Rapid Detection Of Avian Influenza Virus By Diplex RT-PCR And Real-Time RT-PCR

According to the submitted nucleotide sequences of HA gene of H9 subtype avian influenza virus (AIV), a pair of primer (H1 and H2) was synthesized and used to amplify the HA gene of A/Duck/Shanghai/02/99(H9) by RT-PCR using genome RNA as template, the ligation product of the amplified fragment with the linear vector pUCm-T was then transformed into E. coli competent cells. After identified by restrictive endonucleonase and PCR, the recombinant plasmid pUCm-T-HA was sequenced. The BlastN result showed that the cloned HA gene of A/Duck/Shanghai/02/99(H9) has 98% homology to that of A/Duck/Shantou/1605/01(H9N2) and A/Duck/Hong Kong/Y280/97(H9N2).A method of diplex RT-PCR was developed to detect the Avian Influenza Virus (AIV)using a pair of primers (M3 and M4) for matrix(M) gene and another pair of primers (H1 and H2) for hemagglutinin(HA) gene. 17 positive samples were detected by the diplex RT-PCR among 80 clinical samples, which was same as the chicken embryo isolation method.An oligo-nucleotides M5 was designed for probing the M3 and M4 amplified fragment in the real-time RT-PCR for AIV detection. The sensitivity of this assay was up to 0.1EID₅₀, which was higher than the chicken embryo isolation method(1EID₅₀). 80 shares of gorge and cloaca swabs collected from the chicken which were infected by H9 subtype AIV were tested by the real-time RT-PCR. The results showed that the virus can be isolated and detected from all the gorge swabs in the 7th-8th day, while it can not be detected from all the corresponding cloaca swabs. It suggests that the virus content was higher in the gorge than that in the cloaca. The assay was simple and rapid and it could be applied to the clinical detection, the market supervision and export & import quarantine.