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Multi-PCR detection kit capable of simultaneously detecting clostridium perfringens, clostridium hemolyticumbovis and B-type clostridium novyi and application of multi-PCR detection kit

A technology of Clostridium perfringens and Clostridium novyi, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, etc., and can solve the problem of false positives and the inability to simultaneously detect Clostridium hemolyticus and Noviella type B Clostridium, expensive reagents and other issues, to achieve good specificity and sensitivity

Active Publication Date: 2016-11-23
QINGHAI ACAD OF ANIMAL SCI & VETERINARY MEDICINE
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the kit can only detect one type of bacteria, Clostridium perfringens, and cannot detect Clostridium hemolyticus and Clostridium novyi type B at the same time; moreover, this method is prone to produce aerosol, causing genetic contamination in the laboratory, resulting in false positives, Therefore, it is impossible to determine the negative or positive of the sample, the operation method is cumbersome, the reagent is expensive, and it is not suitable for popularization and application.
[0005] At present, there is no report on the simultaneous detection of Clostridium perfringens, Clostridium hemolyticus and Clostridium novyi in the same sample, and there is no commercial kit

Method used

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Embodiment

[0030] 1. Primer design: Download the α-toxin gene of Clostridium perfringens (accession number: L43548) from the website http: / / www.ncbi.nlm.nih.gov / genbank / , use this gene as a template, and use Primer5.0 The software designed a pair of primers: PrimerF1: 5´-GCTAATGTTACTGCCGTTGA-3´, PrimerR1: 5´-CCTCTGATACATCGTGTAAG-3´.

[0031] According to Clostridium hemolyticus flagellin gene FliA(C), FliA(H) (gene accession number: AB058931, AB058939), Clostridium novyi type B flagellin gene FliA (gene accession number: AB058938) designed 3 primers: PrimerF2: 5´-AGAATAAACAGAGCTGGAGATG-3´; PrimerR2: 5´-TTATGCTAACTTTAGCTG CGTC-3´; PrimerR3: 5´-CTGCTGTACCTTCTATGAACC-3´.

[0032]2. Kit assembly: use the primer design software (Primer 5.0) to design the required 5 primers, and then send them to the biological company for chemical synthesis. Dilute the synthesized 5 primers to 10 µM with sterile water and mark them as F1, F2, F3, F4 and F5; purchase 1 bottle (500 µL) of commercial GoTaq DNA ...

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Abstract

The invention relates to a multi-PCR detection kit capable of simultaneously detecting clostridium perfringens, clostridium hemolyticumbovis and B-type clostridium novyi and application of the multi-PCR detection kit, and belongs to the technical field of PCR detection kits. The kit comprises primers PrimerF1 and PrimerR1 which are designed by taking alpha toxin group of the clostridium perfringens as templates, and primers PrimerF2, PrimerR2 and PrimerR3 which are designed by taking flagellin genes FliA(C) and FliA (H) of the clostridium hemolyticumbovis and flagellin gene FliA of the B-type clostridium novyi as templates. The kit is convenient to operate, low in cost and high in specificity, and time and labor are saved.

Description

technical field [0001] The invention belongs to the technical field of PCR detection kits, and in particular relates to a multiplex PCR detection kit capable of simultaneously detecting Clostridium perfringens, Clostridium hemolyticus and Clostridium novyi and its application. Background technique [0002] Clostridium perfringens, Clostridium hemolyticus, and Clostridium novyi type b can cause dysentery and necrotic enteritis in cattle and sheep, and can cause death in animals. These bacteria are sometimes infected alone, sometimes mixed, and it is difficult to make a differential diagnosis clinically, which brings great difficulties to timely prevention and treatment. The current diagnostic methods for these three types of bacterial diseases are mainly: isolation and culture, microscope observation and PCR detection, but the current PCR method can only detect one type of bacteria at a time, and there is no commercial kit, which is time-consuming and laborious. [0003] Suc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6844C12Q1/689C12Q2600/16C12Q2537/143
Inventor 王光华陆艳马利青李秀萍王戈平蔡其刚
Owner QINGHAI ACAD OF ANIMAL SCI & VETERINARY MEDICINE
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