Method for detecting Vibrio parahaemolyticus

[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...

[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...