Cyclospora cayetanensis monitoring in agricultural water

The parasite Cyclospora Cayetanensis is producing illness in people consuming infected produce. Because this pathogen is in very low concentrations on actual produce, which makes it close to impossible to detect, and for prevention reasons, it is more effective to check for its presence in irrigation water, from where it is typically transferred on produce. However, even in water, this parasite is very difficult to detect. It only can be detected by lengthy molecular laboratory procedures such as PCR. One major problem for scientists to develop better and faster detection methods is the fact that there is no antibody or other recognition molecule that would be able to bind to the surface of this intact parasite. We propose to design and synthesize, for the first time, aptamers, molecules that will be able to bind to intact Cyclospora Cayetanensis oocysts, and use them to design simple paper based colorimetric tests that can detect it in the field without the need of sample preparation or specialized laboratories. The paper based test will turn from pink to purple to indicate the water sample being tested is positive for this parasite, making this a very simple and easy to use detection method for Cyclospora Cayetanensis.

Currently, there is a severe lack of standard detection and identification technologies for Cyclospora cayetanensis. There are several obstacle standing in the way of advancement towards technologies for early detection and mitigation of this parasite in agricultural water and produce. These include a lack of commercially available biorecognition elements, such as antibodies, as well as difficulties for researchers to access a source of Cyclospora cayetanensis oocysts. Here, our team propose to integrate SELEX Cyclospora cayetanensis aptamer selection and their integration into a low-cost microfluidic paper based detection devices, which our group previously developed for different analytical targets. The proposed devices will become a solution to some of the major challenges standing in the way of effective detection of C. cayetanensis in agricultural water, namely: (i) the lack of biological recognition elements for this pathogen; (ii) the challenge of low concentration that makes early detection and monitoring decisions difficult and (iii) the lack of low-cost field deployable testing devices that can monitor the presence of Cyclospora cayetanensis oocysts without extensive sample preparation protocols and trained personnel.

1. Design and understand the parameters for the Cyclospora cayetanensis aptamer synthesis via SELEX – systematic evolution of ligands by exponential enrichment. 

2. Design and test a colorimetric microfluidic biosensor platform for the detection of C. cayetanensis intact oocysts spiked in agricultural water (from irrigation ponds around Purdue) and measure parameters critical for achieving biosensing functionality (including multi-replicate ability, selectivity, repeatability, response time, detection limits, and stability).

Cyclospora cayetanensis research in the US is limited due the unavailability of Cyclospora spp. oocyst samples. To date, attempts to grow C. cayetanensis have not succeeded8. Despite such limitations, we have cloned, overexpressed, and purified the C. cayetanensis sporulated oocyst TA4 antigen that is a potential biomarker. The prepared protein has been used for the discovery of aptamers. We have, at present, identified 5 unique aptamer sequences. These aptamers were characterized to determine the binding affinity to the TA4 protein. The binding affinity of the respective aptamers is 92.1–198.2 nM.