College of Food Science and Nutritional Engineering, China Agricultural University, No. 17 Tsinghua Donglu, Beijing 100083, China.
Appl Microbiol Biotechnol. 2012 Sep;95(6):1579-87. doi: 10.1007/s00253-012-4277-8. Epub 2012 Jul 20.
Pseudomonas aeruginosa is a well-known opportunistic pathogen that can cause acute nosocomial necrotizing pneumonia and genetic disorder cystic fibrosis of lung patients. Pathogenic interactions between P. aeruginosa and hosts are often guided by the secreted virulence determinants that interact with specific host targets. Exotoxin A, pyocyanin, elastase, and type III secretion system are the most significant virulence determinants and cause great concern. However, P. aeruginosa in various environments has high genotypic diversity, leading to deficiency of exotoxin genes for some P. aeruginosa strains. In current study, a universal primer-multiplex PCR method (UP-MPCR) was employed for the detection of five significant enterotoxin genes (toxA, phzM, lasB, ExoU, and ExoS) and one internal control gene ecfX in P. aeruginosa. Owing to the application of universal primer (UP), different targeted products have identical amplified efficiency and the sensitivity of multiplex PCR is improved. In addition, the complexity of multiplex PCR system is reduced and the compatibility of primers in a reaction is greatly increased. This UP-MPCR method can detect the presence of five P. aeruginosa enterotoxin genes in a single assay more rapidly and sensitively than conventional methods. In 214 drinking water and environmental isolates, the ExoU, ExoS, phzM, toxA, and lasB genes were detected in 20 (9 %), 180 (84 %), 179 (84 %), 196 (92 %), and 171 (80 %) isolates, respectively.
铜绿假单胞菌是一种众所周知的机会性病原体，可引起急性医院获得性坏死性肺炎和肺囊性纤维化患者的遗传紊乱。铜绿假单胞菌与宿主之间的致病相互作用通常由与特定宿主靶标相互作用的分泌毒力决定因素指导。外毒素 A、绿脓菌素、弹性蛋白酶和 III 型分泌系统是最重要的毒力决定因素，引起了极大的关注。然而，不同环境中的铜绿假单胞菌具有高度的基因型多样性，导致一些铜绿假单胞菌菌株缺乏外毒素基因。在当前的研究中，采用通用引物-多重 PCR 方法（UP-MPCR）检测铜绿假单胞菌中的五个重要肠毒素基因（toxA、phzM、lasB、ExoU 和 ExoS）和一个内部对照基因 ecfX。由于应用了通用引物（UP），不同靶向产物具有相同的扩增效率，并且提高了多重 PCR 的灵敏度。此外，还降低了多重 PCR 系统的复杂性，大大增加了反应中引物的兼容性。与传统方法相比，这种 UP-MPCR 方法可以更快、更灵敏地检测单个试验中五种铜绿假单胞菌肠毒素基因的存在。在 214 份饮用水和环境分离物中，检测到 20 份（9%）、180 份（84%）、179 份（84%）、196 份（92%）和 171 份（80%）分离物中存在 ExoU、ExoS、phzM、toxA 和 lasB 基因。
