title: Universal Primers for Detection and Sequencing of Hepatitis B Virus Genomes across Genotypes A to G.
abstract: Hepatitis B virus (HBV) has been divided into 10 genotypes, A to J, based on an 8% nucleotide sequence divergence between genotypes. The conventional practice of using a single set of primers to amplify a near-complete HBV genome is hampered by its low analytical sensitivity. The current practice of using overlapping conserved primer sets to amplify a complete HBV genome in a clinical sample is limited by the lack of pan-primers to detect all HBV genotypes. In this study, we designed six highly conserved, overlapping primer sets to cover the complete HBV genome. We based our design on the sequences of 5,154 HBV genomes of genotypes A to I downloaded from the GenBank nucleotide database. These primer sets were tested on 126 plasma samples from Malaysia, containing genotypes A to D and with viral loads ranging from 20 to >79,780,000 IU/ml. The overall success rates for PCR amplification and sequencing were >96% and >94%, respectively. Similarly, there was 100% amplification and sequencing success when the primer sets were tested on an HBV reference panel of genotypes A to G. Thus, we have established primer sets that gave a high analytical sensitivity for PCR-based detection of HBV and a high rate of sequencing success for HBV genomes in most of the viral genotypes, if not all, without prior known sequence data for the particular genotype/genome.
authors: Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors: Jack Bee
authors: JB
authors: Chook
authors: Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors: Woon Li
authors: WL
authors: Teo
authors: Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors: Yun Fong
authors: YF
authors: Ngeow
authors: Centre of Excellence for Research in AIDS (CERiA), Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors: Kok Keng
authors: KK
authors: Tee
authors: Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors: Kee Peng
authors: KP
authors: Ng
authors: Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia ros@ummc.edu.my.
authors: Rosmawati
authors: R
authors: Mohamed
mesh_terms: D017931:DNA Primers / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics; D005838:Genotype; D006509:Hepatitis B / Q000821:virology; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods*; D014773:Virology / Q000379:methods*
chemical_list: D017931:DNA Primers