Rapid detection of four diarrheal bacteria by CRISPR-Cas13a combined with recombinase aided amplification

ObjectiveTo establish a rapid， sensitive and specific detection method for 4 diarrheal bacteria （Salmonella， Shigella， Vibrio cholera and Escherichia coli O157：H7） by the Clustered regularly interspaced short palindromic repeats associated protein 13a （CRISPR-Cas13a） combined with recombinant enzyme-mediated isothermal amplification （RAA）， called RAA-Cas13a.MethodsIn this study， the specific primer for RAA and CRISPR RNA （crRNA） of 4 different diarrheal bacteria were designed. The sample nucleic acids were amplified by RAA， and the amplification products were then detected with CRISPR-Cas13a. Compared with real-time quantitative polymerase chain reaction（RT-qPCR）， the sensitivity and specificity of the RAA-Cas13a method were evaluated.ResultsThe established RAA-Cas13a detection method for Shigella， Vibrio cholera and Escherichia coli O157：H7 had the detection limit of 10 copies/μL， the detection limit for Salmonella was 1 copy/μL， and each bacteria did not have cross-reaction with the other ten bacteria. Meantime， the detection of the RT-qPCR and RAA-Cas13a were highly consistent in 200 suspected samples and 40 artificial simulation samples （Kappa=0.927 and 1.000， respectively）.ConclusionThe established RAA-Cas13a detection method has the advantages of high sensitivity and strong specificity. It can quickly detect and screen diarrheal diseases caused by 4 pathogenic bacteria.