Establishment Of Multiplex PCR For H5 H7 Avian Influenza Virus

Based on the H5 and H7 hemagglutinin(HA) gene of the AIV on GenBank,used DNAStar to analyze the two subtypes' homology between different strains,select the highly homologous domains,then A/Chicken/Jilinl/hk/2004(H5N1) was referded to H5 and A/avian/NY/70411-12/00(H7N2) was referded to H7.Through DNAStar to analyze the selected highly homologous domains in the two refered strains,finally, the amplified domains were determined severally.Contrilling primers self dimers,primer pair dimers and primer hairpins strictly.On the end,two primers were carefully designed,P51 P52 and P71 P72,viral RNAs were extracted and RT-PCR amplified H5 and H7 hemagglutinin(HA) gene triumphantly.The ligation product of the amplified fragment with the linear vetor pMD-18T was then transformed into E.coli competent cells.After identified by PCR,the recombinant plasmid and PCR amplified fragment were sequenced.The BlastN result showed that the cloned HA of H5 and H7 have homology to that of H5 and H7 hemagglutinin(HA) gene in GenBank.By single RT-PCR reaction was optimized,H5 subtype was well simplifed at 51℃～55℃dannealing temperature,H7 subtype was at 55℃and detectde rates well.So they have the common reaction procedure at 55℃.The quantity of templates have well simplifed effectiveness at 2.0μl.Primers of H5 subtype were simplifed well at 0.16μM～0.96μM,H7 were at 0.32μM～0.96μM.The concentration of Mg²⁺at 1.5mM,two subtypes were well simplifed singlely.Finally,the optima suitable single RT-PCR reaction procedure and -system were definited.On the basis of single RT-PCR reaction to detect the sensitivities of H5 and H7 subtypes,the results were 10^-4 and 10^-2 respectively.By established single RT-PCR reaction,swine fever virus,avian infectious bronchitis virus,infectious bursal virus,avian infectious laryngotracheitis virus,newcastle disease virus,H9 avian influenza virus were detected,the results were negative.Based on the optimized single RT-PCR,the multiplex RT-PCR was definited through concentration of primers,H5 was 0.32μM and H5 was 0.96μM.Through multiple RT-PCR to detected single template of sensitivity,H5 was 10^-3 lower than single RT-PCR, H7 was 10^-2 and it the same as single RT-PCR.It detected diluted of 10 times reverse transcripted productes of H5 and H7 subtypes.H5 was10^-3 and H7 was10^-2,so the result was the same as detected single template. e.The results indicated that the optimal conditions of the multiple PCR layed a good foundation of sensitive, specialan drapid assay for H5 and H7 subtypes avian influenza virus.