摘要
目的 建立简便的HBVDNA前C区nt1896位点突变的检测方法。方法 采用终点终止法和偏振光检测技术进行点突变的检测。首先对HBVDNA前C区基因进行PCR扩增 ,然后用特异探针与扩增产物中待测核苷酸的下游序列杂交 ,使探针的 3’端可以在DNA聚合酶的作用下 ,依据其互补链上的待测核苷酸连接上一个标有特定荧光素的ddNTP ,然后检测该 3’端带有荧光素探针的偏振光值 ,根据检测得到的荧光素种类及其偏振光的强度可以判定待测点是何种核苷酸。结果 该方法可以检测出HBV基因序列中nt1896的核苷酸类型 ,最低可检出的突变模板量为 1× 10 1拷贝。结论 该技术可以对血清中HBVDNA前C区nt1896位点突变以及其他基因突变进行检测。
Objective To establishe a simple method for detecting the precore mutant HBV (nt1896:G>A). Methods FP-TDI standing for template directed dye terminator incorporation assay with detection by fluorescene polarization(FP) was used. The primary procedure: HBV DNA preC gene was amplified by PCR, the products of PCR were hybridized with probe which is located at the upper steam behind the interesting site and a fluorescence-labeled ddNTP is incorporated into its 3'end, then the fluorescence of probe can be detected by FP since the fluorescein linked to a large molecule has the character of FP. Results The mutant (nt1896:G>A) of HBV DNA precore gene can be detected by FP-TDI. The mutant templates can be detected at least 1×10 1 copy. 125 which is HBV DNA positive serum samples were tested, 53(82.4%) of them were the mutants of HBV DNA precore gene. Conclusions FP-TDI can be used to detect the mutation (nt1896:G>A) of HBV DNA precore gene in serum and also can be used to detect other gene mutation.
出处
《上海医学检验杂志》
北大核心
2003年第6期347-349,共3页
Shanghai Journal of Medical Laboratory Sciences
