Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification  

作　　者：JIANG Han Ji TAN Rong JIN Min YIN Jing GAO Zhi Xian LI Hai Bei SHI Dan Yang ZHOU Shu Qing CHEN Tian Jiao YANG Dong LI Jun Wen 

机构地区：[1]Tianjin Institute of Environmental&Operational Medicine,Tianjin 300050,China

出　　处：《Biomedical and Environmental Sciences》2022年第6期518-527,共10页生物医学与环境科学（英文版）

基　　金：supported by the National Key Research and Development Plan of China[2018YFC1602500];the Natural Science Foundation of Tianjin[19JCZDJC39900]

摘　　要：Objective To establish an ultra-sensitive,ultra-fast,visible detection method for Vibrio parahaemolyticus(VP).Methods We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a(CRISPR/Cas12a)combined with recombinase polymerase amplification and visual detection(CRISPR/Cas12a-VD).Results CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10^(-18)M(single molecule detection)within 30 min without cross-reactivity against other bacteria.When detecting pure cultures of VP,the consistency of results reached 100%compared with real-time PCR.The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10^(2)CFU/g.Conclusion The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods,such as real-time PCR,and has great potential for preventing the spread of pathogens.

关 键 词：Vibrio parahaemolyticus CRISPR/Cas12a-VD Isothermal amplification Recombinase polymerase amplification Visual detection CROSS-REACTIVITY 

分 类 号：R155.5[医药卫生—营养与食品卫生学]