Direct detection of bacterial pathogens in representative dairy products using a combined bacterial concentration-PCR approach.
OBJECTIVE
To develop a simple, rapid method to concentrate and purify bacteria and their nucleic acids from complex dairy food matrices in preparation for direct pathogen detection using polymerase chain reaction (PCR).


RESULTS
Plain non-fat yogurt and cheddar cheese were each seeded with Listeria monocytogenes or Salmonella enterica serovar. Enteritidis in the range of 10(1)-10(6) CFU per 11-g sample. Samples were then processed for bacterial concentration using high-speed centrifugation (9700 g) followed by DNA extraction, PCR amplification, and amplicon confirmation by hybridization. Bacterial recoveries after centrifugation ranged from 53 to >100% and 71 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the non-fat yogurt samples; and from 77 to >100% and 69 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the cheddar cheese samples. There were no significant differences in recovery efficiency at different inocula levels, and losses to discarded supernatants were always <5%, regardless of dairy product or pathogen.


CONCLUSIONS
When followed by pathogen detection using PCR and confirmation by amplicon hybridization, detection limits of 10(3) and 10(1) CFU per 11-g sample were achieved for L. monocytogenes and serovar. Enteritidis, respectively, in both product types and without prior cultural enrichment.


CONCLUSIONS
This study represents progress toward the rapid and efficient direct detection of pathogens from complex food matrices at detection limits approaching those that might be anticipated in naturally contaminated products.
Stevens
K A
KA
Department of Food Science, College of Life Science and Agriculture, North Carolina State University, Raleigh, NC 27695, USA.
Jaykus
L-A
LA
D015139:Blotting, Southern / Q000379:methods; D002611:Cheese / Q000382:microbiology; D015169:Colony Count, Microbial / Q000379:methods; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D003611:Dairy Products / Q000382:microbiology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D005516:Food Microbiology; D005990:Glycerol / Q000494:pharmacology; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization / Q000187:drug effects; D011092:Polyethylene Glycols / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D012477:Salmonella enteritidis / Q000302:isolation & purification*; D012997:Solvents / Q000494:pharmacology; D015014:Yogurt / Q000382:microbiology
D003470:Culture Media; D004269:DNA, Bacterial; D012997:Solvents; D011092:Polyethylene Glycols; D005990:Glycerol; D004229:Dithiothreitol; D004121:Dimethyl Sulfoxide