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GenomeLab eXpress Profiling (GeXP) multiplex quick detection primers and detection method for 6 food-borne pathogens

A food-borne pathogenic bacteria, multiple technology, applied in biochemical equipment and methods, microbial-based methods, microbial determination/testing, etc., to improve detection sensitivity, avoid false positives and false negatives, and avoid false positives and the effect of false negatives

Inactive Publication Date: 2013-11-06
INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, GeXP multiplex PCR technology has not been successfully used to identify Salmonella sp., Listeria monocytogenes, Staphylococcus aureus, Shigella sp. , enterohaemorrhagic Escherichia coli O157: H7 (Escherichia coli O157: H7), Campylobacter jejuni and other six pathogenic bacteria reported in the literature

Method used

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  • GenomeLab eXpress Profiling (GeXP) multiplex quick detection primers and detection method for 6 food-borne pathogens
  • GenomeLab eXpress Profiling (GeXP) multiplex quick detection primers and detection method for 6 food-borne pathogens
  • GenomeLab eXpress Profiling (GeXP) multiplex quick detection primers and detection method for 6 food-borne pathogens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 , design of primers

[0042] The primers for GeXP multiplex PCR detection of 6 kinds of food-borne pathogens are a set of primers for food-borne pathogens. Each pathogenic bacteria primer set consists of two pairs of primers, one pair is specific It is a universal primer, the sequence of the universal primer is the same and the 5' end of the upstream primer is labeled with cy5 fluorescence, and each specific primer contains a sequence that can be combined with the universal primer.

[0043] 1) Design and synthesis of universal primers

[0044] The universal primer is a nucleotide coding sequence of non-biological origin, and the 5' end of the upstream primer is labeled with cy5 fluorescence. Universal primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0045] 2) Design and specificity verification of specific primers

[0046] Select DNA helicase B subunit gene (gyrB) from Salmonella, DNA helicase B subunit gene (gyrB)...

Embodiment 2

[0054] Example 2 , GeXP multiplex PCR detection method for foodborne pathogens

[0055] 1) Strain culture

[0056] Staphylococcus aureus can be cultured in 10% sodium chloride tryptone soybean broth, etc., Listeria monocytogenes can be cultured in Fraser broth, etc., Campylobacter jejuni can be cultured in Brucella broth, etc. It can be cultured by nutrient broth or nutrient agar, etc. Campylobacter jejuni needs to be cultured at 42°C±1°C for 36 hours in microaerophilic conditions, and other strains need to be cultured at 37°C±1°C for 18 hours.

[0057] 2) DNA extraction

[0058] Bacterial DNA extraction commercial kit (OMEGA Bacterial DNA Kit (200)) was used to extract DNA from strain samples. The specific steps are as follows: take 1 mL of the cultured bacterial liquid and centrifuge to obtain the bacterial cells, or obtain the bacterial lawn from the nutrient agar plate. Follow the steps specified in the kit instructions. Obtain the DNA template to be tested.

[0059...

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Abstract

The invention relates to a synchronous detection method for common food-borne pathogens, in particular to a multiplex polymerase chain reaction (PCR) quick detection method for pathogens based on a GenomeLab TMGeXP multiplex gene expression analysis technology, and belongs to the technical field of biology. Amplification of specific primers is combined with amplification of general primers, a high annealing temperature for amplification of the specific primers is adopted in front 16 cycles of PCR amplification, the general primers are generally amplified in large scale in later 16 cycles, and the detection results are automatically read and analyzed by adopting the fragment analysis function of a genetic analysis system according to fluorescent signals of nucleic acid fragment length, so that a plurality of samples can be simultaneously detected, and a competition effect among the primers in common multiplex PCR amplification is effectively avoided. The invention aims to establish a quick method for detecting the food-borne pathogens with high flux, high reliability and high sensitivity.

Description

technical field [0001] The present invention relates to the synchronous detection method of common food-borne pathogenic bacteria, in particular to a kind of Salmonella (Salmonella sp.), Listeria monocytogenes (Listeria monocytogenes), Staphylococcus aureus, Shigella sp., Escherichia coli O157: H7, and Campylobacter jejuni The invention discloses a multiplex PCR rapid detection method for bacteria, which belongs to the field of biotechnology. 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 microorganisms 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/445C12R1/19C12R1/42C12R1/01
Inventor 肖进文刘生峰周蓓莉周庆杨俊李应国王昱聂福平
Owner INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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