乙型肝炎病毒A～D基因型及B1、B2、C1和C2基因亚型巢式聚合酶链反应分型法的建立

Establishment of a nested PCR to identify hepatitis B virus genotypes A-D and subgenotypes B1, B2, C1 and C2

目的建立检测乙型肝炎病毒（HBV）基因型和亚型特异性巢式聚合酶链反应（nPCR）分型法。方法用DNAStar软件比较分析GenBank中登录的A～H8种基因型HBV全基因组序列，用Primer Premier5．0软件进行引物设计，建立nPCR分型法。该法在第一轮扩增基础上，第二轮分为3步扩增：第一步用MixA扩增，检测B、D基因型和C1、C2亚型；第二步用MixB扩增，检测A基因型；第三步用MixC扩增，检测B1和B2亚型。应用该法检测68份慢性HBV感染者的血清样本，并从中随机选取15份样本的PCR产物直接测序，以验证该法的准确性。结果该法检测68份慢性HBV感染者血清样本中，23．53％（16／68）为B2亚型，11．76％（8／68）为C1亚型，48．53％（33／68）为C2亚型，1．47％（1／68）为D型，11．76％（8／68）为B2C2混合型，1．47％（1／68）为C2D混合型，1．47％（1／68）为B2C1D混合型。随机选取15份样本测序分型，结果与PCR法一致。结论nPCR分型法简单快速，具有较高的灵敏度和特异度，可检测A～D基因型和B1、B2、C1、C2亚型。

Objective To establish a hepatitis B virus （HBV） nested PCR （nPCR） for detection of genotypes A-D and subgenotypes B1, B2, C1 and C2. Methods The entire HBV nucleotide sequences of genotypes A-H retrieved from GenBank were compared and analyzed by DNAStar software. The PCR primers were designed by Primer Premier 5.0 software, and the nPCR for genotyping HBV/A-D as well as subgenotyping B1 ,B2 ,C1 and C2 were established. There were 3 steps in the process：step 1 for genotypes B, D and subgenotypes C1,C2 with the amplification of Mix A; step 2 for genotype A with the amplification of Mix B; step 3 for subgenotypes B1 and B2 with the amplification of Mix C in the second- round PCR, based on the first-round amplification procedure. A total of 68 serum samples from patients with chronic HBV infection were detected by nPCR. 15 of 68 sera were selected randomly and their PCR products were directly sequenced to confirm the accuracy of the method. Results Among 68 serum samples of patients with chronic HBV infection detected by the nPCR, 23.53 % （16/68） were infected with B2, 11.76% （8/68） withCl,48.53% （33/68） withC2,1.47% （1/68） with D,11. 76 % （8/68） withB2C2 mix strains,1.47% （1/68） with C2D mix strains and 1.47% （1/68） with B2/C1/D mix strains. The sequencing analysis of the 15 serum samples had the same results as detected by nPCR. Conclusion nPCR is a simple, rapid method and able to detect genotypes A-D and subgenotypes B1, B2, C1 and C2 subtypes of HBV with both high sensitivity and specificity.