Jornal de Técnicas Imunológicas e Doenças Infecciosas

The Establishment of Realtime Fluorescent Quantitative Polymerase chain reaction (PCR) for Detection of Highly Pathogenic Avian Influenza Virus Subtype H5N1

Miao-lian Tan, Yuan-yuan Niu, Wei Shui, Jiancong Lin, Ming Li and Changran Zhang

Highly pathogenic strains of avian influenza virus (AIV), which are influenza A viruses, cause severe disease in domestic poultry and humans. The objective of this study was to establish a fluorescent quantitative RT-PCR assay for detection of highly pathogenic avian influenza virus (AIV) subtype H5N1. The H5 and N1 subtypespecific probe sets were developed based on avian influenza virus sequences detected in China. Two pairs of primers and two fluorescent probes were strictly designed and optimized in a reaction system. According to the amount of plasmid RNA extracted from H5N1 strains, the standard curve DWQBGWDWQBGW of fluorescent quantitative PCR was drawn and all of the specimens were then tested by means of Real-time PCR. The test of highly pathogenic AIV subtype H5N1 was identified to be specific and its sensitivity level was 102~103 copies/reaction. The standard curve was accomplished at 109～105 DNA copies/reaction. It took only three hours from viral RNA extraction through to completion of the test. The assay was easy to carry out and highly reproducible. In conclusion, fluorescent quantitative PCR, described here, provides a rapid, specific and sensitive method to detect not only the H5 but N1 genes as well.