title: Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis.
abstract: Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 104copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries.
authors.affiliation: State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.|Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Mou
authors.affiliation: State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.|Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong, China.
authors.forename: Muhammad Ammar
authors.initials: MA
authors.lastname: Athar
authors.affiliation: State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.|Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong, China.
authors.forename: Yuzhen
authors.initials: Y
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.|Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Xiamen Hospital of Traditional Chinese Medicine, Xiamen, Fujian, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Wu
authors.affiliation: Xiamen Hospital of Traditional Chinese Medicine, Xiamen, Fujian, China.
authors.forename: Zhenxing
authors.initials: Z
authors.lastname: Xu
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 Molecular Pathology, Dow University of Health Sciences, Karachi, Pakistan.
authors.forename: Saeed
authors.initials: S
authors.lastname: Khan
authors.affiliation: Center for Applied Molecular Biology, University of the Punjab, Lahore, Pakistan.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Idrees
authors.affiliation: Department of Molecular Pathology, Liaquat National Hospital, Karachi, Pakistan.
authors.forename: Muhammad Israr
authors.initials: MI
authors.lastname: Nasir
authors.affiliation: State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China yqliao@xmu.edu.cn qgli@xmu.edu.cn.|School of Public Health, Xiamen University, Xiamen, Fujian, China.
authors.forename: Yiqun
authors.initials: Y
authors.lastname: Liao
authors.affiliation: State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China yqliao@xmu.edu.cn qgli@xmu.edu.cn.|Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong, China.
authors.forename: Qingge
authors.initials: Q
authors.lastname: Li
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D003116:Color; D003365:Costs and Cost Analysis; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics*; D024882:Drug Resistance, Viral; D006515:Hepatitis B virus / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D009154:Mutation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D044366:Transition Temperature
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; D012194:RNA-Directed DNA Polymerase