Research Of RT-PCR And Multiplex RT-PCR For Detection Of H5N1 Subtype Of Avian Influenza Virus

Highly pathogenic avian influenza (HPAI) in poultry causes high morbidity and mortality, and it is a List A disease of the Office International des Epizooties. The causative agent is confined to certain isolates of influenza A virus subtypes H5 and H7. During December 2003-February 2004, outbreaks of highly pathogenic avian influenza A (H5N1) among poultry were reported in South Korea, Vietnam, Japan, Cambodia, Thailand, China, Indonesia, Laos. An outbreak of HPAI in commercial poultry not only causes direct disease losses but often results in trade restrictions for the affected country.In this paper, type A-specific and H5, N1 subtype-specific reverse transcriptase -polymerase chain reaction (RT-PCR) assays, based on the matrix, haemagglutinin or neuraminidase gene of H5N1 avian influenza virus, were developed for detection of the influenza type A, H5 or N1 subtype respectively, and further H5/N1, A/H5/N1 specific multiplex RT-PCR assays were also developed for the simultaneous detection and differentiation of H5 and N1 subtypes, or influenza type A, H5 and N1 subtypes respectively. The entire nucleic acid isolation, amplification, and detection procedure was completed within 4~5h. A total of 16 isolates or clinical samples of avian influenza viruses or Newcastle disease virus were detected by the RT-PCR and multiplex RT-PCR assays, and the results of RT-PCR gave an excellent (100%) correlation with the result of the conventional virus isolation method. The assays were sensitive and highly specific, with no cross-reactivity to other subtypes or clinically relevant viruses (Newcastle disease virus). Both A/H5/N1 multiplex RT-PCR and the conventional virus isolation agreed on the H5N1 subtype determination of 80 swab samples obtained from various avian species in Kunming in 2004. These methods therefore provide rapid, simple, cheap, safe, sensitive, type-specific and subtype-specific identification and diagnosis of H5N1 avian influenza virus in clinical samples.