Method for detecting Vibrio parahaemolyticus

A technology of Vibrio hemolyticus and detection kit, which is applied in microorganism-based methods, biochemical equipment and methods, and determination/inspection of microorganisms, etc. , strict technical requirements and other issues, to improve the food safety guarantee system, suppress the occurrence of epidemics, and show obvious results

Inactive Publication Date: 2016-12-07
宁波海洋研究院 +1
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  • Abstract
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  • Application Information

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Problems solved by technology

The national standard uses more traditional microbiological methods for the above food-borne pathogenic bacteria, and these methods are not suitable for rapid detection
On the other hand, emerging immunological methods and molecular biology detection methods are often instrument-dependent, costly, and technically demanding, making these seemingly sensitive, specific, and efficient methods unable to be widely used and marketed. universal

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  • Method for detecting Vibrio parahaemolyticus
  • Method for detecting Vibrio parahaemolyticus
  • Method for detecting Vibrio parahaemolyticus

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Embodiment 1

[0032] Embodiment 1: Screening of primers and probes and establishment of methods

[0033] The present invention designs primers and probes according to the thermolabile hemolysin (thermolabile hemolysin, TLH) coding gene tlh of Vibrio parahaemolyticus:

[0034] The published tlh gene sequence was found through NCBI, and the designed primers were compared by Primer-BLAST, which had good specificity. The gene located at positions 440-608 of the tlh gene sequence was used as the target fragment for real-time RPA primers and probes. design.

[0035] Design 4 sets of primer pairs and 1 probe for optimal primer screening. The primer sequences are fine-tuned sequences, and the sequences that bind to the probes are located in the middle of the amplification regions of the 4 pairs of primers. The primer sets and probe sequences are as follows:

[0036] Forward primer F1-RPA: 5′-CAACATTAGATTTGGCGAACGAGAACGCAGAC-3′(32bp)

[0037] Reverse primer R1-RPA: 5′-TTAAAGATGTTGCCTGTATCAGACAAGCT...

Embodiment 2

[0076] Embodiment 2 is applied to the detection of actual samples

[0077] 1 material

[0078] 9 parts of aquatic products come from the aquatic product circulation market, including 2 parts of sea melon seeds, 2 parts of white sea melon seeds, 2 parts of flower clams, and 3 parts of razor clams (10g net content per part).

[0079] 2 reagents

[0080] 3% sodium chloride alkaline peptone water (3% APW, selective enrichment culture of Vibrio parahaemolyticus), sterile water (nuclease-free)

[0081] 3 sample processing

[0082] The sample processing steps are slightly modified on the basis of "GB 4789.7-2013 Food Safety National Standard for Food Microbiological Examination of Vibrio parahaemolyticus".

[0083] 3.1 Immediately after collection of non-frozen samples, store them in a low-temperature freezer at 7°C to 10°C, and test them as early as possible.

[0084] 3.2 Shellfish take all the contents, including shellfish meat and body fluids. For shellfish, first wash the sh...

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Abstract

The invention provides a method for detecting Vibrio parahaemolyticus. The sequence of the forward primer is SEQ ID NO:1, the sequence of the reverse primer is SEQ ID NO:2, and the sequence of the probe is SEQ ID NO:3. A real-time RPA (recombinase polymerase amplification) process is utilized to detect the food-borne pathogen Vibrio parahaemolyticus, thereby implementing safe, specific, quick, sensitive and simple field quick detection on the Vibrio parahaemolyticus, and further overcoming the defects in the existing traditional detection technique. The method is suitable for field detection, can effectively inhibit the epidemic situation caused by Vibrio parahaemolyticus food poisoning in time, and perfects the food safety control system.

Description

technical field [0001] The invention belongs to the technical field of detection of disease microorganisms, and in particular relates to a method for detecting Vibrio parahaemolyticus. Background technique [0002] Vibrio parahaemolyticus (VP) belongs to Bacteria, Proteobacteria, Gammaproteobacteria, Vibrionales, Vibrionaceae, Vibrio The genus Vibrio is a Gram-negative halophilic bacterium that mainly infects aquatic organisms living in warm, high-salt rivers or sea mouths. It has strong pathogenicity and is the main food-borne pathogen. one of the pathogenic bacteria. If humans eat seafood infected by it by mistake, gastroenteritis reactions such as diarrhea, abdominal pain, intestinal cramps, nausea, vomiting, and watery stools will occur, and in severe cases, clinical reactions such as systemic convulsions and renal failure will occur. The bacterium was first isolated from food poisoning patients by Fujino et al. in Japan. It has flagella, no capsule, and no spores in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/63
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2563/107
Inventor 朱鹏高威芳严小军黄海龙朱竹君李艳荣牟桐
Owner 宁波海洋研究院
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