Multiplex PCR identification of the major Pseudomonas aeruginosa serogroups using specific novel target genes

Abstract

Accurate and efficient identifying Pseudomonas aeruginosa (PA) serotypes is critical for epidemiological monitoring. A multiplex PCR (mPCR) assay was established for rapid identification of major PA serogroups in water. We mined 26 specific target genes of major PA serotypes using pan-genome analysis. Verification by PCR revealed 5 novel target genes: ATCC33349_04445 and ATCC33349_04455 as specific for polyvalent serogroup II, PABCH01_04375 for polyvalent serogroup III, and E6130952_04742 and E6130952_03632 for monovalent serogroup E. Minimum DNA detection limits for primer sets ATCC33349_04445, ATCC33349_04455, PABCH01_04375, E6130952_04742, and E6130952_03632 were 5.9 pg/μL, 5.9 pg/μL, 654 fg/μL, 58 fg/μL, and 58 pg/μL, respectively. Even if the non-target analytes cell concentration reached 109 CFU/mL, the PCR methods had a greater anti-interference ability. A novel 3-mPCR assay was established based on these target genes. Primers targeting PAII-2 (273 bp), PA-III (847 bp), and PAE-2 (100 bp) specific to polyvalent serogroups II and III and monovalent serogroup E were designed. The detection limit was ∼104 CFU/mL for pure cultures and 105 CFU/mL for artificial water samples. mPCR correctly detected the target PA in water samples. These results suggest that novel target genes can help in distinguishing PA serogroups. Our mPCR assay can rapidly and accurately detect PA in aquatic environments.