Development Of A Duplex Real-time RT-PCR Assay For Detection Of H5N6 Avian Influenza Virus

Avian influenza is an acute infectious disease caused by avian influenza virus.At present,H5N6 avian influenza virus replaced H5N1 subtype pandemiced in China.The complex genetic recombination of H5N6 created the threat to the health of human and animals.It is urgent to establish a quickly and accuritied method for the detection of H5N6 avian influenza virus.A duplex real-time RT-PCR assay to detect H5N6 avian influenza virus was established based on the conservative region sequence of HA gene and NA gene,which provided important technical means for clinical diagnosis and disease prevention and control.Test 1:Establishment and Optimization of a duplex real-time RT-PCR assay for detection of H5N6 avian influenza virusAt firest,the full length sequence of HA gene and NA gene from the representative strain of H5N6 virus were downloaded from GenBank,and were alignmented use Mega6.0 software to analyze and determine the conservative region sequence of HA gene and NA gene.Two specific primers and TaqMan probes for H5 gene and N6 gene were designed using Primer Express 3.0software.Different test conditions were used respectively,such as H5 and N6 positive nucleic acid samples used 10^-1,10^-3,10^-5 three concentration gradient,primer concentration with 100nM,200nM,300nM and 400nM,and probe concentration 100nM,150nM,200nM and 250nM,and reverse transcriptional temperature at 42?,45?,50?,55?,and reverse transcriptional time at10min,20min,30min,40min,and fluorescence absorption temperature at 50?,55?,60?,65?,and fluorescence absorption time at 30s,35S,40s,45s.The preferred combinationa conditions to detect H5N6 avian influenza virus were determined based on the lowest Ct value and high fluorescence intensities??Rn?.the optimum working concentration of primers and probes by matrix method.The results showed that the optimum assay conditions were respectively of the primer and probe concentration with 300nM and 150nM,reaction temperature and time at 45?and 30min,fluorescence absorption temperature and time at 60?and 35S.The results indicated that the duplex real-time fluorescence RT-PCR assay for detecting H5N6 avian influenza virus was established with better system and conditions.Test 2:Specificity,sensitivity and stability verification of a duplex real-time RT-PCR assay for the detection of H5N6 avian influenza virusAt first,the positive nucleic acid of the some avian influenza virus was detected with the duplex real-time RT-PCR assay.The results showed that only the H5N6 avian influenza virus was positive from FAM and HEX detection channels,and there were specific amplification curves for H5N1,H5N2 and H5N8 avian influenza viruses only in FAM channel,and other subtype influenza viruses had no amplification curves in the the two channels.The target genes H5 and N6 were amplified and cloned to pcDNAII-H5,pcDNAII-N6 plasmids.The plasmids were quantified and then diluted by 10 times dilution.Different concentrations of plasmids were detected by the duplex RT-PCR assay and were determined the minimum detection limit.The results showed that the low detection limit of H5 gene was 80 copies/?L,the low detection limit of N6 gene was 100copies/?L.The positive nucleic acid with gradient dilution of 10 times(10^-1,10^-2,10^-3)were detected by established assay in the same PCR board with three copies.The positive nucleic acid with gradient dilution of 10 times(10^-1,10^-2,10^-3)were evaluated using developed assay in three PCR board with three copies.The results showed that the coefficient of variation?Ct?obtained from three repeated reactions in the same board was 0.23%⁰.36%,and the coefficient of variation obtained from three boards were 0.27%⁰.49%,which had good reproducibility.The results indicated that the duplex real-time RT-PCR assay could be highly specific,highly sensitive and stable method to detect H5N6 avian influenza virus.Test 3:The comparison of the duplex real-time RT-PCR assay for detecting H5N6 avian influenza virus with the commercialized kits.207 swab specimens collected from the environment of avian influenza live poultry market and chicken and were detected by the duplex RT-PCR assay.At the same time,all samples were detected by the commercialized kits.The results showed that the coincidence rate of the two methods was 100%,which indicated that the duplex real-time fluorescence RT-PCR assay established in this research would be suitable to be used in clinical application.