Farma E, Boeri E, Morsica G, McDermott J, Soldini L, Repetto C M, Ferioli B, Pellizzari G, Molinari F, Molinelli A
Laboratory of Virology, AIDS Center S. Luigi, Hospital S. Raffaele, Milan, Italy.
Clin Exp Rheumatol. 1995 Nov-Dec;13 Suppl 13:S59-61.
Evaluation of the performance of different HCV PCR detection systems for HCV RNA: A nested PCR, considered the reference assay, was compared with two single-step methods (ss-PCR): the first is based on the detection of PCR products by liquid hybridization with a 32P end-labelled probe (isotopic ss-PCR), while the second assay is a colorimetric method (colorimetric ss-PCR) using microwell plate hybridization with a specific nucleic acid probe (Amplicor HCV PCR, Roche Diagnostics Systems).
Sera from 56 patients with suspected hepatitis C infection based on reactive serology or altered liver parameters, and sera from 15 blood donors were tested for HCV RNA: After RNA extraction, the synthesized HCV cDNA was amplified in parallel using isotopic ss-PCR, colorimetric ss-PCR and nested PCR. The products were detected by autoradiography, color development and ethidium bromide fluorescence, respectively.
In order to assess the analytical sensitivity of ss-PCR versus that of nested of PCR, experiments included serial dilutions of positive control samples. Results showed that both methods had an extinction signal at the 1:512 dilution. A comparative analysis of 71 clinical sera samples was obtained using the three protocols and the results clearly documented 100% concordance.
Single step PCR methods for HCV RNA have a sensitivity equal to that of nested PCR and appear more suitable for diagnostic applications. Ss-PCR is safer than nested PCR in terms of both specificity and contamination problems. In particular, the Roche Amplicor HCV PCR assay minimizes sample exposure and management problems.
评估不同丙型肝炎病毒（HCV）PCR检测系统检测HCV RNA的性能：将一种巢式PCR（视为参考检测方法）与两种单步方法（ss-PCR）进行比较，第一种基于用32P末端标记探针进行液相杂交检测PCR产物（同位素ss-PCR），而第二种检测方法是使用与特异性核酸探针进行微孔板杂交的比色法（比色ss-PCR）（Amplicor HCV PCR，罗氏诊断系统公司）。
对56例基于血清学反应阳性或肝功能参数异常而怀疑感染丙型肝炎的患者血清以及15名献血者的血清进行HCV RNA检测：RNA提取后，分别使用同位素ss-PCR、比色ss-PCR和巢式PCR平行扩增合成的HCV cDNA。产物分别通过放射自显影、显色和溴化乙锭荧光检测。
为了评估ss-PCR与巢式PCR的分析灵敏度，实验包括对阳性对照样品进行系列稀释。结果表明，两种方法在1:512稀释度时均有消光信号。使用这三种方法对71份临床血清样本进行了比较分析，结果清楚地表明一致性为100%。
HCV RNA单步PCR方法的灵敏度与巢式PCR相当，似乎更适合诊断应用。就特异性和污染问题而言，ss-PCR比巢式PCR更安全。特别是，罗氏Amplicor HCV PCR检测方法将样本暴露和处理问题降至最低。
