title: [Development of a real-time reverse transcriptase PCR assay for detection of E119V amino acid change in neuraminidase of influenza A (H3N2) using the TaqMan-MGB probe].
abstract: OBJECTIVE
  To develop a rapid duplex Real-time reverse transcription PCR (rRT-PCR) method to detect E119V mutation on neuraminidase (NA) of influenza A(H3N2) subtype with drug resistance to oseltamivir.
  
  
  METHODS
  Twenty-six NA genes of influenza A(H3N2) virus between 2000 and 2012 in GenBank database were selected as the target genes, and specific TaqMan-MGB probe was designed to target the E119V amino acid change in neuraminidase protein. rRT-PCR was then performed and evaluated for the sensitivity, specificity and reproducibility using virus with E119V mutation and clinical samples.
  
  
  RESULTS
  This study described the validation of a highly sensitive and specific duplex rRT-PCR for detection of substitutions leading to the E119V amino acid change in NA protein of influenza A(H3N2). Fluorescence signals could be detected even when diluted a A (H3N2) virus (HA = 8) into 10(-5) and linear correlation between the logarithm of the viral titer with the Ct values was observed. In addition, the assay was highly specific in that there was no cross-react with other respiratory viruses, nor did two TaqMan-MGB probes. E119V substitution in quasispecies with both sensitive and resistant viruses could be detected as well. The limit of detection was 5% for quasispecies with high concentrations and 50% for quasispecies with low concentrations. The average coefficient of variation (CV) for within-run assays was 2.32% and 0.57% for H3N2-119E and H3N2-119V primer/probe sets separately, 1.77% and 0.97% for average CV of between-run assays, which exhibited good repeatability. Sequence analysis of twenty NA genes verified glutamic acid (E) at amino acid site 119, which was in consistent with the results from our rRT-PCR method.
  
  
  CONCLUSIONS
  The assay developed in this study is highly sensitive and specific, and easy to operate; thereby it could be used for identification of A(H3N2) virus with E119V amino acid change in NA protein.
authors: Chinese National Influenza Center, National Institute for Viral Disease Control and Prevention, China.
authors: Xiang
authors: X
authors: Zhao
authors: Wei-juan
authors: WJ
authors: Huang
authors: He-jiang
authors: HJ
authors: Wei
authors: Zhao
authors: Z
authors: Wang
authors: Xi-yan
authors: XY
authors: Li
authors: Yan-hui
authors: YH
authors: Cheng
authors: Min-ju
authors: MJ
authors: Tan
authors: Ning
authors: N
authors: Xiao
authors: Yu
authors: Y
authors: Lan
authors: Jun-feng
authors: JF
authors: Guo
authors: Hong-tao
authors: HT
authors: Sui
authors: Wen-fei
authors: WF
authors: Zhu
authors: Dong-dong
authors: DD
authors: Du
authors: Da-yan
authors: DY
authors: Wang
authors: Yue-long
authors: YL
authors: Shu
mesh_terms: D019943:Amino Acid Substitution; D024882:Drug Resistance, Viral; D053122:Influenza A Virus, H3N2 Subtype / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D009154:Mutation; D009439:Neuraminidase / Q000235:genetics*; D015341:Nucleic Acid Probes; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D015341:Nucleic Acid Probes; D009439:Neuraminidase