Rapid detection of four diarrheal bacteria by CRISPR-Cas13a combined with recombinase aided amplification
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1.College of Pharmacy, Nankai University, Tianjin 300000, China;2.NHC Key Laboratory of Enteric Pathogen Microbiology, Jiangsu Provincial Center for Disease Control and Prevention, Jiangsu Nanjing 210009, China;3.Tianjin International Biomedical Joint Research Institute, Tianjin 300000, China;4.Nanjing Medical University, Jiangsu Nanjing 210029, China;5.Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

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R155

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    Abstract:

    Objective To 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.Methods In 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.Results The 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).Conclusion The 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.

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AN Bailin, SU Xuan, GUO Yue, WANG Xiangxi, GE Yiyue, ZHU Fengcai, CUI Lunbiao. Rapid detection of four diarrheal bacteria by CRISPR-Cas13a combined with recombinase aided amplification[J].中国食品卫生杂志,2023,35(3):381-389.

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History
  • Received:October 12,2022
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  • Online: May 24,2023
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