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http://dx.doi.org/10.3347/kjp.2009.47.3.293

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)
Publication Information
Parasites, Hosts and Diseases / v.47, no.3, 2009 , pp. 293-297 More about this Journal
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
Cryptosporidium parvum; nested PCR; COWP gene; SSU rRNA;
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1 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   DOI   ScienceOn
2 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   DOI   ScienceOn
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   DOI   ScienceOn
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   PUBMED   ScienceOn
5 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   DOI   ScienceOn
6 Petry F, Robinson HA, McDonald V. Murine infection model for maintenance and amplification of Cryptosporidium parvum oocysts. J Clin Microbiol 1995; 33: 1922-1924   PUBMED   ScienceOn
7 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   DOI   ScienceOn
8 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   PUBMED   ScienceOn
9 Tzipori S, Ward H. Cryptosporidiosis: biology, pathogenesis and disease. Microbes Infect 2002; 4: 1047-1058   DOI   ScienceOn
10 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   PUBMED   ScienceOn
11 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   PUBMED   ScienceOn
12 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   PUBMED   ScienceOn
13 Clark DP. New insights into human cryptosporidiosis. Clin Microbiol Rev 1999; 12: 554-563   PUBMED   ScienceOn
14 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   PUBMED   ScienceOn
15 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   DOI   ScienceOn
16 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   DOI   ScienceOn
17 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   DOI   PUBMED   ScienceOn
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   DOI   ScienceOn
19 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