title: Construction and evaluation of a microbiological positive process internal control for PCR-based examination of food samples for Listeria monocytogenes and Salmonella enterica.
abstract: PCR assays for food-borne pathogens are widely available but have had more limited application to food testing compared with their use in clinical laboratories. When testing food samples, false negative PCR results can occur and may be due to interference with target-cell lysis necessary for nucleic acid extraction, nucleic acid degradation and/or direct inhibition of the PCR. Therefore, it is essential to include appropriate controls for the application of PCR to the detection of pathogens in food samples. The purpose of this study was to develop and evaluate a novel internal control (IC), capable of monitoring the complete detection procedure, from DNA extraction through to amplification and detection. A 'positive process' IC was developed for the reliable application of real-time PCR assays for the detection of Salmonella enterica or Listeria monocytogenes in enrichment broths. Two novel real-time 5' nuclease PCR assays for the detection of a 77 bp fragment of the green fluorescent protein (gfp) gene and a 91 bp fragment of the iroB gene of S. enterica were developed. These assays were specific and had detection limits of 5+/-0.88 and 15+/-1.03 CFU per PCR for the gfp and iroB genes respectively. The gfp PCR assay was combined with the iroB PCR assay, and also with a previously published real-time 5' nuclease PCR assay for the detection of the hlyA gene of L. monocytogenes. Duplexed assays were optimised such that the target genes were simultaneously amplified at similar sensitivities to single reactions. The gfp gene was cloned into the chromosome of a non-pathogenic strain of Escherichia coli and the modified strain successfully encapsulated in LENTICULE discs. A single disc was added to 1 ml of standard enrichment broths immediately prior to DNA extraction, and used as an IC for the detection of L. monocytogenes and S. enterica by PCR. This method was evaluated using 393 naturally contaminated food or environmental samples, 267 for the detection of Salmonella spp. and 126 for Listeria spp. PCR inhibition was detected in 29 (8%) extracts, although neither pathogens were detected in these broths by culture. S. enterica was detected by PCR in 43 of 45 (96%) broths that were positive by conventional culture. The iroB gene was also detected in a further 2 broths by PCR alone. L. monocytogenes was detected in 6 broths by both PCR and conventional culture, plus an additional 7 by PCR only. Control strategies such as those described here are important tools for the interpretation of PCR assays by improving the reliability of detection of pathogens in food.
authors.affiliation: Health Protection Agency Food Safety Microbiology Laboratory, Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, United Kingdom.
authors.forename: N M
authors.initials: NM
authors.lastname: Murphy
authors.forename: J
authors.initials: J
authors.lastname: McLauchlin
authors.forename: C
authors.initials: C
authors.lastname: Ohai
authors.forename: K A
authors.initials: KA
authors.lastname: Grant
mesh_terms: D015169:Colony Count, Microbial; D053938:DNA Fragmentation; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D019779:Salmonella enterica / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D004269:DNA, Bacterial