A reverse transcription-PCR for subtyping of the neuraminidase of avian influenza viruses.
To date, nine neuraminidase (NA) subtypes of avian influenza viruses have been identified. In order to differentiate the NA of avian influenza viruses rapidly, a reverse transcription PCR (RT-PCR) was developed. Nine pairs of NA-specific primers for the RT-PCR were designed based on the analysis of 509 complete NA sequences in GenBank. The primers were designed to amplify partial NA genes and each pair is unique to a single NA subtype (N1-N9). By nine RT-PCRs simultaneously in a set of separate tubes, the subtype of NA was determined by subsequent agarose gel electrophoresis and ethidium bromide staining, since only one of the nine RT-PCRs would give a product of expected size for each virus strain. In comparison with the established method of sequence analysis of 101 reference strains or isolates of avian influenza viruses, the RT-PCR method had a sensitivity of 97.3% and a specificity of 91.1% in subtyping avian influenza viruses. These results indicate that the RT-PCR method described below provides a specific and sensitive alternative to conventional NA-subtyping methods.
Qiu
Bao-Feng
BF
Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, China.
Liu
Wu-Jie
WJ
Peng
Da-Xin
DX
Hu
Shun-Lin
SL
Tang
Ying-Hua
YH
Liu
Xiu-Fan
XF
D000818:Animals; D017931:DNA Primers; D004372:Ducks / Q000821:virology*; D009980:Influenza A virus / Q000145:classification* / Q000201:enzymology* / Q000235:genetics; D009439:Neuraminidase / Q000145:classification* / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012367:RNA, Viral; D009439:Neuraminidase