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Journal of Clinical Microbiology, March 2008, p. 1090-1097, Vol. 46, No. 3
0095-1137/08/$08.00+0     doi:10.1128/JCM.02015-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Rapid Differentiation of Influenza A Virus Subtypes and Genetic Screening for Virus Variants by High-Resolution Melting Analysis

Jih-Hui Lin,^1, Ching-Ping Tseng,^2,3, Yen-Ju Chen,⁴ Chy-Yung Lin,⁵ Shy-Shin Chang,^6,7 Ho-Sheng Wu,¹ and Ju-Chien Cheng^4*

Center for Disease Control and Prevention, Taipei, Taiwan, Republic of China,¹ Graduate Institute of Medical Biotechnology,² Clinical Medical Science, Chang Gung University, Taoyuan, Taiwan 333, Republic of China,⁶ Laboratory of Molecular Diagnostics, Department of Clinical Pathology,³ Department of Emergency Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan 333, Republic of China,⁷ Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan 404, Republic of China,⁴ Department of Laboratory Medicine, Changhua Christian Medical Center, Changhua, Taiwan 500, Republic of China⁵

Received 14 October 2007/ Returned for modification 28 November 2007/ Accepted 20 December 2007

We assessed the use of high-resolution melting (HRM) analysis for the rapid identification of influenza A virus subtypes and the detection of newly emerging virus variants. The viral matrix gene was amplified by LightCycler real-time reverse transcription-PCR (RT-PCR) in the presence of the LCGreen I fluorescent dye. Upon optimization of the assay conditions, all the major influenza A virus subtypes, including H1N1, H3N2, H5N1, H7N3, and H9N2, were amplifiable by this method and had a PCR product length of 179 bp. Real-time RT-PCR of in vitro-transcribed H3N2 RNA revealed a standard curve for quantification with a linear range (correlation coefficient = 0.9935) across at least 8 log units of RNA concentrations and a detection limit of 10³ copies of viral RNA. We performed HRM analysis of the PCR products with the HR-1 instrument and used the melting profiles as molecular fingerprints for virus subtyping. The virus subtypes were identified from the high-resolution derivative plot obtained by heteroduplex formation between the PCR products of the viral isolates tested and those of the reference viral isolates. The melting profiles were consistent with minimal interassay variability. Hence, an HRM database and a working protocol were established for the identification of these five influenza A virus subtypes. When this protocol was used to test 21 clinical influenza A virus isolates, the results were comparable to those obtained by RT-PCR with hemagglutinin-specific primer sets. Sequence variants of the clinical isolates (n = 4) were also revealed by our HRM analytical scheme. This assay requires no multiplexing or hybridization probes and provides a new approach for influenza A virus subtyping and genetic screening of virus variants in a clinical virology laboratory.

Published ahead of print on 3 January 2008.

J.-H. Lin and C.-P. Tseng contributed equally to this work and are considered co-first authors.