乙型肝炎病毒逆向点杂交基因分型法建立及应用

目的 利用逆向点杂交技术建立乙型肝炎病毒(HBV)基因分型新方法，通过对HBV DNA阳性血清抽样标本进行基因分型，了解佛山地区HBV基因型分布状态。方法 以HBV基因组的X区序列为主设计分型引物与探针，将活性氨基标记的探针依次固定在尼龙膜上，制成检测膜条。用标记生物素的引物进行HBV DNA扩增，将扩增产物与检测膜条杂交，以POD与TMB显色，判断基因分型结果，通过与基因测序结果比较确定新方法的有效性。从佛山地区HBVDNA阳性患者血清中随机抽取300份，用新建方法进行HBV基因分型检测。结果 新建HBV逆向点杂交基因分型方法可对拷贝数在10^3～10^9／ml之间的300份HBV DNA阳性抽检血清进行基因分型，发现B型147例，占49.0％；C型136例，占45．3％；D型1例，占0．3％；B、C混合型12例，占4．0％；C、D混合型4例，占1．3％；未发现A、E和F型。新方法基因分型结果与测序结果一致。结论 利用逆向点杂交技术可以准确有效和简便经济的进行HBV基因分型，适用于临床检测与流行病学研究；在佛山地区，人群中感染的HBV以B、C型为主。

Establishment of a new HBV genotyping method with PCR-RBD and its application

Objective Using PCR-RDB to establish a new method for HBV genotyping, and to survey the distribution of HBV genotypes in the Foshan area. Methods Biotin-labeled primers for amplification of HBV region X (nt1550-1789) were used to amplify extracted HBV DNA. HBV was genotyped by hybridization of the PCR products with immobilized specific probes (genotype A to F) on C membrane. Color development was achieved by adding POD and TMB. A judgment was made according to color reactions. The reliability of this new method was verified by gene sequencing. 300 samples of HBV DNA-positive sera from the Foshan area were genotyped using this assay. Results Of the 300 sera genotyped by PCR-RBD, 147 (49.0%) cases were genotype B, 136 (45.3%) were genotype C, 1 (0.3%) genotype D, and 12 (4.0%) were mixtures of genotype B and C, and 4 (1.3%) were mixtures of genotype C and D. No genotype A, E or F were found. The results of PCR-RDB genotyping were consistent with the results obtained with sequence analysis. Conclusion This newly established HBV genotyping system proved to be sensitive, specific, precise and economic, and should be suitable for clinical practice and epidemic study. The results of HBV genotyping show that genotype B and C are the predominant genotypes in the Foshan area.