Quantification of Listeria monocytogenes in salads by real time quantitative PCR.
A real time quantitative PCR (RTQ-PCR) was carried out purifying DNA extracts of Listeria monocytogenes using a High Pure Listeria Sample Preparation Kit and quantifying in a LightCycler system with hybridisation probes. A standard curve was constructed with serial dilutions. A range linear relationship, from 10 to 10(5)L. monocytogenes colony forming units (CFU), was observed between threshold cycle (Ct) and logarithmic concentration of the serial dilutions. The assay was linear in a range from 10 to 10(5)L. monocytogenes CFU and the coefficient of determination (r2) was >0.98. RTQ-PCR presented an efficiency of >85%. The accuracy of the PCR-based assay, expressed as % bias, ranged from 9% to 26% and the precision, expressed as % CV, ranged 9-22%. Intraday and interday variabilities were studied at 10(2) CFU/g and resulted in 12% and 14%, respectively. The proposed RTQ-PCR method and classical cultural methods were applied to analyse 77 salads from restaurants in Valencia (Spain). All culture positive samples were also RTQ-PCR positive.
Berrada
H
H
University of Valencia, Faculty of Pharmacy, Laboratory of Bromatology and Toxicology, Burjassot, Valencia, Spain.
Soriano
J M
JM
Picó
Y
Y
Mañes
J
J
D015169:Colony Count, Microbial; D003257:Consumer Product Safety; D004269:DNA, Bacterial / Q000032:analysis*; D005504:Food Analysis / Q000379:methods; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D006801:Humans; D018545:Lactuca / Q000382:microbiology*; D016014:Linear Models; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D008954:Models, Biological; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013030:Spain
D004269:DNA, Bacterial