采用竞争性定量PCR检测幽门螺杆菌cagA基因

Quantitative detection of the cagA of Helicobacter pylori by competitive PCR

目的建立竞争性定量 PCR 检测幽门螺杆菌 cagA 基因的方法。方法以重组 PCR 将乳糖操纵子中 Lac 阻抑蛋白特异性结合序列(21bp)重组入 cagA 基因397bp 的片段中构建内参标模援(rfcagA)。体外克隆并表达具有双功能的 GST-LacI 融合蛋白,其一端只有 GST 酶活性,另一端可特异性地与重组内参标模板结合。在定量 PCR 中,以 rfcagA 为内参标模板,pMC3 或 Hp 基因组 cagA 作为竞争性模板,PCR 产物的5′-端标记了生物素,可与包被了亲和素的微孔板结合,只有 rfcagA 的 PCR 产物可与融合蛋白结合而显色。结果 GST 底物的显色程度与样本的 ca-gA 含量呈负相关,当反应体系中的起始模板量为10-10~5拷贝,循环次数小于等于20次。原始模板数以指数方式增长,并建立了原始模板的考贝数与 A_(340)值同的标准曲线。用该方法检测12份已知菌株中的 cagA 含量,8份 cagA 阳性,cagA 基因的拷贝数为6.3×10^(10)-2.14×10^(11)/L,特异性为100%,敏感度达可检出 cagA 基因2倍的差别。结论这是一个特异、敏感及很实用的定量检测 cagA 基因的方法。

Objective To establish a quantitative PCR(QPCR)for cagA+Hp using internal standard.Method The lac re- pressor specific combining sequence(the lac operator,21bp)was inserted into the cagA gene fragment(fcagA,397bp)by overlaping extention PCR,and the recombanant was subsequently cloned into vector pUC19 to construct pUC19/rfcagA. By cloning lacI repressor gene into the expression vector(pGEX-4T-3),transforming the E.coli(JM109)with the plasmid pGEX-4T-3/lacI,and inducing the bacterium with IPTG,we got the fusion protein(GST-lacI)Activity tests showed that GST had an activity of 105IU/g,and the LacI could conjugate to rfcagA.In the competitive PCR,rfcagA was used as the internal standard template,pMC3 that contained most part of cagA from the 5'-end or Hp cagA was used as the competi- tive one.Since one primer of the PCR was labelled with biothin at the 5'-end,the PCR products could conjugate to the microplate wall that coated with avidin.When the GST-LacI was added to the walls,only the products which contain the lac operator sequence could combine with it,and the GST could catalyze its substrates(CDNB and GSH)to form a sub- stance that had the most ultraviolet(340nm)absorbance.Results when the copies of the internal standard was fixed,a standard curve of A340 against the logarithmic value of the competitive templates was established.So the copies of the ca- gA gene in a sample could be determined according to the A340 value and the standard curve.Using this method,we found 8 out of the 12 bacteria cultures were positive with cagA gene copies from 6.3×1010 to 2.14×1011/L.The coin- cident rate was 100%.It could also identify the copies of cagA gene with two times difference.Conclusion This is a spe- cific,sensitive and applicable method for quantitative detetion of cagA gone.