Visual Detection of Salmonella by Combined Application of G-quadruplex and As-PCR Methods
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(1.College of Food Science and Technology, Hebei Agricultural University, Hebei Bioinformatic Utilization and Technological Innovation Center for Agricultural Microbes, Baoding 071000;2.Hebei of Biology and Food Science, Hebei Normal University for Nationalities, Chengde 067000, Hebei;3.Affiliated Hospital of Hebei University, Baoding 071000, Hebei)

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    Abstract:

    Salmonella(SALM) is a foodborne pathogen that often transmit via food as media, so rapid detection is the key to controlling the pathogen. In this study, Salmonella genomic DNA was used as templates, the amplification conditions of asymmetric PCR(As-PCR) were optimized. The results demonstrated that the maximum concentration of single-strand DNA (ssDNA) could be obtained when the final concentration of the excess primer (HF) was 0.4 μmol/L, the ratio of the final concentration of HF to GR was 10∶1, the annealing temperature was 58 ℃ and the amplification cycle was 40 cycles. When ssDNA with G-quadruplex sequence interacted with 40 μmol/L hemins for 60 min, the peroxidase activity of G-quadruplex was the highest. On this basis, the visual detection of the invH gene of Salmonella was realized by combining As-PCR with G-quadruplex technology. The method could detect Salmonella genomic DNA in a range of 2 pg/μL-258 ng/μL, and the logarithm of its concentration had a good linear relationship with the absorbance value at 421 nm (y = 0.0691x + 0.3085, R2 = 0.9729). The detection limit of Salmonella for contaminated milk samples was 35 CFU/mL, showing that this method had the advantages of simple operation with a high sensitivity and providing new technical support for the detection of these pathogenic microorganisms.

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  • Received:May 13,2022
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  • Online: June 25,2023
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