多重引物PCR测序方法分析河北青县乙肝HBsAg阳性者的S基因特点

S Gene Characteristics in HBsAg Positive People in Qingxian County of Hebei Province with Multiple Primer PCR Sequencing Method

目的分析河北青县地区乙肝HBsAg阳性者的S基因特点。方法乙肝血清标志物酶联免疫检测试剂检测乙肝五项血清标志物,乙肝荧光PCR核酸定量试剂检测HBV病毒载量,利用多重PCR的方法,扩增乙肝S基因,序列分析其基因型、血清型特点,并分析不同标志物组合中乙肝病毒载量变化。结果在101份乙肝HBsAg阳性者中,主要有4种血清学模式:A(HBsAg+,HBeAg-,HBeAb-,HBcAb+)、B(HBsAg+,HBeAg-,HBeAb+,HBcAb+)、C(HBsAg+,HBeAg+,HBeAb-,HBcAb+)和D(HBsAg+,HBeAg+,HBeAb-,HBcAb-),比例分别为15.8%(16/101)、50.5%(51/101)、28.7%(29/101)和5.0%(5/101)。荧光定量PCR法HBV核酸阳性率为86%(87/101),多重PCR方法的核酸阳性率为94%(95/101),两者无显著统计学意义(χ2=3.55,P>0.05)。A、B、C和D血清学模式的病毒载量分别为:1.6×104、8.5×102、1.8×107和1.5×108 IU·mL-1。该地区以C基因型为主占91%(86/95),B基因型仅9%(9/95);血清型以adr为主占80%(76/95),adw血清型19%(18/95),ay血清型1%(1/95)。不同血清学模式下乙肝的基因型和血清型分布没有显著差异,血清模式e抗原阳性人群的病毒载量显著高于e抗原阴性人群,e抗原阳性人群的年龄低于e抗原阴性人群(P<0.01)。结论多重PCR方法与荧光定量PCR方法的灵敏度相当,可以用于乙肝的S基因序列分析,青县地区C基因型乙肝病毒占绝对优势,应引起重视。

Objective To analyze the S gene characteristics of HBsAg positive people in Qingxian County of Hebei Province. Methods Hepatitis B virus （HBV） related serological markers were detected with corresponding ELISA kits. The DNA levels of HBV were detected with HBV fluorescent quantitative PCR reagent. S genes of HBV were amplified with multiplex primer PCR. Genotypes and serotypes of HBV were identified through sequence analysis, and the relationship between HBV serological marker patterns and HBV DNA levels was analyzed. Results In 101 HBV infected persons, there were mainly 4 serological patterns： A （HBsAg@, HBeAg--, HBeAb--, HBcAb＋）, B （HBsAg＋, HBeAg-, HBeAb2-, HBcAb ＋）, C （HBsAg ＋, HBeAg ＋, HBeAb-, HBcAh ＋） and D （HBsAg＋, HBeAg4-, HBeAb-, HBcAb-）. The proportions of each pattern were 15.8% （16/101）, 50.5% （51/101）, 28.7% （29/101） and 5% （5/101）, respectively. The positive rate of HBV DNA was 86% （87/101） by fluorescent quantitative PCR assay, and 94% （95/101） by multiplex primer PCR method. There was no statistical significance （Z2 =3.55, P〉0.05） between the two methods. The geometric mean viral loads of A, B, C and D serological patterns were： 1.6）〈 104 , 8. 5）〈 10^2 , 1.8）〈 10^7 and 1.5 ）〈 10^8 IU ·mL^-1. Genotype C was the dominant genotype in this area, accounting for 91% （86/95）, with genotype B accounting for 9% （9/95）. Serotype adr was the dominant serotype in this area, serotypes adr, adw and ay accounted for 80% （76/95）, 19% （18/95） and 1% （1/95）, respectively. The distribution of genotypes and serotypes of HBV in Qingxian did not differ significantly in different serological patterns. The HBV DNA levels were higher in e antigen positive population than those in e antigen negative population. Furthermore, the ages of the patients were much younger than in the e antigen negative population （P〈0.01）. Conclusion Sequence analysis of S gene with multiplex primer PCR in this study has the same sensitivity compared with the fluorescent quantitative PCR assay, which can be used for analysis of the S gene characteristics of HBV. It should be noted that the dominant genotype of HBV in Qingxian County is genotype C.