单核细胞增生李斯特氏菌与副溶血性弧菌RPA-CRISPR/Cas12a检测方法的建立

Establishment of CRISPR/Cas12a System Combined with Recombinase Polymerase Amplification Method for Detection of Listeria monocytogenes and Vibrio parahaemolyticus

本研究建立了快速检测单核细胞增生李斯特氏菌(Listeria monocytogenes,LM)与副溶血性弧菌(Vibrio parahaemolyticus,VP)的重组酶聚合酶等温扩增(Recombinase polymerase amplification,RPA)方法。根据单核细胞增生李斯特氏菌hlyA基因和副溶血性弧菌tlh基因设计合成引物和CRISPR RNA(cr RNA),合成荧光标记ssDNA探针,建立RPA-CRISPR/Csa12a检测方法。对建立方法的特异性、灵敏度进行检测,并通过人工污染样本对方法进行验证。结果显示,建立的RPA-CRISPR/Cas12a方法可特异性检出单核细胞增生李斯特氏菌和副溶血性弧菌靶标基因,且不与大肠埃希氏菌、金黄色葡萄球菌、沙门氏菌、伊氏李斯特菌等常见致病菌发生交叉反应;灵敏度试验结果显示,对于质粒标准品,副溶血性弧菌检出限为27 copies/μL,单增李斯特菌检出限为21 copies/μL;人工污染样品检测结果显示,本方法可以准确快速地检出样品中的单核细胞增生李斯特氏菌和副溶血性弧菌。该方法特异性良好,灵敏度高,不依赖温度循环装置,可实现采样现场快速检测,提升检测效率。

This study established a RPA-CRISPR/Cas12a method for detecting Listeria monocytogenes and Vibrio parahaemolyticus.Primers and CRISPR RNA(crRNA)were designed and synthesized based on the hly A gene sequence of L.monocytogenes and the tlh gene sequence of V.parahaemolyticus,respectively.Fluorescently labeled ssDNA probes were synthesized to establish the RPA-CRISPR/Cas12a detection method.The specificity and sensitivity of the established method were tested,and the method was verified by artificial contaminated samples.The results showed that the established RPA-CRISPR/Cas12a method could specifically detect the target genes of Listeria monocytogenes and Vibrio parahaemolyticus without crossreaction with common pathogens including Escherichia coli,Staphylococcus aureus,Salmonella and Listeria eiderii.Sensitivity test results showed that for plasmid standards,the detection limit for Vibrio parahaemolyticus was 27 copies/μL,and for Listeria monocytogenes,it was 21 copies/μL.Artificially contaminated sample detection results showed that the method could accurately and rapidly detect Listeria monocytogenes and Vibrio parahaemolyticus in samples.The method has good specificity,high sensitivity,does not require a temperature cycling device,and can achieve rapid detection on-site and improved detection efficiency.