GeXP analysis method for synchronous detection of vibrio cholera and vibrio parahaemolyticus in food

A technology for Vibrio hemolyticus and Vibrio cholerae, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and the determination/inspection of microorganisms, which can solve the problems of simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus, etc. , to avoid competitive effects, high sensitivity, and improve sensitivity

Inactive Publication Date: 2017-03-15
INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

So far, there has been no literature report on the simultaneous detection of Vibrio cholerae (Vibrio cholerae) and Vibrio parahemolyticus (Vibrio Parahemolyticus) using GeXP technology

Method used

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  • GeXP analysis method for synchronous detection of vibrio cholera and vibrio parahaemolyticus in food
  • GeXP analysis method for synchronous detection of vibrio cholera and vibrio parahaemolyticus in food

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Embodiment 1

[0026] Embodiment 1, primer design

[0027] The ompW gene of Vibrio cholerae and the toxR gene of Vibrio parahaemolyticus were selected as specific genes for amplification, and specific primers were designed using eXpressDesigner. The primer sequences and product sizes are shown in Table 1.

[0028] Table 1

[0029]

[0030] The universal primers are a pair of non-biological oligonucleotide sequences, and the 5' ends of the upstream primers are labeled with cy5 fluorescence. The sequences are shown in Table 2.

[0031] Table 2

[0032] Primer name Primer sequence (5'-3') SEQ ID NO.5 GACACTGTGATGTCTTAT SEQ ID NO.6 GTTGCTGAGTGATATCCCT

[0033] The above primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

Embodiment 2

[0034] Example 2, using the GeXP method to simultaneously detect Vibrio cholerae and Vibrio parahaemolyticus in aquatic products (yellow croaker).

[0035] Take 25g of samples to be tested from different parts of yellow croaker and inoculate them in 225mL of 3% sodium chloride alkaline peptone water, place them in a biochemical incubator at 37°C±1°C and incubate for 18-24 hours.

[0036] Nucleic acid extraction: Take 1.5 mL of the enrichment culture solution and place it in a centrifuge tube, centrifuge at 12,000 rpm for 3 minutes and discard the supernatant. A general-purpose commercial Gram-negative bacterial DNA extraction kit was selected, and the nucleic acid of the culture was extracted according to the steps specified in the kit instruction manual, and stored at -70°C for future use.

[0037]Configuration and amplification parameters of the PCR amplification system: 25 μL reaction system, containing 0.2 μL each of specific primers SEQ ID NO.1 and SEQ ID NO.2 (0.1 mM), S...

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Abstract

The invention belongs to the field of biological detection technology and relates to a GeXP analysis method for synchronous detection of vibrio cholera and vibrio parahaemolyticus in food. Primers used in the detection comprise a pair of vibrio cholera specific primers, a pair of vibrio parahaemolyticus specific primers and a pair of universal primers. The method comprises the following steps: carrying out enrichment culture on a sample to be detected, extracting nucleic acid, carrying out PCR amplification by the use of a specific primer pair and a fluorescently-labeled primer, detecting the amplification product by the use of a genetic analysis system, contrasting with standard molecular weight, and judging whether there is vibrio cholera and vibrio parahaemolyticus contamination in the detected sample according to detection atlas. Through verification, the detection system has characteristics of high specificity, good stability, short detection period, etc.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a method for detecting Vibrio cholerae (Vibrio cholerae) and Vibrio parahaemolyticus (Vibrio Parahaemolyticus) based on GenomeLabTM GeXP (GenomeLabeXpress Profiling) multiple gene expression analysis technology. Background technique [0002] Food safety issues and related foodborne diseases have become a hot spot in the field of public health. Strengthening the research on the detection technology of pathogenic bacteria in food has important practical significance. According to the statistics of the World Health Organization (WHO), 1.5 million people died of diarrhea and other diseases worldwide in 2005, 70% of which were caused by food-borne factors. . The situation is even worse in developing countries, where an estimated 270 million cases of diarrhea and its associated cases occur each year, killing 2.4 million children under the age of five. In recent years, food...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/63
CPCC12Q1/6851C12Q1/689C12Q2600/16C12Q2531/113C12Q2537/143C12Q2565/125Y02A50/30
Inventor 刘生峰杨俊谭志张雷李贤良王国民聂福平王昱
Owner INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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