Abstract
Due to recent outbreaks of cyclosporiasis associated with consumption of fresh berries, producers are demanding modern microbiological tools for the rapid and accurate identification of the human pathogen Cyclospora cayetanensis in berries and environmental samples. The aim of the present work was to develop a molecular tool based on a PCR approach for the rapid and accurate detection of C. cayetanensis. A nested PCR assay was validated for the amplification of a 294 bp size region of the 18S rRNA gene from C. cayetanensis. The limit of detection for the nested PCR assay was validated using 48 berry samples spiked with ~0, 10, 100, and 1000 oocyst per gram of sample. With this assay, it was possible to detect as few as 1 oocyst per gram of berry, in a 50 g sample. Sanger DNA sequencing and phylogenetic analysis were carried out to confirm the presence of C. cayetanensis in berry (n = 17) and soil (n = 5) samples. The phylogenetic analysis revealed that the C. cayetanensis sequences obtained from Mexico clustered within a group recovered from China, Peru, Guatemala-Haiti, and Japan. The PCR protocol designed in the present study could be an important tool for the rapid and accurate detection of this human pathogen in environmental and food samples.
Highlights
The consumption of raspberries (Rubus idaeus), blackberries (Rubus sp.), and blueberries (Vaccinium corymbosum) has increased worldwide in recent years because these fruits are considered an important source of antioxidant compounds [1,2]
The presence of C. cayetanensis in berries is directly associated with the presence of the parasite in soil [5,6,7]
To improve improvesensitivity sensitivity in PCR-based detection of C. cayetanensis, twowere strategies were in PCR-based detection of C. cayetanensis, two strategies implemented; implemented; first,approach a nested was PCRdesigned approachbecause was designed because this pathogen is found at low first, a nested PCR
Summary
The consumption of raspberries (Rubus idaeus), blackberries (Rubus sp.), and blueberries (Vaccinium corymbosum) has increased worldwide in recent years because these fruits are considered an important source of antioxidant compounds [1,2]. Consumption of berries is associated with a risk of acquiring foodborne parasites, such as Cyclospora cayetanensis [1,3]. In the USA and Canada, 4250 cyclosporiasis cases have been linked to consumption of raspberries, blackberries, and strawberries [1]. In the UK, 43 cases of cyclosporiasis were linked to the consumption of fresh strawberries and raspberries [4]. The presence of C. cayetanensis in berries is directly associated with the presence of the parasite in soil [5,6,7]. It is fundamental that producers monitor the presence of this pathogen on farms and packing facilities
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