针对副溶血性弧菌toxR基因的RPA-CRISPR/Cas13a荧光快速检测方法的建立及应用

Establishment and application of RPA-CRISPR/Cas13a fluorescent rapid assay targeting Vibrio parahaemolyticus tox R gene

目的探讨并建立非专业实验室场景下恒温、快速及简便的检测副溶血性弧菌的方法.方法本研究针对副溶血性弧菌toxR基因设计特异性引物和成簇规律间隔短回文重复序列(CRISPR)RNA(crRNA),建立基于重组酶聚合酶扩增(RPA)结合CRISPR及其相关蛋白13a(CRISPR-Cas13a)的反应体系,采用自配十二烷基硫酸钠(SDS)核酸快速提取试剂提取样本全基因组,并结合荧光法实现检测结果的可视化判读.结果利用副溶血性弧菌菌株ATCC 17802和其他3种非副溶血性弧菌(溶藻弧菌、河流弧菌和梅氏弧菌)以及3种临床上常见腹泻致病菌(金黄色葡萄球菌、大肠埃希菌和铜绿假单胞菌)对RPA-CRISPR/Cas13a荧光法的特异性进行验证,结果显示特异性为100.00%;对副溶血性弧菌基因组DNA进行倍比稀释并检测,该方法最低检出限为102 copies/μl.最后,将建立的方法应用于野生型副溶血性弧菌检测,检测结果与TaqMan定量聚合酶链式反应(TaqMan-qPCR)检测结果及基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)鉴定结果一致.结论本研究建立了一种针对toxR基因的RPA-CRISPR/Cas13a检测方法,具有简便、快速、特异性强、结果判读可视化等优点,为非专业实验室场景下的副溶血性弧菌快速检测提供了较好工具.

OBJECTIVE To explore a constant,rapid and simple method for detection of Vibrio parahemolyticus in non-specialized laboratory settings.METHODS Specific primers and clustered regular interval short palindrome re-peating sequence(CRISPR)RNA(crRNA)were designed in targeting toxR gene of V.parahemolyticus so as to establish a reaction system based on recombinase polymerase amplification(RPA)and its related protein 13a(CRISPR-Cas13a).The whole genome was extracted from the samples by sodium dodecyl sulfate(SDS)nucleic acid rapid extraction reagent,and the visualized interpretation of the detection results was carried out by fluores-cence method.RESULTS The specificity of the RPA-CRISPR/Cas13a fluorescence method was verified by using V.parahemolyticus ATCC 17802 strains and 3 species of non-V.parahemolyticus(Vibrio alginolyticus,Vibrio fluvialis and Vibrio metschnikovi)as well as 3 common species of pathogens causing diarrhea(Staphylococcus aureus,Escherichia coli and Pseudomonas aeruginosa),and the result showed that the specificity was 100.00%.The genome DNA of the V.parahemolyticus was subjected to serial dilutions and detected,and the minimum de-tectable limit of the method was 102 copies/μl.The established methods was applied for detection of wild typeⅤ.parahemolyticus,and the detection result was consistent with the result of TaqMan quantitative polymerase chain reaction(TaqMan-qPCR)and the result of matrix-assisted laser desorption ionization-time-of-flight(MALDI-TOF)mass spectrometry.CONCLUSION The established RPA-CRISPR/Cas13a detection method targeting the toxR gene is simple,rapid and has high specificity and visualized interpretation of result,and it facilitates the rapid de-tection of V.parahemolyticus in non-specialized laboratory settings.