Comparison of nested PCR-restriction fragment length polymorphism with mutiple-PCR by type-specific primers for HBV genotyping and subgenotyping

Objective: To compare the efficiency between nested PCR with type-specific primers and PCR-RFLP method and improved multiplex-PCR method in HBV genotyping and subgenotyping.Methods: The nested PCR with PCR-RFLP method and the improved multiplex-PCR method were used to identify HBV genotypes and subgenotypes of 100 sera of patients with HBV infection from three hospitals in Dali.Results: The discrepancy between the nested PCR with PCR-RFLP method and the improved multiplex-PCR method was 50%（27/54）.Of 100 sera from patients with HBV infection determined by the nested PCR with PCR-RFLP,the proportion of genotype B,C,and B+C were 41%（41/100）,25%（25/100）and 34%（34/100）;subgenotype Bj,Ba,Cs and Ce were 7.3%（3/41）,92.7%（38/41）,84%（21/25） and 12%（3/25）respectively.Only one of genotype C was unidentified 4%（1/25）.By the improved multiplex-PCR,genotype B,C,and B+C were 33.3%（18/54）,13%（7/54） and 9.3%（5/54）;subgenotypeBa and Cs were 11%（2/18） and 28.5%（2/7）.The detections rate of PCR-RFLP were 100% in genotype and 98.5% in subgenotype,which were significantly higher than that of multiplex-PCR 55.6% and 16%（P＜0.05）.Conclusion: The nested PCR with PCR-RFLP is more reliable and simple to identify HBV genotypes and subgenotypes.