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

Authors : Fadime EROGLU

Pages : 194-202

Doi:10.5798/dicletip.1313299

View : 27 | Download : 31

Publication Date : 2023-06-12

Article Type : Research Paper

Abstract :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 insert ignore into journalissuearticles values(Integrated DNA technologies, Coralville, USA); software program” and “Multiple sequence alignment use a computer software Primer Express™Software v3.0.1 Lience insert ignore into journalissuearticles values(ThermoFisher Scientific, Waltham, USA);”. The primer-probes designed in the study were spore-forming Cryptosporidium spp. insert ignore into journalissuearticles values(ATCC®87715™);, Cyclospora spp. insert ignore into journalissuearticles values(ATCC®PRA-3000SD™);, Cystoispora spp. insert ignore into journalissuearticles values(KF648871);, Encephalitozoon spp. insert ignore into journalissuearticles values(FJ026010); and Sarcocystis spp. insert ignore into journalissuearticles values(ATCC®CCL-70); parasites were amplified with DNA isolates from the American Type Culture Collection insert ignore into journalissuearticles values(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 insert ignore into journalissuearticles values(Ct); ±25.7, Standard curve insert ignore into journalissuearticles values(R2 );: ±0,993, and Efficiency insert ignore into journalissuearticles values(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.
Keywords : Spore forming intestinal parasites, pentaplex real time PCR, rapid diagnostic