Establishment And Application Of Rpa Detection Methods For Riemerella Anatipestifer And Pasteurella Multocida

The market for waterfowl in China is expanding and the stocking of commercial ducks is rising year on year.With the increase in waterfowl farming,various waterfowl diseases are also occurring,among which the duck diseases Riemerella anatipestifer and Pasteurella multocida pose a serious risk to the duck industry.Currently,most bacterial diseases occur clinically as mixed infections,of which RA and Pm mixed infections are more common,showing no obvious symptoms and difficult to identify based on lesion characteristics alone,and specific,rapid and sensitive detection methods need to be established.Recombinase polymerase amplification is a new isothermal amplification technique that allows amplification reactions to be completed in just 20 min at a constant temperature of 37°C to 42°C.The lateral flow dipstick allows direct visualisation of nucleic acid amplification results.In this study,single RPA-LFD and dual RPA-LFD assays for the detection of RA and Pm were developed using recombinant enzyme polymerase amplification in combination with lateral flow strips and successfully applied to clinical samples.The details of the study are as follows:1.Establishment and application of RPA-LFD detection method for Riemerella anatipestifer.Based on the published sequence of RA ompA gene on Gen Bank,PCR amplification primers were designed and the RA genome was extracted for PCR amplification to construct the positive standard plasmid pMD19-T-ompA.Four sets of(ompA-F1/R1,ompA-F2/R2,ompA-F3/R3,ompA-F4/R4)RPA Basic primers with pre-amplified fragment lengths of 211 bp,410 bp,378 bp and 322 bp,respectively,using the ompA gene as the target gene;A probe(RA-P)was designed according to the requirements of LFD,and the RPA-LFD method for the detection of RA was established after primer screening and optimisation of reaction conditions.The results showed that the ompA-F2/R2 primer amplification was the most efficient,RA-P was able to bind normally to the test strip for colour development,the best reaction temperature was 37°C and the reaction time was 15 min.The method was negative for E.coli,SE,Pm,Staphylococcus,GPV,DPV and MDPV with a sensitivity of 1.83×10~1copies/μL.The RA detection rate for clinical samples was 10.93%(7/64),with a100%compliance rate with the PCR method.It shows that the established RA-RPA-LFD method is fast,sensitive and specific and can be applied to the detection of clinical samples of RA.2.Establishment and application of RPA-LFD detection method for pasteurella multocida from ducks.Based on the Pm kmt1 gene sequence published on Gen Bank,PCR amplification primers were designed and the Pm genome was extracted for PCR amplification to construct the positive standard plasmid pMD19-T-kmt1.Four sets of(kmt1-F1/R1,kmt1-F2/R2,kmt1-F3/R3,kmt1-F4/R4)RPA Basic primers with pre-amplified fragment lengths of 174 bp,254 bp,226 bp and 333 bp,respectively,using the kmt1gene as the target gene;A probe(Pm-P)was designed according to the requirements of LFD,and the RPA-LFD method for detecting Pm was established after primer screening and optimisation of reaction conditions.The results showed that kmt1-F3/R3 amplification was the most efficient,Pm-P could bind normally to the test strip for colour development,the best reaction temperature was 37°C and the reaction time was 15 min.The method was negative for E.coli,SE,RA,Staphylococcus,GPV,DPV and MDPV with a sensitivity of 1.50×10~1copies/μL.The Pm detection rate for clinical samples was 4.68%(3/64),with 100%compliance with the PCR method.It shows that the established Pm-RPA-LFD method is fast,sensitive and specific and can be applied to the detection of Pm in clinical samples.3.Establishment and application of dual RPA-LFD detection method for Riemerella anatipestifer and Pasteurella multocida.The primer sets(RA:ompA-F2/R2,Pm:kmt1-F3/R3)and probes(RA-P,Pm-P)were used as primers and probes for the dual RPA-LFD method,and a dual RPA-LFD method was established based on the RPA-LFD technique that can detect RA and Pm simultaneously.The results show that the method takes only about 20 min to complete amplification,does not cross-react with E.coli,SE,Staphylococcus,GPV,DPV and MDPV,and has a minimum detection limit of 10~2copies/μL.The results showed that the established dual RPA-LFD method detected 10.93%(7/64)of RA positivity and4.68%(3/64)of Pm positivity,which were consistent with the results of the RA-RPA-LFD,Pm-RPA-LFD and normal PCR methods.It shows that the established dual RPA-LFD method has the advantages of high sensitivity,high specificity and easy and rapid operation,and can be used for the differentiation of clinical cases of RA and Pm single infections and mixed infections.