Rapid differentiation of influenza A virus subtypes and genetic screening for virus variants by high-resolution melting analysis

Jih Hui Lin, Ching Ping Tseng, Yen Ju Chen, Chy Yung Lin, Shy Shin Chang, Ho Sheng Wu, Ju Chien Cheng

研究成果: 雜誌貢獻文章

61 引文 (Scopus)

摘要

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-I 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.

原文英語
頁(從 - 到)1090-1097
頁數8
期刊Journal of Clinical Microbiology
46
發行號3
DOIs
出版狀態已發佈 - 三月 1 2008
對外發佈Yes

指紋

Influenza A virus
Genetic Testing
Freezing
Viruses
Polymerase Chain Reaction
Reverse Transcription
RNA
H1N1 Subtype Influenza A Virus
Virology
Viral Genes
Viral RNA
Hemagglutinins
Dermatoglyphics
Fluorescent Dyes
Limit of Detection
Databases

ASJC Scopus subject areas

  • Microbiology (medical)

引用此文

Rapid differentiation of influenza A virus subtypes and genetic screening for virus variants by high-resolution melting analysis. / Lin, Jih Hui; Tseng, Ching Ping; Chen, Yen Ju; Lin, Chy Yung; Chang, Shy Shin; Wu, Ho Sheng; Cheng, Ju Chien.

於: Journal of Clinical Microbiology, 卷 46, 編號 3, 01.03.2008, p. 1090-1097.

研究成果: 雜誌貢獻文章

Lin, Jih Hui ; Tseng, Ching Ping ; Chen, Yen Ju ; Lin, Chy Yung ; Chang, Shy Shin ; Wu, Ho Sheng ; Cheng, Ju Chien. / Rapid differentiation of influenza A virus subtypes and genetic screening for virus variants by high-resolution melting analysis. 於: Journal of Clinical Microbiology. 2008 ; 卷 46, 編號 3. 頁 1090-1097.
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