Detection and quantification of the iap gene of Listeria monocytogenes andListeria innocua by a new real-time quantitative PCR assay

A real-time quantitative polymerase chain reaction (PCR) assay for direct d etection and enumeration of Listeria monocytogenes and Listeria innocua was developed and applied to artificially contaminated milk samples. The lap g ene present in both species was used as a target for amplification of a 175 -bp (L. monocytogenes) and a 309-bp (L. innocua) fragment. To ensure that L . monocytogenes and L. innocua are specifically detectable, tests were carr ied out using 42 L. monocytogenes strains and 33 L. innocua strains belongi ng to different serovars. Specificity was also confirmed using 22 bacterial strains not belonging to the genus Listeria, including closely related bac teria. In addition to specificity, the reported assay is characterized by a wide dynamic range of quantification and a high sensitivity, as we could d etect as few as six copies of the iap gene per PCR using purified DNA as te mplate. When applied to direct detection and quantification of L. monocytog enes in milk, the more rapid real-time quantitative PCR assay was as sensit ive as the traditional plate count method, bur real-time quantitative PCR-d erived iap gene copy numbers were one to two logs higher than colony-formin g units obtained by the plate count method, (C) 2001 editions scientifiques et medicales Elsevier SAS.