Establishment Of Real-time RT-PCR For The Detection Of H5 And H7 Subtypes Avian Influenza Virus

Avian influenza(AI) is a worldwide zoonotic disease caused by influenza A virus, which has a extensive host range. It mainly infects aquatic birds, also can infect mammals and humans. In general, avian influenza virus(AIV) is harmless to humans, but it is changed the after the occurrence of H5N1 subtype avian influenza virus infection in humans since 1997. There also have been different subtypes of avian influenza virus infection cases since then, especially for H7N9 influenza virus epidemic in China, 2003, which not only brought huge losses in the poultry industry, also caused a lot of human infections. Therefore, timely diagnosis research on influenza virus, to prevent the flu pandemic, has the vital significance to reduce harm to the animal and human life safety. Hence, rapid and accurate method for the identification of influenza virus is needed to facilitate clinical care and infection control.Real-time RT-PCR is a novel molecular nucleic acid detection method. Based on the previous work platform, this study established a real-time RT-PCR method for the detection of H5 and H7 subtypes avian influenza virus. Primers and probes were designed and synthesized according to HA and NA genes of H7N9 subtype and HA gene of H5N1 subtype sequences, and the study established H5 and influenza A virus duplex and H7N9 and influenza A virus multiplex real-time RT-PCR methods. Two methods showed good specificity. In the sensitivity test, their detection limits can reach 10-5 dilution. In the repeatability experiment, the intra- and inter-assay coefficients of variation were all less than 2 %. Through the analysis of test results of a large number of clinical samples, the real-time RT-PCR assays had high accuracy.Veterinary biological material products can be used as the reference material for the determination of the value of the products or verify the accuracy of diagnosis methods for biological products of biological potency of characteristics of animal disease diagnostic reagents and test microbes and their products qualitatively or quantitatively. Inactivated H5N1 and H7N9 subtype virus were developed as standard materials in this study. In the heat stability test, the material were well kept for a certain period of time at the temperature of-20℃, 4℃ and 37℃. In the repeated freeze-thaw test, after repeated 16 times freeze-thaw, the standard materials still showed good stability. The results of sterility test met the requirements, and standard materials were inactivated fully, so they can be used as reference in RT-PCR and real-time RT-PCR detection.