巢氏PCR-RFLP和多重PCR检测HBV基因型、基因亚型方法的比较
Comparison of nested PCR-restriction fragment length polymorphism with mutiple-PCR by type-specific primers for HBV genotyping and subgenotyping
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摘要: 目的:建立HBV基因型和亚型的分型方法,并分析相关的两种方法特异性和敏感性。方法:采用型特异性引物的巢氏PCR-RFLP和六种主要的HBV型特异性引物和亚型特异性引物的多重PCR方法,分别检测了100例患者标本。结果:两法不一致率为50%(27/54)。型特异性引物巢氏PCR-RFLP法检测出B基因型41例(41%),C基因型25例(25%),B+C基因型34例(34%),B j亚型3例(7.3%),Ba亚型为38例(92.7%),Cs亚型为21例(84%),Ce亚型为3例(12%),1例C型未分出亚型(4%)。多重PCR法检出B型18例(33.3%),C型7例(13%),B+C混和型5例(9.3%),B2亚型2例(11%),C1亚型2例(28.5%)。巢氏PCR-RFLP法型检出率100%亚型检出率98.5%,多重PCR法型检出率55.6%,亚型检出率仅为16%,前者高于后者(P<0.05)。结论:巢氏PCR-RFLP鉴定HBV基因型、基因亚型较多重PCR敏感性高,重复性好,但耗时长,费用高。Abstract: 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.
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