铜绿假单胞菌荧光实时定量PCR标准品的构建

Construction of Reference Standards for Detecting Pseudomonas aeruginosa with Fluorescence Real-time Quantitative PCR

构建了荧光实时定量PCR标准品以检测水中的铜绿假单胞菌。利用所报道的铜绿假单胞菌gyrA基因为目的基因设计引物,通过培养细胞,煮沸法裂解细胞后做PCR,电泳,胶回收,然后与pMD19-TVector连接并转化到感受态大肠杆菌中;氨苄青霉素筛选出白色菌落,菌落PCR及测序鉴定其特异性,根据OD值确定浓度,制备梯度浓度参考标准品,制作标准曲线,检测水样品中铜绿假单胞菌的菌量。

The reference standards were constructed for detecting Pseudomonas aeruginosa in water with Fluorescence Real-time Quantitative PCR. Primers for specific PCR amplification were designed based on the published sequence of the gyrA gene of P. aeruginosa. The following procedures were taken,including cell culture, PCR, agarose gel eleetrophoresis and gel extraction. The purified DNA product was then linked with pMD19-T Vector to construct recombined plasmids and transformed into competent Escherichia coli. Target plasmids in white colonies selected by arnpicillin screening were identified by colony PCR and DNA sequencing. The concentration was identified by OD value and the copy numbers were calculated by value of OD in A260. The purified plasmid DNA was diluted into serial gradient concentrations as standard to plot a standard curve for Fluorescence Real-time Quantitative PCR to predict the load of P. aeruginosa contaminant in water.