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Method for detecting vibrio parahemolyticus through combination of unlabelled fluorescent PCR (Polymerase Chain Reaction) and HRMA (High Resolution Melting Analysis)

A technology for labeling fluorescent and hemolytic vibrio, which is applied in the field of microbial detection, can solve the problems of cumbersome operation, short reagent storage period, and complicated operation, and achieve the effects of avoiding cross-contamination, shortening the detection cycle, and simplifying the detection process

Active Publication Date: 2013-07-03
邳州市博睿投资管理有限公司
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  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional detection methods have disadvantages such as cumbersome operation, long time consumption, and easy missed detection.
Molecular detection methods include conventional PCR methods, but conventional PCR requires electrophoresis after amplification, which is complicated to operate and easily causes pollution
However, the probe method PCR has the disadvantages of high detection cost and short shelf life of reagents.

Method used

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  • Method for detecting vibrio parahemolyticus through combination of unlabelled fluorescent PCR (Polymerase Chain Reaction) and HRMA (High Resolution Melting Analysis)
  • Method for detecting vibrio parahemolyticus through combination of unlabelled fluorescent PCR (Polymerase Chain Reaction) and HRMA (High Resolution Melting Analysis)
  • Method for detecting vibrio parahemolyticus through combination of unlabelled fluorescent PCR (Polymerase Chain Reaction) and HRMA (High Resolution Melting Analysis)

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Effect test

Embodiment 1

[0066] Use the method of non-labeled fluorescent PCR of the present invention in conjunction with HRMA to detect Vibrio parahaemolyticus in aquatic products:

[0067] DNA extraction

[0068] Weigh 25 g of the sample by aseptic operation, add it to 225 mL of 3.5% NaCl BPW, and incubate at 36±1°C for 18 hours. Take 1.5 mL of the culture and centrifuge at 10,000 rpm for 2 min; add 500 μL of TE buffer to the precipitate, repeatedly pipette to resuspend it, add 30 μL of 10% SDS and 15 μL of proteinase K, mix well, and incubate at 37°C for 1 h; L NaCl, mix well, then add 80ul CTAB / NaCl solution, mix well and then incubate at 65°C for 10min; add an equal volume of phenol / chloroform / isoamyl alcohol and mix well, centrifuge for 4-5min, transfer the supernatant to a Add 0.6-0.8 times the volume of isopropanol to a new tube, mix gently until the DNA precipitates, and the precipitate can be centrifuged slightly; after the precipitate is washed with 1mL of 70% ethanol, the ethanol is disc...

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Abstract

The invention discloses a method for detecting vibrio parahemolyticus through combination of unlabelled fluorescent PCR and HRMA, which is characterized by including the following steps: firstly, a primer pair is designed according to a TLH (thermolabile hemolysin) gene of the vibrio parahemolyticus; secondly, after DNA (Deoxyribonucleic acid) samples are extracted, unlabelled fluorescent PCR amplification is performed by utilizing the designed primer pair; and thirdly, an amplification curve of a PCR amplification product and the HRMA are analyzed by application software. The purposes of the method are to overcome defects of the prior art and provide a method, which is simple, convenient and fast to operate, accurate in detection result and low in usage cost, for detecting the vibrio parahemolyticus through the combination of the unlabelled fluorescent PCR and the HRMA.

Description

technical field [0001] The invention relates to a method for detecting Vibrio parahaemolyticus by combining non-labeled fluorescent PCR with high-resolution melting curve (HRM) analysis, and belongs to the field of microbial detection. Background technique [0002] Vibrio parahemolyticus (Bibrio Parahemolyticus) is a halophilic bacterium belonging to the genus Vibrio in the family Vibrio, Gram stain negative, and is a polymorphic bacillus or slightly curved Vibrio. Eating food containing this bacteria can cause food poisoning, also known as halophilic bacteria food poisoning, mainly from seafood. Clinically, the main symptoms are acute onset, abdominal pain, vomiting, diarrhea and watery stool. Vibrio parahaemolyticus is a marine bacterium mainly derived from seafood such as fish, shrimp, crab, shellfish and seaweed. The disease mostly occurs in coastal areas in summer and autumn, often causing collective disease. In recent years, due to the air transportation of seafood,...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
CPCY02A50/30
Inventor 蔡先全邱德义柏建山邓艳伍朝晖简志华张宪臣蔡仁杰
Owner 邳州市博睿投资管理有限公司
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