Clostridium perfringens enterotoxin positive bacteria dual fluorescent quantitative PCR rapid detection kit

A clostridium perfringens, detection kit technology, applied in the direction of determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problem of non-involving Clostridium perfringens exotoxin cpa and enterotoxin cpe diagnostic technology and other problems, to achieve the effect of fast detection speed, high accuracy and low false positive rate

Active Publication Date: 2015-04-22
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] In the prior art, there are few literatures on Clostridium perfringens antigen, antibody detection and fluorescent qu

Method used

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  • Clostridium perfringens enterotoxin positive bacteria dual fluorescent quantitative PCR rapid detection kit
  • Clostridium perfringens enterotoxin positive bacteria dual fluorescent quantitative PCR rapid detection kit
  • Clostridium perfringens enterotoxin positive bacteria dual fluorescent quantitative PCR rapid detection kit

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[0030] Example 1

[0031] Composition and preparation of double fluorescence quantitative PCR rapid detection kit for Clostridium perfringens enterotoxin positive

[0032] 1. Reagent composition:

[0033] EasyTaq enzyme (5U / μL), dNTPs (10mM), were purchased from Promega Company; PCR primer pairs cpa01, cpa02 and cpe01, cpe02, and probes cpap, cpep were synthesized by Shanghai Sangon Bioengineering Company;

[0034] 2. Reagent preparation

[0035] A) Fluorescence quantitative reaction solution: 1×PCR Buffer (containing Mg 2+), dNTPs 0.5mM, Taq enzyme 2U, upstream primer cpa01 and downstream primer cpa02 of Clostridium perfringens exotoxin cpa 0.4 μM each, fluorescent probe cpap is 0.4 μM, upstream of Clostridium perfringens enterotoxin cpe The primer cpe01 and the downstream primer cpe02 were each 0.4 μM, and the fluorescent probe cpep was 0.4 μM. Among them, the primer sequences of exotoxin cpa, upstream primer cpa01: 5'-GATTTGTAAGGCGCTTATTTGT-3', downstream primer cpa02: ...

Example Embodiment

[0039] Example 2

[0040] Clostridium perfringens enterotoxin-positive double fluorescence quantitative PCR rapid detection primers and methods of using the kit

[0041] 1. Processing of samples

[0042] The tested sample is a tissue or liquid sample: according to the national standard method, aseptically take 10g (mL) of the sample and put it into 90mL of 0.5% buffered peptone water, shake and mix well, and then inoculate the sample liquid into TSC medium for enrichment, 37 Anaerobic culture at ℃ for 24h.

[0043] 2. The processing of the tested samples adopts the rapid boiling method to extract bacterial genomic DNA, which is used as a template for fluorescence quantitative PCR reaction. Pick a single colony on the TSC medium, put it in 200 μl of sterile deionized water, heat and boil for 15 min, take out the test tube, centrifuge at 10,000 rpm for 5 min, and take the supernatant as the template for the fluorescence quantitative PCR reaction.

[0044] 3. Fluorescence quan...

Example Embodiment

[0055] Example 3

[0056] Application of primers and kits for rapid detection of Clostridium perfringens enterotoxin-positive double fluorescence quantitative PCR

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Abstract

The invention relates to a fluorescent quantitative PCR rapid detection kit for specifically detecting clostridium perfringens enterotoxin positive bacteria infection. The detection kit comprises (a) a fluorescent quantitative reaction solution; (b) clostridium perfringens exotoxin cpa positive and clostridium perfringens enterotoxin cpe positive quality control substances; and (c) a negative quality control substance, wherein the fluorescent quantitative reaction solution comprises 2 pairs of primers and one pair of probes, namely a forward primer cpa01 and a reverse primer cpa02 of clostridium perfringens exotoxin cpa and a fluorescence probe cpap, as well as a forward primer cpe01 and a reverse primer cpe02 of clostridium perfringens enterotoxin cpe and a fluorescence probe cpep. The detection kit disclosed by the invention has the advantages that positive clostridium perfringens of the enterotoxin cpe can be rapidly diagnosed; the specificity is high, the sensitivity is high, and the false positive rate is low; and moreover, the detection speed is high, and only one and a half hours are needed.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR detection kit for specifically detecting the infection of Clostridium perfringens enterotoxin-positive bacteria, which belongs to the field of bacterial nucleic acid detection and is suitable for rapid detection of Clostridium perfringens enterotoxin-positive bacteria in clinical and scientific research. Qualitative and quantitative detection. Background technique [0002] Clostridium perfringens (Clostridium perfringens) is an important zoonotic pathogen, widely present in soil, water, human and animal intestines in the environment. The pathogenicity of the bacterium is mainly based on its ability to produce various toxins. So far, at least 15 types of Clostridium perfringens toxins have been found, except for the four types of lethal toxins α, β, In addition to ε and ι toxins, all types of Clostridium perfringens contain exotoxin cpα, and important new bacterial toxins closely related to human ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q2537/143C12Q2563/107C12Q2561/113
Inventor 张蓉蓉艾地云张腾飞罗青平邵华斌温国元王红琳汪宏才罗玲
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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