Novel specific molecular target for vibrio parahaemolyticus and rapid detection method thereof

A technology of Vibrio hemolyticus and a primer set, applied in the field of microbial testing, can solve the problems of human health hazards, difficult to meet the actual detection, unable to achieve accurate screening of Vibrio parahaemolyticus, etc., to reduce the detection cost, The results are easy to determine and the specificity is good.

Active Publication Date: 2020-05-15
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the virulence genes of Vibrio parahaemolyticus show different degrees of homology with other species of the Vibrio family. It is harmful to human health, so the precise screening of Vibrio parahaemolyticus cannot be well achieved through virulence gene targets
According to our current reports, there are very few gene targets...

Method used

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  • Novel specific molecular target for vibrio parahaemolyticus and rapid detection method thereof
  • Novel specific molecular target for vibrio parahaemolyticus and rapid detection method thereof
  • Novel specific molecular target for vibrio parahaemolyticus and rapid detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Discovery of new molecular targets specific to Vibrio parahaemolyticus

[0046] According to the GenBank database and the whole genome DNA sequence of Vibrio parahaemolyticus self-tested by our team, bioinformatics analysis was performed; 23 non-essential genes unique to Vibrio parahaemolyticus strains were screened, and 20 parahaemolytics were obtained by further analysis and screening Specific gene fragments of Vibrio sex, the nucleotide sequences of the gene fragments are shown in SEQ ID NO. 1-20.

Embodiment 2

[0047] Example 2 Rapid detection method of Vibrio parahaemolyticus

[0048] 1) Primer design

[0049] A specific PCR amplification primer set (including forward primer and reverse primer) was designed according to the sequence SEQ ID NO. 1-20 in Example 1. The sequence of the primer set is shown in Table 1 below.

[0050] Table 1 Specific PCR detection primer set

[0051]

[0052]

[0053]

[0054] 2) The method for identifying Vibrio parahaemolyticus, the steps are as follows:

[0055] S1DNA template preparation: the test strains are respectively enriched and cultured in LB liquid medium, and the bacterial genomic DNA is extracted separately using a commercial bacterial genomic DNA extraction kit as the template to be tested;

[0056] S2PCR amplification: use one of the primer sets 1-20 to perform PCR amplification on the DNA of the test sample

[0057] ①PCR detection system:

[0058]

[0059] ②PCR amplification program:

[0060]

[0061] S3: Take the PCR amplified product and perform gel ...

Embodiment 3

[0063] Example 3 Results of specificity evaluation of PCR detection method

[0064] Twelve strains of Vibrio parahaemolyticus, 2 strains of non-target Vibrio (Vibrio fluvialis and Vibrio vulnificus), 9 strains of non-parahaemolytic Vibrio such as Staphylococcus aureus and Salmonella enteritidis were detected by PCR according to the method of Example 2. , Where the S1 DNA template is prepared by extracting the genomic DNA of each bacteria. Set a blank control. The template of the blank control is an aqueous solution without genome.

[0065] The strains and test results of the bacteria used are shown in Table 2 below. In the table, the "+" in the test result column indicates positive and "-" indicates negative. The results of PCR product electrophoresis are as follows figure 1 As shown; among them, (1) to (20) respectively correspond to the primer sets with serial numbers 1 to 20 in Example 1, M is 2000Maker, C is a blank control, and 1 to 23 respectively correspond to the strains i...

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Abstract

The invention discloses a novel specific molecular target for vibrio parahaemolyticus and a rapid detection method thereof. The method provided by the invention provides 20 novel specific molecular detection targets for identifying the vibrio parahaemolyticus; a corresponding primer group can be designed according to target molecules; and whether the vibrio parahaemolyticus exists or not can be obtained by carrying out PCR on a to-be-detected object and analyzing an electrophoresis result of a PCR product. Compared with the prior art, the rapid detection method provided by the invention can beused for detecting certain strains insensitive to biochemical reaction and making up the defect of biochemical identification; meanwhile, more specific molecular targets of the vibrio parahaemolyticus are provided; the defect of insufficient detection discrimination of common virulence gene targets is made up; and the method is more practical. The rapid detection method provided by the inventionalso has the advantages of simple operation, good detection result specificity and simple result determination, and is of great significance to the identification of vibrio parahaemolyticus in food.

Description

Technical field [0001] The invention belongs to the technical field of microbial testing, and specifically relates to a new specific molecular target of Vibrio parahaemolyticus and a rapid detection method thereof. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a common food-borne pathogen, and it is the primary factor that causes food poisoning in my country. Due to the halophilic nature of Vibrio parahaemolyticus, it is mainly distributed in salt-rich environments such as coastal and marine waters, and is attached to cod, sardines, mackerel, flounder, octopus, shrimp, crab, clams, lobster, and crayfish , Scallops, oysters and other marine life growth. If people eat uncooked food contaminated by Vibrio parahaemolyticus, it will cause food poisoning. The incubation period is generally 1 to 4 days, causing fever (approximately 27% of poisoned patients) and nausea (71%) , Vomiting (32%), abdominal pain (82%), diarrhea (98%) and other acute gast...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/63
CPCC12Q1/689Y02A50/30
Inventor 吴清平相欣然叶青华张菊梅庞锐雷涛王涓古其会韦献虎张友雄曾海燕
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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