多杀性巴氏杆菌重组酶聚合酶扩增快速诊断方法的建立OA北大核心

The purpose of this study is to establish a sensitive,specific,rapid and visualized clinical diagnostic method based on recombinase polymerase amplification(RPA)and lateral flow im-munochromatography strip(LFD)technology.Specific primers and probes are designed for RPA amplifica-tion and nfo RPA amplification based on species specific Kmt Ⅰ gene of P.multocida.The optimal primers were screened,the reaction system of RPA was optimized,and the optimal reaction conditions were de-termined by agarose gel electrophoresis and lateral flow immunochromatography strip analyzing.The results showed that Kmt Ⅰ could be well detected at 30 ℃ within 20 minutes,and the minimum number of plasmid copies that could be detected was 2.26 × 101 copies/µL.There was no cross-reaction with Es-cherichia coli,Staphylococcus aureus,Salmonella typhimurium,Salmonella cholerae,and Pseudomonas aeruginosa,which showed good specificity.15 clinical samples of Zi Geese were detected RPA basic,RPA-LFD and ordinary PCR,and the results are consistent,with a positive detection rate of 100％and a compliance rate of 100％.In conclusion,RPA basic and RPA-LFD rapid detection methods with high sensi-tivity and specificity,simple operation,low instrument and site requirements for P.multocida detec-tion were established in this study,which provides reliable technical support for the point-of-care testing and epidemiological investigation of established.