Detection of Cryptosporidium parvum Oocysts on Fresh Produce Using DNA Aptamers

Abstract

There are currently no standard methods for the detection of Cryptosporidium spp., or other protozoan parasites, in foods, and existing methods are often inadequate, with low and variable recovery efficiencies. Food testing is difficult due to the low concentrations of parasites, the difficulty in eluting parasites from some foods, the lack of enrichment methods, and the presence of PCR inhibitors. The main objectives of the present study were to obtain DNA aptamers binding to the oocyst wall of C. parvum, and to use the aptamers to detect the presence of this parasite in foods. DNA aptamers were selected against C. parvum oocysts using SELEX (Systematic Evolution of Ligands by EXponential enrichment). Ten rounds of selection led to the discovery of 14 aptamer clones with high affinities for C. parvum oocysts. For detecting parasite-bound aptamers, a simple electrochemical sensor was employed, which used a gold nanoparticle-modified screen-printed carbon electrode. This aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and the anti- C. parvum aptamer. Square wave voltammetry was employed to quantitate C. parvum in the range of 150 to 800 oocysts, with a detection limit of approximately 100 oocysts. The high sensitivity and specificity of the developed aptasensor suggests that this novel method is very promising for the detection and identification of C. parvum oocysts on spiked fresh fruits, as compared to conventional methods such as microscopy and PCR.

Fig 1. Schematic representation of an electrochemical detection protocol adopted for this study.

A hybrid of a thiol-modified primer and aptamer was self-assembled onto a gold nanoparticles-modified screen-printed carbon electrode (GNPs-SPCE). Binding of the Cryptosporidium parvum oocyst to the immobilized aptamer causes an increase in the redox current, measured by square wave voltammetry.

Fig 2. Flow cytometric analysis of the binding affinities between 3 x 10⁵ C. parvum oocysts and 300nM 56-FAM labeled aptamer pools.

A control experiment was performed using the native DNA library instead of aptamer pools.

Fig 3. Affinity analyses of aptamer clones by square wave voltammetry.

(A) Square wave voltammograms of developed aptasensors based on 14 aptamer sequences (R1–4 → R8–6) obtained before (violet curve) and after binding of 3,000 Cryptosporidium parvum oocysts (pink curve), whereas a control experiment is performed using an aptasensor based on the ssDNA library. All measurements were carried out after incubating the developed aptasensors with the oocysts in DPBS for 1 h at 25°C. Square wave voltammograms were carried out in the range of-400 to 800 mV with a step potential of 4 mV, amplitude of 5 mV, and frequency of 10 Hz. Electrochemical measurements were performed in PBS (pH 7.4), containing 2.5 mM of K₄[Fe(CN)₆] and 2.5 mM of K₃[Fe(CN)₆]. (B) Plot of the aptamer sequence vs. the change in current intensity (ΔI) obtained after incubation of the developed respective aptasensors with 3,000 oocysts.

Fig 4. Limit of detection of the aptasensor.

(A) Square wave voltammograms obtained after incubating the R4–6 aptamer-based sensors with (a) 0, (b) 100, (c) 200, (d) 300, (e) 400, (f) 500, (g) 600, (h) 700, and (i) 800 Cryptosporidium parvum oocysts. (B) Calibration plot of the change in current intensity (ΔI) vs. number of oocysts. (C) Calibration plot of the change in potential (ΔE) vs. number of oocysts.

Fig 5. Selectivity and specificity of the aptasensor.

(A) Square wave voltammograms of the selectivity experiments performed by incubating the R4–6 aptamer-based sensor with (a) buffer alone, (b) 700 C. parvum oocysts, and (c) 1,000 G. duodenalis cysts, and (d) 5.1 mg/mL HSA. (B) Plot of ΔI and (C) ΔE vs. the tested target.

Fig 6. Detection of C. parvum in fruit concentrates.

(A) Square wave voltammograms of the selectivity experiments performed by incubating the R4–6 aptamer-based sensor with (a) buffer alone, (b) 300 Cryptosporidium parvum oocysts, and (c) 700 C. parvum oocysts, in pineapple and mango concentrates. (B) Plot of ΔI vs. the tested target. All measurements were repeated three times with separate electrodes (p < 0.005).