Establishment Of A Rapid Detection Method For Identification Of H5、H7 And H9 Subtypes Of Avian Influenza Virus

Avian influenza (AI) caused by type A influenza virus is a disease and infection of poultry. There are many subtypes of avian influenza virus (AIV), which have a wide spectrum of host, mutate and recombine easily. In recent years, AI breaks out more frequently and wordwidely than before, resulting in not only a loss to poultry industry, but also a great threat to human health. Currently, H5, H7 and H9 subtype of AI is the most important disease to poultry industry.Therefore, there is an urgent need to establish a method for rapid detection of avian influenza virus. In this study, we use one step multiplex RT-PCR technology to establish a rapid diagnosis method for H5, H7 and H9 subtypes of avian influenza virus. In addition, we also prepared monoclonal antibodies against H5N1 and H7N9 subtypes of avian influenza viruses. Further, we developed a colloidal gold test strip for detecting H7N9 AIV.1 Establishment of a single-step multiplex RT-PCR for detecting H5, H7 and H9 subtypes of avian influenza virusesOne pair of primer was designed based on the conservative region of AIV M gene, which was specific for type A AIV. Three pairs of primers for identification of subtype were designed based on the HA genes of H5, H7 and H9 subtype AIV. The single-step RT-PCR was established with the four pairs of primers. The specificity and sensitivity of the method were determined. The results showed that the specific amplicons of M gene and HA genes of H5, H7, H9 subtype were 300 bp,173 bp,525 bp and 378 bp in size, consistent with the expected size. No fragment was not amplified from H1, H4, H6, H10, and H11 subtype AIVs, NDV, and IBV by the single-step RT-PCR. This method had high sensitivity with a minimum detection concentration of 10 ng/mL viral RNA. When the method was also applied to detect 30 samples, the RT-PCR results was consistent with HA test after virus isolation.2 Development of monoclonal antibodies against H5N1 and H7N9 subtype AIVTwo representative AIV strains C18 and JT corresponding H5N1 and H7N9 subtype were chosen as antigen to prepare the monoclonal antibodies. The viruses purified by sucrose density gradient centrifugation were used to immunize 6-8 weeks old Balb/c mice. Positive clones were screened by indirect ELISA and haemagglutination inhibition (HI), and ten monoclonal antibody hybridoma cell lines were obtained.4 McAbs against H5 AIV were named as 1C4,5E10,1G11, 6F4.6 McAbs against H7 AIV were named as 3D8,5D4,3E4,4F4,6C5,3G5. Respectively, specificity results showed that the ten monoclonal antibodies had no reaction with H9, NDV and IBV viruses. The results of HI showed that the McAbs 5E10,1G11 had HI titer against AIV strain C18, and the McAbs 4F4,6C5 had HI titer against AIV strain JT. The results of western-blot showed that the McAbs 1C4 and 6F4 reacted with AFV strain C18, respectively, with 55-KD specific band. The results of indirect immunofluorescent assay revealed that the McAbs 1C4,5E10,1G11 and 6F4 had specific fluorescent reaction to AIV strain C18, at the same time, the McAbs 1C4 and 6F4 had specific fluorescent reaction to ATV strain SY, LK, HA from different branches virus of H5 AIV, the McAbs 1C4 and 6F4 had a broad-spectrum reaction against AIV. The McAbs 4F4 and 6C5 had specific fluorescent reaction to AIV strain JT.3 Development of a Colloidal gold test strip for detecting H7N9 AIVNanometer colloidal gold was prepared by reducing gold chloride with sodium citrate. We made the test strip in sandwich format. The monoclonal antibody 6C5 labeled with colloidal gold was diluted to 10 times and coated onto the conjugate pad, Anti-chicken polyclonal antibody was dispensed on the bottom of the NC membrane as the test line with a concentration of 1 mg/mL, while the goat anti-mouse IgG was dispensed at the upper position as the control line with a concentration of 1.5 mg/mL. So two band colours will be visible when the samples contain H7N9 antigen, otherwise, a negative sample will result in the formation of just one band colour on control line. The results showed that the test strip had specificity to H7N9 with no cross-reaction with other subtypes of avian influenza virus and other virus that can cause disease in poultry. As for detection spectrum, the test strip detected all of the H7N9 subtypes AIVs used in this experiment. When allantoic fluids of H7N9 AIVs were used as the antigens, the limitation of detection was 24 of the hemagglutination units.