State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, P. R. China.
Zystein, LLC., Fayetteville, Arkansas, 72703, USA.
J Microbiol. 2020 Aug;58(8):668-674. doi: 10.1007/s12275-020-0084-6. Epub 2020 Jun 25.
A multiplex polymerase chain reaction (mPCR) with propidium monoazide (PMA) and internal amplification control (IAC) for the simultaneous detection of waterborne pathogens Salmonella spp., Pseudomonas aeruginosa, Bacillus cereus, and Escherichia coli O157:H7, was developed. This PMA-IAC-mPCR assay used four new specific primers based on the genes for invA, ecfX, cesB, and fliC, respectively. A 16S rRNA primer was chosen for IAC to eliminate false negative results. The photosensitive dye, propidium monoazide (PMA) was used to exclude signals from dead bacteria that could lead to false positive results. In pure culture, the limits of detection (LOD) were 10 CFU/ml for P. aeruginosa, 10 CFU/ml for both Salmonella spp. and E. coli O157:H7, and 10 CFU/ml for B. cereus, respectively. In addition, with a 6-8 h enrichment of all four bacteria that were combined in a mixture that was spiked in water sample matrix, the LOD was 3 CFU/ml for Salmonella spp., 7 CFU/ml for E. coli O157:H7, 10 CFU/ml for B. cereus and 2 CFU/ml for P. aeruginosa. This PMA-IAC-mPCR assay holds potential for application in the multiplex assay of waterborne pathogens.
建立了一种基于聚合酶链反应（PCR）的多重探针实时荧光定量检测方法，用于同时检测水中的病原菌沙门氏菌、铜绿假单胞菌、蜡样芽孢杆菌和大肠杆菌 O157:H7。该方法使用了四个新的特异性引物，分别针对 invA、ecfX、cesB 和 fliC 基因。16S rRNA 引物被选为内部扩增对照（IAC），以消除可能导致假阴性结果的死菌信号。光敏染料吖啶橙（PMA）被用于排除可能导致假阳性结果的死菌信号。在纯培养物中，铜绿假单胞菌、沙门氏菌和大肠杆菌 O157:H7 的检测限（LOD）分别为 10 CFU/ml、10 CFU/ml 和 10 CFU/ml，蜡样芽孢杆菌的检测限为 10 CFU/ml。此外，在将这四种细菌混合并在水样基质中进行 6-8 小时富集后，沙门氏菌、大肠杆菌 O157:H7、蜡样芽孢杆菌和铜绿假单胞菌的 LOD 分别为 3 CFU/ml、7 CFU/ml、10 CFU/ml 和 2 CFU/ml。这种 PMA-IAC-mPCR 检测方法具有应用于水中病原菌多重检测的潜力。
