A single-step polymerase chain reaction for combined gene detection and epidemiological typing of Listeria monocytogenes

Abstract

A variant of the random amplification of polymorphic DNA (RAPD) technique, referred to as the ‘combined gene detection and epidemiological typing’ (COGEDET) method, was employed in the development of a one-step PCR assay for simultaneous identification and typing of Listeria monocytogenes. An arbitrary (RAPD) primer was used in combination with a species specific gene primer set. Four gene primers, HLYA, PRFA, ISP and FL2A, and six RAPD primers were screened for their discriminatory abilities.Amplification conditions were optimized for both PCR techniques to obtain a clear gene-specific band in a background of RAPD generated amplification products, by testing primer concentrations, annealing temperatures and different DNA extraction methods. Efforts to increase the discriminatory power of this modified RAPD by lowering the annealing temperature to 25°C were unsuccessful, as more complex banding profiles were observed with the annealing temperature of 35°C. Additional efforts to enhance the complexity of banding patterns by testing gene primer and RAPD primer concentrations showed that the ISP gene primer/RAPD primer combination of 20 p Mμl−1for each primer was optimal. Three RAPD primers (UBC 155, UBC 156 and Listra) in conjunction with the ISP gene primers provided for species-specific gene detection and discriminatory typing of L. monocytogenes. Overall, the COGEDET-RAPD method appears promising as a sensitive and reproducible method for the combined gene detection and typing of L. monocytogenes.