Combined PCR-oligonucleotide ligation assay for detection of dairy cattle-derived Cyclospora sp.

A rapid and sensitive assay for the detection of Cyclospora species in dairy cattle faecal specimens has been developed. The method utilizes a nested PCR to amplify a 168-bp DNA fragment of the 18S rRNA gene of cattle-derived Cyclospora sp. and an enzyme-linked immunosorbent assay (ELISA)-based oligonucleotide ligation assay (OLA) to detect the amplified product. In this study, the OLA technique was compared with conventional gel electrophoresis for the detection of amplified product. In evaluating the PCR-OLA for Cyclospora sp. and non-Cyclospora parasites, A405 reading value for Cyclospora species was significantly higher than those for non-Cyclospora control. At known concentrations of purified amplicons from cattle-derived Cyclospora sp., the OLA was able to detect more than 0.5 ng of the amplified DNA. Of 168 clinical specimens collected from four dairy cattle farms, 6 were positive by both PCR-gel electrophoresis and the PCR-OLA procedure, and 2 were positive only by PCR-OLA, indicating the PCR-OLA procedure was more sensitive than the common way with gel electrophoresis. The results indicated that the PCR-OLA is simple, rapid and suitable in clinical detection of cattle-derived Cyclospora species.