High-throughput real-time reverse transcription PCR quantitation of hepatitis C virus RNA
High-throughput real-time reverse transcription PCR quantitation of hepatitis C virus RNA
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DOI：
10.1128/jcm.37.2.327-332.1999
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发表时间：
1999-02-01
影响因子：
9.4
通讯作者：
Guardia, J
中科院分区：
文献类型：
--
作者：
Martell, M;Gómez, J;Guardia, J
我们描述了一种快速和可重复的方法来评估血清样本中的丙型肝炎病毒（HCV）载量。该方法结合了Taqman技术（Roche）和ABI Prism 7700 （Perkin Elmer）实时序列检测系统。我们优化了一种含有双标记荧光探针的单管逆转录pcr (RT-PCR)，用于定量HCV的5‘非编码区（5’ NCR）。探针在5‘端包含一个荧光报告，在3’端包含一个荧光猝灭剂。使用这种探针结合Tag聚合酶的5‘-3’核酸酶活性，可以通过检测PCR指数阶段过程中释放的荧光报告基因来直接定量PCR产物。为了在含有未知数量的样品中准确定量HCV拷贝数，我们使用了HCV合成5' NCR RNA标准物的连续稀释，该标准物以前是用同位素示踪剂定量的。该方法具有5对数动态范围(10（3)至10(7)）。标准曲线的回归系数平均为0.98。阈值周期的组内和组间变异系数分别为1%和6.2%。用该方法对79例感染患者血清中的RNA样本进行了定量分析。将结果与其他定量方法获得的结果（Quantiplex 2.0分支dna测定法和国家遗传研究所的Superquant测定法）进行比较，发现所有结果都具有显著的相关性。平均值也具有统计学上的可比性。总之，实时HCV RNA定量的高灵敏度、简单性和可重复性，允许筛选大量样本，结合其宽动态范围，使该方法特别适用于治疗期间的病毒载量监测和治疗计划的定制。
We describe a rapid and reproducible method for assessment of the hepatitis C virus (HCV) load in serum samples. The method combines Taqman technology (Roche) and the ABI Prism 7700 (Perkin Elmer) real-time sequence detection system. We have optimized a single-tube reverse transcription-PCR (RT-PCR) that contains a dual-labeled fluorogenic probe to quantify the 5' noncoding region (5' NCR) of HCV. The probe contains a fluorescent reporter at the 5' end and a fluorescent quencher at the 3' end. The use of such a probe combined with the 5'-3' nuclease activity of Tag polymerase allows direct quantitation of the PCR product by the detection of a fluorescent reporter released in the course of the exponential phase of the PCR. For accurate quantitation of the number of copies of HCV in samples containing unknown quantities, we have used serial dilutions of a synthetic 5' NCR RNA standard of HCV that was previously quantified with an isotopic tracer. The method has a 5-log dynamic range (10(3) to 10(7)). The coefficient of regression of the standard curve was, on average, 0.98. The intra-assay and the interassay coefficients of variation of the threshold cycle were 1% and 6.2%, respectively. Seventy-nine RNA samples from the sera of infected patients were quantified by this method. Comparison of the results with those obtained by other quantitation methods (the Quantiplex 2.0 branched-DNA assay and the Superquant assay from the National Genetics Institute) revealed a significant correlation with all of the results. The mean values were also statistically comparable. In conclusion, the high sensitivity, simplicity, and reproducibility of the real-time HCV RNA quantitation which allows the screening of large numbers of samples, combined with its wide dynamic range, make this method especially suitable for monitoring of the viral load during therapy and tailoring of treatment schedules.