Nakatani, Sueli M.Santos, Carlos A.Riediger, Irina N.Krieger, Marco A.Duarte, Cesar A. B.Lacerda, Marco A.Biondo, Alexander W.Carilho, Flair J.Ono-Nita, Suzane K.2020-05-192020-05-192010-04-13Nakatani SM, Santos CA, Riediger IN, Krieger MA, Duarte CAB, Lacerda MA, et al. (2010) Development of Hepatitis C Virus Genotyping by Real-Time PCR Based on the NS5B Region. PLoS ONE 5(4): e10150. https://doi.org/10.1371/journal.pone.0010150https://hdl.handle.net/1805/22801Hepatitis 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.en-USAttribution 4.0 InternationalPolymerase chain reactionGenotypingHepatitis C virusUntranslated regionsBrazilRNA extractionReverse transcriptionPhylogenetic analysisArticle