CN103789426A - Real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for Gram positive bacteria - Google Patents

Real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for Gram positive bacteria Download PDF

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CN103789426A
CN103789426A CN201410027285.8A CN201410027285A CN103789426A CN 103789426 A CN103789426 A CN 103789426A CN 201410027285 A CN201410027285 A CN 201410027285A CN 103789426 A CN103789426 A CN 103789426A
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gram positive
quantitative pcr
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positive organism
positive bacteria
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杜立中
尚世强
舒强
陶然
李伟
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Zhejiang University ZJU
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Abstract

The invention provides a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for Gram positive bacteria. The detection kit consists of Gram positive bacteria DNA (Deoxyribonucleic Acid) leaching liquor, quantitative PCR reaction liquid, a standard, a positive reference substance, a negative reference substance, a specification and a box, wherein the quantitative PCR reaction liquid comprises a PCR buffer solution, MgC12, dNTPs, heat-resistant DNA polymerase, an upstream amplification primer, a downstream amplification primer and a Gram positive bacteria fluorescent probe. By adopting the real-time fluorescent quantitative PCR detection kit developed by taking a Gram positive bacteria 16SrRNA (Ribonucleic Acid) gene as a target gene, all Gram positive bacteria can be detected easily, conveniently, rapidly and universally, Gram positive bacteria with positive detection results can be quantified accurately, the universality and sensitivity of Gram positive bacteria detection are improved, and a basis can be laid for early clinical diagnosis of all Gram positive bacterial infection.

Description

Gram positive organism real-time fluorescence quantitative PCR detection kit
Technical field
The invention belongs to biological technical field, relate to fluorescent quantificationally PCR detecting kit, be specifically related to the diagnostic kit of gram positive organism in a kind of real-time fluorescence quantitative PCR detection blood samples of patients, cerebrospinal fluid, hydrothorax, ascites equal samples.
Background technology
Gram positive organism is the important pathogen of community and hospital infection.In recent years, gram positive organism infects and is increasing year by year trend, as gram positive organism dominate gradually in the pathogenic bacteria of the infectious diseases such as hospital acquired septicemia.Along with extensive, the irrational application of microbiotic, the gram positive organism of Multiple Classes of Antibiotics resistance is also increased sharply, as methicillin-resistant staphylococcus aureus (MRSA), penicillin resistance pneumococcus (PRSP), vancomycin-resistant enterococcus (VRE) etc.Gram positive organism is made in early days, is detected and identify accurately, can be in time for clinician provides reliable etiology information, to the rational antibacterial therapy of patient, reduce persister generation significant.The method that clinical diagnosis gram positive organism infects at present comprises routine blood test, c reactive protein, hemoculture, focus secretory product smear and cultivation, pathogenic bacteria antigen or antibody test etc., but exist take long, easily pollute, positive rate is low, poor specificity, can not quantitatively wait shortcoming, cannot meet clinical early stage, fast, the needs that infect of Accurate Diagnosis gram positive organism.
Along with developing rapidly of Protocols in Molecular Biology, polymerase chain reaction (polymerase chain reaction, PCR) technology, the detection that the Real-Time Fluorescent Quantitative PCR Technique particularly rising is in recent years pathogenic micro-organism provides new direction.The ultimate principle of Real-Time Fluorescent Quantitative PCR Technique is to utilize 5 '-3 ' excision enzyme nucleic acid activity of Taq enzyme, designs fluorescence double-tagging probe on regular-PCR basis, and two ends are mark fluorescent reporter group (R) and quenching group (Q) respectively; When probe keeps complete, the fluorescent signal of R group is suppressed by Q group, once probe is cut off, the restraining effect of Q group disappears, and the fluorescent signal of R group just can be detected.This technology is by realizing the Real-Time Monitoring to product amount in PCR process to the detection of fluorescent signal, and can accurate calculation original template amount, except having quantitatively accurately, detect the advantage such as quick, maximum advantage is to adopt complete stopped pipe to detect, save the aftertreatment to PCR product, avoided crossed contamination.
Bacterial 16 S rRNA gene is the corresponding DNA sequence dna of rRNA of encoding on bacterial chromosome, this gene has two large features: (1) multiple copied: 16S rRNA gene is present in all bacterial chromosome genomes with the multiple copied form of 3-7 copy, makes that the detection of this gene is had to higher susceptibility; (2) many information: 16S rRNA gene is made up of variable region and conserved regions, conserved regions is that all bacteriums are total, variable region can be used for the somatotype of different bacterium.The 16S rRNA gene sequencing of current nearly all pathogenic bacteria completes, and selects 16S rRNA gene to become just gradually the gold standard of discrimination of bacteria, classification as the target sequence of bacterial gene classification.
The present invention is based on above-mentioned research background, with common conserved sequence design pcr amplification primer and fluorescent probe in gram positive organism 16S rRNA gene, developed and can be used for the real-time fluorescence quantitative PCR test kit that all gram positive organisms detect.This test kit can not only detect all gram positive organisms pointedly, and can carry out accurate quantitative analysis to the gram positive organism infecting, and has the features such as easy, quick, efficient, is applicable to early stage, quick diagnosis that clinical all gram positive organisms infect.
Summary of the invention
The object of this invention is to provide a kind of real-time fluorescence quantitative PCR detection kit of gram positive organism, be made up of gram positive organism DNA extract, quantitative PCR reaction solution, standard substance, positive reference substance, negative control product, specification sheets and box body, wherein quantitative PCR reaction solution contains PCR damping fluid, MgCl2, dNTPs, hot resistant DNA polymerase, upstream amplimer, downstream amplimer and gram positive organism fluorescent probe.
Pcr amplification primer sequence is:
Upstream amplimer (SEQ ID No:1): 5 '-CAACGCGAAGAACCTTACC-3 ';
Downstream amplimer (SEQ ID No:2): 5 '-ACGTCATCCCCACCTTCC-3 '.
Fluorescent probe and fluorescent marker are:
Gram positive organism fluorescent probe (SEQ ID No:3): 5 '-FAM-TGACGACAACCATGCACCACC-BHQ-1-3 '.
Standard substance sequence is (SEQ ID No:4): CAACGCGAAG AACCTTACCA AATCTTGACA TC CTTTGACA ACTCTAGAGA TAGAGCCTTC CTCTTCGGGG GACAAAGTGA CAGGTGG TGC ATGGTTGTCG TCAGCTCGTG TCGTGAGATG TTGGGTTAAG TCCCGCAACG AG CGCAACCC TTAAGCTTAG TTGCCATCAT TAAGTTGGGC ACTCTAAGTT GACTGCC GGT GACAAACCGG AGGAAGGTGG GGATGACGT.
Gram positive organism DNA extract contains 10mmol/L Tri-HCl, 5mmol/L disodium ethylene diamine tetraacetate (pH7.6) and 0.5% sodium lauryl sulphate.
Positive reference substance is streptococcus aureus deactivation DNA sample, and negative control product are sterile water for injection sample.
Test kit of the present invention should be stored in-20 ℃, reduces multigelation as far as possible.
Test kit using method of the present invention:
Each detection all should be set up positive control and negative control, and standard substance are 1 × 10 with aseptic deionized water dilution 2-1 × 10 9copy/ml.
The extraction of gram positive organism DNA: gather fresh whole blood 1ml and put in the aseptic vacuum test tube of Sodium Citrate anti-freezing, after mixing, getting whole blood 50 μ l adds equivalent DNA extract and puts 100 ℃ and boil 10 minutes, 12000 revs/min centrifugal 5 minutes, get 5 μ l supernatants as pcr template.In inoculum, cerebrospinal fluid, hydrothorax, ascites equal samples, the extracting method of gram positive organism DNA is the same.
Pcr amplification detects: on quantitative real time PCR Instrument, carry out, each PCR reaction system cumulative volume is 50 μ l, comprising 45 μ l PCR reaction solutions and 5 μ l templates (the gram positive organism DNA of extraction, standard substance, positive or negative contrast).PCR reaction conditions: 94 ℃ of 5 minutes denaturations, 94 ℃ 20 seconds → 60 ℃ 60 seconds, totally 40 circulations.After setting completes, preserve file, working procedure.
Fluorescent quantitation report the test: 1. detect the amplification curve of sample without increased logarithmic phase or CT(threshold cycle, the fluorescent signal in each reaction tubes reaches sets thresholding required cycle number) value >=40 are gram positive organism feminine gender; 2. detecting sample CT value≤35 and amplification curve, to have obvious increased logarithmic phase be the gram positive organism positive; 3. detect sample 35 < CT value < 40, need re-start DNA extraction and PCR detection to sample.According to typical curve, calculate the gram positive organism quantity (copy/ml) in each sample.
The real-time fluorescence quantitative PCR test kit that the present invention develops take gram positive organism 16S rRNA gene as target gene, the all gram positive organisms of easy, quick, the general detection of energy, and can carry out accurate quantitative analysis to the gram positive organism of the detected result positive, improved versatility and the susceptibility of gram positive organism identification and detection, the early diagnosis that can be clinical all gram positive organism infection provides foundation.
Accompanying drawing explanation
Fig. 1 is the structural representation of test kit of the present invention.
Fig. 2 is reference culture streptococcus aureus fragment sequence.
Embodiment
The present invention is described further with accompanying drawing in conjunction with the embodiments.Should be appreciated that, these embodiment, only for illustration purpose, limit the scope of the invention and be not used in.
Embodiment 1
Referring to Fig. 1, gram positive organism real-time fluorescence quantitative PCR detection kit provided by the invention, formed by gram positive organism DNA extract (1), quantitative PCR reaction solution (2), standard substance (3), positive reference substance (4), negative control product (5), specification sheets (6) and box body (7), the packing of quantitative PCR reaction solution single tube, arranged.
Wherein quantitative PCR reaction solution contains PCR damping fluid, MgCl 2, dNTPs, hot resistant DNA polymerase, upstream amplimer, downstream amplimer and gram positive organism fluorescent probe.
Pcr amplification primer sequence is:
Upstream amplimer (SEQ ID No:1): 5 '-CAACGCGAAGAACCTTACC-3 ';
Downstream amplimer (SEQ ID No:2): 5 '-ACGTCATCCCCACCTTCC-3 '.
Fluorescent probe and fluorescent marker are:
Gram positive organism fluorescent probe (SEQ ID No:3): 5 '-FAM-TGACGACAACCATGCACCACC-BHQ-1-3 '.
Standard substance sequence is (SEQ ID No:4): CAACGCGAAG AACCTTACCA AATCTTGACA TC CTTTGACA ACTCTAGAGA TAGAGCCTTC CTCTTCGGGG GACAAAGTGA CAGGTGG TGC ATGGTTGTCG TCAGCTCGTG TCGTGAGATG TTGGGTTAAG TCCCGCAACG AG CGCAACCC TTAAGCTTAG TTGCCATCAT TAAGTTGGGC ACTCTAAGTT GACTGCC GGT GACAAACCGG AGGAAGGTGG GGATGACGT.
Gram positive organism DNA extract contains 10mmol/L Tri-HCl, 5mmol/L disodium ethylene diamine tetraacetate (pH7.6) and 0.5% sodium lauryl sulphate.
Positive reference substance is streptococcus aureus deactivation DNA sample, and negative control product are sterile water for injection sample.
Test kit of the present invention should be stored in-20 ℃, reduces multigelation as far as possible.
Embodiment 2 gram positive organism real-time fluorescence quantitative PCR detection kits detect the strain of clinical common gram positive organism separation and Culture
(1) material:
Selection pathogenic micro-organism comprises: experimental group: clinical 25 bacterial strains of 5 Pseudomonas of common gram positive organism that cause septicemia and purulent meningitis; Control group: clinical 17 Pseudomonas of gram-negative 25 bacterial strains, human cytomegalic inclusion disease virus, Epstein-Barr virus, hepatitis B virus, Cryptococcus neoformans, Candida albicans and human genome DNAs that cause septicemia and purulent meningitis.Above material provides by Medical College of Zhejiang Univ.'s attached children's hospital Bacteriology Room and infectious laboratory.
(2) design of primer and probe is with synthetic:
From GenBank, screen the 16S rRNA gene order of different gram positive organisms, these sequences of application MegAlign software analysis, find the conservative fragments between different gram positive organism kinds, analyze the rule of organic evolution tree, at gram positive organism conservative region screening universal primer and general probe, entrust the Shanghai biological company limited of raw work synthetic.
Upstream amplimer sequence is: 5 '-CAACGCGAAGAACCTTACC-3 ';
Downstream amplimer sequence is: 5 '-ACGTCATCCCCACCTTCC-3 ';
Gram positive organism fluorescent probe sequence is: 5 '-FAM-TGACGACAACCATGCACCACC-BHQ-1-3 '.
(3) preparation of examination criteria product:
With above-mentioned primer amplification reference culture streptococcus aureus, PCR product length is 229bp.PCR product through identify and purifying after insert pGEM-T-Easy cloning vector with cloning system, extracting recombinant clone plasmid, carries out sequence verification, measures concentration and is converted into copy number/volume the positive colony of recombinating with carrier primer M13R.
Result: through order-checking, above-mentioned standard product conform to expection completely, and standard substance fragment sequence is streptococcus aureus, referring to Fig. 2.
(4) real-time fluorescence quantitative PCR of clinical common gram positive organism separation and Culture strain detects:
To experimental group 25 strain gram positive organism, the detection of single tube real-time fluorescence quantitative PCR is carried out in clinical separation and Culture strain, and result is all positive, and CT value is (referring to table 1) between 14-23; The clinical separation and Culture strain of control group 25 strain gram-negative bacteria, human cytomegalic inclusion disease virus, Epstein-Barr virus, hepatitis B virus, Cryptococcus neoformans, Candida albicans and all negative (referring to table 2) of human genome DNA's detected result.
Table 1 gram positive organism real-time fluorescence quantitative PCR detection kit detects common gram positive organism result
Figure BDA0000459836400000041
Table 2 gram positive organism real-time fluorescence quantitative PCR detection kit detects control sample result
The present invention is described in conjunction with most preferred embodiment, but is reading after foregoing of the present invention, and those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the scope of the application's appended claims institute limit equally.
<110> Zhejiang University
<120> gram positive organism real-time fluorescence quantitative PCR detection kit
<160> 4
<210> 1
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> detects the general upstream primer sequence of gram positive organism
<400> 1
CAACGCGAAGAACCTTACC 19
<210> 2
<211> 18
<212> DNA
<213> artificial sequence
<220>
<223> detects the general downstream primer sequence of gram positive organism
<400> 2
ACGTCATCCCCACCTTCC 18
<210> 3
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> detects gram positive organism fluorescent probe sequence
<400> 3
TGACGACAACCATGCACCACC 21
<210> 4
<211> 229
<212> DNA
<213> artificial sequence
<220>
<223> is according to the gram positive organism fluorescent quantitation examination criteria product sequence of gram positive organism 16S rRNA gene order design
<400> 4
CAACGCGAAG AACCTTACCA AATCTTGACA TCCTTTGACA ACTCTAGAGA TAGAGCCTTC 60
CTCTTCGGGG GACAAAGTGA CAGGTGGTGC ATGGTTGTCG TCAGCTCGTG TCGTGAGATG 120
TTGGGTTAAG TCCCGCAACG AGCGCAACCC TTAAGCTTAG TTGCCATCAT TAAGTTGGGC 180
ACTCTAAGTT GACTGCCGGT GACAAACCGG AGGAAGGTGG GGATGACGT 229

Claims (4)

1. a gram positive organism real-time fluorescence quantitative PCR detection kit, formed by gram positive organism DNA extract (1), quantitative PCR reaction solution (2), standard substance (3), positive reference substance (4), negative control product (5), specification sheets (6) and box body (7), the packing of quantitative PCR reaction solution single tube, arranged, wherein quantitative PCR reaction solution contains PCR damping fluid, MgCl 2, dNTPs, hot resistant DNA polymerase, upstream amplimer, downstream amplimer and gram positive organism fluorescent probe;
Pcr amplification primer sequence is:
Upstream amplimer: 5 '-CAACGCGAAGAACCTTACC-3 ';
Downstream amplimer: 5 '-ACGTCATCCCCACCTTCC-3 ';
Gram positive organism fluorescent probe: 5 '-FAM-TGACGACAACCATGCACCACC-BHQ-1-3 ';
Standard substance sequence is: CAACGCGAAG AACCTTACCA AATCTTGACA TCCTTTGACA ACTCTAGAGA TAGAGCCTTC CTCTTCGGGG GACAAAGTGA CAGGTGGTGC ATGGTTGTCG TCAGCTCGTG TCGTGAGATG TTGGGTTAAG TCCCGCAACG AGCGCAACCC TTAAGCTTAG TTGCCATCAT TAAGTTGGGC ACTCTAAGTT GACTGCCGGT GACAAACCGG AGGAAGGTGG GGATGACGT.
2. a kind of gram positive organism real-time fluorescence quantitative PCR detection kit according to claim 1, it is characterized in that the 5mmol/L disodium ethylene diamine tetraacetate that gram positive organism DNA extract contains 10mmol/L Tri-HCl, pH 7.6 and 0.5% sodium lauryl sulphate.
3. a kind of gram positive organism real-time fluorescence quantitative PCR detection kit according to claim 1, is characterized in that, positive reference substance is streptococcus aureus deactivation DNA sample, and negative control product are sterile water for injection sample.
4. a kind of gram positive organism real-time fluorescence quantitative PCR detection kit according to claim 1, is characterized in that, test kit of the present invention should be stored in-20 ℃, reduces multigelation as far as possible.
CN201410027285.8A 2014-01-21 2014-01-21 Real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for Gram positive bacteria Pending CN103789426A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101144775B (en) * 2007-08-31 2010-05-26 浙江大学 Bacteria real-time fluorescence quantitative polymerase chain reaction detection reagent kit
CN101200765B (en) * 2007-11-27 2010-11-24 浙江大学 Gram bacteria retest real-time fluorescent quantitative PCR detection reagent kit and use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101144775B (en) * 2007-08-31 2010-05-26 浙江大学 Bacteria real-time fluorescence quantitative polymerase chain reaction detection reagent kit
CN101200765B (en) * 2007-11-27 2010-11-24 浙江大学 Gram bacteria retest real-time fluorescent quantitative PCR detection reagent kit and use

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AGGARWAL P: "bistro-primer-tool to design and validate specific pcr primer pairs for phylogenetic analysis", 《MARQUETTE UNIVERSITY E-PUBLICATIONS@MARQUETTE》, 31 December 2011 (2011-12-31), pages 53 *

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Application publication date: 20140514