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

• Published in: Zhōnghuá yùfáng-yīxué zázhì Vol. 47; no. 5; p. 448
• Main Authors: Zhao, Xiang, Huang, Wei-juan, Wei, He-jiang, Wang, Zhao, Li, Xi-yan, Cheng, Yan-hui, Tan, Min-ju, Xiao, Ning, Lan, Yu, Guo, Jun-feng, Sui, Hong-tao, Zhu, Wen-fei, Du, Dong-dong, Wang, Da-yan, Shu, Yue-long
• Format: Journal Article
• Language: Chinese
• Published: China 01.05.2013
• Subjects: Amino Acid Substitution; Drug Resistance, Viral; Influenza A Virus, H3N2 Subtype - drug effects; Influenza A Virus, H3N2 Subtype - enzymology; Influenza A Virus, H3N2 Subtype - genetics; Mutation; Neuraminidase - genetics; Nucleic Acid Probes; Reverse Transcriptase Polymerase Chain Reaction - methods
• ISSN: 0253-9624

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. 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. 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