Detection of Fecal Contamination of Strawberries by Digital PCR (dPCR)

An estimated 48 million cases of foodborne illness occur annually in the US resulting in 3,000 deaths. These diseases result from fecal contamination through various routes from human and animal sources of fruits and vegetables. Methods to detect fecal contamination include collection of contaminated water on a filter or adding contaminated samples to a nutrient containing petri dish and culturing the bacteria. An alternative is real-time PCR, measures the increase in the quantity of DNA by measuring the change in intensity of a fluorescent signal after each cycle of amplification. Digital PCR offers a quicker, more reliable, and sensitive method, dPCR estimates the absolute number of target molecules through statistical methods rather than relying on the number of amplification cycles to determine the initial amount of template molecule. dPCR plates contain wells that are split into thousands of nano wells, samples are partitioned into these individual reactions and each one is analyzed after end-point PCR cycling for the presence/absence of fluorescent signal to determine the absolute number of molecules in a sample without using a reference sample or standard curve. Bacteroidales have been used as indicators of fecal contamination in water because they are limited to warm-blooded animals, are key components of intestinal microflora, and do not proliferate in the environment. They are commensal bacteria that are found at high levels in the small intestines of humans and other higher mammals and represent 30-40% of total fecal bacteria. Systems developed for qPCR are transferrable to dPCR; dPCR also provides an improved estimate of the relative quantity of human or bacterial contamination and is more resistant to inhibition. Assays designed to detect all bacteroides have proven to yield sensitivities as low as 230 fg via dPCR for initial detection, can further be speciated using species specific mtDNA assays, initial work was done differentiating dogs, cows, pigs.