型特异性引物巢式PCR快速检测HBV基因型及其临床意义

目的　应用型特异性引物巢式PCR检测HBV基因型并初步了解山东地区HBV基因型的分布状况。方法　随机选择山东地区HBVDNA阳性患者共 178例 ,其中无症状携带者 (ASC) 5 3例 ,慢性活动性肝炎 (CH) (包括轻、中、重度 ) 87例 ,肝硬化(LC) 38例 ,先通过外引物进行第一轮PCR ,然后 ,将 6对型特异性引物分到两个反应体系 (MixA和MixB)中分别对第一轮扩增产物进行第二轮PCR ,根据阳性结果判断出样本的基因型。结果　C基因型 85例 (47.8% ) ,B基因型 6 3例 (35 .4 % ) ,B C混合基因型 30例 (16 .8% )。C基因型在活动性肝病 (CH及LC)中占优势 ,而ASC组则是以B基因型为主 ,差异有显著性 (P <0 .0 1)。C基因组中HBeAg阳性率、HBVDNA定量及ALT水平均较B基因组为高 (P <0 .0 0 1或P <0 .0 0 5 ) ,而抗HBe阳性率则在B基因型组显著升高 (P <0 .0 0 5 )。结论　山东地区存在C、B及B C混合基因型 ,并以C基因型为主。型特异性引物巢式PCR敏感性高 ,特异性强 ,可以快捷准确地检测HBV基因型。

Rapid genotyping for hepatitis B virus by nested-PCR using type-specific primers and its clinical significance

Objective To genotype rapidly for HBV by nested-PCR using type-specific primers and investigate the distribution of HBV genotypes in Shandong Province.Methods HBV genotypes were determined by nested-PCR analysis in randomly selected 178 HBV DNA positive patients, including 53 of asymptomatic HBV carriers (ASC),87 of chronic active hepatitis (mild,moderate and severe) and 38 of liver cirrhosis in Shandong Province.In the first PCR the samples were amplified by outer primers,then the products of the first PCR were amplified by 6 inner primers in MixA and MixB respectively in the second PCR.Results Of the 178 patients 85 (47.8%) were genotype C,63 (35.4%) were genotype B, and 17 (16.8%) were mixed genotype B and C.There was a statistical significance for the distributions of genotype B between active liver diseases and ASC(P<0.01).HBV-DNA copies,HBeAg positive rates and ALT levels were higher in genotype C than those in genotype B (P<0.001 and P<0.005,respectively).However,the anti-HBe titers were significantly higher in genotype B than those in genotype C (P<0.005).Conclusion HBV genotype B, C and mixed B and C exist in Shandong Province,and genotype C is the major genotype.The nested-PCR using type-specific primers for HBV genotyping is proved to be sensitive and specific for rapid determination of genotypes of HBV,so it can be used in clinical diagnosis and epidemiological study for HBV genotypes.