The Multiplex RT-PCR Assay For Detecting AMPV,IBV,NDV And The Expression Of AMPV N Protein And The Development And Application Of Indirect Elisa Method

Avian metapneumovirus(a MPV)mainly causes upper respiratory tract infection in chickens and turkeys,resulting in decreased egg production and egg quality in laying hens.AMPV has also been found to be pathogenic to Muscovy duck and other poultry in recent years.AMPV has a low mortality with single infection,but it can lead to weakened immunity,which can promote secondary infection and increase mortality.AMPV has been reported in all countries except Oceania and is a serious threat to the poultry industry worldwide.Serological investigation showed that a MPV infection was prevalent in the flocks of chicken farms in many regions including Guangxi,and the flocks of different breeds,genders and usages were seriously infected.The serum positive rate of some breeding flocks even reached 100%.The virus is difficult to isolate,and the clinical symptoms and anatomical lesions of a MPV infection are similar to those of infectious bronchitis virus(IBV)and Newcastle disease Virus(NDV)infection.In addition,it was frequently accurred that the mixed infection of a MPV,IBV and NDV.Therefore,it is important to establish diagnostic methods for simultaneous detection of a MPV,IBV and NDV and to understand the prevalence of the disease in chickens.AMPV RT-PCR detection method was previously established by our team.On this basis,the establishment and application of the multiplex RT-PCR method for detecting a MPV,IBV and NDV were conducted in this study.At the same time,the conserved region of a MPV N protein with good hydrophilicity and antigenicity was expressed.And then the universal indirect ELISA method for detection of a MPV specificity antibodies was established using the purified N protein as coating antigens.The details are as follows.?.The establishment and application of a multiplex RT-PCR method for detection of a MPV,IBV and NDVA pair of specific primers were designed according to the F gene sequence of NDV published in Genbank.A multiplex RT-PCR method for simultaneous detection of a MPV,IBV and NDV was established with the previously designed a MPV and IBV universal primers.The amplification conditions were optimized,and the results of specificity test,sensitivity test and repeatability test showed the feasibility of the method.The results showed that the multiplex RT-PCR method could specifically amplify a MPV,IBV and NDV,while the amplification results of IBDV,ILTV,REV,ALV,MDV,MDPV,DHAV,FADV,DTMUV,E.coli and Salmonella were all negative.The established multiplex RT-PCR method has a minimum detection limit of 1 ng RNA.The repeatability test results show that the method has good repeatability.220 clinical samples were dectected by the established method,and the positive rate of a MPV,IBV and NDV were 10.45%(23/220),56.4%(124/220)and 2.7%(6/220),respectively.In this study,an efficient,rapid,sensitive and specific multiplex RT-PCR detection method for a MPV,IBV and NDVwas established,providing a strong technical support for the simultaneous rapid diagnosis and epidemic monitoring of three common respiratory diseases in poultry production. ?.Prokaryotic expression of avian metapneumonovirus N proteinAccording to bioinformatics software,the conservative amino acid sequence,the secondary structure and B cell antigen sites of a MPV N protein were predicted.Partial N gene protein fragment with conservatism,hydrophilicity and good antigenicity was selected,recombined with the expression vector p ET32a-1,transfored into E.coli DE3 cells and then induced expression by IPTG.A large quantities N protein was expressed after optimizing conditions,and purified by NiNTA resin.SDS-PAGE electrophoresis and Western blot analysis results showed that the N protein was successfully expressed.The successful expression of a MPV N protein in the prokaryotic expression system has laid a foundation for the development of a MPV diagnostic techniques and the development of genetically engineered vaccine.?.Establishment and application of indirect ELISA method based on N protein for avian metapneumonovirusThe purified recombinant N protein was used as the coating antigen.After a series of conditions optimization,an indirect ELISA method based on the N protein was preliminarily established.The established ELISA method did not showed positive results for dectetion of NDV,IBDV,AIV,ALV,MDV,IBV positive sera.The maximum coefficient of variation in the in-batch repeatability test was 6.20%,and that in the inter-batch repeatability test was 8.25%,which were all less than 10%.The N protein indirect ELISA method and commercial kit were used to detect 326 serum samples in parallel.The results showed that the coincidence rate between the established N protein indirect ELISA method and the commercial kits was 95.4%.960 serum samples from chickens of large-scale breeding companies in Guangxi were detected by the established indirect ELISA,and the positive rate of a MPV antibody was 94.06%.Therefore,this study laid a foundation for the development of a MPV ELISA detection kit,and established a technical platform for the early diagnosis and epidemiological investigation of a MPV.In summary,an efficient,rapid,sensitive and specific multiplex RT-PCR detection method for a MPV,IBV and NDV was established in this study.The conserved,antigenic,hydrophilic universal N protein was selected and expressed.An indirect ELISA method was established to provide a good technical platform for rapid diagnosis and epidemic monitoring of mixed infections of a MPV,IBV and NDV,and lays a solid technical foundation for early diagnosis and epidemiological investigation of a MPV based on the N protein.