A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection

文献类型： 外文期刊

第一作者： Gong, Jiansen

作者： Gong, Jiansen;Dou, Xinhong;Gong, Jiansen;Dou, Xinhong;Zhang, Di;Fu, Lixia;Dong, Yongyi;Wu, Kun;Wang, Chengming

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关键词： Salmonella Indiana; RPA; CRISPR/Cas12b; one pot; one step

期刊名称：MICROORGANISMS （ 影响因子：4.5； 五年影响因子：4.8 ）

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年卷期： 2024 年 12 卷 3 期

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收录情况： SCI

摘要： Salmonella enterica serovar Indiana (S. Indiana) is among the most prevalent serovars of Salmonella and is closely associated with foodborne diseases worldwide. In this study, we combined a recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein Cas12b (CRISPR/Cas12b)-based biosensing in a one-pot platform to develop a novel one-step identification method for S. Indiana infection diagnosis. The entire RPA-CRISPR/Cas12b reaction can be completed at 41 degrees C within 1 h without the need for specific instruments. The optimal concentrations of Cas12b and single-guide RNA (sgRNA) for the reaction were the same at 250 nM. The single-stranded DNA (ssDNA) reporter 8C-FQ (5 '-/6-FAM/CCCCCCCC/BHQ1/-3 ') presented the best performance in the reaction compared with the other reporters. The limit of detection (LoD) of the RPA-CRISPR/Cas12b assay was 14.4 copies per reaction. As for specificity, we successfully identified four S. Indiana strains among twenty-two Salmonella strains without any false-positive results, presenting 100% accuracy for S. Indiana, and no cross-reactions were observed in eight other pathogens. Moreover, a total of 109 chicken carcasses were classified by the S. Indiana RPA-CRISPR assay and PCR methods from three processing points, including 43 post-shedding, 35 post-evisceration, and 31 post-chilling. There were 17 S. Indiana-positive samples identified during the whole processing step, consisting of nine post-shedding, five post-evisceration, and three post-chilling. The corresponding S. Indiana-positive rates of post-shedding, post-evisceration, and post-chilling were 20.93% (9/43), 14.29% (5/35), and 9.68% (3/31), respectively. Results from the S. Indiana one-step RPA-CRISPR/Cas12b assay were totally in agreement with those obtained using a traditional culture method, demonstrating 100% agreement with no false-positive or false-negative results observed. Altogether, the RPA-CRISPR/Cas12b assay developed in this study represents a promising, accurate, and simple diagnostic tool for S. Indiana detection.

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