分级定量PCR检测血清HBV DNA

Grading quantitative PCR to measure serum HBV DNA levels

目的利用竞争ＰＣＲ法建立分级测定ＨＢＶＤＮＡ含量的定量方法．方法设立３个标准竞争模板（１０２ｃｏｐ，１０４ｃｏｐ，１０６ｃｏｐ），分别和恒量的待测模板混合，分别进行４０，３０，２０次循环的ＰＣＲ扩增，最后凝胶电泳分离待测模板和竞争模板的ＰＣＲ产物，测定两者的荧光强度的比值，计算待测模板的初始量．结果对乙型肝炎血清ＨＢＶＤＮＡ定量表明，１２份ＨＢｅＡｇ阳性血清，ＨＢＶＤＮＡ的血清浓度在６×１０７～１×１０１１ｃｏｐ／Ｌ，而１２份ＨＢｅＡｂ阳性血清只有７份可检出ＨＢＶＤＮＡ，它们的血清浓度均在３×１０８ｃｏｐ／Ｌ以下，其余５份用该ＰＣＲ方法，检测不到．

AIM To set up a new method of measuring serum HBV DNA levels with grading quantitative PCR. METHODS Three standard competition DNA templates (10 2 cop, 10 4 cop, 10 6 cop) were mixed separately with stable sample DNA templates, and then PCR with 40, 30 and 20 cycles was carried out. Then the PCR products of the sample DNA templates were separated from that of the competition DNA complates, and the fluorescence intensity ratio of them were measured. The initial contents of the sample DNA templates were calculated. RESULTS The serum HBV DNA contents of the 12 samples of HBeAg positive serum ranged from 6×10 7 to 1×10 11 cop/L . Only 7 of the 12 HBeAb positive serum could be detected and their HBV DNA were all below 3×10 8 cop/L and the other 5 samples HBV DNA could not be detected with PCR. CONCLUSION This method can be used to measure the HBV DNA and other pathogenic DNA.