Okamoto M, Baba M, Kodama E, Sekine K, Takagi T, Kasukawa R, Shigeta S
Department of Microbiology, Fukushima Medical College, Japan.
J Med Virol. 1993 Sep;41(1):6-10. doi: 10.1002/jmv.1890410103.
A multi-targeted "hemi-nested" PCR (M-PCR) assay in which the primer pairs derived from the 5' non-coding (5'NC) and the nonstructural protein 3 (NS3) regions of HCV genome were concurrently used for amplification in order to compare the sensitivity and specificity of polymerase chain reaction (PCR) with different primer pairs in detecting hepatitis C virus (HCV) genome. Sera from patients with virus-associated liver diseases were examined for the presence of HCV RNA by the M-PCR method following reverse transcription to cDNA. The amplified products derived from both the 5'NC and the NS3 regions were detected in 28 (70%) of the 40 HCV RNA-positive samples. However, 12 samples (30%) were devoid of the signal of NS3-derived product. Sensitivity tests using serial dilutions of HCV RNA revealed that the 5'NC-derived band was still detectable in the 10(5)-fold diluted sample by the M-PCR method, yet the NS3-derived band could hardly be detected in the 10(4)-fold diluted sample. Thus, as previously demonstrated by a single-targeted "nested" PCR assay, the present study using the M-PCR assay has clearly shown that the 5'NC-derived primers are more sensitive and specific than the NS3-derived primers in detecting HCV RNA.
一种多靶点“半巢式”PCR（M-PCR）检测方法，其中源自丙型肝炎病毒（HCV）基因组5'非编码（5'NC）区和非结构蛋白3（NS3）区的引物对同时用于扩增，以便比较聚合酶链反应（PCR）中不同引物对在检测丙型肝炎病毒（HCV）基因组时的敏感性和特异性。对病毒相关性肝病患者的血清进行逆转录为cDNA后，采用M-PCR方法检测HCV RNA的存在情况。在40份HCV RNA阳性样本中，有28份（70%）检测到源自5'NC区和NS3区的扩增产物。然而，有12份样本（30%）未检测到NS3区衍生产物的信号。使用HCV RNA系列稀释液进行的敏感性试验表明，通过M-PCR方法在10（5）倍稀释的样本中仍可检测到5'NC区衍生的条带，但在10（4）倍稀释的样本中几乎检测不到NS3区衍生的条带。因此，正如先前通过单靶点“巢式”PCR检测所证明的那样，本研究使用M-PCR检测清楚地表明，在检测HCV RNA时，5'NC区衍生的引物比NS3区衍生的引物更敏感、更特异。
