一种新型的双靶实时逆转录-聚合酶链反应检测试剂盒，用于检测丙型肝炎病毒 RNA，以装甲 RNA 作为内对照。

BACKGROUND

The hepatitis C virus (HCV) genome is extremely heterogeneous. Several HCV infections can not be detected using currently available commercial assays, probably because of mismatches between the template and primers/probes. By aligning the HCV sequences, we developed a duplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using 2 sets of primers/probes and a specific armored RNA as internal control. The 2 detection probes were labelled with the same fluorophore, namely, 6-carboxyfluorescein (FAM), at the 5' end; these probes could mutually combine, improving the power of the test.

RESULTS

The limit of detection of the duplex primer/probe assay was 38.99 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. All HCV genotypes in the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques could be detected. In the testing of 109 serum samples, the performance of the duplex real-time RT-PCR assay was identical to that of the COBAS AmpliPrep (CAP)/COBAS TaqMan (CTM) assay and superior to 2 commercial HCV assay kits.

CONCLUSIONS

The duplex real-time RT-PCR assay is an efficient and effective viral assay. It is comparable with the CAP/CTM assay with regard to the power of the test and is appropriate for blood-donor screening and laboratory diagnosis of HCV infection.

背景

丙型肝炎病毒（HCV）基因组具有极高的异质性。目前可用的商业检测方法无法检测到几种 HCV 感染，可能是由于模板和引物/探针之间不匹配。通过对齐 HCV 序列，我们使用 2 组引物/探针和特定的装甲 RNA 作为内部对照，开发了一种双工实时逆转录聚合酶链反应（RT-PCR）检测方法。这 2 个检测探针在 5'端用相同的荧光团，即 6-羧基荧光素（FAM）进行标记；这些探针可以相互结合，提高了检测的效力。

结果

双工引物/探针检测方法的检测下限为 38.99IU/ml。该检测方法的灵敏度显著提高，而特异性不受影响。可检测到核酸扩增技术 HCV RNA 基因分型面板中的所有 HCV 基因型。在对 109 份血清样本的检测中，双工实时 RT-PCR 检测方法的性能与 COBAS AmpliPrep（CAP）/COBAS TaqMan（CTM）检测方法相同，优于 2 种商业 HCV 检测试剂盒。