CN103571939B - Fluorescent quantitative polymerase chain reaction (PCR) method for detecting tubercle bacillus infection from clinical specimen - Google Patents

Fluorescent quantitative polymerase chain reaction (PCR) method for detecting tubercle bacillus infection from clinical specimen Download PDF

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CN103571939B
CN103571939B CN201310068046.2A CN201310068046A CN103571939B CN 103571939 B CN103571939 B CN 103571939B CN 201310068046 A CN201310068046 A CN 201310068046A CN 103571939 B CN103571939 B CN 103571939B
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primer seq
group
forward primer
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CN103571939A (en
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张凤民
宋武琦
黄颖
陈小贝
李爱梅
钱均
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Harbin Engineering University
Harbin Medical University
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    • C12Q2545/10—Reactions characterised by their quantitative nature the purpose being quantitative analysis
    • C12Q2545/114—Reactions characterised by their quantitative nature the purpose being quantitative analysis involving a quantitation step

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Abstract

The invention relates to a fluorescent quantitative PCR detection method for detecting tubercle bacillus infection from a clinical specimen. The fluorescent quantitative PCR detection method is used for detecting small RNA (Ribonucleic Acid) of tubercle bacillus in the clinical specimen. The method comprises the following steps: 1, extracting total RNA in the clinical specimen; 2, performing reverse transcription by taking the total RNA extracted in the step 1 to synthesize cDNA (complementary Deoxyribonucleic Acid); 3, performing fluorescent quantitative PCR detection aiming at a tubercle bacillus small RNA detection primer and cDNA synthesized in the step 2.

Description

The fluorescence quantifying PCR method of tubercle bacillus affection is detected from clinical samples
Technical field
The present invention relates to the fluorescence quantifying PCR method detecting tubercle bacillus affection from clinical samples.
Background technology
In prior art, conventional tubercle bacillus affection detection method CSF acid-fast bacilli smear method and mycobacterium tuberculosis cultivate detection method.The weak point that CSF acid-fast bacilli smear method exists is that positive rate is low, about 10%, and needs bacterium>=10 in sample 4/ ml just can find, poor specificity.The weak point that mycobacterium tuberculosis cultivates detection method existence is complicated operation, and incubation time is longer, need 4 ~ 8 weeks, and positive rate is low, how 20 ~ 30%.
Summary of the invention
Goal of the invention of the present invention is to provide a kind of fluorescence quantifying PCR method detecting tubercle bacillus affection from clinical samples, and simple to operate, consuming time short, positive rate is high.
Realize the technical scheme of the object of the invention:
From clinical samples, detect a fluorescence quantifying PCR method for tubercle bacillus affection, it is characterized in that: with fluorescent quantitative PCR detection method, the tiny RNA of tubercule bacillus in clinical samples is detected.
Step one: extract the total serum IgE in clinical samples;
Step 2: with the total serum IgE extracted in step one for template reverse transcription synthesis cDNA;
Step 3: detect for tubercule bacillus tiny RNA the cDNA synthesized in primer and step 2 and carry out fluorescence quantitative PCR detection.
Preferably, in step 3, quantitative fluorescent PCR reaction system comprises 2 times of concentrated Fluorescence PCR damping fluids, tubercule bacillus tiny RNA detects the cDNA template of synthesizing in primer, step 2.
Preferably, in step 3, quantitative fluorescent PCR reaction conditions is, 95 DEG C 10 seconds, 60 DEG C 20 seconds, repeat 50 circulations; Gather fluorescent brightness value during 60 DEG C of extensions, product slowly heats up through 65 DEG C-95 DEG C, detects fluorescent brightness simultaneously, draws solubility curve; According to solubility curve and standard comparison, judge that whether sample reaction product is correct, contrast with normal people's sample, the clinical samples that tiny RNA content obviously raises is tuberculosis infection patient.
Preferably, in step 3, quantitative fluorescent PCR reaction system is: 2 times of concentrated quantitative fluorescent PCR reaction mixture 10 microlitres, and tubercule bacillus tiny RNA detects primer 1 microlitre, and cDNA template 1 microlitre synthesized in step 2, adds deionized water and supplement volume to 20 microlitre.
It can be following group of primer wherein one group that tubercule bacillus tiny RNA detects primer:
First group: forward primer SEQ ID NO.1:5 '-TCGTCGTCGACGCTTAGGTA-3 '
Reverse primer SEQ ID NO.2:5 '-TGGGTTGGACGACTCGACGT-3 '
Second group: forward primer SEQ ID NO.3:5 '-ATCGGTGCCGGACCAGTTCA-3 '
Reverse primer SEQ ID NO.4:5 '-AGCTGGTACTTCGCACCGGA-3 '
3rd group: forward primer SEQ ID NO.5:5 '-CGGCCTGCGTATGACCCATA-3 '
Reverse primer SEQ ID NO.6:5 '-AGGCGTTGTTGGCCACTTAT-3 '
4th group: forward primer SEQ ID NO.7:5 '-CGGATAGCCCCGTGTTGTTG-3 '
Reverse primer SEQ ID NO.8:5 '-CTGGGTCCCCTCCCACCAGC-3 '
5th group: forward primer SEQ ID NO.9:5 '-ATAGAGGACGGAGTCGGTGA-3 '
Reverse primer SEQ ID NO.10:5 '-AGAAACTTCGCCTCGAGGTA-3 '
6th group: forward primer SEQ ID NO.11:5 '-CCACGTACTCGATCCCGTTG-3 '
Reverse primer SEQ ID NO.12:5 '-ACTTGGGGGGATTGGGAGGT-3 '
7th group: forward primer SEQ ID NO.13:5 '-AACTCCCCCAGGGCTGGAAG-3 '
Reverse primer SEQ ID NO.14:5 '-CGTGGGTATACCGAGGTCCA-3 '
8th group: forward primer SEQ ID NO.15:5 '-CTCGGCCATAGCCGAGGGTG-3 '
Reverse primer SEQ ID NO.16:5 '-AGGGACGACCCCCGCCAGGG-3 '
9th group: forward primer SEQ ID NO.17:5 '-CGGGACTCCTGAGAAGGATC-3 '
Reverse primer SEQ ID NO.18:5 '-CGCACGTCAGCCCCACCCGT-3 '
The beneficial effect that the present invention has:
The present invention's fluorescent quantitative PCR detection method detects the tiny RNA of tubercule bacillus in clinical samples, and peculiar design is carried out to quantitative fluorescent PCR reaction system and reaction conditions, tubercule bacillus tiny RNA detection primer, detect simple to operate, consuming time short, positive rate is high (can reach 60%-70%), be convenient to extensive examination detect.The tiny RNA of tubercule bacillus is free to be present in outside bacterium, and sample easily obtains, and cultivates without the need to tubercule bacillus, safer, quick, greatly shortens detection time; The tiny RNA of tubercule bacillus can resist the Degradation of RNA enzyme, and Absorbable organic halogens exists, and effectively improves the susceptibility of detection.The present invention, can low-level tubercule bacillus tiny RNA in highly sensitive detection sample to the peculiar design of quantitative fluorescent PCR reaction system and reaction conditions.The present invention is according to the tiny RNA feature of tubercule bacillus, and detect primer to tubercule bacillus tiny RNA and design, sequence is optimized more.
Accompanying drawing explanation
Fig. 1 is each group of primer pair mycobacterium tuberculosis infection patients blood plasma Samples detection fluorescent quantitative PCR result figure, and every bar curve represents a kind of fluorescence results of product of primer;
Fig. 2 is the fluorescent quantitative PCR result figure of U6 gene and G2 tiny RNA in the blood plasma of normal people and tuberculosis infection patient;
Fig. 3 is the solubility curve figure of quantitative fluorescent PCR product;
Fig. 4 is content and normal people's comparision contents figure of tiny RNA in tuberculosis infected students blood plasma.
Embodiment
Obtain clinical samples: gather peripheral blood in patients 3-5 milliliter, 1000-1500 rev/min, horizontal centrifugal 10 minutes.In careful absorption upper plasma 200 microlitre to aseptic 1.5 milliliters of centrifuge tubes.Other liquid samples, through 13000 revs/min, centrifugal 10 minutes, are carefully drawn in supernatant liquid 200 microlitre to aseptic 1.5 milliliters of centrifuge tubes.
Step one: the total serum IgE extracting clinical samples.
In 200 microlitre clinical samples, add 500 microlitre phenol guanidine RNA extracting solutions, fully mix, room temperature leaves standstill 5 minutes.Add 300 microlitre chloroforms, mix latter 12000 revs/min centrifugal 10 minutes.In the aseptic 2 milliliters of centrifuge tubes of careful absorption supernatant liquid body to.Add 900 L acetone, adsorb with pellosil after mixing.Protein wash rinses once, and 100% ethanol and 80% ethanol rinse once successively, after ethanol volatilization, with TE or deionized water eluted rna, collects with aseptic 1.5 milliliters of centrifuge tubes, is the total serum IgE in clinical samples.
Step 2: with the total serum IgE extracted in step one for template reverse transcription synthesis cDNA.
Reaction system: total serum IgE gets 10 microlitres, 9 bases are primer 1 microlitre immediately, dNTP1 microlitre, reversed transcriptive enzyme 100 unit, 5 times of concentration response damping fluid 4 microlitres, and 0.1M dithiothreitol (DTT) 1 microlitre, deionized water supplements volume to 20 microlitres.
Reaction conditions: 50 DEG C one hour, 75 DEG C after 15 minutes, cryopreservation, is cDNA.
Step 3: detect for tubercule bacillus tiny RNA the cDNA synthesized in primer and step 2 and carry out fluorescence quantitative PCR detection.
Quantitative fluorescent PCR reaction system: 2 times of concentrated quantitative fluorescent PCR reaction mixture 10 microlitres, tubercule bacillus tiny RNA detects primer 1 microlitre, and cDNA template 1 microlitre synthesized in step 2, adds deionized water and supplement volume to 20 microlitre.
Quantitative fluorescent PCR reaction conditions: 95 DEG C 10 seconds, 60 DEG C 20 seconds, repeat 50 circulations.Fluorescent brightness value is gathered during 60 DEG C of extensions.Product slowly heats up through 65 DEG C-95 DEG C, detects fluorescent brightness simultaneously, draws solubility curve.
Result judges: according to solubility curve and standard comparison, judges that whether sample reaction product is correct, contrasts with normal people's sample, and that tiny RNA content obviously raises is tuberculosis infection patient.
It can be following group of primer wherein one group that tubercule bacillus tiny RNA detects primer:
First group: forward primer SEQ ID NO.1:5 '-TCGTCGTCGACGCTTAGGTA-3 '
Reverse primer SEQ ID NO.2:5 '-TGGGTTGGACGACTCGACGT-3 '
Second group: forward primer SEQ ID NO.3:5 '-ATCGGTGCCGGACCAGTTCA-3 '
Reverse primer SEQ ID NO.4:5 '-AGCTGGTACTTCGCACCGGA-3 '
3rd group: forward primer SEQ ID NO.5:5 '-CGGCCTGCGTATGACCCATA-3 '
Reverse primer SEQ ID NO.6:5 '-AGGCGTTGTTGGCCACTTAT-3 '
4th group: forward primer SEQ ID NO.7:5 '-CGGATAGCCCCGTGTTGTTG-3 '
Reverse primer SEQ ID NO.8:5 '-CTGGGTCCCCTCCCACCAGC-3 '
5th group: forward primer SEQ ID NO.9:5 '-ATAGAGGACGGAGTCGGTGA-3 '
Reverse primer SEQ ID NO.10:5 '-AGAAACTTCGCCTCGAGGTA-3 '
6th group: forward primer SEQ ID NO.11:5 '-CCACGTACTCGATCCCGTTG-3 '
Reverse primer SEQ ID NO.12:5 '-ACTTGGGGGGATTGGGAGGT-3 '
7th group: forward primer SEQ ID NO.13:5 '-AACTCCCCCAGGGCTGGAAG-3 '
Reverse primer SEQ ID NO.14:5 '-CGTGGGTATACCGAGGTCCA-3 '
8th group: forward primer SEQ ID NO.15:5 '-CTCGGCCATAGCCGAGGGTG-3 '
Reverse primer SEQ ID NO.16:5 '-AGGGACGACCCCCGCCAGGG-3 '
9th group: forward primer SEQ ID NO.17:5 '-CGGGACTCCTGAGAAGGATC-3 '
Reverse primer SEQ ID NO.18:5 '-CGCACGTCAGCCCCACCCGT-3 '

Claims (2)

1., with the reagent that fluorescent quantitative PCR detection method detects the tiny RNA of tubercule bacillus in clinical sample, it is characterized in that: it comprises:
One, the reagent of the total serum IgE in clinical samples is extracted;
Two, with the reagent of the total serum IgE extracted for template reverse transcription synthesis cDNA;
Three, tubercule bacillus tiny RNA detects primer and the cDNA of synthesis is carried out to the reagent of fluorescence quantitative PCR detection;
Wherein, tubercule bacillus tiny RNA detects the combination that primer is following group of primer:
First group: forward primer SEQ ID NO.1:5'-TCGTCGTCGACGCTTAGGTA-3'
Reverse primer SEQ ID NO.2:5'-TGGGTTGGACGACTCGACGT-3'
Second group: forward primer SEQ ID NO.3:5'-ATCGGTGCCGGACCAGTTCA-3'
Reverse primer SEQ ID NO.4:5'-AGCTGGTACTTCGCACCGGA-3'
3rd group: forward primer SEQ ID NO.5:5'-CGGCCTGCGTATGACCCATA-3'
Reverse primer SEQ ID NO.6:5'-AGGCGTTGTTGGCCACTTAT-3'
4th group: forward primer SEQ ID NO.7:5'-CGGATAGCCCCGTGTTGTTG-3'
Reverse primer SEQ ID NO.8:5'-CTGGGTCCCCTCCCACCAGC-3'
5th group: forward primer SEQ ID NO.9:5'-ATAGAGGACGGAGTCGGTGA-3'
Reverse primer SEQ ID NO.10:5'-AGAAACTTCGCCTCGAGGTA-3'
6th group: forward primer SEQ ID NO.11:5'-CCACGTACTCGATCCCGTTG-3'
Reverse primer SEQ ID NO.12:5'-ACTTGGGGGGATTGGGAGGT-3'
7th group: forward primer SEQ ID NO.13:5'-AACTCCCCCAGGGCTGGAAG-3'
Reverse primer SEQ ID NO.14:5'-CGTGGGTATACCGAGGTCCA-3'
8th group: forward primer SEQ ID NO.15:5'-CTCGGCCATAGCCGAGGGTG-3'
Reverse primer SEQ ID NO.16:5'-AGGGACGACCCCCGCCAGGG-3'
9th group: forward primer SEQ ID NO.17:5'-CGGGACTCCTGAGAAGGATC-3'
Reverse primer SEQ ID NO.18:5'-CGCACGTCAGCCCCACCCGT-3'.
2. reagent according to claim 1, is characterized in that: the cDNA template that quantitative fluorescent PCR reaction system comprises 2 times of concentrated Fluorescence PCR damping fluids, tubercule bacillus tiny RNA detects primer, synthesis.
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CN104313127B (en) * 2014-09-02 2016-05-18 哈尔滨医科大学 A kind of kit for quick specific detection mycobacterium tuberculosis infection and uses thereof
CN105671188B (en) * 2016-04-07 2020-01-24 深圳月曜生命科技有限公司 Molecular marker for diagnosing mycobacterium tuberculosis infection, primer set and application
CN112725484A (en) * 2021-01-21 2021-04-30 哈尔滨医科大学 Mycobacterium tuberculosis sRNA real-time fluorescence quantitative PCR standard substance and application thereof

Citations (1)

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Publication number Priority date Publication date Assignee Title
CN101974630A (en) * 2010-10-28 2011-02-16 复旦大学 Fluorescent quantitative RT-PCR detection method of M.tuberculosis-complex

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101974630A (en) * 2010-10-28 2011-02-16 复旦大学 Fluorescent quantitative RT-PCR detection method of M.tuberculosis-complex

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Title
A Genome-Wide Identification Analysis of Small Regulatory RNAs in Mycobacterium tuberculosis by RNASeq and Conservation Analysis;Danilo Pellin et al.;《PLoS ONE》;20120328;第7卷(第3期);e32723 *
Identification of small RNAs in Mycobacterium tuberculosis;Kristine B. et al.;《Molecular Microbiology》;20090710;第73卷(第3期);第400页表1，第402页右栏20-21行 *

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