SYBR Green Based Real-Time Reverse Transcription PCR for Typing and Subtyping of All Hemagglutinin and Neuraminidase genes of Avian Influenza Viruses and Comparison to Standard Serological Subtyping Tests

KENJI TSUKAMOTO; PANEI CARLOS JAVIER; MAKIKO SHISHIDO; DAIGO NOGUCHI; JOHN PEARCE; HYUN-MI KANG; OK MI JEONG; YOUN-JEONG LEE; KOJI NAKANISHI; TAKAYOSHI ASHIZAWA

JOURNAL OF CLINICAL MICROBIOLOGY

AMER SOC MICROBIOLOGY

Año: 2011 p. 37 - 45

0095-1137

Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes, and mixed base positions per primer were set within five, and concentration of each primer set was optimized empirically. Also, 30 cycles´ amplification of one-tenth dilution of cDNAs from cultured viruses effectively reduced minor cross or non-specific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected with average sensitivities of NP, HA, and NA genes as 101.5, 102.3, and 103.1 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA and 99% NA genes were genetically subtyped, while only 45 % of HA genes and 74 % of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles´ amplification: approximately 70 % of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that the rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples.