竞争性RT-PCR法及对大肠杆菌acrA基因mRNA表达水平的定量研究

Quantitative competitive RT-PCR and quantitative detection of Escheriia coli acrA-mRNA

摘要:

建立了用于检测大肠杆菌(Escherichia coli)ATCC25922的acrA基因mRNA表达水平的定量竞争性RT-PCR(QC-RT-PCR)体系。PCR合成目标片段的突变型片段(321bp)作为内标准模板(Internal standard,IS),与目标片段一致的片段(389bp)作为目标模板(Target standard,TS),优化两种模板共扩增体系;梯度稀释IS与等量大肠杆菌cDNA样本共扩增,扫描电泳条带,软件分析数据。结果表明,引物设计合适,以IS和TS为模板实现共扩增,产物(321bp和389bp)通过1.5%琼脂糖凝胶电泳有效分离;梯度稀释IS与cDNA共扩增产物出现亮度梯度电泳条带;获得一元回归曲线y=-0.345+0.097x(相关系数r=0.959,标准差s=0.05997)。该研究成功构建内标准模板,优化的共扩增PCR体系实现了对大肠杆菌ATCC25922中acrA基因mRNA表达水平的检测,具有简便、高效、敏感度高等优点。

Abstract:

A Quantitative detection assay of acrA-mRNA of Escherichia coli ATCC25922 was developed by Quantitative Competitive RT-PCR. Target Standard(TS) which was same as target-templete acrA was amplified by PCR with P1 and P2 as primers. Internal Standard (IS) which was shorter 68bp then target-templete acrA was amplified by temperature-gradient-PCR with P1 and P3P2 as primers, whose annealing temperatures ranged from 55～65℃, and the most suitable annealing temperature was acquired at 56℃. Both TS and IS were largely amplified by PCR as above and extracted to store. Co-amplification with both TS and IS as templetes was optimized by temperature-gradient-PCR with P1 and P2 as primers, whose annealing temperatures were ranged from 55～65℃, and then the most suitable annealing temperature was also acquired at 56℃. Then co-amplification optimized as above was did again but with both cDNA of Escherichia coli (with target-templete acrA-cDNA copies unknown) and IS(10-fold serial dilution, and with IS copies known) as templates. The electrophoresis bands were photographed and analysed with UVIband and each band area was acquired, then linear regression analysis was did with SPSS11.5 and CurveExpert1.3 and a competitive curve was drawn as y=－0.345+0.097x. Results revealed that the two kinds of product electrophoresis bands of co-amplification, whose templates were both 10-fold diluted IS and cDNA, could be distinguished clearly in 1.5% agarose gel because of 68bp discrepancy, and showed lighteness dimming gradually with IS copies 10-fold diluting. With the competitive curve, the copies of acrA-mRNA in sample could be counted accurately and easily.