Risk assessment of false-positive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells.
Real-time PCR technology is increasingly used for detection and quantification of pathogens in food samples. A main disadvantage of nucleic acid detection is the inability to distinguish between signals originating from viable cells and DNA released from dead cells. In order to gain knowledge concerning risks of false-positive results due to detection of DNA originating from dead cells, quantitative PCR (qPCR) was used to investigate the degradation kinetics of free DNA in four types of meat samples. Results showed that the fastest degradation rate was observed (1 log unit per 0.5 h) in chicken homogenate, whereas the slowest rate was observed in pork rinse (1 log unit per 120.5 h). Overall results indicated that degradation occurred faster in chicken samples than in pork samples and faster at higher temperatures. Based on these results, it was concluded that, especially in pork samples, there is a risk of false-positive PCR results. This was confirmed in a quantitative study on cell death and signal persistence over a period of 28 days, employing three different methods, i.e. viable counts, direct qPCR, and finally floatation, a recently developed discontinuous density centrifugation method, followed by qPCR. Results showed that direct qPCR resulted in an overestimation of up to 10 times of the amount of cells in the samples compared to viable counts, due to detection of DNA from dead cells. However, after using floatation prior to qPCR, results resembled the viable count data. This indicates that by using of floatation as a sample treatment step prior to qPCR, the risk of false-positive PCR results due to detection of dead cells, can be minimized.
Wolffs
Petra
P
Applied Microbiology, Lund Institute of Technology, Lund University, SE-221 00 Lund, Sweden. pwolffs@uoguelph.ca
Norling
Börje
B
Rådström
Peter
P
D000818:Animals; D052160:Benzothiazoles; D016923:Cell Death / Q000502:physiology; D002499:Centrifugation, Density Gradient; D002645:Chickens; D015169:Colony Count, Microbial; D004247:DNA / Q000032:analysis / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry; D003959:Diamines; D005189:False Positive Reactions; D005516:Food Microbiology / Q000592:standards*; D008460:Meat / Q000382:microbiology*; D009930:Organic Chemicals / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D011804:Quinolines; D013552:Swine; D015009:Yersinia Infections / Q000382:microbiology / Q000517:prevention & control*; D015008:Yersinia enterocolitica / Q000254:growth & development*
D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA