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Kit and detection method for absolute quantitative detection of vibrio parahaemolyticus

A hemolytic Vibrio, absolute quantitative technology, applied in the field of molecular biology, can solve the problems of time-consuming, high concentration, complex background bacteria, etc., achieve high accuracy and repeatability, simplify the operation process, and shorten the detection time.

Inactive Publication Date: 2018-05-11
BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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AI Technical Summary

Problems solved by technology

However, the MNP method is a method for probabilistically estimating the bacterial concentration, and there are many disadvantages in practical applications: 1) It needs complex gradient dilution and detection repetition of the sample, and the accuracy of dilution will directly affect the quantitative results; Bacteria enrichment culture is not only time-consuming, but also other potential miscellaneous bacteria in the sample may interfere with the identification of target bacteria due to excessive proliferation; 3) It still needs to go through complicated processes such as selective plate separation and biochemical identification, and the detection time generally takes 3 to 5 days; 4) Vibrio parahaemolyticus in food often exists in a "viable non-culturable state (VBNC)", and the MPN method cannot effectively detect this part of the bacteria, thus affecting the accuracy of the test results
At present, the quantitative analysis of this technology for microorganisms is still in its infancy. Related studies such as human herpesvirus, methicillin-resistant Staphylococcus aureus, Chlamydia trachomatis, parasite Cryptosporidium in clinical samples, and Shigella in bovine feces samples Escherichia coli with toxins, and plant pathogenic bacteria that cause amylovora blight and potato brown rot, etc., have not yet seen relevant reports that can be used for the quantitative detection of Vibrio parahaemolyticus in food
Since the food matrix is ​​mostly rich in nucleic acid amplification inhibitors such as protein, fat, and pectin, and the background bacteria are complex and have a high concentration, the effectiveness and practicability of ddPCR for the quantitative analysis of Vibrio parahaemolyticus in food is still unknown. Broad development space and application prospects

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  • Kit and detection method for absolute quantitative detection of vibrio parahaemolyticus
  • Kit and detection method for absolute quantitative detection of vibrio parahaemolyticus
  • Kit and detection method for absolute quantitative detection of vibrio parahaemolyticus

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Embodiment 2

[0047] Example 2 ddPCR primer, probe design, screening and specificity verification

[0048] In order to achieve the specific detection and absolute quantitative analysis of Vibrio parahaemolyticus, we selected a species-specific and highly conserved single-copy β-lactamase coding gene blaCARB (GenBank no.NG_048735.1 ) as the target sequence, sequence analysis and alignment were performed by NCBI online tools, and 4 pairs of primer and probe combinations were designed using Prime Express software V3.0 (ABI, Foster City, CA, USA). The sequences are shown in Table 1. The 5' end of the probe is labeled with FAM, and the 3' end is labeled with BHQ. Primers / probes were synthesized by Beijing Liuhetong Economic and Trade Co., Ltd.

[0049] First, the qPCR method was used to screen the 4 pairs of primers and probes that had been designed and synthesized. Through the detection of the genomic DNA of Vibrio parahaemolyticus (ATCC17802), it was found that all the primer probes could ef...

Embodiment 3

[0051] Embodiment 3 detects the ddPCR kit of vibrio parahaemolyticus

[0052] The kit mainly includes:

[0053] (1) Primers and probes for detecting Vibrio parahaemolyticus (the sequences of the primers and probes are shown in SEQ ID No.1-3), synthesized by Beijing Liuhetong Economic and Trade Co., Ltd.;

[0054] (2) Positive control: Genomic DNA of Vibrio parahaemolyticus ATCC 17802, 1000 copies / μL;

[0055] (3) ddPCR master mix, purchased from BioRad, USA;

[0056] (4) microdroplet generating oil, purchased from BioRad Company of the United States;

[0057] (5)) Droplet generation card, purchased from BioRad, USA;

[0058] (6) Twin Tec Semi-Skirted 96-well plate, purchased from Eppendorf, Germany;

[0059] (7) Aluminum foil heat-sealing film, purchased from American BioRad Company.

Embodiment 4

[0060] The establishment of embodiment 4 detection method

[0061] (1) Extract the DNA of the genome of the sample to be tested: use the Bacterial Genome DNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd., article number: DP302) to extract the DNA of each dilution of the bacterial liquid, and the extraction steps are carried out according to the kit instructions, and finally the nucleic acid is dissolved In 50 μL TE buffer;

[0062] (2) Configure the ddPCR reaction system, see Table 2 for details;

[0063] (3) Droplet generation: Add 20 μL of ddPCR reaction master mix and 70 μL of droplet generation oil to the 8-well droplet generation card, and place it in a droplet generator (QX100, Bio-Rad, Pleasanton, CA) to generate droplet;

[0064] (4) Amplification reaction: Slowly transfer the generated water-in-oil droplets (40 μL) to a 96-well plate, seal the film and place on a PCR machine (GeneAmp 9700, Applied BioSystems, Foster City, CA) for amplification Reaction, ...

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Abstract

The invention discloses a kit and a detection method for absolute quantitative detection of vibrio parahaemolyticus, and belongs to the field of molecular biology. The present invention provides a setof primers and probe for the absolute quantitative detection of the Vibrio parahaemolyticus. The nucleotide sequences of the primers are as shown in SEQ ID No. 1 and SEQ ID No. 2. The nucleotide sequence of the probe is as shown in SEQ ID No. 3. The invention also provides a method for the absolute quantitative detection of the Vibrio parahaemolyticus by use of a micro-droplet digital polymerasechain reaction. The method has strong specificity, high sensitivity, good repeatability, strong operability and easy standardization.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to a primer / probe and a kit for the absolute quantitative detection of Vibrio parahaemolyticus, more specifically, to a droplet digital polymerization for the absolute quantitative detection of Vibrio parahaemolyticus Enzyme chain reaction (droplet digital polymerase chain reaction, ddPCR) kit and detection method. Background technique [0002] Vibrio parahaemolyticus is a halophilic Gram-negative bacterium that widely exists in seawater and seafood, and is the most common food poisoning pathogen in coastal areas of China. Its pathogenicity is closely related to the amount of bacteria in food, and the total number of Vibrio parahaemolyticus is an important indicator often used to evaluate the degree of food contamination. For example, China's "National Food Safety Standard Limits of Pathogenic Bacteria in Food (GB 29921-2013)" clearly states that the minimum limit of Vibrio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/6844C12Q1/06C12N15/11C12R1/63
CPCC12Q1/6844C12Q1/6851C12Q1/689C12Q2527/125C12Q2563/159C12Q2537/143
Inventor 魏海燕马丹魏咏新曾静周熙成张西萌付溥博汪琦徐蕾蕊李丹刘莉赵晓娟
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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