Optimization and validation of methods for isolation and real-time PCR identification of protozoan oocysts on leafy green vegetables and berry fruits

• Processing methods were optimized for isolation of protozoan oocysts from produce
• A qPCR assay with melting curve analysis (qPCR MCA) was validated for oocyst detection
• 3-5 oocysts per gram of produce were reliably detected with the optimized methods

Leafy green vegetables and berry fruits have vastly different physical and biochemical characteristics, are typically consumed raw with minimal washing, and are potential transmission vehicles for food-borne disease caused by protozoan parasites such as Cryptosporidium, Cyclospora and Toxoplasma. Validation of a generic oocyst isolation and detection method applicable to each leafy green and berry type is required to provide reliable laboratory support for surveillance programs and as necessary for disease outbreak investigations. The objectives of the current study were to optimize and validate the performance of these methods for the isolation of protozoan oocysts from several types of leafy greens and berries. Eimeria papillata oocysts were used as a surrogate for coccidia of public health concern to spike produce samples. An artificial stomacher or orbital shaker was used, followed by centrifugation, to isolate and concentrate oocysts, respectively, and a qPCR melt curve analysis (qPCR MCA) was used for detection and identification. Processing methods, wash buffers and storage conditions were evaluated and optimized for five types of berries (blackberries, blueberries, cranberries, raspberries and strawberries), five types of herb (cilantro, dill, mint, parsley, thyme) and green onions. Blackberries, cranberries, raspberries and strawberries were most effectively washed by orbital shaking with an elution solution, while glycine buffer was more effective for blueberries. Stomaching with a glycine buffer was optimal for oocyst recovery in leafy herbs with soft stems, while aromatic woody-stemmed herbs such as thyme required orbital shaking to minimize the release of PCR inhibitors. Oocyst recovery from green onions was highest when processed by orbital shaking with elution solution. Oocyst recovery rates ranged from 4.1–12% for berries and 5.1–15.5% for herbs and green onions. As few as 3 oocysts per gram of fruit, or 5 oocysts per gram of herbs or green onions could reliably be detected using the optimized isolation methods and qPCR MCA.