HBV拉米夫定耐药株及BCP、Pre—C突变株芯片检测方法的应用研究

Construction and appfication of a genechip method for detection of hepatitis B virus lamivudine-resistant mutants and basal core promotor/Pre-C mutants

目的构建针对HBV拉米夫定耐药株及c区启动子（basal core promoter，BCP）、前C区（Pre-C）突变株，多位点突变的基因芯片检测方法。并与DNA测序法比较，以了解该芯片的灵敏度、特异性、稳定性等性能并初步应用于临床实践。方法该基因芯片能对HBVDNA及P区（DNA多聚酶区）：180、204、207三个拉米夫定耐药突变位点；C区启动子（basal core promoter，BCP）及前C区（Pre—c）：nt1896、nt1899、nt1862、nt1764、nt17625个突变热点，共8个HBV突变位点进行检测，并用测序法对该基因芯片进行验证。结果①检测HBVDNA方面，两种方法检测结果100％相符。②检测突变位点方面：总体统计32份阳性血清共有256（32×8）个突变位点。两种方法检测结果有251个突变位点完全相符；5个突变位点不完全相符，基因芯片法检测为混合型，测序法检测为野生型或突变型之一。结论结果提示该基因芯片和DNA测序法检测结果阳性率无差异，特异性与DNA测序法相当，检测混合株有更大优势。

Objective The objective of this research is to constract a clinic-usable genechip method for detection of hepatitis B virus lamivudine-resistant mutants and basal core promotor/Pre-C mutants, compare this method with DNA sequencing to investigate this genechip＇s character （sensity, specificity, stability and practicability in clinic） and apply it in clinic. Methods This genechip detection method can detect the DNA and 8 mutative site of HBV, include 3 lamivudine-resistant mutation site（ No. 180, 204, 207 site in DNA polymerase gene） ,5 HBeAg escape-related mutation site（nt 1896, 1899, 1862, 1764, 1762 site in BCP/Pre-C region）. The results of genechip method was verified by DNA sequncing. Results In detecting HBV DNA, the results of genechip were agree with 100% of the results of DNA sequencing. In detecting HBV mutants, 251 sites （in 32 samples ,256 sites） showed the same results using both methods, and only 5 sites were not completely match（ P 〉 0.05）. In these 5 sites, genechip methods got multi-infection results, but sequencing got single-infection results. Conclusion These results suggest that genechip method has the same positive rate and almost these same specificity with DNA sequencing method, and is better than DNA sequencing method in detecting multi-infected HBV strains.