Loop-mediated isothermal amplification method for detecting salmonella in food and reagent kit

[0042] 1. Design and synthesize primers

[0043] The HisJ gene sequence of Salmonella was searched in GenBank, and its homology was compared, and the homology was above 99%. Primers were designed referring to Salmonella typhimurium ATCC 14028s (accession CP001363.1) in GenBank. Use the PrimerExplorer V4 software of Japan Rongyan Chemical Co., Ltd. to design according to the default settings ( http: / / primerexplorer.jp / elamp4.0.0 / index.html ). The primer design region of the loop-mediated isothermal amplification is located in the Salmonella genome HisJ gene 2,516,325-2,516,524nt ( figure 1 ). A set of four primers SALFIP, SALBIP, SALF3 and SALB3C were synthesized in Dalian Bao Biological Company according to the PAGE purity level.

[0044] 2. Loop-mediated isothermal amplification reaction procedure and reaction system establishment

[0045] By selecting the primer concentration, magnesium sulfate concentration, betaine concentration, etc., the optimal reaction syste

[0072] Detection of Salmonella in food by loop-mediated isothermal amplification using His J as target gene

[0073] 1. Sample processing and bacterial enrichment

[0074] 200 supermarket food samples (chicken, pork and vegetables) were collected and the samples were first washed in sterile sample bags using 100ml 0.1% BPW. After the sample was fully washed, the washing solution was poured into a 250ml Erlenmeyer flask, and incubated in a shaker at 37°C at a constant temperature of 150rpm for 24 hours. Next, transfer 1 ml of BPW culture into 9 ml of malachite green medium and incubate at 42° C. at 180 rpm for 24 hours.

[0075] 2. Extraction of DNA

[0076] First the bacterial culture was centrifuged at 8,000×g for 5 minutes, the pellet was suspended and washed with PBS. Then centrifuge at 8,000×g to obtain the pellet and resuspend the bacteria in 100 μl ultrapure water. Bacteria were lysed using the boiling method, that is, the bacterial suspension was boiled at 100°C for 10