USE OF A FLUOROGENIC PROBE IN A PCR-BASED ASSAY FOR THE DETECTION OF LISTERIA-MONOCYTOGENES

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the t arget has been formatted, The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Tag DNA pol ymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, t he two fluorophores interact such that the emission of the reporter dy e is quenched, During amplification, the probe is hydrolyzed, relievin g the quenching of the reporter and resulting in an increase in its fl uorescence intensity, This change in reporter dye fluorescence is quan titative for the amount of PCR product and, under appropriate conditio ns, for the amount of template, We have applied the fluorogenic 5' nuc lease PCR assay to detect L. monocytogenes, using an 858-bp amplicon o f hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay co mponents and cycling parameters. With crude cell lysates, the total as say could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10( 5) CFU.