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A duplex-PCR detection kit based on Vibrio parahemolyticus TDH

A detection kit, a technology for Vibrio hemolyticus, applied in microorganism-based methods, microbial determination/inspection, microorganisms, etc., can solve problems such as sensitivity, specificity, limitation of anti-interference ability, etc., to eliminate miscellaneous bacteria Effects of interference, saving labor costs and material consumption

Inactive Publication Date: 2018-12-18
SHANGHAI MUNICIPAL CENT FOR DISEASE CONTROL & PREVENTION
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  • Abstract
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  • 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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  • A duplex-PCR detection kit based on Vibrio parahemolyticus TDH
  • A duplex-PCR detection kit based on Vibrio parahemolyticus TDH
  • A duplex-PCR detection kit based on Vibrio parahemolyticus TDH

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Embodiment

[0038] 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:

[0039] 1.1 Establishment of DPCR system

[0040] 1.1.1 Primer design and DPCR system construction

[0041] 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.

[0042]

[0043]

[0044] 1.1.2 Optimization of double PCR reaction condit...

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Abstract

A duplex-PCR detection kit based on Vibrio parahemolyticus TDH is disclosed. The kit includes a direct hemolysin gene detection primer pair having sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and athermolabile hemolysin gene detection primer pair having sequences shown as SEQ ID NO.3 and SEQ ID NO.4. The kit can eliminate interferences caused by other microbes, and can replace detection of a plurality of virulence genes of Vibrio parahemolyticus. Whether a sample contains Vibrio parahemolyticus or not is determined by tlh screening, and whether a sample contains ''pandemic florae'' of Vibrio parahemolyticus or not is determined by tdh1_368 screening, thus differentiating epidemic florae from non-epidemic florae of Vibrio parahemolyticus in one time and facilitating increase in sensitivity and specificity of conventional epidemic flora detection. The kit is simple and effective.

Description

technical field [0001] The invention relates to a direct hemolysin gene (TDH) double PCR detection kit based on Vibrio parahaemolyticus. Background technique [0002] Vibrio parahemolyticus (VP) has now become a foodborne 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 parahaemolyticus The proportion of food poisoning phenomenon in China is as high as 60%, which is much higher than other ty...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12R1/63
CPCC12Q1/689C12Q2600/16
Inventor 陈敏陈洪友张曦宋元君屠丽红庄源
Owner SHANGHAI MUNICIPAL CENT FOR DISEASE CONTROL & PREVENTION
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