Abstract
Specific genotypes of hepatitis B virus (HBV) are increasingly recognized for their clinical significance and association with particular viral mutations. Although many HBV genotyping methods exist, there has been no standardized or commercially available method for direct molecular typing of the HBV genome. A newly available line probe assay (INNO-LiPA HBV Genotyping assay; Innogenetics N.V., Ghent, Belgium) that allows the identification of HBV genotypes A to G was assessed by comparison with pre-S1/pre-S2 sequence analysis of the isolates in 188 serum specimens. All seven genotypes were detected by the line probe assay (LiPA), and complete concordance between LiPA and sequence analysis was observed for 152 specimens (81%). LiPA was able to detect 19 mixed genotype infections not detected by amplicon sequencing, which for the most part were confirmed by cloning and sequencing of the pre-S1/pre-S2 amplicon. Four specimens had discrepant results between the two methods, and five specimens had indeterminate results by LiPA. The HBV DNA in four specimens was unable to be amplified by the nested INNO-LiPA HBV DR amplification primers; however, the HBV DNA in six specimens unable to be genotyped by sequencing was clearly genotyped by LiPA. The INNO-LiPA HBV Genotyping assay appears to be useful for the rapid genotyping of HBV, particularly for the sensitive detection of mixed genotype infections.
Highlights
A newly available line probe assay (INNO-LiPA hepatitis B virus (HBV) Genotyping assay; Innogenetics N.V., Ghent, Belgium) that allows the identification of HBV genotypes A to G was assessed by comparison with pre-S1/pre-S2 sequence analysis of the isolates in 188 serum specimens
All seven genotypes were detected by the line probe assay (LiPA), and complete concordance between LiPA and sequence analysis was observed for 152 specimens (81%)
The present study evaluated the INNO-LiPA HBV Genotyping assay by testing 188 serum specimens positive for HBV DNA that had been genotyped by sequencing and phylogenetic analysis of the pre-S1/pre-S2 region of the HBV genome
Summary
If the 277-bp product was not initially detected, the extracted DNA was amplified by nested PCR with outer primers that produce a 479-bp product (sense primer, 5Ј-TCACCATATTCTTGGGAACAAGA-3Ј; antisense primer, 5Ј-TTCCTGAACTGGAGCCACCA-3Ј) [15], followed by second-stage amplification with the pre-S primers described above. The extracted DNA was amplified by nested PCR according to the instructions of the manufacturer (Innogenetics) for amplification of the HBsAg region to provide a biotinylated product. The HBV genomic region amplified extends from nucleotides 415 to 824 for the outer primers and nucleotides 456 to 798 for the nested inner primers (the numbering is based on the sequence with GenBank accession number AY128092) These procedures have been described previously [17]. Specimens showing discrepant genotypes between sequencing and LiPA were further analyzed by cloning of the pre-S region amplicon used for sequencing and phylogenetic analysis. Insert sequence data were aligned with GenBank pre-S1/pre-S2 sequences, as described above, to determine the genotype of each individual clone
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