Multiplex PCR-Based Assay for Species Identification of Enviromental Strains of Pseudomonas

Indianapolis, IN

Pseudomonas is a genus composed of gram-negative, rod shaped bacteria that inhabit the natural environment where they are often found in soil, water, and leaf litter. A multiplex PCR-based approach was developed for species identification and direct detection of Pseudomonas strains. A selective set of primers using the rpoD sequence was designed for each Pseudomonas strain used. The specificity of each primer set was tested on four different species of Pseudomonas – P. aeruginosa, P. fluorescens, P. putida, and P. syringae. In order to test these strains, bacteria were grown on LANS, and DNA extracted from LBNS overnight cultures. The bacteria were then placed in a multiplex PCR mixture and later visualized using ethidium bromide agarose gel electrophoresis. A PCR gradient was used to determine the correct annealing temperature for the primers. The primer sets for each organism showed specificity for the coinciding organism. With proper optimization, this assay can be used to identify and determine unknown environmental strains of Pseudomonas.