Laboratório Central do Estado (LACEN-PR), São José dos Pinhais, Paraná, Brazil.
PLoS One. 2010 Apr 13;5(4):e10150. doi: 10.1371/journal.pone.0010150.
Hepatitis C virus (HCV) genotyping is the most significant predictor of the response to antiviral therapy. The aim of this study was to develop and evaluate a novel real-time PCR method for HCV genotyping based on the NS5B region.
METHODOLOGY/PRINCIPAL FINDINGS: Two triplex reaction sets were designed, one to detect genotypes 1a, 1b and 3a; and another to detect genotypes 2a, 2b, and 2c. This approach had an overall sensitivity of 97.0%, detecting 295 of the 304 tested samples. All samples genotyped by real-time PCR had the same type that was assigned using LiPA version 1 (Line in Probe Assay). Although LiPA v. 1 was not able to subtype 68 of the 295 samples (23.0%) and rendered different subtype results from those assigned by real-time PCR for 12/295 samples (4.0%), NS5B sequencing and real-time PCR results agreed in all 146 tested cases. Analytical sensitivity of the real-time PCR assay was determined by end-point dilution of the 5000 IU/ml member of the OptiQuant HCV RNA panel. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotypes 1b and 2b, and 500 IU/ml for genotype 1a.
CONCLUSIONS/SIGNIFICANCE: The total time required for performing this assay was two hours, compared to four hours required for LiPA v. 1 after PCR-amplification. Furthermore, the estimated reaction cost was nine times lower than that of available commercial methods in Brazil. Thus, we have developed an efficient, feasible, and affordable method for HCV genotype identification.
丙型肝炎病毒 (HCV) 基因分型是抗病毒治疗反应的最重要预测因素。本研究旨在开发和评估一种基于 NS5B 区的新型 HCV 基因分型实时 PCR 方法。
方法/主要发现：设计了两个三重反应体系，一个用于检测基因型 1a、1b 和 3a；另一个用于检测基因型 2a、2b 和 2c。该方法的总灵敏度为 97.0%，检测了 304 个测试样本中的 295 个。通过实时 PCR 基因分型的所有样本均与使用 LiPA 版本 1（探针在线测定法）分配的相同类型。尽管 LiPA v. 1 无法对 295 个样本中的 68 个（23.0%）进行亚分型，并且对 295 个样本中的 12 个（4.0%）的结果与实时 PCR 分配的结果不同，但 NS5B 测序和实时 PCR 结果在所有 146 个测试病例中均一致。通过 OptiQuant HCV RNA 面板的 5000 IU/ml 成员进行终点稀释来确定实时 PCR 测定的分析灵敏度。检测到的基因型 3a 的下限为 125 IU/ml，基因型 1b 和 2b 为 250 IU/ml，基因型 1a 为 500 IU/ml。
结论/意义：与 PCR 扩增后 LiPA v. 1 所需的四个小时相比，执行该测定的总时间为两个小时。此外，估计的反应成本比巴西现有商业方法低九倍。因此，我们已经开发出一种高效、可行且负担得起的 HCV 基因型鉴定方法。
