竞争性定量聚合酶链反应检测幽门螺杆菌cagA基因

Quantitative detection of cagA of helicobacter pylori by competitive PCR

目的 建立竞争性聚合酶链反应 (PCR)定量检测幽门螺杆菌 (Hp)细胞毒素相关蛋白基因A(cagA)的方法。方法 以重组PCR将乳糖操纵子中乳糖调节蛋白 (LacI)的特异性结合序列 (2 1bp)插入cagA基因的一段序列 (40 0bp)中得到重组cagA基因片段 (rfcagA)。体外克隆并表达谷胱甘肽转硫酶 LacI(GST LacI)的融合蛋白 ,其一端具有GST活性 ,另一端可特异性地与rfcagA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cagA含量呈负相关 ,当反应体系中的起始模板量为 10～ 10 5拷贝 ,循环次数小于或等于 2 0次 ,原始模板数以指数方式增长 ,并建立了原始模板的对数与A3 40 值间的标准曲线。用该方法检测 14份已知菌液中的cagA ,9份阳性 ,5份阴性 ,符合实际情况 ;cagA的拷贝数为 6 3× 10 10 ～ 2 14× 10 11/L。结论 这是一项特异、敏感及实用的定量检测cagA基因的技术。

Objective To establish a method for quantitative detection of cytotoxin associated gene A (cagA) positive Helicobacter pylori (cagA + Hp) by competitive polymerase chain reaction. Methods The lactose regulatory protein (LacI) specific combining sequence (the lactose operator, 21 bp) was inserted into the cagA fragment (fcagA, 400 bp) by overlapping extension PCR. By cloning lactose regulatory gene (lacI) into pGEX 4T 3, transforming E.coli JM109 with pGEX 4T 3/lacI, and inducing the bacterium with isopropyl β D thiogalactopyranoside (IPTG), we got the active fusion protein of glutathione s transferase (GST) and LacI. In the competitive PCR, rfcagA was used as the internal standard template and pMC3 that contained most part of cagA from the 5′ end or Hp cagA as the competitive one. Since one primer of the PCR was labelled with biotin 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 containing the lactose operator sequence could combine with it, and the GST could catalyze 1 chloro 2,4 dinitrobenzene and glutathione to form a substance that had the maximal ultraviolet(340nm) absorbance. Results When the copies of the internal standard was fixed (100 copies), a standard curve of A 340 against the logarithmic value of the competitive templates was established. Using this method, we found that, among the 14 bacteria cultural media, 9 were cagA positive with the copies from 6.3×10 10 /L to 2.14× 10 11 /L. The coincidentce rate was 100%. Conclusion This is a specific,sensitive and applicable method for quantitative detection of cagA.