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模板指导的末端碱基掺入反应结合荧光偏振检测技术(templatedirectdye-terminatorincorporationwithfluorescence-polarization,TDI-FP)是SNP检测新技术.应用TDI-FP方法分析中国陕西HPV16阳性宫颈组织HPV16E7基因第647位核苷酸A→G热点突变(即A647G),首先在HPV16阳性的91例宫颈癌及49例正常/宫颈炎妇女宫颈DNA标本中,PCR扩增含647位点在内的HPV16E7部分基因,然后将紧邻647位点5′端的寡核苷酸探针与PCR产物内的模板杂交,并延伸一个与647位点碱基互补的荧光标记碱基:TAMRA-ddTTP或R110-ddCTP.用荧光偏振仪读取荧光偏振(FP)值,根据升高的相应FP值判断647位点碱基.结果表明,宫颈组织HPV16E7A647G的总体检出率为35.71%(50/140).宫颈癌组的A→G突变率为42.86%(39/91),显著高于正常/宫颈炎组22.45%(11/49)的突变率(x2=5.778,P=0.016),两组间的OR值为2.59(95%CI=1.17￣5.71).提示TDI-FP可用于HPV有意义点突变的分析;我国陕西地区妇女HPV16A647G突变率及其对宫颈癌的警示性与其他地区相比有明显差异,该地区携带此突变病毒株的妇女患宫颈癌的风险可能较高.
TDI-FP is a novel technique for SNP detection using template-directed end incorporation reaction.And 647th nucleoside of HPV16E7 gene in Shaanxi Province of China with TDI-FP method Acid A → G hot spot mutation (A647G), first in HPV16-positive in 91 cases of cervical cancer and 49 cases of normal / cervicitis women cervical DNA samples, PCR amplification of 647 sites including HPV16E7 part of the gene, and then immediately The oligonucleotide probe at the 5 ’end of the 647 site hybridizes to the template within the PCR product and extends a fluorescently labeled base that is base complementary to the 647 site: TAMRA-ddTTP or R110-ddCTP Read with a Fluorescence Polarimeter Fluorescence polarization (FP) values of 647 base pairs were determined based on the corresponding FP value.Results showed that the overall detection rate of HPV16E7A647G in cervical tissues was 35.71% (50/140) .The A → G mutation rate Was 42.86% (39/91), which was significantly higher than that of the normal / cervicitis group (22.45%, 11/49) (x2 = 5.778, P = 0.016) 1.17 ~ 5.71) .This indicated that TDI-FP could be used to analyze the point mutation of HPV. In Shaanxi province, women H The PV16A647G mutation rate and its alertness to cervical cancer are significantly different from those in other regions where women with this mutant strain may have a higher risk of developing cervical cancer.