Detection and Differentiation of Coccidian Oocysts by Real-Time PCR and Melting Curve Analysis

Abstract

Rapid and reliable detection and identification of coccidian oocysts are essential for animal health and foodborne disease outbreak investigations. Traditional microscopy and morphological techniques can identify large and unique oocysts, but they are often subjective and require parasitological expertise. The objective of this study was to develop a real-time quantitative PCR (qPCR) assay using melting curve analysis (MCA) to detect, differentiate, and identify DNA from coccidian species of animal health, zoonotic, and food safety concern. A universal coccidia primer cocktail was designed and employed to amplify DNA from Cryptosporidium parvum, Toxoplasma gondii, Cyclospora cayetanensis, and several species of Eimeria, Sarcocystis, and Isospora using qPCR with SYBR Green detection. MCA was performed following amplification, and melting temperatures (T(m)) were determined for each species based on multiple replicates. A standard curve was constructed from DNA of serial dilutions of T. gondii oocysts to estimate assay sensitivity. The qPCR assay consistently detected DNA from as few as 10 T. gondii oocysts. T(m) data analysis showed that C. cayetanensis, C. parvum, Cryptosporidium muris, T. gondii, Eimeria bovis, Eimeria acervulina, Isospora suis, and Sarcocystis cruzi could each be identified by unique melting curves and could be differentiated based on T(m). DNA of coccidian oocysts in fecal, food, or clinical diagnostic samples could be sensitively detected, reliably differentiated, and identified using qPCR with MCA. This assay may also be used to detect other life-cycle stages of coccidia in tissues, fluids, and other matrices. MCA studies on multiple isolates of each species will further validate the assay and support its application as a routine parasitology screening tool.

Application of a qPCR Assay with Melting Curve Analysis for Detection and Differentiation of Protozoan Oocysts in Human Fecal Samples from Dominican Republic

“…The reaction mix was loaded into clear-well PCR plates (catalogue no. HSP9611; Bio-Rad) and contained 1 + SsoFast EvaGreen Supermix (Bio-Rad), 400 nM Crypto-F, Crypto-R, Cyclo-F, Cyclo-R, and Sarco-R, 800 nM Toxo-F, 5 100 ng/μL bovine serum albumin (Sigma-Aldrich, Oakville, Canada), 2 μL DNA template, and sterile dH 2 O to a final volume of 25 μL. Standard curves for all qPCR runs consisted of DNA from diluted E. papillata oocysts (10 5 -10 1 ).…”

“…Melt and standard curves were generated by the CFX Manager Software (version 2.0; Bio-Rad) as described previously. 5 Oocyst-positive samples were identified by comparison of the test sample melt curves with the melt curves of the protozoan controls included in each run. Previously published conventional PCR assays targeting internal transcribed spacer 2 (ITS-2), 5.8S + ITS-2, and Cryptosporidium oocyst wall protein (COWP) regions [8][9][10] were used for additional confirmation of the positive samples of C. cayetanensis, C. belli, and Cryptosporidium spp., respectively.…”

“…We recently reported on the development of a real-time quantitative polymerase chain reaction assay with melting curve analysis (qPCR-MCA) using a universal coccidia primer cocktail for use in detection and differentiation of a wide range of coccidia species of public and veterinary health concern. 5 Using genomic DNA (gDNA) from pure isolates of coccidia, we showed that C. cayetanensis, Cryptosporidium parvum, Cryptosporidium muris, Toxoplasma gondii, Eimeria bovis, Eimeria acervulina, Cystoisospora suis, and Sarcocystis cruzi could be identified by unique melting curves and differentiated based on a specific melting temperature (Tm). However, the assay had not been validated for use on field matrices, such as food, water, or feces, where PCR inhibitors and background DNA are commonly present and could interfere with detection and identification of species.…”

“…5 The SSU rDNA region targeted by the primer cocktail was selected, because it is highly variable within the subclass, which allows for many coccidia species to be differentiated based on their Tm, and the identification can be confirmed by sequencing of the PCR products. Primers to amplify this region, originally designed by Morgan and others, 15 have also been modified by others and used in real-time PCR to detect and differentiate Cryptosporidium spp.…”

“…As a result, the oocyst quantification aspect of this assay is not entirely accurate; however, because it was designed to target multiple coccidia species, its power is not in quantification but species differentiation and identification. 5 There are several multiplex real-time PCR assays targeting diarrhea-causing parasitic protozoa, including G. duodenalis, Cryptosporidium spp., and E. histolytica [20][21][22] ; however, this qPCR-MCA assay is the only published method for detection, differentiation, and identification of a wide range of protozoan oocyst species that are important for human and animal health. The current method requires only a single channel real-time instrument and does not use probes, and therefore, it is simpler and may be more cost-effective to run than other multiplex real-time PCR assays used for diarrhea-causing protozoa.…”