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An evaluation of primers amplifying DNA targets for the detection of Cryptosporidium spp. using C. parvum HNJ-1 Japanese isolate in water samples

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Abstract

The performance of polymerase chain reaction (PCR) procedures for the detection of Cryptosporidium parvum HNJ-1 strain (genotype II) oocysts purified from mice using published protocols was evaluated. Oocysts were concentrated from fecal samples of infected severe combined immunodeficiency (SCID) mice by sucrose flotation and were then purified by immunomagnetic separation method. The genotype of C. parvum was established as type II by restriction fragment length polymorphism (RFLP) analysis. Water samples were spiked with different numbers of oocysts, determined by limiting dilution. Genomic DNA was extracted and used for PCR assays targeting various Cryptosporidium species genes (Beta-Tubulin, COWP, 70 kDa HSP, SSU rRNA, ITS1, TRAP-C1 and TRAP-C2 gene). DNA from oocyst numbers of more than 1 × 104 was detected using each of the primers. However, when using lower oocyst numbers, the tools based on 9 of the 16 different primer assays gave sufficient results. Assays using the remaining seven primers gave less than satisfactory results. A new primer set, named VKSS-F1/2 and VKSS-R1/2, that target the 18 SSU rRNA gene of C. parvum was constructed and applied.The VKSS-F1/2 and VKSS-R1/2 assays amplified DNA isolated from spiked samples in 206 of 211 trials (97.6%). This illustrates the difficulty of detecting low numbers of Cryptosporidium spp. oocysts by molecular methods when working with environmental samples.

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Acknowledgement

This study was supported by institutional funds of the Center of Anatomy, Medical School of the University of Cologne, by the University of Cologne Investitionsfonds ‘Lehre & Forschung’ 2006, and by the Grant-in-Aid for Young Scientists, Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and from the 21st Century COE Program (A-1), Ministry of Education, Sports, Science, and Technology of Japan. We would like to thank to thank Professor Juergen Koebke (Center of Anatomy, Institute II, Medical School, University of Cologne), coordinator for international affairs, for his support on this collaboration. The authors would like to thank Professor Arwid Daugschies and Dr Michael Najdrowski, Veterinary School, University of Leipzig, Germany for kindly providing one bovine isolate.

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  1. Medical and Molecular Parasitology Laboratory, University of Cologne, Medical School, Center of Anatomy, Institute II, Cologne, Germany

    Anna Susanne Leetz & Isaia Sotiriadou

  2. Civil Mining and Environmental Engineering, The University of Wollongong, Wollongong, NSW 2522, Australia

    Jerry Ongerth

  3. National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan

    Panagiotis Karanis

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  1. Anna Susanne Leetz
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  2. Isaia Sotiriadou
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  3. Jerry Ongerth
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  4. Panagiotis Karanis
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Correspondence to Panagiotis Karanis.

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Leetz, A.S., Sotiriadou, I., Ongerth, J. et al. An evaluation of primers amplifying DNA targets for the detection of Cryptosporidium spp. using C. parvum HNJ-1 Japanese isolate in water samples. Parasitol Res 101, 951–962 (2007). https://doi.org/10.1007/s00436-007-0567-y

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