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 10¹ CFU/ml for P. aeruginosa, 10² CFU/ml for both Salmonella spp. and E. coli O157:H7, and 10³ 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.

中文翻译：

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使用PMA和mPCR结合检测环境水中的沙门氏菌，铜绿假单胞菌，蜡状芽孢杆菌和大肠杆菌O157：H7。

开发了一种具有单叠氮化丙锭（PMA）和内部扩增对照（IAC）的多重聚合酶链反应（mPCR），用于同时检测水性病原体沙门氏菌，铜绿假单胞菌，蜡状芽孢杆菌和大肠杆菌O157：H7。此PMA-IAC-mPCR分析分别基于invA，ecfX，cesB和fliC的基因使用了四种新的特异性引物。一16S rRNA基因选择了IAC引物以消除假阴性结果。光敏染料一叠氮化丙锭（PMA）用于排除死细菌的信号，这些信号可能导致假阳性结果。在纯培养中，铜绿假单胞菌的检出限（LOD）为10 ¹ CFU / ml，沙门氏菌属均为10 ² CFU / ml 。和大肠杆菌O157：H7，蜡样芽胞杆菌分别为10 ³ CFU / ml 。另外，将所有四种细菌的6-8小时富集在掺入水样品基质的混合物中后，沙门氏菌的LOD为3 CFU / ml，大肠杆菌O157为7 CFU / ml ： H7，10 CFU / ml用于蜡状芽孢杆菌和铜绿假单胞菌2 CFU / ml 。该PMA-IAC-mPCR测定具有在水生病原体多重测定中应用的潜力。