Detection Of Brucella Infection Based On CRISPR/Cas12a System

Objective: To establish and optimize a rapid and accurate diagnosis method for Brucella infection and a diagnostic method for distinguishing wild strains from S2 vaccine strains,by combining the Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)and its associated protein,Cas12 a,also known as the CRISPR/Cas12 a system,with the Recombinase Polymerase Amplification(RPA)technique.Methods: The specific RPA primers were designed using highly conversed gene bp26,and the gene upstream sequence of sugar ABC transporter periplasmic sugar-binding protein in wild strain and S2 vaccine strain of Brucella for designing specific RPA primers.Specific cr RNAs,fluorescent reporter probe,and nucleic acid detection test strip probe were designed based on the detection principle of the CRISPR/Cas12 a system.A rapid diagnostic method for detecting Brucella infection and distinguishing wild strain from S2 vaccine strain was established using the combination of CRISPR/Cas12 a and RPA,and the target sequences were detected through three different methods: real-time fluorescence quantitative detection system,fluorescence imaging instrument,and nucleic acid detection test strip.The sensitivity and specificity of the established method in detecting Brucella infection were further analyzed.The established method was compared with regular RPA and fluorescence quantitative RPA in terms of sensitivity,and its consistency with q PCR was also compared in actual sample detection.Results: The RPA-CRISPR/Cas12 a rapid nucleic detection method established and optimized can detect Brucella DNA in patient serum samples within 30 min.The sensitivity of the three detection methods were as follow: 10 copies/μL(RPA-CRISPR/Cas12a-real-time fluorescence quantification),10 copies/μL(RPA-CRISPR/Cas12a-fluorescence imaging),and100 copies/μL(RPA-CRISPR/Cas12a-test strip),all of which were higher than regular RPA and fluorescence quantitative RPA.The method showed strong specificity with non-specific amplification observed,and no antigen cross-reaction with other bacteria such as Yersinia enterocolitica O:9,Escherichia coli O157,Salmonella enterica serovar Urbana O:30,and Francisella tularensis.The detection results of actual samples were consistent with the positivity rate based on the q PCR detection method.Conclusion: In this study,the established RPA-CRISPR/Cas12 a detection method possesses characteristics such as rapidity,simplicity,high sensitivity,and strong specificity,providing a new nucleic acid detection method for the early,accurate,and rapid diagnosis of Brucella infection and the differentiation of wild strain and S2 vaccine strain in infected bacteria.Particularly,fluorescence imaging and test strip detection provide cost-effective tools for nucleic acid rapid detection of Brucella infection and auxiliary differential diagnosis of occupational diseases in countries and regions with limited resources and high incidence of brucellosis.