The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus
The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus
复制标题
DOI：
10.1007/s10096-008-0556-9
复制
发表时间：
2008-12-01
影响因子：
4.5
通讯作者：
Herra, C.
中科院分区：
文献类型：
--
作者：
Clancy, A.;Crowley, B.;Herra, C.
实时聚合酶链反应（PCR）是检测丙型肝炎病毒（HCV）的一个有利选择。采用ABI PRISM 7500序列检测系统，开发了一种实时逆转录酶PCR（RT-PCR）检测方法，作为检测HCV的定性诊断筛查方法。设计引物和探针以靶向丙型肝炎病毒基因组的5 '-非翻译区。第二种异源探针检测法用于检测福辛犬瘟热病毒（PDV）的血凝素基因，并用作内部对照。还使用ABI PRISM（TM）6100 Nucleic Acid PrepStation实施半自动HCV提取方法。HCV检测试剂盒被优化为定性单重RT-PCR检测试剂盒，同时对靶标和内部质控品进行平行检测。将检测结果（n=200）与COBAS AMPDECOR（TM）HCV Test v2.0检测结果进行比较。该检测方法具有较高的灵敏度（99%）、特异性（100%）和可接受的检测限（LOD）（100 IU/ml）。同时检测HCV和内部控制的定性多重检测的发展表明同样高的灵敏度和特异性。这种敏感的实时检测可能被证明是一种有价值的方法，用于检测HCV。
Real-time polymerase chain reaction (PCR) represents a favourable option for the detection of hepatitis C virus (HCV). A real-time reverse transcriptase PCR (RT-PCR) assay was developed as a qualitative diagnostic screening method for the detection of HCV using the ABI PRISM 7500 Sequence Detection System. The primers and probe were designed to target the 5'-untranslated region of the hepatitis C viral genome. A second heterologous probe assay was developed for the detection of the haemagglutinin gene of phocine distemper virus (PDV) and was used as an internal control. A semi-automated HCV extraction method was also implemented using the ABI PRISM (TM) 6100 Nucleic Acid PrepStation. The HCV assay was optimised as a qualitative singleplex RT-PCR assay with parallel testing of the target and internal control. The assay results (n=200) were compared to the COBAS AMPLICOR (TM) HCV Test v2.0 assay. The assay demonstrated a high rate of sensitivity (99%), specificity (100%) and an acceptable limit of detection (LOD) of 100 IU/ml. The development of a qualitative multiplex assay for the simultaneous detection of HCV and internal control indicates the same high rates of sensitivity and specificity. This sensitive real-time assay may prove to be a valuable method for the detection of HCV.