Investigation On The Multiplex Reverse Transcription-PCR Amplification And DNA Microarray For AIV Subtypes

All the diseases and infection caused by avian influenza virus(AIV) are called avian influenza.Especially high pathogenic avian influenza(HPAI) can infect and cause disease in humans with a fatal illness.Not only HPAI bring a big economical loss to international trade, but also it threat badly human.AIV included twenty-five subtypes, with no cross-protection each other. In order to develop rapid, sensitive, specific identification and subtypeing, DNA microarray for AIV were studied on this paper.The paper has two parts, one is the study on the multiplex reverse transcriptase- PCR(RT-PCR) of AIV. The other is the primary study on the subtying of AIV using DNA microarray method. The main experiments and results are as follows:(1)Based on the conserved region sequences of the AIV, twenty-five pairs of primers were carefully designed. The primers were synthesized. Viral RNA of H1, H3, H5, H6, H7, H9, N1and N2 were extracted and performed multiplex RT-PCR amplication. The target genes of other subtypes AIV were obtained by overlapping PCR. The results showed that target gene of 17 AIV subtypes were achieved successfully. Then they were random divided into three groups and develop multiplex RT-PCR respectively. Reference virual RNA of AIV run multiplex RT-PCR amplication except H16, N7 subtypes, H11, H13 and N6 subtypes cDNA bands were dim, the other were brighten bands. The sensitivity and specificity of the method were evaluated by using NDV, IBV, ARV, IBDV, MDV, FPV, the results showed that the method was very specific and sensitive. the sensitivity of H5N1 is up to 247 PFU. The method was also used to detect 395 avian field samples from more than 20 regions of 4 different provinces, and the detection results was consistent with the conventional virus isolation method.(2)Multiplex asymmetric RT-PCR were performed using universal primers, and special primers. Based on the conserved region sequences of the AIV, subtyping probes and quality control probes were designed and synthesized. After spotting on the glassslide, the microarray has been developed by application of several control and gene-specific probes of AIV. The specificity of the gene microarray were evaluated by using NDV, IBV, ARV, IBDV, MDV, FPV. Gene-specific probes of AIV indicates good fluorescence signal in their location, with no cross-reaction to other subtype or clinical relvant virus. the results showed that the method was very specific.The sensitivity of the cDNAs is 2.5 ng.