A PCR Method Based on 16S rRNA Sequence for Simultaneous Detection of the Genus Listeria and the Species Listeria monocytogenes in Food Products

Abstract

The genus Listeria comprises six closely related species, of which only Listeria monocytogenes is a human pathogen. The rapid and sensitive detection of L. monocytogenes is important in the food industry as well as in medical diagnosis. In this study, a PCR-based method for the rapid, specific, and sensitive detection of L. monocytogenes in food products was developed. The PCR is based on DNA sequences and primer pairs that are found within the 16S subunit of the rRNA gene and are specific to the Listeria genus and to L. monocytogenes within the Listeria genus. The primers for the Listeria genus and for L. monocytogenes were used in the same reaction mix for their simultaneous detection. In addition, a pair of bacterial primers universal to any bacterial DNA at the 16S subunit of the rRNA gene were developed as a positive control. For the detection of Listeria and L. monocytogenes in food products, the method includes selective enrichment for Listeria followed by DNA extraction and a specific PCR reaction. The method detects 1 to 5 CFU in a 25-g sample in < or = 24 h. It can be easily incorporated into the routine screening of diverse food products and readily adapted for clinical use.

Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target

“…Some of the assays were quantitative and did not require enrichment but they were generally less sensitive than those including an enrichment step (Koo and Jaykus, 2003;Rodriguez-Lazaro et al, 2004b). Reported sensitivities range from 1000 CFU/ml (Bhagwat, 2003) to 1-5 CFU per 25 ml/g (Somer and Kashi, 2003). The reported real-time PCR assays employed a range of fluorescent detection chemistries including SYBR Green and TaqMan or 5 0 exonuclease technologies.…”

“…Recently conventional and real-time PCR assays have been developed for the detection of L. monocytogenes in foods (Nogva et al, 2000;Hein et al, 2001;Bhagwat, 2003;Koo and Jaykus, 2003;Somer and Kashi, 2003;D'Agostino et al, 2004;Rodriguez-Lazaro et al, 2004b;Wang et al, 2004;Rudi et al, 2005). Many are based on the detection of virulence genes in L. monocytogenes and have been tested in a variety of foods.…”

“…Previous studies have described PCR and real-time PCR assays combined with culture enrichment for the specific identification of L. monocytogenes in foods in a shorter time than can be achieved by standard culture methods alone (Jothikumar et al, 2003;Somer and Kashi, 2003;D'Agostino et al, 2004;Nguyen et al, 2004;Kawasaki et al, 2005;Rudi et al, 2005). In this study, the development of a qualitative real-time PCR assay for the LightCycler employing fluorescence resonance energy transfer (FRET) hybridization probe technology (Wittwer et al, 1997) targeting the ssrA gene of L. monocytogenes and an internal amplification control (IAC) is described.…”

Vibrio cholerae Strain Typing and Phylogeny Study Based on Simple Sequence Repeats

“…High-quality DNA extraction of pure cultures was used throughout the study (30,76). Briefly, a loop full of V. cholerae colonies was washed with SSC buffer (0.15 M NaCl, 15 mM sodium citrate [pH 8.0]).…”

Diagnosis of Listeria monocytogenes Meningoencephalitis by Real-Time PCR for the hly Gene

“…Several molecular methods have been developed in the food industry (12,23,26,37,43), based on the amplification of several specific genes of L. monocytogenes (iap, hly, prfA, actA) (27,33,36,41). Some of these assays are sensitive enough to be valuable for the detection and quantification of L. monocytogenes in the environment and food products (23,27,33,37,41,43). However, the results are highly dependent on the method used, the chosen target to be amplified, and the complexity of the food matrix product.…”