and the reagent kit comprises a PCR reaction liquid tube 1, a standard 2, a positive control 3, a negative control 4, a lysis buffer extracting 5, an operating manual 6 and a packing box body 7, wherein, the box body 7 is provided with a container hole, where the PCR reaction liquid tube 1, the standard 2, the positive control 3, the negative control 4 and the lysis buffer extracting 5 are respectively arranged, the PCR reaction liquid tube is subpackaged by a single tube, and arranged in matrix. The reagent kit can be used in the bacteria DNA detecting. The invention achieves the purpose of the universal detection to the common bacteria by applying the real time fluorescence quantitative PCR technology, to effectively solve the problem that the PCR method is only used to detect unitary pathogenic bacteria, the common bacterial infection is detected versatilely, the versatility and the sensibility of the bacteria detecting are advanced, and a basis of the premature pathogen detecting of the bacterial infection disease is provided.
Description
Technical field
The invention belongs to biological technical field, relate to quantitative PCR detection kit, be specifically related to the kit that a kind of real-time fluorescence quantitative PCR bacterial detection sexuality is caught DNA of bacteria in youngster's blood, cerebrospinal fluid, hydrothorax, the ascites equal samples.
Background technology
Bacterial 16 S rRNA gene is the corresponding dna sequence dna of coding rRNA on the bacterial chromosome.This gene has following two big characteristics: (1) multicopy: 16S rRNA encoding gene is present in all bacterial chromosome genomes with the multicopy form, and each bacterium contains 5～10 copies approximately, and this feasible detection to this gene has susceptibility.(2) many information: 16S rRNA encoding gene inner structure is made up of variable region and conserved region.Conserved region is that all bacteriums are common, and the title of " molecular fossil " is arranged.Can be used for the general detection of clinical common bacteria according to universal primer and the general probe of the various bacteriums of conserved regions design.At present, the 16S rRNA gene sequencing of nearly all pathogen is finished.Therefore 16S rRNA gene has become comparatively ideal clinical bacteria infection detection target-gene sequence, becomes the goldstandard that bacterium and other pathogen are differentiated, classified gradually.
The ultimate principle of real time fluorescent quantitative technology: utilize 5 of Taq enzyme '-3 ' excision enzyme nucleic acid activity, on the regular-PCR basis, design fluorescence double-tagging probe, fluorescence report group (R) and quenching group (Q) on the mark respectively.When this probe was kept perfectly, the fluorescence signal of R group was suppressed by the Q group, in case probe is cut off, inhibiting effect disappears, and the fluorescence signal of R group just can be detected.This method adopts complete stopped pipe to detect, and has saved the product postprocessing to PCR, has avoided cross pollution, and result's judgement is finished by computing machine, has simplified operation steps, and has increased result's reliability.
Septicemia (Septicemia) is meant pathogen and the caused clinical syndrome of toxin intrusion blood flow thereof.The activation and the release of inflammatory mediator are often arranged in the course of disease, cause high heat, shiver, tachycardia, be short of breath, a series of clinical symptoms such as fash and mind change, severe patient can cause shock, DIC and MOF.Even give suitable antibiotic therapy, case fatality rate is still higher.Children particularly the neonate because self resistibility is poor, skin thin tender, birth back umbilical region does not heal etc. all easily suffers from septicemia.If children's septicemia can not judge in early days, in time, treatment up hill and dale, can cause purulent meningitis and take place, influence developing infantile intelligence and grow, cause children's permanent disability and dead.
Children's septicemia lacks specificity clinical symptoms and early stage lab index reliably, and early diagnosis is very difficult.At present existing kinds of experiments chamber method is applied in the diagnosis of children's septicemia, as blood culture, routine blood test, acute phase protein such as CRP, focus secretion is cultivated and smear, pathogen Detection of antigen such as counter immunoelectrophoresis (CIE), and molecular Biological Detection such as methods such as restriction endonuclease analysis (RFLP), dna probe.Fluorescence quantitative PCR method is the biology techniques that rose in recent years, has characteristics such as accuracy height, favorable reproducibility, has been widely used in numerous areas such as gene expression research, pathogen detection, snp analysis, is one of present research focus in detection with application.
Along with the develop rapidly of Protocols in Molecular Biology and the widespread use in medical research thereof, the new method of setting up bacterial infection diseases such as rapid and reliable diagnosis children's septicemia and purulent meningitis has become and may and have been attempted.
Summary of the invention
The purpose of this invention is to provide bacteria real-time fluorescence quantitative polymerase chain reaction detection reagent kit, this quantification kit is by quantitative PCR reactant liquor, standard items, reference substance, extracting lysate, operational manual, Packaging box body are formed, Packaging box body is provided with container hole, place PCR reactant liquor pipe, standard items, positive reference substance, negative control product, extracting lysate respectively, PCR reactant liquor pipe is by single tube packing, arranged.
Wherein the quantitative PCR reactant liquor contains PCR damping fluid, MgCl
2, dNTPs, Bacteria Detection with upstream primer, detect with downstream primer, fluorescence probe, hot resistant DNA polymerase (Taq archaeal dna polymerase) and form.The extracting lysate contains 10mmol/L Tri-HCl, and pH 7.6,5mmol/L disodium ethylene diamine tetraacetate (EDTA), 0.5% lauryl sodium sulfate (SDS).The quantitative PCR reactant liquor must be with 0.22 μ m membrane filtration pre-service.
Wherein:
The upstream primer sequence is: 5`-CAACGCGAAGAACCTTACC-3`
The downstream primer sequence is: 5 '-ACGTCATCCCCACCTTCC-3 '.
The fluorescence probe sequence is: 5 '-FAM-TAAGTCCCGCAACGAGCGCAA-TAMRA-3 '.
The standard items sequence is:
CAACGCGAAG?AACCTTACCT?GGTCTTGACA?TCCACAGAAC?TTTCCAGAGA
TGGATTGGTG?CCTTCGGGAA?CTGTGAGACA?GGTGCTGCAT?GGCTGTCGTC
AGCTCGTGTT?GTGAAATGTT?GGGTTAAGTC?CCGCAACGAG?CGCAACCCTT
ATCCTTTGTT?GCCAGCGGTC?CGGCCGGGAA?CTCAAAGGAG?ACTGCCAGTG
ATAAACTGGA?GGAAGGTGGG?GATGACGT
Reference substance is divided into positive reference substance and negative control product, and negative control is the sterile water for injection sample, and positive control is an Escherichia coli deactivation DNA sample.
This quantitative reagent is stored in-20 ℃, reduces multigelation as far as possible.
Second purpose of the present invention provides the application of this kit in bacterial detection DNA.
This product is selected the amplified target zone of clinical common bacteria 16S rRNA gene high conservative for use, and design bacterium universal primer and general TaqMan fluorescence probe utilize the real-time fluorescence quantitative PCR technology, and real-time quantitative detects the application of clinical sample DNA of bacteria.
Kit using method of the present invention:
Each detection all should be set up positive control and negative control.Standard items are 1 * 10 with the aseptic deionized water dilution
2-1 * 10
9Copy/ml.
DNA of bacteria is extracted: gather fresh whole blood 1ml and be positioned in the aseptic vacuum test tube that contains Chinese holly edge acid sodium anti-freezing liquid collecting, mixing is got extracting lysate (the 10mmol/L Tri-HCl pH7.65mmol/L EDTA that whole blood 50ul adds equivalent, 0.5%SDS) put 100 ℃ and boil 10min, the centrifugal 5min of 12000r/min gets 5 μ l supernatants as pcr template.Bacterium liquid, cerebrospinal fluid, hydrothorax, ascites equal samples method are the same.
Augmentation detection: on quantitative real time PCR Instrument, carry out cumulative volume 50 μ l, 45.0 μ l PCR reactant liquors wherein, 5 μ l test sample (extracting product, standard items, positive or negative contrast).Reaction conditions: 94 ℃ of pre-sex change of 5min, 94 ℃ of 20sec, 60 ℃ of 60sec are a circulation, circulate altogether 40 times.
Fluorescent quantitation result report: 1. CT value (threshold cycle, the period that fluorescence signal in each reaction tube is experienced when reaching the thresholding of setting) it is negative to equal 40.0 sample, 2. the sample of CT value≤35, testing result is positive, 3. the CT value is ash value zone between 35～40, and the back of reforming is negative greater than 38 values.According to the typical curve that is obtained, calculate the amount (copy number/ml) of bacterium in each sample to be measured.
Usefulness of the present invention is: this method utilization real-time fluorescence quantitative PCR technology has realized the purpose to the general detection of clinical common bacteria.Solve the problem that PCR method in the past can only be used to detect single pathogen effectively, common bacterial infection has been carried out general detection, improved the versatility and the susceptibility of Bacteria Detection, improved the bacillary and abacterial ability distinguished.For the early stage pathogen detection of bacterial infection provides foundation.
Description of drawings
Fig. 1 is a structural representation of the present invention.
Fig. 2 is Escherichia coli standard items fragment sequences.
Fig. 3 is that bacteria real-time fluorescence quantitative PCR standard items detect.
Fig. 4 is a bacteria real-time fluorescence quantitative PCR typical curve.
Embodiment
The present invention is described further with specific embodiment in conjunction with the accompanying drawings.Should be appreciated that these embodiment only are used for illustration purpose, and are not used in the restriction scope of the invention.
Referring to Fig. 1, bacteria real-time fluorescence quantitative polymerase chain reaction detection reagent kit provided by the invention is made up of PCR reactant liquor 1, standard items 2, positive reference substance 3, negative control product 4, extracting lysate 5, operational manual 6, Packaging box body 7.Box body 7 is provided with container hole, places PCR reactant liquor pipe 1, standard items 2, positive reference substance 3, negative control product 4, extracting lysate 5 respectively, and PCR reactant liquor pipe is by single tube packing, arranged.
Wherein the quantitative PCR reactant liquor contains PCR damping fluid, MgCl
2, dNTPs, Bacteria Detection with upstream primer, detect with downstream primer, fluorescence probe, hot resistant DNA polymerase (Taq archaeal dna polymerase) and form.The extracting lysate contains 10mmol/L Tri-HCl, and pH 7.6,5mmol/L disodium ethylene diamine tetraacetate (EDTA), 0.5% lauryl sodium sulfate (SDS).
One, material:
Clinical common bacteria isolated strains staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli provide by this laboratory cultures; PCR related reagents such as pGEM-T-Easy cloning system, Taq archaeal dna polymerase, dNTP are available from U.S. Promega company, and sequencing reagent, 377 type sequenators, PE-5700 type quantitative PCR instrument are U.S. Perkin Elmer company product.
Two, primer and probe design and synthetic:
Select clinical 50 bacterial strains of 22 kinds of Pseudomonas of common bacteria that cause septicemia and purulent meningitis (change brain), wherein removing from office blue yin and yang attribute bacterial strain respectively is 25 strains (seeing Table 1).Use its 16S rRNA gene order of MEGALIGN software analysis, seek the conservative fragments between the different bacterium kind, analyze the rule of biological evolution tree, according to the principle of design of primer and probe, at these bacterium conservative region screening universal primer and general probe, therefrom select best a pair of combination.
The upstream primer sequence is: 5`-CAACGCGAAGAACCTTACC-3`
The downstream primer sequence is: 5 '-ACGTCATCCCCACCTTCC-3 ' is synthetic by the living worker in Shanghai company.
The fluorescence probe sequence is: 5 '-FAM-TAAGTCCCGCAACGAGCGCAA-TAMRA-3 '.Precious biotech company is synthetic by Dalian.
The clinical common bacteria that causes septicemia and purulent meningitis (change brain) of table 1
Three, examination criteria product preparation:
With above-mentioned primer amplification reference culture Escherichia coli, the PCR product is 228bp, the PCR product promptly inserts the pGEM-T-Easy cloning vector with cloning system after electrophoresis detection, extracting recombinant clone plasmid carries out sequence verification with carrier primer M13R to the positive colony of recombinating.Measure concentration and be converted into (copy number/volume).
The result: through order-checking, above-mentioned design standards product conform to expection fully, and standard items fragment sequence wherein is an Escherichia coli, referring to Fig. 2.
Four, clinical common bacterial strain gradient dilution control test:
From the nutrient culture media of three kinds of common clinical separation strains (staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli), scrape the bacterial plaque that takes a morsel respectively with aseptic cotton carrier, be put in separately in the test tube of the sterilized water that contains 2ml, use to be made into the liquid of 0.5 titre than turbid instrument (being equivalent to concentration is 10
8CFU/m1).Respectively these three kinds of bacterial strains are carried out 10 successively
-1Inferior, 10
-2Inferior, 10
-3Inferior, 10
-4Inferior, 10
-5The power dilution respectively gets 10
-5Inferior, 10
-4Inferior, 10
-3Power dilution 5ul fluorescent quantitation and draw 37 ℃ of cultivations overnight of dull and stereotyped bacterium is counted the clump count of double dish next day.
Table 2 plate count and fluorescence quantitative PCR detection result contrast
One, sample detects:
Choose in Dec, 2003～2005 year Dec, I am institute's Neonatal Ward and the clinical neonate (age 1d～28d that is in hospital that doubts to bacterial infection (predisposing factor and the clinical manifestation [3] of bacterial infection are all arranged) of NICU, man's 420 examples, woman's 410 examples, premature's 65 examples wherein, all the other are the term infant) 830 examples, it is main that the predisposing factor of these 830 routine neonate's infants and clinical manifestation mainly contain premature, jaundice, pneumonia, enteritis, heating, infectious shock etc.Select neonate's 30 routine samples of being in hospital because of noninfectious disease the same period in contrast.Each 1～2ml of every routine venous blood samples inspects blood culture and the genetic test of bacterial 16 S rRNA quantitative fluorescent PCR respectively by ready samples.
Two, sample detection result
The standard items testing result is seen Fig. 3.The typical curve that detects is seen Fig. 4.
830 routine patient's blood specimen quantitative fluorescent PCRs and blood culture testing result are as follows:
Table 316SrRNA gene by fluorescence quantitative PCR (FQ-PCR) and blood culture Bacteria Detection result contrast
Bacterial 16 S rRNA gene by fluorescence quantitative PCR detects positive rate 5.18% (43/830), blood culture with positive bacteria rate 2.41% (20/830), the former is apparently higher than the latter, and difference has statistical significance P＜0.01, and 20 routine sample fluorescence quantitative PCR detection all positive (table 3) of blood culture with positive bacteria.30 routine noninfectious disease infant fluorescence quantitative PCR detection and microbe growth are all negative.If with blood culture in contrast, the diagnostic sensitivity of bacterial fluorescence quantifying PCR method is 100%, and specificity is 97.16%, and correctly diagnosing index is 0.972.
The present invention is described in conjunction with most preferred embodiment, yet after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The sequence that the present invention relates to:
＜110〉Zhejiang University
＜120〉bacteria real-time fluorescence quantitative polymerase chain reaction detection reagent kit and application thereof
<160>4
<210>1
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉bacterial detection 16S rRNA upstream region of gene universal primer sequence
<400>1
CAACGCGAAGAACCTTACC 19
<210>2
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉bacterial detection 16S rRNA gene downstream universal primer sequence
<400>2
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the general fluorescence probe sequence of bacterial detection 16S rRNA gene
<400>3
TAAGTCCCGCAACGAGCGCAA 21
<210>4
<211>228
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the fluorescent quantitation examination criteria product sequence that designs according to bacterial 16 S rRNA gene order
<400>4
CAACGCGAAG?AACCTTACCT?GGTCTTGACA?TCCACAGAAC?TTTCCAGAGA?TGGATTGGTG 60
CCTTCGGGAA?CTGTGAGACA?GGTGCTGCAT?GGCTGTCGTC?AGCTCGTGTT?GTGAAATGTT 120
GGGTTAAGTC?CCGCAACGAG?CGCAACCCTT?ATCCTTTGTT?GCCAGCGGTC?CGGCCGGGAA 180
CTCAAAGGAG?ACTGCCAGTG?ATAAACTGGA?GGAAGGTGGG?GATGACGT 228
Claims (1)
1. bacteria real-time fluorescence quantitative polymerase chain reaction detection reagent kit, it is characterized in that by PCR reactant liquor (1), standard items (2), positive reference substance (3), negative control product (4), extracting lysate (5), operational manual (6), Packaging box body (7) is formed, Packaging box body (7) is provided with container hole, place PCR reactant liquor pipe (1) respectively, standard items (2), positive reference substance (3), negative control product (4), extracting lysate (5), wherein positive reference substance (3) is an Escherichia coli deactivation DNA sample, negative control product (4) are the sterile water for injection sample, PCR reactant liquor (1) is by single tube packing and arranged, and wherein the quantitative PCR reactant liquor contains the PCR damping fluid, MgCl
2, dNTPs, Bacteria Detection with upstream primer, detect with downstream primer, fluorescence probe, hot resistant DNA polymerase, wherein:
The upstream primer sequence is: 5`-CAACGCGAAGAACCTTACC-3`,
The downstream primer sequence is: 5 '-ACGTCATCCCCACCTTCC-3 ',
The fluorescence probe sequence is: 5 '-FAM-TAAGTCCCGCAACGAGCGCAA-TAMRA-3 ',
The standard items sequence is:
CAACGCGAAG?AACCTTACCT?GGTCTTGACA?TCCACAGAAC?TTTCCAGAGA
TGGATTGGTG?CCTTCGGGAA?CTGTGAGACA?GGTGCTGCAT?GGCTGTCGTC
AGCTCGTGTT?GTGAAATGTT?GGGTTAAGTC?CCGCAACGAG?CGCAACCCTT
ATCCTTTGTT?GCCAGCGGTC?CGGCCGGGAA?CTCAAAGGAG?ACTGCCAGTG
ATAAACTGGA?GGAAGGTGGG?GATGACGT
The extracting lysate contains the 10mmol/L Tri-HCl of pH 7.6,5mmol/L disodium ethylene diamine tetraacetate, 0.5% lauryl sodium sulfate; The quantitative PCR reactant liquor must be with 0.22 μ m membrane filtration pre-service.
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| CN200710070629A CN101144775B (en) | 2007-08-31 | 2007-08-31 | Bacteria real-time fluorescence quantitative polymerase chain reaction detection reagent kit |
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| CN101144775B true CN101144775B (en) | 2010-05-26 |
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Cited By (1)
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| CN103789426A (en) * | 2014-01-21 | 2014-05-14 | 浙江大学 | Real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for Gram positive bacteria |
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| CN101649318B (en) * | 2009-07-28 | 2011-07-06 | 鲁辛辛 | Pretreatment method for pathogenic bacteria gene identification in clinical samples |
| CN102321740A (en) * | 2011-08-03 | 2012-01-18 | 河北省健海生物芯片技术有限责任公司 | Identification technology and assay kit for infectious bacteria |
| CN103789425A (en) * | 2014-01-21 | 2014-05-14 | 浙江大学 | Gram-negative bacteria real-time quantitative PCR (polymerase chain reaction) detection kit |
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| CN106755301A (en) * | 2016-11-18 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of real-time fluorescence quantitative PCR kit |
| CN109439737A (en) * | 2019-01-09 | 2019-03-08 | 赵志军 | A kind of kit and its diagnostic device for cerebrospinal fluid difficulty pathogen diagnosis |
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