高灵敏度巢式PCR方法扩增HBV全长基因组

Amplification of Complete Hepatitis B Virus Genome by a Sensitive Nested PCR

探讨一种用于乙型肝炎病毒（HBV）基因组扩增的灵敏度高、广谱性好的巢式聚合酶链式反应（nested PCR）方法，适用于较低病毒拷贝数标本的HBVDNA扩增．通过设计两套通用巢式引物。实现对各基因型（A，B，C，D，E，G）HBV均具有较高的扩增效率；采用两步法分段扩增HBV基因组，对两段目的产物进行测序拼接获得HBV的全基因序列．应用本方法对4组病毒拷贝量（IU／mL）分别为：〉1．0×10^4，1．0×10^3～1．0×10^，2．0×10^2～1．0×10^3，〈2．0×10^2的100份血清标本进行全长基因组扩增，其中75份获得阳性结果，各组阳性率依次为：100％，66．7％，60％，25％．有效扩增灵敏度为2．0×10^2～1．0×10^3IU／mL；而一步法只在病毒拷贝量〉1．0×10^4 IU／mL的标本组中获得15份阳性结果，总的阳性率仅为15％，说明该方法扩增灵敏度明显高于一步法．因此。本方法为HBV流行病学调查研究HBV基因组序列提供了更高的灵敏度，可在流行病学调查中发挥重要作用．

A broad and high-sensitive nested PCR method was developed to amplify complete hepatitis B virus （HBV） genome.Two sets of nested primers were designed to amplify two overlapped fragments on HBV genome （Fragment A： nt1825-nt3215/0-702; Fragment B：ntS03-1823）. The two fragments covered complete viral genome. Serial dilutions of six HBV plasmids with different genotype （genotype A, B, C, D, E and G） , the HBV DNA IU of four groups was： 〉 1.0 ×10^4 , 1.0 × 10^3-1. 0× 10^4 , 2.0 × 10^2-1. 0 ×10^3 , 〈2.0 × 10^2 IU/mL,and a total of 100 HBsAg（＋） clinical serum specimens were tested by the new method for evaluating its performance. The results demonstrated that the average lower detection limit of the new method for the HBV plasmids with different genotype was 2.0× 10^2-1. 0 × 10^3 IU/mL. Among 100 clinical serum specimens, complete HBV genomes were successfully obtained in 75 specimens （75 %） using the new nested PCR method,while were only obtained in 15 specimens （15 %） using previously described one-step method. The detection sensitivity of the new method was associated with viral load of specimens and was significantly higher than previous on-step method. In conclusions, the presented new method supplied a sensitive way to complete HBV genome sequencing and could important role in functional and epidemic research for hepatitis B virus.