Identification of Spore-Forming Intestinal Parasites with Pentaplex Real-Time PCR

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Objective: Spore-forming intestinal parasites; Cryptosporidium spp., Cyclospora spp., Cytoisospora spp., Encephalitozoon spp., and Sarcoystis spp. are very common in immunocompromised patients, but these parasites are overlooked by healthcare proffesionals. It was aimed to develop a new pentaplex real-time PCR panel for the identification of spore-forming intestinal parasites in this study. Methods: Primer-probes for pentaplex real-time PCR were designed using the “PrimerQuest Tool (Integrated DNA technologies, Coralville, USA) software program” and “Multiple sequence alignment use a computer software Primer Express™Software v3.0.1 Lience (ThermoFisher Scientific, Waltham, USA)”. The primer-probes designed in the study were spore-forming Cryptosporidium spp. (ATCC®87715™), Cyclospora spp. (ATCC®PRA-3000SD™), Cystoispora spp. (KF648871), Encephalitozoon spp. (FJ026010) and Sarcocystis spp. (ATCC®CCL-70) parasites were amplified with DNA isolates from the American Type Culture Collection (ATCC) and then these primer-probes were validated with 232 DNA samples obtained from the stools of the patient samples. Results: It was found that Cycle Threshold (Ct) ±25.7, Standard curve (R2 ): ±0,993, and Efficiency (E): %96,1 according to the results of multiplex real-time PCR analysis. Similar results were found in pentaplex real-time PCR analysis of DNA isolates of stool samples. When the pentaplex real-time PCR results of DNA samples isolates from stool samples were compared with the positivie predictive value results of traditional methods, it was found that the pentaplex results were higher. Conclusion: The new designed pentaplex real-time PCR panel can be used in the diagnosis of spore-forming intestinal parasites, which are very common in immunocompromised patients. Thus, the diagnosis of five different parasites can be made faster, more economically and faster with a single reaction.

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