Parasites, Hosts and Diseases

The Korea Society for Parasitology and Tropical Medicine (대한기생충학·열대의학회)
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Comparative Sensitivity of PCR Primer Sets for Detection of Cryptosporidium parvum

  • Yu, Jae-Ran (Department of Environmental and Tropical Medicine, Konkuk University, School of Medicine) ;
  • Lee, Soo-Ung (Department of Environmental and Tropical Medicine, Konkuk University, School of Medicine) ;
  • Park, Woo-Yoon (Department of Radiation Oncology, College of Medicine, Chungbuk National University)
  • Published : 2009.09.30
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Abstract

Improved methods for detection of Cryptosporidium oocysts in environmental and clinical samples are urgently needed to improve detection of cryptosporidiosis. We compared the sensitivity of 7 PCR primer sets for detection of Cryptosporidium parvum. Each target gene was amplified by PCR or nested PCR with serially diluted DNA extracted from purified C. parvum oocysts. The target genes included Cryptosporidium oocyst wall protein (COWP), small subunit ribosomal RNA (SSU rRNA), and random amplified polymorphic DNA. The detection limit of the PCR method ranged from $10^3$ to $10^4$ oocysts, and the nested PCR method was able to detect $10^0$ to $10^2$ oocysts. A second-round amplification of target genes showed that the nested primer set specific for the COWP gene proved to be the most sensitive one compared to the other primer sets tested in this study and would therefore be useful for the detection of C. parvum.

Keywords

References

  1. Clark DP. New insights into human cryptosporidiosis. Clin Microbiol Rev 1999; 12: 554-563
  2. Tzipori S, Ward H. Cryptosporidiosis: biology, pathogenesis and disease. Microbes Infect 2002; 4: 1047-1058 https://doi.org/10.1016/S1286-4579(02)01629-5
  3. Coupe S, Sarfati C, Hamane S, Derouin F. Detection of Cryptosporidium and identification to the species level by nested PCR and restriction fragment length polymorphism. J Clin Microbiol 2005; 43: 1017-1023 https://doi.org/10.1128/JCM.43.3.1017-1023.2005
  4. Johnson DW, Pieniazek NJ, Griffin DW, Misener L, Rose JB. Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples. Appl Environ Microbiol 1995; 61: 3849-3855
  5. Wu Z, Nagano I, Matsuo A, Uga S, Kimata I, Iseki M, Takahashi Y. Specific PCR primers for Cryptosporidium parvum with extra high sensitivity. Mol Cell Probes 2000; 14: 33-39 https://doi.org/10.1006/mcpr.1999.0280
  6. Xiao L, Escalante A, Yang C, Sulaiman I, Escalante AA, Montali RJ, Fayer R, Lal AA. Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Appl Environ Microbiol 1999; 65: 1578-1583
  7. Spano F, Puri C, Ranucci L, Putignani L, Crisanti A. Cloning of the entire COWP gene of Cryptosporidium parvum and ultrastructural localization of the protein during sexual parasite development. Parasitology 1997; 114: 427-437 https://doi.org/10.1017/S0031182096008761
  8. Pedraza-Diaz S, Amar C, McLauchlin J. The identification and characterization of an unusual genotype of Cryptosporidium from human faeces as Cryptosporidium meleagridis. FEMS Microbiol Lett 2000; 189: 189-194 https://doi.org/10.1111/j.1574-6968.2000.tb09228.x
  9. Xiao L, Limor J, Morgan UM, Sulaiman IM, Thompson RC, Lal AA. Sequence differences in the diagnostic target region of the oocyst wall protein gene of Cryptosporidium parasites. Appl Environ Microbiol 2000; 66: 5499-5502
  10. Yang S, Healey MC. The immunosuppressive effects of dexamethasone administered in drinking water to C57BL/6N mice infected with Cryptosporidium parvum. J Parasitol 1993; 79: 626-630 https://doi.org/10.2307/3283395
  11. Petry F, Robinson HA, McDonald V. Murine infection model for maintenance and amplification of Cryptosporidium parvum oocysts. J Clin Microbiol 1995; 33: 1922-1924
  12. McCluskey BJ, Greiner EC, Donovan GA. Patterns of Cryptosporidium oocyst shedding in calves and a comparison of two diagnostic methods. Vet Parasitol 1995; 60: 185-190 https://doi.org/10.1016/0304-4017(95)00790-4
  13. Webster KA, Smith HV, Giles M, Dawson L, Robertson LJ. Detection of Cryptosporidium parvum oocysts in faeces: comparison of conventional coproscopical methods and the polymerase chain reaction. Vet Parasitol 1996; 61: 5-13 https://doi.org/10.1016/0304-4017(95)00811-X
  14. Rochelle PA, De Leon R, Stewart MH, Wolfe RL. Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water. Appl Environ Microbiol 1997; 63: 106-114
  15. Gobet P, Buisson JC, Vagner O, Naciri M, Grappin M, Comparot S, Harly G, Aubert D, Varga I, Camerlynck P. Detection of Cryptosporidium parvum DNA in formed human feces by a sensitive PCRbased assay including uracil-N-glycosylase inactivation. J Clin Microbiol 1997; 35: 254-256
  16. Mayer CL, Palmer CJ. Evaluation of PCR, nested PCR, and fluorescent antibodies for detection of Giardia and Cryptosporidium species in wastewater. Appl Environ Microbiol 1996; 62: 2081-2085
  17. Balatbat AB, Jordan GW, Tang YJ, Silva JJ. Detection of Cryptos-poridium parvum DNA in human feces by nested PCR. J Clin Microbiol 1996; 34: 1769-1772
  18. Lee SU, Joung M, Ahn MH, Huh S, Song H, Park WY, Yu JR. CP2 gene as a useful viability marker for Cryptosporidium parvum. Parasitol Res 2008; 102: 381-387 https://doi.org/10.1007/s00436-007-0772-8
  19. Lantz PG, Matsson M, Wadstro_m T, Radstro_m P. Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR. J Microbiol Methods 1997; 28: 159-167 https://doi.org/10.1016/S0167-7012(97)00979-2

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