Rapid reverse transcription-PCR detection of hepatitis C virus RNA in serum by using the TaqMan fluorogenic detection system
Rapid reverse transcription-PCR detection of hepatitis C virus RNA in serum by using the TaqMan fluorogenic detection system
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DOI：
10.1128/jcm.34.12.2933-2936.1996
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发表时间：
1996-12-01
影响因子：
9.4
通讯作者：
Gallagher, M
中科院分区：
文献类型：
--
作者：
Morris, T;Robertson, B;Gallagher, M
我们描述了一种新的基于荧光探针的 PCR 测定（TaqMan；Perkin EImer Corp./Applied Biosystems，福斯特城，加利福尼亚州）用于检测血清和血浆中丙型肝炎病毒 RNA 的应用。该测定可通过监测染料标记寡核苷酸探针荧光的增加，在 PCR 完成后几分钟内直接检测特定 PCR 产物。我们通过将巢式 PCR 获得的结果与 TaqMan PCR 获得的结果进行比较来评估该测定。测试样品包括来自实验感染的黑猩猩的两个单独稀释系列的血浆样品以及来自社区获得性丙型肝炎病毒患者的一组 48 份血清样本。通过使用分支DNA（bDNA）信号放大测定（Quantiplex HCV RNA测定；Chiron Corp.，Emeryville，CA）测定每个黑猩猩血浆样品中的HCV RNA的量。两种 PCR 检测均显示出相似的检测水平，并且可以可靠地检测每个样本的 13 个 bDNA 基因组当量。我们发现两次 PCR 检测结果与社区获得的面板的总体一致性为 88%，当通过巢式 PCR 重新测试差异样本时，一致性达到 100%。与巢式 PCR 相比，TaqMan 的主要优点是速度快、通量高，并且由于消除了第二轮扩增而减少了假阳性结果的机会。
We describe the application of a new fluorogenic probe-based PCR assay (TaqMan; Perkin EImer Corp./Applied Biosystems, Foster City, Calif.) for the detection of hepatitis C virus RNA in serum and plasma. This assay allows for the direct detection of specific PCR products within minutes of completion of the PCR by monitoring the increase in fluorescence of a dye-labeled oligonucleotide probe. We evaluated this assay by comparing the results obtained by nested PCR with those obtained by TaqMan PCR Test samples included two separate dilutions series of plasma samples from experimentally infected chimpanzees and a panel of 48 serum specimens from patients with community-acquired hepatitis C virus. The quantity of HCV RNA in each chimpanzee plasma sample was determined by using branched DNA (bDNA) signal amplification assay (Quantiplex HCV RNA assay; Chiron Corp., Emeryville, Calif.). Both PCR assays demonstrated similar levels of detection and could reliably detect 13 bDNA genome equivalents per sample. We found an overall concordance of 88% between results of the two PCR assays with the community-acquired panel, which resolved to 100% when discrepant samples were retested by nested PCR. TaqMan compared favorably with nested PCR with key advantages of speed, increased throughput, and decreased opportunity for false-positive results because of elimination of second-round amplification.