重组酶聚合酶扩增结合侧向流动试纸条技术快速可视化检测嗜麦芽窄食单胞菌方法的建立与应用OACSTPCD

Objective:To develop a rapid and visual detection method of Stenotrophomonas maltophilia(S.maltophilia)by recombinant polymerase amplification(RPA)combined with lateral flow strip(LFS).Methods:RPA primers were designed based on specific sequences(NC_010943.1)of S.maltophilia.Through basic RPA reaction and agarose gel electrophoresis,the best primer pair was selected according to the amplification performance and the formation of cross dimers.According to the best primer pair,the probe and modified-primer were designed.Base mismatches were introduced into the primer and probe to eliminate false positive signals,and then the RPA-LFS reaction system was established.The optimal reaction conditions of RPA-LFS were optimized based on the color of the test line.The specificity of the method was identified by detecting 12 common clinically pathogens and 12 S.maltophilia of clinical origin.The sensitivity of this method was tested by diluting the genome template.Kappa analysis and clinical application evaluation of RPA-LFS were performed by detecting 108 clinical samples,comparing with qPCR and culture-biochemical method.Results:The RPA-LFS detection method can complete the amplification process by reacting for 8 min under 37℃,and the results can be observed on the LFS within 1 min.This method had high sensitivity and the limit of detection was 1.107 CFU/reaction.In addition,the RPA-LFS method had high specificity and no cross-reaction with other pathogens.Compared with qPCR,the RPA-LFS method showed the same accuracy.The results of RPA-LFS exhibited a high degree of consistency with the culture-biochemical method,with a kappa index of 0.972.Conclusion:This study has established a rapid and accurate RPA-LFS dectection method for identifying S.maltophilia,which is independent of the precision instruments and the professional technician.The implementation of this method can provide valuable information for timely formulation of appropriate reasonable antibacterial treatment plans,and shows promise in clinical application.