Volume 89, Issue 5
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645



A quantitative polymerase chain reaction assay with melt curve analysis (qPCR-MCA) was applied for the detection of protozoan oocysts in 501 human fecal samples collected in Dominican Republic. Samples were subjected to qPCR using universal coccidia primers targeting 18S rDNA to detect oocysts followed by MCA to identify oocyst species based on amplicon melting temperature. Putative positive samples were also tested by conventional PCR and microscopy. (×3), (×3), (×5), (×1), (×1), and (×9) were detected by qPCR-MCA and confirmed by sequencing. This assay consistently detected 10 copies of the cloned target fragment and can be considered more efficient and sensitive than microscopy flotation methods for detecting multiple species of oocysts in human feces. The qPCR-MCA is a reliable protozoan oocyst screening assay for use on clinical and environmental samples in public health, food safety and veterinary programs.


Article metrics loading...

The graphs shown below represent data from March 2017
Loading full text...

Full text loading...



  1. Gerba CP, Rose JB, Haas CN, , 1996. Sensitive populations: who is at the greatest risk? Int J Food Microbiol 30: 113123.[Crossref] [Google Scholar]
  2. Mansfield LS, Gajadhar AA, , 2004. Cyclospora cayetanensis, a food- and waterborne coccidian parasite. Vet Parasitol 126: 7390.[Crossref] [Google Scholar]
  3. Murphy SC, Hoogestraat DR, SenGupta DJ, Prentice J, Chakrapani A, Cookson BT, , 2011. Molecular diagnosis of cystoisosporiasis using extended-range PCR screening. J Mol Diagn 13: 359362.[Crossref] [Google Scholar]
  4. Shields JM, Olson BH, , 2003. PCR-restriction fragment length polymorphism method for detection of Cyclospora cayetanensis in environmental waters without microscopic confirmation. Appl Environ Microbiol 69: 46624669.[Crossref] [Google Scholar]
  5. Lalonde LF, Gajadhar AA, , 2011. Detection and differentiation of coccidian oocysts by real-time PCR and melting curve analysis. J Parasitol 97: 725730.[Crossref] [Google Scholar]
  6. Gajadhar AA, , 1994. Host specificity studies and oocyst description of a Cryptosporidium sp. isolated from ostriches. Parasitol Res 80: 316319.[Crossref] [Google Scholar]
  7. Levine ND, Levine ND, , 1985. Laboratory diagnosis of protozoan infections. , ed. Veterinary Protozoology. Ames, IA: Iowa State University Press, 365386. [Google Scholar]
  8. Lalonde LF, Gajadhar AA, , 2008. Highly sensitive and specific PCR assay for reliable detection of Cyclospora cayetanensis oocysts. Appl Environ Microbiol 74: 43544358.[Crossref] [Google Scholar]
  9. Spano F, Putignani L, McLauchlin J, Casemore DP, Crisanti A, , 1997. PCR-RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene discriminates between C. wrairi and C. parvum, and between C. parvum isolates of human and animal origin. FEMS Microbiol Lett 150: 209217.[Crossref] [Google Scholar]
  10. Taniuchi M, Verweij JJ, Sethabutr O, Bodhidatta L, Garcia L, Maro A, Kumburu H, Gratz J, Kibiki G, Houpt ER, , 2011. Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples. Diagn Microbiol Infect Dis 71: 386390.[Crossref] [Google Scholar]
  11. Collins RF, Edwards LD, , 1981. Prevalence of intestinal helminths and protozoans in a rural population segment of the Dominican Republic. Trans R Soc Trop Med Hyg 75: 549551.[Crossref] [Google Scholar]
  12. Green ST, McKendrick MW, Mohsen AH, Schmid ML, Prakasam SFR, , 2000. Two simultaneous cases of Cyclospora cayetanensis enteritis returning from the Dominican Republic. J Travel Med 7: 4142.[Crossref] [Google Scholar]
  13. Weitzel T, Wichmann O, Mühlberger N, Reuter B, Hoof HD, Jelinek T, , 2006. Epidemiological and clinical features of travel-associated cryptosporidiosis. Clin Microbiol Infect 12: 921924.[Crossref] [Google Scholar]
  14. Kar S, Gawlowska S, Daugschies A, Bangoura B, , 2011. Quantitative comparison of different purification and detection methods for Cryptosporidium parvum oocysts. Vet Parasitol 177: 366370.[Crossref] [Google Scholar]
  15. Morgan UM, Constantine CC, Forbes DA, Thompson RC, , 1997. Differentiation between human and animal isolates of Cryptosporidium parvum using rDNA sequencing and direct PCR analysis. J Parasitol 83: 825830.[Crossref] [Google Scholar]
  16. Hadfield SJ, Robinson G, Elwin K, Chalmers RM, , 2011. Detection and differentiation of Cryptosporidium spp. in human clinical samples by use of real-time PCR. J Clin Microbiol 49: 918924.[Crossref] [Google Scholar]
  17. Gajadhar AA, Marquardt WC, Hall R, Gunderson J, Ariztia-Carmona EV, Sogin ML, , 1991. Ribosomal RNA sequences of Sarcocystis muris, Theileria annulata and Crypthecodinium cohnii reveal evolutionary relationships among apicomplexans, dinoflagellates, and ciliates. Mol Biochem Parasitol 45: 147154.[Crossref] [Google Scholar]
  18. Morgan JAT, Morris GM, Wlodek BM, Byrnes R, Jenner M, Constantinoiu CC, Anderson GR, Lew-Tabor AE, Molloy JB, Gasser RB, Jorgensen WK, , 2009. Real-time polymerase chain reaction (PCR) assays for the specific detection and quantification of seven Eimeria species that cause coccidiosis in chickens. Mol Cell Probes 23: 8389.[Crossref] [Google Scholar]
  19. Le Blancq SM, Khramtsov NV, Zamani F, Upton SJ, Wu TW, , 1997. Ribosomal RNA gene organization in Cryptosporidium parvum . Mol Biochem Parasitol 90: 463478.[Crossref] [Google Scholar]
  20. Verweij JJ, Blange RA, Templeton K, Schinkel J, Brienen EAT, van Rooyen MAA, van Lieshout L, Polderman AM, , 2004. Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR. J Clin Microbiol 42: 12201223.[Crossref] [Google Scholar]
  21. Haque R, Roy S, Siddique A, Mondal U, Rahman SMM, Mondal D, Houpt E, Petri WA, , 2007. Multiplex real-time PCR assay for detection of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp. Am J Trop Med Hyg 76: 713717. [Google Scholar]
  22. Stark D, Al-Qassab SE, Barratt JLN, Stanley K, Roberts T, Marriott D, Harkness J, Ellis JT, , 2011. Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples. J Clin Microbiol 49: 257262.[Crossref] [Google Scholar]

Data & Media loading...

  • Received : 26 Feb 2013
  • Accepted : 02 Jun 2013
  • Published online : 06 Nov 2013

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error