title: Newly emerging mutations in the matrix genes of the human influenza A(H1N1)pdm09 and A(H3N2) viruses reduce the detection sensitivity of real-time reverse transcription-PCR.
abstract: New variants of the influenza A(H1N1)pdm09 and A(H3N2) viruses were detected in Taiwan between 2012 and 2013. Some of these variants were not detected in clinical specimens using a common real-time reverse transcription-PCR (RT-PCR) assay that targeted the conserved regions of the viral matrix (M) genes. An analysis of the M gene sequences of the new variants revealed that several newly emerging mutations were located in the regions where the primers or probes of the real-time RT-PCR assay bind; these included three mutations (G225A, T228C, and G238A) in the A(H1N1)pdm09 virus, as well as one mutation (C163T) in the A(H3N2) virus. These accumulated mismatch mutations, together with the previously identified C154T mutation of the A(H1N1)pdm09 virus and the C153T and G189T mutations of the A(H3N2) virus, result in a reduced detection sensitivity for the real-time RT-PCR assay. To overcome the loss of assay sensitivity due to mismatch mutations, we established a real-time RT-PCR assay using degenerate nucleotide bases in both the primers and probe and successfully increased the sensitivity of the assay to detect circulating variants of the human influenza A viruses. Our observations highlight the importance of the simultaneous use of different gene-targeting real-time RT-PCR assays for the clinical diagnosis of influenza.
authors.affiliation: Centers for Disease Control, Taipei, Taiwan, Republic of China.
authors.forename: Ji-Rong
authors.initials: JR
authors.lastname: Yang
authors.forename: Chuan-Yi
authors.initials: CY
authors.lastname: Kuo
authors.forename: Hsiang-Yi
authors.initials: HY
authors.lastname: Huang
authors.forename: Fu-Ting
authors.initials: FT
authors.lastname: Wu
authors.forename: Yi-Lung
authors.initials: YL
authors.lastname: Huang
authors.forename: Chieh-Yu
authors.initials: CY
authors.lastname: Cheng
authors.forename: Yu-Ting
authors.initials: YT
authors.lastname: Su
authors.forename: Feng-Yee
authors.initials: FY
authors.lastname: Chang
authors.forename: Ho-Sheng
authors.initials: HS
authors.lastname: Wu
authors.forename: Ming-Tsan
authors.initials: MT
authors.lastname: Liu
mesh_terms: D020137:Base Pair Mismatch; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D025202:Molecular Diagnostic Techniques / Q000379:methods; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics; D009154:Mutation; D015345:Oligonucleotide Probes / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013624:Taiwan; D014763:Viral Matrix Proteins / Q000235:genetics*
chemical_list: D017931:DNA Primers; C488936:M1 protein, Influenza A virus; D050505:Mutant Proteins; D015345:Oligonucleotide Probes; D012367:RNA, Viral; D014763:Viral Matrix Proteins