两种型特异性引物聚合酶链反应法检测乙型肝炎病毒基因型和亚型的评价

Evaluation of two type-specific primers PCR genotyping methods of hepatitis B virus

目的比较并评价本室创建的方法和日本Naito等建立的乙型肝炎病毒（HBV）型特异性引物聚合酶链反应（PCR）基因分型法。方法分别用新方法和Naito法检测系列稀释的含有A、D基因型和B1、C2亚型HBV基因组的质粒及不同浓度比例的B1／C2亚型混合质粒，评价两种方法的灵敏度和特异度。此外，用该两种方法分别对深圳市、河北邯郸市、新疆乌鲁木齐市送检的113份HBV DNA阳性慢性乙型肝炎患者的血清标本进行HBV基因分型，并对该两种分型方法检测结果不一致的血清样本，用PCR产物直接测序法分析。结果两种方法对单一型别的质粒样本灵敏度无差异，但新方法对B／C混合型质粒样本的灵敏度及对各型别质粒样本的特异度明显优于Naito法。该两种方法检测单一基因型血清标本的灵敏度无差异，但新方法对B／C混合型血清样本的检出率更高。该两种方法检测结果总符合率为83．2％（94／113），不一致率为16．8％（19／113）。从两法分型不一致的19份血清标本中，选取15份以PCR产物直接测序法进行分析，结果与新方法检测结果完全一致，而与Naito法不一致。结论本室建立的HBV型特异性引物-PCR基因分型法具有较高灵敏度，其特异度也明显优于目前临床上常用的Naito法，适用于HBV基因型和基因亚型的临床及流行病学研究。

Objective To compare and evaluate two type-specific primers PCR genotyping methods of hepatitis B virus （HBV） which were established by Naito et al （Naito method） and our lab （new method）. Methods The two genotyping methods were applied for detecting the plasmids containing the HBV genomes of genotype A or D or subgenotype B1 or C2 and the plasmids mixed with different proportion of subgenotypes B1 and C2. In addition, the genotypes of 113 serum samples of patients with chronic HBV infection from Shenzhen, Handan and Urumqi cities of China were identified by the two methods, respectively. The results were verified by PCR product based sequencing. Results The sensitivity of the two methods showed no difference when they were applied to detect the plasmids containing the HBV genomes of genotype A or D or subgenotype B1 or C2. While detecting the plasmids mixed with different proportion of subgenotypes B1 and C2, the sensitivity of the new method was superior than that of Naito method. Meanwhile, the specificity of the new method was obviously superior than that of Naito method. Both of the two methods were highly sensitive in identification of HBV genotypes of serum samples with a single genotype. However, the new method showed more sensitive in identification of the B/C mix strains than that of Naito method. The total coincidence rate between the two methods was 83.2% （94/113）, with the discrepancy of 16. 8% （ 19/113 ）. Fifteen of the 19 inconsistent genotypes by the two methods were selected and their PCR products were sequenced directly. The sequencing results were identical with that of the new methods, but not with that of the Naito method. Conclusion The new method is more sensitive, and its specificity is superior to the Naito method. It could be used for clinical and epidemiological studies on HBV genotype and subgenotype in China.