Isolation And Identification Of APMV-6 Hubei Strain And Development Of SYBR Green ? Real-time PCR Assay For Detection

APMV-6 is a member of Mononegavirales,Paramyxoviridae,Paramyxovirinae,Avulavirus.Epidemiological investigation showed that APMV-6 strains could be isolated from different kinds of waterfowl and 9-11 days old chicken embryo in the experiment.APMV-6 can cause mild respiratory symptoms in birds and lower egg production.There is little research on avian paramyxovirus type 6 at home and abroad.At present,there are only 9 complete sequences of APMV-6 strains in GenBank.Therefore,it is necessary to analyze the prevalence and biological characteristics of APMV-6 and to establish an efficient detection method for APMV-6.First of all,we collected samples from five regions of China.After virus culture was carried out on the samples,the positive samples were sequenced using common PCR.After the positive sample was verified as APMV-6,the genome was amplified and sequenced.Then the samples identified as APMV-6 were subcultured,and the hemagglutination of each generation virus was tested by erythrocyte agglutination test.Finally,a fluorescence quantitative PCR method for the detection of SYBR Green I was established.Evaluate its sensitivity,specificity,repeatability,etc.An APMV-6 strain named Hubei strain was isolated from 11 368 samples.The genomic length of Hubei strain is 16 236bp,which conforms to the six-base principle of avian paramyxovirus.The genetic evolution analysis of Hubei strain showed that it belonged to subtype 1a of gene 1.In the hemagglutination test of Hubei strain,it was found that there was no hemagglutination of Hubei strain in F1 and F2,but occurred in F3,this phenomenon does not accord with the biological characteristics of avian paramyxovirus with hemagglutination.According to the Avian paramyxovirus 6 strain M gene sequence available in Gen-Bank(Accession number:KF267717).Taking advantage of Oligo7 to design a pair of primer,and the recombinant positive plasmids were used as the standard samples to establish the detection method of APMV-6 SYBR Green real-time reverse transcription PCR.There was a good liner relationship with the liner correlation and the amplification efficiency of the standard curve were 0.998 after optimizing the detection method.It was specific and sensitive to detect Avian paramyxovirus 6,the minmum detection limit was 2.78×102 copies/?L and there was no specific reaction with NDV,APMV-4,APMV-13 and GPV.The detection rate was 18%higher than the conventional detection method.In this experiment,APMV-6 Hubei strain was isolated successfully,and nucleotide sequence is 16 236bp.A fluorescence quantitative PCR detection method for avian parvivirus type 6 is established.