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Real-time fluorescent quantitative PCR nucleic acid sequences and kit for detecting Clostridium botulinum type A

A Clostridium botulinum and nucleic acid sequence technology, applied in the field of molecular detection of Clostridium botulinum, can solve the problems of time-consuming, labor-intensive sensitivity, low sensitivity, etc., and achieve the effect of sensitive response and good specificity

Inactive Publication Date: 2018-12-18
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection methods for Clostridium botulinum mainly include immunological methods such as isolation and culture, mouse animal experiments, indirect ELISA for toxins, and molecular biology methods such as ordinary PCR. Most of them have the disadvantages of time-consuming, laborious and low sensitivity

Method used

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  • Real-time fluorescent quantitative PCR nucleic acid sequences and kit for detecting Clostridium botulinum type A
  • Real-time fluorescent quantitative PCR nucleic acid sequences and kit for detecting Clostridium botulinum type A
  • Real-time fluorescent quantitative PCR nucleic acid sequences and kit for detecting Clostridium botulinum type A

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 Detects the design of special primers and probes for Clostridium botulinum type A

[0035] According to the Clostridium botulinum type A toxin gene sequence published on GenBank, use the CLUSTLAW software to perform multiple alignments of the Clostridium botulinum toxin A gene, select its stable conserved region as the detection target sequence, and design a pair of Primers and a probe, wherein the sequence of primers A-F is shown in SEQ ID No: 1; the sequence of primers A-R is shown in SEQ ID No: 2; the sequence of probes A-P is shown in SEQ ID No: 3. The 5' ends of the probes A-P are labeled with the reporter fluorophore FAM, and the 3' ends are labeled with the quencher fluorophore BHQ1. Finally, blast comparison with GenBank to determine the specificity of primers and probes, and then handed over to Shanghai Sangon Biotechnology Co., Ltd. for synthesis.

Embodiment 2

[0036] Embodiment 2 detects the composition of the kit of the fluorescent quantitative PCR of Clostridium botulinum type A

[0037] (1) qPCR reaction system components, including upstream primers A-F shown in sequence SEQ ID No: 1, downstream primers A-R shown in sequence SEQ ID No: 2 and probes A-P shown in sequence SEQ ID No: 3 , wherein the 5' end of the probe A-P is labeled with the reporter fluorescent group FAM, and the 3' end is labeled with the quencher fluorescent group BHQ1; the qPCR reaction system also includes qPCRSuperMix, the qPCR SuperMix contains dNTPs, Taq DNA polymerase and PCR reaction buffer. qPCR SuperMix was purchased from Quanshijin Biological Company.

[0038] (2) Negative control, using TE buffer, the composition is as follows: 10mM Tris-HCl, 1mM EDTA

[0039] (3) Positive control, Clostridium botulinum type A recombinant plasmid standard.

[0040] (4) Dilution buffer. Choose TransNGS from Quanshijin Biotechnology Co., Ltd. TM Library Dilution B...

Embodiment 3

[0041] Construction and preparation of embodiment 3 plasmid standard

[0042] A-F and A-R were used as primers for PCR amplification. The target product fragment was purified and recovered by gel cutting and ligated to pMD-18T vector, transformed into JM109 competent cells, screened positive clones, verified by PCR and sequenced for final confirmation.

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Abstract

The invention provides real-time fluorescent quantitative PCR nucleic acid sequences and a kit for detecting Clostridium botulinum type A. The nucleic acid sequences comprise an upstream primer A-F, adownstream primer A-R and a probe A-P. As the primers and the probe provided by the invention are used for detecting Clostridium botulinum type A, detection accuracy reaches 100%; and when the primers and the probe are used for amplification of other 25 intestinal pathogenic bacteria and common bacterial DNAs, no specific amplification curve appears, so the primers and the probe have good specificity. According to a fluorescence quantitative PCR standard curve constructed on the basis of a recombinant plasmid standard substance, it is determined that the detection sensitivity to Clostridium botulinum type A is 5.04*10<2> copies / [mu]l. In a fluorescent quantitative PCR detection system, when the concentration of a recombinant plasmid containing the A toxin gene is in the range of 10<2>-10<9> copies / [mu]l, the Ct values of amplification reactions and template concentrations are in good linear relationship. The real-time fluorescent quantitative PCR nucleic acid sequences and the kit ofinvention have the characteristics of sensitive reaction, high specificity, rapidity, convenience, high throughput, etc., and provide an effective detection means for screening, large-scale quarantineand epidemiological investigation of Clostridium botulinum type A.

Description

technical field [0001] The invention relates to molecular detection of Clostridium botulinum, in particular to a real-time fluorescent quantitative PCR nucleic acid sequence and a kit for detecting Clostridium botulinum type A. Background technique [0002] Clostridium botulinum ( C. botulinum ) is a Gram-positive, anaerobic, flagellated, acapsulated, spore-shaped, elliptical stubby bacillus. It was first discovered from sausage by Belgian scholar Van Ermengemcong in 1896, and confirmed that it can produce exotoxin-botulinum toxin (Botulinum toxin), causing human and animal poisoning. This bacterium is widely present in the sediments of soil, oceans and lakes, as well as in the intestines, feed and food of mammals, birds and fish. Clostridium botulinum cannot grow in living organisms. When the environment is anaerobic and has proper nutrition, it can grow and reproduce to produce botulinum toxin. When humans and animals eat food and feed containing this toxin, symptoms of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/689C12Q1/04C12N15/11
CPCC12Q1/6851C12Q1/689C12Q2563/107C12Q2531/113C12Q2545/113Y02A50/30
Inventor 徐雪芳黄英叶长芸
Owner ICDC CHINA CDC
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