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改变退火温度对甲基特异性PCR检验指标有效性的影响

The influence on clinical values by optimizing the annealing-temperature of methylation specific PCR
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摘要 目的探讨政变退火温度对甲基特异性聚合酶链反应PCR检验指标有效性的影响。方法将经测序证实的RASSF1A甲基化标本分别作为完全甲基化(100%M)和完全末甲基化的标本(0%M),按1:3体积比例混合,作梯度PCR扩增,摸索适当的退火温度。选取单一电泳条带的温度为优化的退火温度,并以这些温度检测70例标本[35对,以同一患者的肿瘤组织(K)、癌旁组织(N)为1对],比较其在各温度情况时的扩增效果。结果以上指标依年龄(70岁以L/70岁以下)不同出现不同的变化趋热;似然比分析数值均介于0.1—10之间;70岁以下组阳性预测值大多〉70岁以上组;若定义64℃以上温度为高温段,63.3℃以下温度为低温段,则低温段70岁以下组阴性预测值〉70岁以上组,高温段70岁以卜组〉70岁以下组。结论与普通PCR类似,甲基特异性PCR可以通过调接退火温度来改变其俭测效能,但温度变化间隔最好不要〈1℃;应用甲基特异性PCR检测RASSF1A甲基化可以作为膀胱肿瘤早期诊断的辅助方法,在较低年龄组的应用价值高于高年龄组,尤其是在排除肿瘤方面。 Objective To investigate the influence of the effectiveness for methylation specific polymerase chain reaction (PCR) by optimizing its annealing-temperature,so as to improve its resuhs better coincidence to clinical diagnosis. Methods A standard sample of RASSF1A methylation identified by sequence was set as following:firstly,prepare fully methylated sample authenticated by bisulfite-sequence (100% M), and the fully unmethylated sample(0% M); Secondly,mix them with volume ratio 1 : 3 ;Then run gradient PCR with the range of lower 5℃ and upper 5℃ just to the reference annealing temperature used by former research articles. Choose the annealing-temperatures that only occur one special desired bound after electrophoresis. After this,use these temperatures as optiomal annealing-temperatures to run methylation specific PCR in all samples. Finally,compare their sensitivities, specificities, predictive values and likelihood ratios. Results 70 bladder cancer tissue samples with normal (N) and tumor (K) in couple were detected,which 36 (18 couples) were under 70 years old (group A) and 34 (17 couples) were above 70 (group B). After analysis with the mentioned index, we couht see:the indexes changed with ages;the values of index of likelihood ratios were between 0.1-10;the positive predictive values,group B 〉 group A;when 64℃ was defined upper annealing-temperature (UAT) and 63.3℃ as lower annealing-temperature (LAT) ,group B 〉 group A in LAT, group A 〉 group B in UAT. Conclusions As normal PCR,the ehange range of methylation specific PCR should not more than 1℃. There is no evidence that we can deteet bladder cancer only by methylation specific PCR,but it still shows enough value in the group of youn- ger age,especially in the diagnosis for eliminating cancer.
出处 《检验医学》 CAS 北大核心 2006年第6期629-632,共4页 Laboratory Medicine
关键词 DNA甲基化 聚合酶链反应 甲基特异性 预测值 似然比 DNA methylation Polymerase chain reaction Methylation specific Predictive value Likelihood ratios
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参考文献6

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二级参考文献15

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