A combined PCR and selective enrichment method for rapid detection of Listeria monocytogenes

Fitter, Stephen; Heuzenroeder, M N; Thomas, Connor

doi:10.1111/j.1365-2672.1992.tb04968.x

Cited by 59 publications

(26 citation statements)

References 29 publications

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“…Oligonucleotide specific L. monocytogenes primers for the PCR assay typing were selected based on the published nucleotide sequence of the actA gene (Cai, Kabuki, Kuaye, Cargioli, Chung, Nielsen, & Wiedmann, 2002), the hlyA gene (Fitter, Heuzenroeder, & Thomas, 1992) and iap gene (Fureer, Candrian, Horfelein, & Luethy, 1991). The pair of primers 01 (5 0 -GCTGATTTAAGAGATA-GAGGAACA-3 0 ), 02 (5 0 -TTTATGTGGTTATTTGCTGTC-3 0 ) (Alpha DNA, Montreal, CA) were used to amplify a 827 bp DNA fragment of the actA gene (Cai et al, 2002).…”

Section: Dna Extraction and Pcr Conditionmentioning

confidence: 99%

Prevalence of Listeria spp. and antibiotic susceptibility of Listeria monocytogenes isolated from raw chicken and ready-to-eat chicken products in Jordan

2011

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Section: Dna Extraction and Pcr Conditionmentioning

confidence: 99%

Prevalence of Listeria spp. and antibiotic susceptibility of Listeria monocytogenes isolated from raw chicken and ready-to-eat chicken products in Jordan

2011

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“…A simple means intended to favor the detection of viable cells and, at the same time, lower the detection limit is to include an enrichment step prior to DNA extraction. This approach has been reported mainly for the qualitative detection of pathogens in food (15,19,47,52,60). In this study, sensitive detection could be achieved with samples containing freshly inoculated cells of Salmonella spp.…”

Section: Vol 69 2003 Pcr and Rt-pcr In Organic Waste 4623mentioning

confidence: 71%

Evaluation of the Use of PCR and Reverse Transcriptase PCR for Detection of Pathogenic Bacteria in Biosolids from Anaerobic Digestors and Aerobic Composters

Burtscher

Wuertz

2003

Appl Environ Microbiol

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A PCR-based method and a reverse transcriptase PCR (RT-PCR)-based method were developed for the detection of pathogenic bacteria in organic waste, using Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and Staphylococcus aureus as model organisms. In seeded organic waste samples, detection limits of less than 10 cells per g of organic waste were achieved after one-step enrichment of bacteria, isolation, and purification of DNA or RNA before PCR or RT-PCR amplification. To test the reproducibility and reliability of the newly developed methods, 46 unseeded samples were collected from diverse aerobic (composting) facilities and anaerobic digestors and analyzed by both culture-based classical and newly developed PCR-based procedures. No false-positive but some false-negative results were generated by the PCR-or RT-PCR-based methods after one-step enrichment when compared to the classical detection methods. The results indicated that the level of activity of the tested bacteria in unseeded samples was very low compared to that of freshly inoculated cells, preventing samples from reaching the cell density required for PCR-based detection after one-step enrichment. However, for Salmonella spp., a distinct PCR product could be obtained for all 22 nonamended samples that tested positive for Salmonella spp. by the classical detection procedure when a selective two-step enrichment (20 h in peptone water at 37°C and 24 h in Rappaport Vassiliadis medium at 43°C) was performed prior to nucleic acid extraction and PCR. Hence, the classical procedure was shortened, since cell plating and further differentiation of isolated colonies can be omitted, substituted for by highly sensitive and reliable detection based on nucleic acid extraction and PCR. Similarly, 2 of the 22 samples in which Salmonella spp. were detected also tested positive for Listeria monocytogenes according to a two-step enrichment procedure followed by PCR, compared to 3 samples that tested positive when classical isolation procedures were followed. The study shows that selective two-step enrichment is useful when very low numbers of bacterial pathogens must be detected in organic waste materials, such as biosolids. There were no falsepositive results derived from DNA of dead cells in the waste sample, suggesting that it is not necessary to perform RT-PCR analyses when PCR is combined with selective enrichment. Large numbers of added nontarget bacteria did not affect detection of Salmonella spp., L. monocytogenes, and Y. enterocolitica but increased the detection limit of Staphylococcus aureus from <10 to 10 4 CFU/g of organic waste. Overall, the detection methods developed using seeded organic waste samples from one waste treatment facility (WTF) needed to be modified for satisfactory detection of pathogens in samples from other WTFs, emphasizing the need for extensive field testing of laboratory-derived PCR protocols. A survey of 13 WTFs in Germany revealed that all facilities complied with the German Biowaste Ordinance, which mandates that t...

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“…and aminopeptidase [15] . PCR was found to be specific, but not very sensitive, since preenrichment in selective media was generally necessary [16] ; furthermore, some substances contained in the food samples could negatively influence the activity of the Taq DNA polymerase, with the possibility of obtaining false negative results [17,18] . Another important defect of this technique is its intrinsic failure in differentiating live from dead cells and in giving a quantitative record of the listeria contained in food samples [19] ; in fact, a quantitative determination of the listeria content in a food sample is required for monitoring the real risk of infection caused by contaminated food ingestion [20,21] .…”

Section: Introductionmentioning

confidence: 99%

Isolation and Identification of Listeria monocytogenes in Processed Meat by a Combined Cultural-molecular Method

Ingianni¹,

Quartuccio²,

Madeddu³

et al. 2007

American J. of Infectious Diseases

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Abstract:The isolation and identification of Listeria monocytogenes in processed meat samples by a combined cultural-molecular method is described. It allows the identification of Listeria strains by means of a hybridization technique with a specific DNA probe directed to the listerial internalin gene. The specificity of this method was found to be 100% and sensitivity was as low as 1 CFU/2.5 g of food sample. A total of 278 meat samples were tested in comparison with PCR and conventional cultural assays. A total of 42 (15.4%) L. monocytogenes were detected. PCR analysis gave 3 false negative results and culture failed to detect the Listeria in 5 cases. With this cultural-molecular method the identification and quantitative detection of L. monocytogenes were achieved within 36 hours and no false positive or negative tests were obtained, thus fitting most food industry requirements.

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A combined PCR and selective enrichment method for rapid detection of Listeria monocytogenes

Cited by 59 publications

References 29 publications

Prevalence of Listeria spp. and antibiotic susceptibility of Listeria monocytogenes isolated from raw chicken and ready-to-eat chicken products in Jordan

Prevalence of Listeria spp. and antibiotic susceptibility of Listeria monocytogenes isolated from raw chicken and ready-to-eat chicken products in Jordan

Evaluation of the Use of PCR and Reverse Transcriptase PCR for Detection of Pathogenic Bacteria in Biosolids from Anaerobic Digestors and Aerobic Composters

Isolation and Identification of Listeria monocytogenes in Processed Meat by a Combined Cultural-molecular Method

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