BACKGROUND: Fecal contamination in fresh produce is a public health concern because it may contain human pathogens. We introduced host-specific quantitative real-time polymerase chain reaction (qPCR) assays for the rapid detection and identification of fecal contamination sources from humans and farm animals (cow, pig, chicken) in fresh produce. Each composite fecal sample was spiked on lettuce at two contamination levels (0.2 mg or 2 mg feces g−1), followed by qPCR assays for detecting each host-specific genetic marker: BoBac (cow); PF163 (pig); CP3-49 (chicken); and HF183 and gyrB (human). Two commercial DNA extraction kits were compared to evaluate DNA recovery yields and removal of PCR inhibition. Sketa2 assay was conducted to assess the presence of PCR inhibition in the contaminated lettuce.
RESULTS: All the qPCR assays yielded reliable detection from contaminated lettuce (2 mg feces g−1), where their target gene numbers were 1.5–5.0 × 103 (HF183), 0.8–2.2 × 103 (gyrB), 0.6–1.6 × 103 (BoBac), 1.6–3.0 × 103 (CP3-49) and 1.1–2.2 × 103 (PF163) copies g−1 of lettuce. Among the two extraction kits, QIAamp DNA Stool Kit resulted in 2–3 times higher sensitivity and 20% less PCR inhibition than the PowerFood™ kit.
CONCLUSION: This study provides information on the optimized host-specific qPCR assay in identifying sources of fecal contamination in fresh produce and is useful for tracking the contamination source and improving agricultural practice. © 2012 Society of Chemical Industry