TY - JOUR
T1 - Rapid detection of Listeria monocytogenes in dairy samples utilizing a PCR-based fluorogenic 5' nuclease assay
AU - Cox, T.
AU - Frazier, C.
AU - Tuttle, J.
AU - Flood, S.
AU - Yagi, L.
AU - Yamashiro, C. T.
AU - Behari, R.
AU - Paszko, C.
AU - Cano, R. J.
PY - 1998
Y1 - 1998
N2 - 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.
AB - 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.
KW - Dairy products
KW - Detection
KW - Fluorogenic
KW - Listeria monocytogenes
KW - PCR
UR - http://www.scopus.com/inward/record.url?scp=0032426865&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032426865&partnerID=8YFLogxK
U2 - 10.1038/sj.jim.2900578
DO - 10.1038/sj.jim.2900578
M3 - Article
AN - SCOPUS:0032426865
SN - 1367-5435
VL - 21
SP - 167
EP - 174
JO - Journal of Industrial Microbiology and Biotechnology
JF - Journal of Industrial Microbiology and Biotechnology
IS - 3
ER -