基因芯片技术检测乙型肝炎病毒基因型

Detection of hepatitis B virus genotype by gene chip

目的建立乙型肝炎病毒(HBV)基因分型检测芯片方法,并检测其特异性、敏感性和稳定性。方法收集HBV各基因型序列,并对比分析后,设计7支探针,制成HBV基因型检测芯片;对芯片检测的实验条件进行探索,分析其检测特性。结果实验条件优化为杂交温度39℃,试验统计结果判定为荧光信号软件输出值在30以上为阳性,30以下为阴性。正常对照组的健康人和非HBV的常见病毒芯片检测结果均为阴性,实际应用检测30份HBV DNA阳性临床留取的样本,结果 19份为C型,11份为B型,抽查2份标本测序观察,结果与芯片检测结果完全一致。用HBV基因B型、C型标准品考察,检测符合率为100%,芯片检测的最低病毒拷贝数为104拷贝/ml,芯片检测的总变异度为9%、芯片内变异度为2%,芯片能同时检测不同基因型的重叠感染。用加速试验评价芯片的稳定性,结果检测荧光信号值有所下降,但下降后的信号值未低于检测临界值30,试剂盒性能较为稳定。结论使用HBV基因型检测芯片进行HBV基因分型操作简便、敏感性高、特异性好、方法可靠。

Objective To set up a method based on gene chip for determination of HBV genotype.Methods Seven gene fragments were screened from GenBank as probes dotted on chips to make HBV genotyping gene chips,many examinatorial experiments were done to evaluate the quality of this gene chip.Results Hybridization temperature was defined at 39℃ based on experiments,distinguished the test result by statistic software：fluorescence signal value above 30 was positive,or was negative.HBV-DNA negative serum samples and containing ordinary virus except HBV serum samples were tested by this chip,all of them was negative.The results of determination of 30 clinical HBV-DNA positive serum samples showed that 19 cases were type C,11 cases type B.The results of equence analysis of 2 cases among them was in accordance with that tested by gene chips.Standard serum of type B and type C were used to examine the gene chip： the result of standard serum test showed a coincidence rate of 100%,and the gene chip could test the minimum virus quantity was 104 copy/ml,gene chip had the total CV 9% and work function character for all tests was stable.Conclusions HBV genotyping gene chip was sensitive and specific for HBV genotype determination,it can be used as a highly efficient,accurate method for determination of HBV genotype.