title: Rapid detection of HCV genotyping 1a, 1b, 2a, 3a, 3b and 6a in a single reaction using two-melting temperature codes by a real-time PCR-based assay.
abstract: The genotype of the hepatitis C virus (HCV) is an important indicator for antiviral therapeutic response. We hereby described development of a rapid HCV genotyping approach that enabled the identification of the six most common HCV subtypes of Asia, i.e., 1a, 1b, 2a, 3a, 3b, and 6a, in a single reaction. Using two dual-labeled, self-quenched probes that target the core region of the HCV genome, the exact subtype could be accurately identified by two-melting temperature codes determined from the two respective probes in a real-time PCR assay. Analytical sensitivity studies using armored RNA samples representing each of the six HCV subtypes showed that 5 copies/reaction of HCV RNA could be detected. The assay was evaluated using 244 HCV-positive serum samples and the results were compared with sequencing analysis. Of the 224 samples, subtype 3a (127, 52.3%) was the dominant, followed by 1b (51, 20.9%), 3b (47, 19.3%), 2a (8, 3.3%), 6a (4, 1.6%) and the least was subtype 1a (1, 0.4%). Moreover, 6 (2.5%) mixed infection samples were also detected. These results were fully concordant with sequencing analysis. We concluded that this real-time PCR-based assay could provide a rapid and reliable tool for routine HCV genotyping in most Asian countries.
authors.affiliation: The State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China; Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong 518057, China.
authors.forename: Muhammad Ammar
authors.initials: MA
authors.lastname: Athar
authors.affiliation: The State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China; Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong 518057, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Xu
authors.affiliation: The State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
authors.forename: Xiaoting
authors.initials: X
authors.lastname: Xie
authors.affiliation: Xiamen Hospital of Traditional Chinese Medicine, Xiamen 361005, Fujian, China.
authors.forename: Zhenxing
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Division of Health Biotechnology, National Institute for Biotechnology & Genetic Engineering, P.O. Box 577, Faisalabad, Punjab, Pakistan.
authors.forename: Vakil
authors.initials: V
authors.lastname: Ahmad
authors.affiliation: Department of Pathology, Quid-e-Azam Medical College, Bahawalpur, Punjab, Pakistan.
authors.forename: Zulfiqar
authors.initials: Z
authors.lastname: Hayder
authors.affiliation: Department of Pathology, Quid-e-Azam Medical College, Bahawalpur, Punjab, Pakistan.
authors.forename: Syed Sajid
authors.initials: SS
authors.lastname: Hussain
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361005, Fujian, China. Electronic address: yqliao@xmu.edu.cn.
authors.forename: Yiqun
authors.initials: Y
authors.lastname: Liao
authors.affiliation: The State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China; Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong 518057, China. Electronic address: qgli@xmu.edu.cn.
authors.forename: Qingge
authors.initials: Q
authors.lastname: Li
mesh_terms: D001208:Asia; D060005:Genotyping Techniques / Q000379:methods*; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D044366:Transition Temperature
keywords: Fluorescence melting curve analysis; Genotyping; Hepatitis C virus; Mixed infection