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PMA-qPCR detection method of vibrio parahaemolyticus

A technology of Vibrio hemolyticus and a detection method, applied in the field of inspection and quarantine, can solve the problem of rare detection of Vibrio parahaemolyticus, and achieve the effects of shortening detection time, accurate detection means and simple operation

Inactive Publication Date: 2020-08-07
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are few studies using the above method for the detection of Vibrio parahaemolyticus

Method used

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  • PMA-qPCR detection method of vibrio parahaemolyticus
  • PMA-qPCR detection method of vibrio parahaemolyticus
  • PMA-qPCR detection method of vibrio parahaemolyticus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Select control bacteria Escherichia coli, Vibrio cholerae, Vibrio vulnificus and three different strains of Vibrio parahaemolyticus respectively, inoculate into 225mL of 3% NaCl alkaline peptone water, and incubate at 37°C for 6h-18h with shaking at 180rpm.

[0023] 1 mg of PMA dye was dissolved in 200 μL of dimethyl sulfoxide (20% DMSO) and mixed evenly to obtain a stock solution with a concentration of 10 mM, which was stored in a -20° C. refrigerator in the dark. The PMA stock solution was diluted 10 times to a concentration of 1 mM for use as a working solution, and stored in a -4°C refrigerator away from light.

[0024] The bacterial solution was centrifuged at 5000×g for 5 min, washed 2-3 times with phosphate buffered saline (PBS), and the bacteria were resuspended in PBS for later use. Add PMA working solution to the resuspended bacteria solution, the final concentration is 4 μM (in the dark environment), after mixing well, keep away from light for 5 minutes, sha...

Embodiment 2

[0033] Viable bacteria of Vibrio parahaemolyticus were respectively selected as a sample, and inactivated bacteria and water were used as a control group, and the specific method steps were the same as in Example 1. The experimental results show that the Vibrio parahaemolyticus living bacteria group has qPCR amplification, and the result is positive, and the inactivated bacteria group and the control group have no Ct value, which is negative (such as figure 2 ). It shows that this method can effectively detect the live bacteria of Vibrio parahaemolyticus.

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Abstract

The invention provides a PMA-qPCR (Polymethyl Acrylate-Quantitative Polymerase Chain Reaction) detection method of vibrio parahaemolyticus. According to the detection method, PMA is adopted to pretreat bacterial liquid, so that PMA is crosslinked with thallus DNA; then thallus DNA is extracted to serve as a template for qPCR amplification; and whether a sample contains vibrio parahaemolyticus or not is judged according to a collected fluorescence signal Ct value. The method is high in detection sensitivity, high in specificity and short in consumed time, interference of dead bacterium DNA canbe effectively eliminated, and the effect of specific detection of living bacteria is achieved; and the method can be used for rapid detection of vibrio parahaemolyticus in food.

Description

technical field [0001] The invention belongs to the field of inspection and quarantine, and relates to a detection method of Vibrio parahaemolyticus, in particular to a PMA-qPCR detection method of Vibrio parahaemolyticus. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus) is the main pathogenic bacteria causing gastrointestinal tract infection, widely exists in seawater, sediments and various seafood. V. parahaemolyticus infection can cause diarrhea, vomiting, abdominal cramps, and, in rare cases, fever. The relevant staff of my country's foodborne disease monitoring website investigated more than 8,000 cases of food poisoning in its monitoring areas (16 provinces) in 14 years, and found that microbial pathogens are still the main cause of foodborne diseases in my country (accounting for 30% to 40%). %). In the past 10 years, Vibrio parahaemolyticus food poisoning caused by cross-contamination of meat or aquatic products has replaced Salmonella...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/63
CPCC12Q1/686C12Q1/689C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 陈光哲曾德新周昊谢青轩沈嘉敏
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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