Premium
Real‐time PCR detection of L isteria monocytogenes in infant formula and lettuce following macrophage‐based isolation and enrichment
Author(s) -
Day J.B.,
Basavanna U.
Publication year - 2015
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12674
Subject(s) - listeria monocytogenes , biology , microbiology and biotechnology , infant formula , listeria , isolation (microbiology) , detection limit , macrophage , food microbiology , food science , chromatography , bacteria , chemistry , biochemistry , in vitro , genetics
Abstract Aims To develop a rapid detection procedure for L isteria monocytogenes in infant formula and lettuce using a macrophage‐based enrichment protocol and real‐time PCR . Methods and Results A macrophage cell culture system was employed for the isolation and enrichment of L. monocytogenes from infant formula and lettuce for subsequent identification using real‐time PCR . Macrophage monolayers were exposed to infant formula and lettuce contaminated with a serial dilution series of L. monocytogenes . As few as approx. 10 CFU ml −1 or g −1 of L. monocytogenes were detected in infant formula and lettuce after 16 h postinfection by real‐time PCR . Internal positive PCR controls were utilized to eliminate the possibility of false‐negative results. Co‐inoculation with Listeria innocua did not reduce the L. monocytogenes detection sensitivity. Intracellular L. monocytogenes could also be isolated on Listeria selective media from infected macrophage lysates for subsequent confirmation. Conclusions The detection method is highly sensitive and specific for L. monocytogenes in infant formula and lettuce and establishes a rapid identification time of 20 and 48 h for presumptive and confirmatory identification, respectively. Significance and Impact of the Study The method is a promising alternative to many currently used q‐ PCR detection methods which employ traditional selective media for enrichment of contaminated food samples. Macrophage enrichment of L. monocytogenes eliminates PCR inhibitory food elements and contaminating food microflora which produce cleaner samples that increase the rapidity and sensitivity of detection.