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Published on: September 5, 2013
A universal primer multiplex PCR method for typing of toxinogenic Pseudomonas aeruginosa
Hui Shi1, Quoclinh Trinh, Wentao Xu
1College of Food Science and Nutritional Engineering, China Agricultural University, No. 17 Tsinghua Donglu, Beijing 100083, China.
Abstract:
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.
Insights
A new universal primer-multiplex PCR method rapidly detects five key Pseudomonas aeruginosa virulence genes. This approach enhances sensitivity and efficiency for identifying pathogenic strains in environmental samples.
Area of Science:
- Microbiology
- Molecular Biology
- Environmental Science
Background:
- Pseudomonas aeruginosa is an opportunistic pathogen causing severe lung infections, including cystic fibrosis complications.
- Virulence factors like Exotoxin A, pyocyanin, elastase, and type III secretion system are crucial for P. aeruginosa pathogenicity.
- Genotypic diversity in P. aeruginosa strains can lead to the absence of specific exotoxin genes, complicating detection.
Purpose of the Study:
- To develop and validate a universal primer-multiplex PCR (UP-MPCR) method for simultaneous detection of five significant enterotoxin genes (toxA, phzM, lasB, ExoU, ExoS) in P. aeruginosa.
- To improve the speed, sensitivity, and efficiency of detecting P. aeruginosa virulence genes compared to conventional methods.
- To assess the prevalence of these virulence genes in environmental and drinking water isolates.
Main Methods:
- A universal primer-multiplex PCR (UP-MPCR) assay was designed to target five enterotoxin genes and one internal control gene (ecfX) in P. aeruginosa.
- The UP-MPCR method utilizes universal primers to ensure identical amplification efficiency for all targeted products.
- The assay was applied to 214 drinking water and environmental isolates of P. aeruginosa.
Main Results:
- The UP-MPCR method demonstrated increased sensitivity and reduced complexity compared to traditional multiplex PCR.
- Prevalence rates in 214 isolates were: toxA (92%), phzM (84%), lasB (80%), ExoU (9%), and ExoS (84%).
- The study successfully detected the presence of multiple P. aeruginosa enterotoxin genes in a single assay.
Conclusions:
- The developed UP-MPCR method offers a rapid, sensitive, and efficient tool for detecting key P. aeruginosa virulence genes.
- This method facilitates the identification of pathogenic P. aeruginosa strains in environmental settings.
- The high prevalence of certain virulence genes highlights the potential public health concern associated with environmental P. aeruginosa.
