Development of rapid and easy detection of Salmonella in food matrics using RPA-CRISPR/Cas12a method
2022
Liu, Li | Zhao, Gang | Li, Xiangmei | Xu, Zhenlin | Lei, Hongtao | Shen, Xing
Salmonella species are common foodborne pathogenic bacteria. At present, most detection methods for Salmonella are unsuitable for on-site applications because they require large instruments or complicated procedures. This study developed a novel method of the on-site detection for Salmonella in food by combining the CRISPR/Cas12a system with recombinant polymerase amplification (RPA). The optimal concentration ratio of Cas12a enzyme, CRISPR RNA (crRNA) and FQ-probe is 1 : 1: 2.5. The detection limit of the RPA–CRISPR/Cas12a method was 1 × 10⁻⁴ ng/μL for genomic DNA (gDNA), and 10² CFU/mL for bacterial liquid. The method exhibited no cross-reactivity to 4 common pathogenic bacteria. A simple boiling method was used to pretreat chicken and egg samples to meet on-site testing requirements. The detection limits of chicken and egg samples were 10³ CFU/mL initially and could reach 10² CFU/mL and 10¹ CFU/mL, respectively, after 3 h enrichment at 37 °C. The sensitivity of the RPA–CRISPR/Cas12a method was comparable to that of the quantitative polymerase chain reaction (qPCR) technique. In addition, the operation of this method is simpler and faster than qPCR. Thus, the method is applicable to the on-site detection for Salmonella in food.
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