Comparative evaluation of INNO-LiPA HBV assay, direct DNA sequencing and subtractive PCR-RFLP for genotyping of clinical HBV isolates.
Genotypes (A to H) of hepatitis B virus (HBV) influence liver disease progression and response to antiviral therapy in HBV-infected patients. Several methods have been developed for rapid genotyping of HBV strains. However, some of these methods may not be suitable for developing countries. The performance of INNO-LiPA HBV Genotyping assay (LiPA), direct DNA sequencing and subtractive PCR-RFLP of genotype-specific HBV genome regions were evaluated for accurately determining the HBV genotypes by analyzing sera (n = 80) samples from chronic HBV patients. Both, LiPA and DNA sequencing identified 63, 4 and 13 HBV strains as belonging to genotype D, genotype A and mixed genotype A and D, respectively. On the contrary, the PCR-RFLP-based method correctly identified all 4 genotype A but only 56 of 63 genotype D strains. Seven genotype D strains yielded indeterminate results. DNA sequence comparisons showed that a single nucleotide change in the target region generated an additional restriction site for Nla IV that compromised the accuracy of this method. Furthermore, all the mixed genotype A and D strains were identified only as genotype A strains. The data show that the PCR-RFLP-based method incorrectly identified some genotype D strains and failed to identify mixed genotype infections while LiPA and DNA sequencing yielded accurate results.
Ali
Maisa M
MM
Department of Microbiology, Health Sciences Center, Kuwait University, Kuwait. maisa_ali@ymail.com
Hasan
Fuad
F
Ahmad
Suhail
S
Al-Nakib
Widad
W
D001483:Base Sequence; D015342:DNA Probes / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005838:Genotype; D006509:Hepatitis B / Q000821:virology*; D006515:Hepatitis B virus / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics
D015342:DNA Probes; D004279:DNA, Viral; D014764:Viral Proteins