定量检测Hp cagA基因内参标模板的构建及测序

Construction and sequencing of an internal standard template for quantitative PCR detection of cagA Hp

目的 构建竞争性定量PCR检测幽门螺杆菌(Hp)cagA基因的内参标模板并做序列测定。 方法 根据cagA基因及Lac Ⅰ蛋白的特异性结合部位的序列,设计二对PCR引物(cagAp1,cagAp2,cagAp3,cagAp4)。其中cagAp1及cagAp3用于扩增cagA基因2593～2788位碱基间196bp的片段,cagAp2及cagAp4用于扩增2789～2993位碱基之间204bp的核酸片段;在cagAp1的5′-端引入了BamHⅠ的限制性酶切位点,在cagAp2的5′-端结合了Sal Ⅰ的限制性酶切点。在cagAp3的5′-端引入Lac Ⅰ蛋白的特异性结合序列(21bp),在cagAp4的5′-端结合了cagAp3之5′-端21个碱基的反义DNA序列。利用重组PCR,将乳糖操纵子中Lac Ⅰ蛋白的特异性结合序列重组入cagA基因400Lp的片段中,得到cagA基因片段的重组体(rfcagA)。将重组rfcagA定向克隆入pUC19进行序列测定。 结果 用DNA序列分析仪双向测定rfcagA的序列,其含400bp的cagA片段及Lac Ⅰ蛋白特异性结合部位的序列(21bp),一级结构完全正确。 结论 内参标模板rfcagA构建,对竞争性定量PCR检测Hp cagA基因的方法的建立具有重要价值。

AIM To construct an internal standard template for quantitative PCR detection of cagA+ Hp. METHODS Two pairs of oligonucleotide primers were designed and synthesized according to the sequences of cagA and lac operator. The primers were cagAp1, cagAp2, cagAp3 and cagAp4. The cagAp1 and cagAp3 were used to amplify a 196 bp cagA fragment from positions 2593 to 2788, and the cagAp2 and cagAp4 to amplify a 204 bp cagA fragment from positions 2789 to 2992. At the 5'-end of cagAp1 or cagAp2, there was a restriction endo-nuclease site (BamH Ⅰ or Sal Ⅰ), which was favorable for the cloning of the recombinant PCR products. At the 5'-end of cagAp3 or cagAp4, there was the lac repressor specific combining sequences (21 bp) or its antisense sequences. By overlapping extension PCR, the lac repressor specific combining sequences were inserted into the cagA gene fragment (fcagA. 400 bp) and the recombinant (rfcagA) was subsequently cloned into pUC19 to generate pUC19/ rfcagA for sequencing.RESULTS Sequencing from both ends showed that rfcagA did contain the 400 bp cagA gene fragment and lac repressor specific combining sequences (21 bp). CONCLUSION The construction of an internal standard template (rfcagA) has a high value for quantitative PCR detection of cagA+ Hp.