Lin Jih-Hui, Tseng Ching-Ping, Chen Yen-Ju, Lin Chy-Yung, Chang Shy-Shin, Wu Ho-Sheng, Cheng Ju-Chien
Center for Disease Control and Prevention, Taipei, Taiwan, Republic of China.
J Clin Microbiol. 2008 Mar;46(3):1090-7. doi: 10.1128/JCM.02015-07. Epub 2008 Jan 3.
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 10(3) 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.
我们评估了高分辨率熔解曲线（HRM）分析在快速鉴定甲型流感病毒亚型和检测新出现病毒变体方面的应用。在LCGreen I荧光染料存在的情况下，通过LightCycler实时逆转录聚合酶链反应（RT-PCR）扩增病毒基质基因。优化检测条件后，包括H1N1、H3N2、H5N1、H7N3和H9N2在内的所有主要甲型流感病毒亚型均可通过该方法扩增，且PCR产物长度为179 bp。体外转录的H3N2 RNA的实时RT-PCR显示了一条用于定量的标准曲线，其线性范围（相关系数 = 0.9935）跨越至少8个对数单位的RNA浓度，检测限为10³份病毒RNA拷贝。我们使用HR-1仪器对PCR产物进行了HRM分析，并将熔解曲线用作病毒亚型分型的分子指纹。通过测试病毒分离株与参考病毒分离株的PCR产物之间形成异源双链获得的高分辨率导数图来鉴定病毒亚型。熔解曲线一致，测定间变异性最小。因此，建立了一个HRM数据库和工作方案来鉴定这五种甲型流感病毒亚型。当使用该方案检测21株临床甲型流感病毒分离株时，结果与使用血凝素特异性引物组的RT-PCR结果相当。我们的HRM分析方案还揭示了临床分离株（n = 4）的序列变体。该检测无需多重检测或杂交探针，为临床病毒学实验室中甲型流感病毒亚型分型和病毒变体的基因筛查提供了一种新方法。
