Identification of commensal Pseudomonas aeruginosa isolates using duplex PCR targeting the oprL and algD genes

Abstract

Background: Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous bacterium that can be found in most moisture places, such as soil, water, food, plants, and animals, including humans. Due to genetic flexibility among strains, there is no standard molecular identification for P. aeruginosa from different sources. In this study, monoplex and duplex PCR targeting oprL, algD and nfxB were assessed for the identification of commensal P. aeruginosa. Methods: Twenty-nine commensal Pseudomonas isolates, including 16 P. aeruginosa isolates and 13 P. aeruginosa-like isolates, were used in the study. First, monoplex PCR targeting oprL, algD and nfxB using published primers was carried out to test their ability to detect commensal Pseudomonas isolates. Then, two new primer pairs targeting oprL were designed (oprL-pp1 and oprL-pp2) and used for oprL-algD duplex PCR to check for the improvement of commensal P. aeruginosa detection. Result and conclusion: AlgD or nfxB monoplex PCR had the same sensitivity of 93.75% and specificity of 100%, while oprL PCR using published primers was more sensitive (100%) but less specific (0%). Duplex PCR yielded high sensitivity and specificity in detecting P. aeruginosa. Both oprL-pp1/algD and oprL-pp2/algD duplex-PCR had 93.75% sensitivity (15/16 P. aeruginosa isolates) and 100% specificity (0/13 P. aeruginosa-like isolates). In addition, oprL-pp2 primers were more specific than oprL-pp1 primers, with only 2 of 13 P. aeruginosa-like isolates detected, while oprL-pp1 primers detected all P. aeruginosa-like isolates. Compared to the monoplex PCR that only targeted the oprL gene, the duplex PCR utilizing oprL-pp2 and algD primers identified 15/16 P. aeruginosa isolates (93.75% specificity). Additionally, the duplex PCR used in this study was negative for non-Pseudomonas species, including E. coli, V. cholera, V. parahaemolyticus, and S. aureus. In conclusion, our duplex PCR targeting oprL and algD could be a valuable tool for commensal P. aeruginosa screening.