Published ahead of print 23 October 2013, doi: 10.1128/JCM.02467-13 J. Clin. Microbiol. January 2014 vol. 52 no. 1 76-82

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

Ji-Rong Yanga,
Chuan-Yi Kuoa,
Hsiang-Yi Huanga,
Fu-Ting Wua,
Yi-Lung Huanga,
Chieh-Yu Chenga,
Yu-Ting Sua,
Feng-Yee Changa,b,
Ho-Sheng Wua,c and
Ming-Tsan Liua

aCenters for Disease Control, Taipei, Taiwan, Republic of China
bDepartment of Internal Medicine, National Defense Medical Center, Taipei, Taiwan, Republic of China
cSchool of Medical Laboratory Science and Biotechnology, Taipei Medical University, Taipei, Taiwan, Republic of China

A. J. McAdam, Editor

+ Author Affiliations

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.