Rapid detection of Listeria monocytogenes in dairy samples utilizing a PCR-based fluorogenic 5' nuclease assay

T. Cox, C. Frazier, J. Tuttle, S. Flood, L. Yagi, C. T. Yamashiro, R. Behari, C. Paszko, R. J. Cano

Research output: Contribution to journalArticle

28 Scopus citations

Abstract

The presence of Listeria monocytogenes as a dairy food contaminant is a lethal threat to dairy industrialists; therefore, products tainted with L. monocytogenes must be quickly detected and removed from production. This fluorogenic PCR-based assay was developed to rapidly detect L. monocytogenes contamination in dairy samples before a final product is distributed. The detection method employed uses a PCR primer pair and a fluorogenic TaqMan probe which bind to a region of a virulence determinant gene specific to L. monocytogenes. As the DNA target is amplified, the 5' nuclease activity of Taq DNA polymerase hydrolyzes the internal fluorogenic probe creating a change in fluorescence that can be monitored and automatically analyzed with a fluorometer. Sensitivity studies indicated a lower detection limit of under 10 CFU for pure culture extracts and spiked dairy enrichments. A study was performed on 266 dairy product samples obtained from Central California dairy production plants. Eighty-three of these samples were artificially spiked with both high and low concentrations of L. monocytogenes before an overnight enrichment in TSB/LiCl/colostin sulfate/moxalactam media. DNA from enriched samples was obtained using a rapid Chelex extraction specifically designed for dairy sample enrichments and automated analysis. The extraction was followed by the fluorogenic PCR assay and measurement of fluorescence increase. The assay was completed within 24 h, with an observed 95.2% sensitivity, 96.7% specificity, 92.9% positive predictive value, 97.8% negative predictive value, and 96.2% accuracy. According to specificity studies, five other bacterial species cross-reacted with the fluorogenic 5' nuclease PCR. However, only one of these strains (Listeria grayi) was able to grow in the enrichment medium employed, and was not isolated from any of the 266 dairy product enrichments evaluated in this study. Therefore, this method provides a rapid, sensitive, and automatable analysis alternative to standard culture techniques for the detection of Listeria monocytogenes in dairy samples.

Original languageEnglish (US)
Pages (from-to)167-174
Number of pages8
JournalJournal of Industrial Microbiology and Biotechnology
Volume21
Issue number3
DOIs
StatePublished - 1998

Keywords

  • Dairy products
  • Detection
  • Fluorogenic
  • Listeria monocytogenes
  • PCR

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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    Cox, T., Frazier, C., Tuttle, J., Flood, S., Yagi, L., Yamashiro, C. T., Behari, R., Paszko, C., & Cano, R. J. (1998). Rapid detection of Listeria monocytogenes in dairy samples utilizing a PCR-based fluorogenic 5' nuclease assay. Journal of Industrial Microbiology and Biotechnology, 21(3), 167-174. https://doi.org/10.1038/sj.jim.2900578