Application of dual PCR (polymerase chain reaction) detection kit in identification of vibrio parahaemolyticus

A detection kit, a technology for Vibrio hemolyticus, which is used in the determination/inspection of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc., can solve the problems of sensitivity, specificity, and limitations of anti-interference ability.

Inactive Publication Date: 2018-12-21
SHANGHAI MUNICIPAL CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, these PCR detection methods have more or less limitations in the sensitivity, specificity and anti-interference ability of experiments.

Method used

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  • Application of dual PCR (polymerase chain reaction) detection kit in identification of vibrio parahaemolyticus
  • Application of dual PCR (polymerase chain reaction) detection kit in identification of vibrio parahaemolyticus
  • Application of dual PCR (polymerase chain reaction) detection kit in identification of vibrio parahaemolyticus

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Embodiment

[0034]This embodiment relates to a dual PCR detection kit and detection system based on direct hemolysin gene (TDH) and thermolabile hemolysin (TLH) of Vibrio parahaemolyticus. Specifically include the following steps:

[0035] 1.1 Establishment of DPCR system

[0036] 1.1.1 Primer design and DPCR system construction

[0037] Specific primers were designed with reference to the sequence of the specific coding gene tlh and the coding gene tdh1_368 of the pandemic strain of Vibrio parahaemolyticus, as shown in Table 1. The primer sequences were compared with the GenBank database online by BLAST, and no non-specific binding to other substances was found. The target band sizes of tlh and tdh1_368 are: 450bp and 208bp, respectively. Through the verification of primerBLAST, the DPCR system can simultaneously recognize the sequence of the pandemic tdh1_368 and the non-pandemic Vibrio parahaemolyticus.

[0038]

[0039]

[0040] 1.1.2 Optimization of double PCR reaction condi...

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Abstract

The invention discloses application of a dual PCR (polymerase chain reaction) detection kit in non-diagnostic and non-therapeutic identifying and distinguishing of prevalence floras from non-prevalence floras of vibrio parahaemolyticus. The detection kit comprises direct hemolysin gene detection primer pairs of which the sequences are shown as SEQ ID NO.1 and SEQ ID NO.2, and tlh (thermolabile hemolysin) detection primer pairs of which the sequences are shown as SEQ ID NO.3 and SEQ ID NO.4. The dual PCR detection kit is adopted to remove infectious microbe interference, replaces multiple vibrio parahaemolyticus virulence genes for detection, and screens whether vibrio parahaemolyticus exists or not in a sample through tlh and whether big prevalence floras of vibrio parahaemolyticus exist or not in the sample at the tdhl-368, so that the prevalence floras and the non-prevalence floras of vibrio parahaemolyticus are distinguished through one-time amplification, the improvement on the detection sensitivity and the specificity of conventional epidemic strains is facilitated, and the dual PCR detection kit is simple, convenient and effective.

Description

technical field [0001] The invention relates to the use of a direct hemolysin gene (TDH) double PCR detection kit based on Vibrio parahaemolyticus in non-diagnostic and non-therapeutic identification and differentiation of Vibrio parahaemolyticus. Background technique [0002] Vibrio parahemolyticus (VP) has now become a food-borne pathogen of particular concern in China and even the world. It mainly causes food poisoning through human consumption of water sources and aquatic products contaminated by this bacteria. It can cause acute gastroenteritis, and severe cases can cause sepsis and even endanger human life. While affecting human health, it also restricts the development of aquaculture industry. The data checked from the China Foodborne Disease Surveillance Network shows that the proportion of food poisoning incidents caused by Vibrio parahaemolyticus has been higher than that of other types of pathogenic Vibrio, and in some coastal cities caused by Vibrio parahaemolyt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143Y02A50/30
Inventor 陈洪友张曦陈敏宋元君屠丽红庄源
Owner SHANGHAI MUNICIPAL CENT FOR DISEASE CONTROL & PREVENTION
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