Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR

Abstract

a b s t r a c tA rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24 h incubation in half-Fraser broth, 4 h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1-5 CFU/ 25 g food sample and can be performed in 2 working days compared to up to 7 days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n ¼ 175) and controls (n ¼ 31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food.

Real-Time PCR Assay To Differentiate Listeriolysin S-Positive and -Negative Strains ofListeria monocytogenes

“…To address the extensive labor and time required for the detection and identification of L. monocytogenes by conventional means, a number of rapid and highly sophisticated realtime PCR-based technologies have been developed (4,44,48,50,51). More specifically, such technologies are attractive due to the fact that culture-based methods, which typically involve selective enrichment, culturing on selective media, and a battery of biochemical tests and serology, can take from 5 to 10 FIG.…”

Development and validation of a rapid real-time PCR based method for the specific detection of Salmonella on fresh meat

“…Conserved regions at the extremities flank divergent sequences, making the gene an ideal target for nucleic acid diagnostics (O'Grady et al, 2008). The ssrA gene has been demonstrated as a suitable diagnostic target for the detection of Listeria monocytogenes in enriched food samples (O'Grady et al, 2008(O'Grady et al, , 2009). There was limited heterogeneity in the ssrA gene between genera of the Enterobacteriaceae family, making specific assay design for Salmonella challenging.…”

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

“…In some cases, higher amounts of microorganisms are measured with qPCR analyses than with classical agar counts, which may be explained by the fact that DNA from dead cells can also be amplified. In order to lower the detection levels of pathogens, it is possible to perform culture enrichment of the food samples before qPCR (Rossmanith et al, 2006;Chiang et al, 2007;Karns et al, 2007;O'Grady et al, 2009;Omiccioli et al, 2009 Table 1. Examples of applications of qPCR for the quantification or detection of microbial populations in dairy products.…”