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Composition for detecting O1 group vibrio cholerae, kit and detection method

A technology of Vibrio cholerae and composition, which is applied in the field of specific detection composition for O1 group Vibrio cholerae, can solve the problems of false positive results, insufficient specificity of primers, false negative results, etc., so as to reduce biological hazards and save The effect of manpower and time

Inactive Publication Date: 2010-07-07
蔡剑平
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the primers are not specific enough, it may lead to false positive results or false negative results of the detection
In addition, for the detection of toxigenic Vibrio cholerae, detection of a single gene is often not enough to determine its pathogenicity
In 2003, Fukushima et al. established a multiplex fluorescent real-time PCR detection system using SYBR fluorescent dyes (Hiroshi F, Yoshie T, Ryotaro S. Duplex real-time SYBR Green PCR assays for detection of 17 species of foodorwaterbornepathogensinstools. J.Clin.Microbiol, 2003, 41:5134-5146), although the detection time and workload have been shortened, due to the single target site detection of non-toxin genes, when used to detect pathogenic bacteria, the detection results have certain limitations. false positive rate of

Method used

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  • Composition for detecting O1 group vibrio cholerae, kit and detection method
  • Composition for detecting O1 group vibrio cholerae, kit and detection method
  • Composition for detecting O1 group vibrio cholerae, kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 adopts the detection of common PCR method

[0043] Extraction of Bacterial Genomic DNA

[0044] The genomic DNA of each bacteria was extracted (MiniBEST kit from TAKARA Company). Use ultraviolet spectrophotometer to measure the purity and concentration of each bacterial genome DNA, O1 group Vibrio cholerae genome DNA was diluted to 1×10 with TE buffer 5 pg / μl to 1×10 -3 pg / μl gradient standard, the genome DNA of other 19 kinds of other intestinal pathogenic bacteria or common pathogenic bacteria in hospital infection was diluted to 5×10 with TE buffer 4 pg / μl, all kinds of genomic DNA were aliquoted in small quantities and stored at -20°C for future use.

[0045] Construction and preparation of plasmid standards

[0046] The target plasmid was constructed by TA cloning technique. After the molecular weight of the amplified fragment was confirmed by electrophoresis, the PCR products of rfb-O1 and ctxA specific sequences were connected to the pMD18-T vec...

Embodiment 2

[0049] Embodiment 2 adopts fluorescence real-time quantitative PCR reaction method to detect

[0050] Using the same bacterial genomic DNA and plasmid standard as in the examples, the difference is that the PCR method used is fluorescent real-time quantitative PCR, and the probes designed and synthesized above are used.

[0051] Real-time fluorescence duplex PCR reaction conditions

[0052] The reaction system is 25 μl, take 2.5 μl of 10×PCR buffer, 2.0 μl of each dNTP of 2.5 mmol / L, 0.5 μl of each 25 μmol / L primer, 0.5 μl of each 5 μmol / L probe, 1 U of Blend Taq plus DNA polymerase, template DNA 10ul, add sterilized water to 25μl of the final system. PCR cycle parameters: pre-denaturation at 94°C for 2min, denaturation at 94°C for 10s, extension at 60°C for 30s, a total of 45 cycles of reaction. After the amplification was completed, the data was analyzed under the same conditions to determine the Ct value of each sample.

[0053] The evaluation of the detection sensitivit...

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Abstract

The invention relates to a composition for detecting O1 group vibrio cholerae, a kit and a detection method. The composition comprises a specific primer aiming at O antigen encoding genes rfb-O1 of toxigenic O1 group vibrio cholerae and nosotoxin genes ctxA secreting cholera toxin. The composition and the kit can delicately detect the toxigenic O1 group vibrio cholerae existing in a sample, wherein the detection lower limit is 1.0*102 copies per reaction system, and the detection sensitivity to a genome DNA (deoxyribonucleic acid) is 1.0*10-1 pg per reaction system.

Description

technical field [0001] The invention relates to a composition, a kit and a detection method for the specific detection of Vibrio cholerae, in particular to a composition, a kit and a detection method for the specific detection of O1 group Vibrio cholerae. Background technique [0002] Cholera is a severe intestinal infectious disease caused by Vibrio cholerae. The clinical symptoms of patients often show a large amount of rice soup-like excretion, resulting in rapid dehydration. If left untreated, it can lead to hypovolemic shock, acidosis, and even death. Vibrio cholerae can be divided into several groups according to serotypes, but not all groups can cause epidemics. Among them, there are mainly two serotypes that can cause cholera epidemics: O1 group and O139 group (Kaper JB, Morris JG, Levine MM. Cholera . Clin. Microbiol. Rev, 1995, 8: 48-86). Therefore, in the detection of Vibrio cholerae, its serotyping is an important part of effective detection. [0003] As far ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10
CPCY02A50/30
Inventor 蔡剑平杨辉龚成吴丽娟胡继红汪仕奎
Owner 蔡剑平
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