Establishment And Application Of Universal And Duplex (H5 And H9 Subtypes) Real-time RT-PCR For Detection Of Avian Influenza Viruses

Avian influenza virus(AIV)is one of the common pathogens causing respiratory disease and even death in poultry,which poses huge economic losses to the poultry industry and seriously hampering its development.There are currently known multiple subtypes of AIV,In recent years,H5,H7,H9 subtypes of avian influenza viruses cocirculated in poultry in China.At the same time,new recombinant AIVs continue to emerge,such as H9N9 and H10N3.The low pathogenic H9N2 subtype AIV is widespread and the dominant AIV subtype,and has the higher transmission efficacy in chickens.H5N8 has replaced H5N6 as the dominant the H5 subtype AIV.These bring the huge challenge to the surveillance and control of avian influenza.Molecular biology methods are commonly used to detect avian influenza viruses including real-time PCR,but this is usually designed for the detection of one subtype of avian influenza virus.However,H5,H7 and H9 subtypes AIV are more common in chickens and ducks,while other subtypes of AIV continually emerged and disseminated in avian population,such as H3,H5Nx,H6 and H10 subtypes of AIV.Therefore,a universal and H5-H9 duplex real-time RT-PCR were in urge need to be developed to detect all subtypes and common subtypes of AIVs.Real-time RT-PCR based on TaqMan-MGB probe is a highthroughput multiplex influenza virus detection assay with high sensitivity,specificity,simplicity of operation.In this study,one-step TaqMan-MGB Real-time RT-PCR assays were developed to detect all subtypes and H5-H9 subtypes of avian influenza viruses,and identify and differentiate common H5 and H9 subtypes and other subtypes of AIV,providing a reference for rapid detection of avian influenza viruses.1.Establishment of universal and duplex(H5 and H9 subtypes)Real-time RT-PCR for detection of avian influenza virusesIn this study,we developed a universal real-time RT-PCR assay for rapid detection of avian influenza viruses based on probes of the NP gene,and a duplex Real-time RTPCR for simultaneous detection of HA gene of H5 and H9 subtypes AIV s.Fluorescence signals corresponding to H1-H11 subtypes(no H12-H16 subtypes AIV are currently available in our laboratory)were detected by the universal Real-time RT-PCR;the detection limit of the universal Real-time RT-PCR was 1 copy/?L and is highly sensitive;the variability tests of intra-and inter-assay showed excellent reproducibility with<1%of the coefficient of variation.A significant amplification curve was observed by the duplex Real-time RT-PCR for H5 and H9 subtypes of AIVs,while no fluorescence signals corresponding to H1-H11 subtypes as well as other avian viruses such as IBV,ILTV and NDV;the detection limit reached 5 copies/?L;the variability tests of intra-and inter-assay showed excellent reproducibility.We compared Real-time RT-PCR assay with chicken embryo isolation to detect AIV in the mixed laryngeal/cloacal swab samples collected from live poultry markets.There is 90.48%of compliance rate between Real-time RT-PCR assay and chicken embryo isolation.The detection rates of H5 and H9 and other subtypes AIV by Real-time RT-PCR were higher than those by chicken embryo isolation.In conclusion,the Real-time RT-PCR established in this study are specific,sensitive and reproducible,with higher detection rates than isolation in the chicken embryonated eggs method,and are suitable for the rapid detection of large quantities of samples,and have good prospects for application in the detection of avian influenza viruses.2.Application of universal and duplex(H5 and H9 subtypes)Real-time RT-PCR for detection of avian influenza viruses157 samples were tested,The AIV detection rate was 26.75%,the detection rate of H9 subtype AIV was 21.02%,and the detection rate of H5 subtype AIV was 3.18%.The detection rate of AIV in free-range chickens was higher than that in commercial chickens.the infection rate was relatively high in chickens less than 3 months of age,respectively,among which H9 subtype AIV was dominant.AIVs can be detected throughout the year.Although there is no obvious seasonality,the positive rate of AIV is relatively high in August,September and October,respectively.It was common for H9 subtype AIV in these months.AIV positive samples mainly distributed in Jiangsu and Anhui provinces and was dominant for H9 AIV.In summary,it is clear that avian influenza is still prevalent in the poultry industry.The Real-time RT-PCR established in this study can be used as a reference technique to monitor avian influenza viruses and provide support for detection of AIVs,prevention and control of avian influenza in the poultry industry.