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.
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 PCR (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; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10(1.5), 10(2.3), and 10(3.1) 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the 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 of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples.
Research Team for Zoonotic Diseases, National Institute of Animal Health, Tsukuba, Ibaraki, Japan. ktsukamo@affrc.go.jp
Kenji
K
Tsukamoto
Carlos Javier
CJ
Panei
Panei Carlos
PC
Javier
Makiko
M
Shishido
Daigo
D
Noguchi
John
J
Pearce
Hyun-Mi
HM
Kang
Ok Mi
OM
Jeong
Youn-Jeong
YJ
Lee
Koji
K
Nakanishi
Takayoshi
T
Ashizawa
D000818:Animals; D052160:Benzothiazoles; D001717:Birds; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D005838:Genotype; D006389:Hemagglutinins, Viral / Q000235:genetics*; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D005585:Influenza in Birds / Q000175:diagnosis / Q000821:virology*; D009439:Neuraminidase / Q000235:genetics*; D009930:Organic Chemicals / Q000378:metabolism; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D014764:Viral Proteins / Q000235:genetics*
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D006389:Hemagglutinins, Viral; D009930:Organic Chemicals; D011804:Quinolines; D014764:Viral Proteins; C458881:hemagglutinin fusogenic peptide, influenza virus; C098022:SYBR Green I; C487630:NA protein, influenza A virus; D009439:Neuraminidase