Establishment Of Detection Methods For Avian Metapneumovirus Subgroup B

Avian metapneumovirus was first described in South Africa in the1970s, which mainlycauses upper respiratory tract or central nervous system diseases in turkeys and chickens, suchas Turkey rhinotracheitis and Swollen head syndrome. Apart from Australasia, all majorpoultry raising regions of the world have reported the presence of aMPV. Avianmetapneumoviruses has been further classified into four subtypes(A, B, C, D) based onnucleotide sequence divergence in the attachment glycoprotein (G) gene and antigenicdifferences (Juhasz and Easton,1994). Subtypes A and B are reported in Europe, Asia, andthe Middle East countries while subtypes C and D are respectively reported in the UnitedStates and France.In1999, the virus was successfully separated and reported in China for the first time,which provided evidence for the existence of aMPV in our country. Serology investigation byGuo in2008showed that the infection of aMPV can be100%in breeding chickens inShandong Province. Subtype A was detected in39%positive from chicken samples by RT-PCR in the same year, while subtype B was negative. For further comprehension of the aMPVepidemic and theory basis of prevention and control, the research focus on the establishmentof the diagnosis method. This research content was divided into two parts:1.Establishment of RT-LAMP system for rapid detection of Avian metapneumovirussubgroup BA rapid diagnostic method of reversetranscription loop-mediated isothermal amplify-cation(RT-LAMP)for Avian metapneumovirus(aMPV)subgroup B was established with a setof special primers designed for aMPV F gene with Primer Explorer V4. And the reactionsystem and reaction conditions were optimized in the reaction time, temperature, concentra-tion of various components and other aspects. The fragment of Avian metapneumo-virus(aMPV) subgroup B F gene could be specifically amplified by the RT-LAMP at63℃within1h and the amplification results of H9N2avian influenza virus(AIV), Newcastledisease virus (NDV), Infectious bronchitis virus(IBV), infectious bursal disease virus (IBDV)were negative. The amplification products could be observed by naked-eye. The detectionlimit of the system was found to be1×102copies/μL plasmid sample. The positive rate oftwenty chicken samples suspected Avian metapneumovirus was10%. The results showed thatthe RT-LAMP diagnosticmethod for Avian metapneumovirus (aMPV) subgroup B appeared to be fast, accurate, highly sensitive and specific for the purpose and it could be applied to theclinical diagnosis of the relevant disease.2.Development and application of SYBR GreenⅠ real-time quantitative PCR technique forAvian metapneumovirus subgroup BA pair of specific primers was designed according to the Avian metapneumovirussubgroup B strain (JN224985.1) F conserved gene sequence available in GenBank. The target214bp fragment was amplified in RT-PCR experiments, and inserted into pMD18-T vectorafter being purified. Then the restructured vectors were transformed to E.coli DH5α. AfterPCR identifying, enzyme cutting and sequencing, positive plasmid was extracted as templateto establish SYBR Green Ⅰfluorescence quantitative PCR standard curve. Sensitivity,reproducibility and specificity of the method were determined. The results indicated thatstandard curve established by recombinant plasmid showed a good linear relationshipbetween threshold cycle and templat concentration, the Tm was83.0~83.5℃and the sensitivedegree is7.9×102copies per μL, and the quantitative PCR was highly reproducibility andmore specificity than traditional PCR. A SYBR Green Ⅰ fluorescent quantitative RT-PCRassay for detecting F gene of aMPV subgroup B was developed for the basis of the earlydetection and quantitative analyzing of the aMPV infection degree.