Fig. 3. SYBR Green I melting curve analysis of Cp492 amplicons from one C. parvum type 1 isolate (TU502) and three type 2 isolates (TAMU, 7C, CISD). Because the differences in the melting temperature are determined by the length of the amplicon and by the base composition, melting temperatures do not always correlate with amplicon length or gel mobility. In the example shown, isolate TU502 has a lower melting temperature than isolate CISD in spite of the fact that TU502 has the highest number of repeats. F1, SYBR Green fluorescence measured at 530 nm. The negative value of the first derivative of F1 over the temperature is plotted on the y-axis.
pipet and repeating the aspiration. As with all procedures involving ether, buildup of pressure should be prevented by periodically venting the tubes while performing the extraction.
5. Because of the relatively high cost of the immunomagnetic products, it is often possible to reduce the volume of immunomagnetic beads and adjust the buffer volume proportionally. Unless we expect to extract a large number of oocysts, say more than 107, the volume of beads and buffers can be reduced to one-half (50-^L bead suspension) or one-third (30-^L bead suspension) of the recommended volumes. For routine genetic analyses, this method will recover sufficient oocysts for extracting DNA and performing multiple PCR assays.
6. Several modifications to the standard amplification program were attempted, but the amplification curves obtained with (AT) microsatellites remained flat in comparison with those amplified from nonrepeat sequences or repeats containing C or G residues. This observation indicates suboptimal amplification. In addition, melting curves obtained from (AT) microsatellites frequently display multiple peaks. We assume that this is caused by partial melting of the AT-rich region at a lower temperature than that of the sequences flanking the repeat. If this assumption is confirmed, (AT) repeats may not be suitable for real-time PCR typing. Additional microsatellites containing repeats of G and/or C residues are being evaluated as alternative markers.
7. The general applicability of SYBR Green I real-time PCR for allele discrimination is still being investigated. Preliminary results indicate that AT microsatellites are not efficiently amplified. Repeats containing G and C nucleotides appear to amplify better, but rela tively large differences in repeat length (6 bp or more) may only result in relatively small differences in melting temperature. This observation contrasts with those from real-time PCR methods based on internal fluorescent probes, which can generate measurable and reproducible melting profiles on the basis of single-nucleotide polymorphisms (30,31).
1 Barwick, R. S., Levy, D. A., Craun, G. F., Beach, M. J., and Calderon R. L. (2000) Surveillance for waterborne-disease outbreaks—United States, 1997-1998. MMWR 49,1-35.
2 McLauchlin. J., Amar, C., Pedraza-Diaz, S., and Nichols, G. L. (2000) Molecular epidemiological analysis of Cryptosporidium spp. in the United Kingdom: results of genotyping Cryptosporidium spp. in 1,705 fecal samples from humans and 105 fecal samples from livestock animals. J. Clin. Microbiol. 38, 3984-3990.
3 Hunter, P. R. and Nichols, G. (2002) Epidemiology and clinical features of Cryptosporidium infection in immunocompromised patients. Clin. Microbiol. Rev. 15, 145-154.
4 Morgan, U. M., Sargent, K. D., Elliot, A., and Thompson, R. C. (1998) Cryptosporidium in cats—additional evidence for C. felis. Vet. J. 156, 159-161.
5 Lindsay, D. S., Upton, S. J., Owens, D. S., Morgan, U. M., Mead, J. R., and Blagburn, B. L. (2000) Cryptosporidium andersoni n. sp. (Apicomplexa: Cryptosporiidae) from cattle, Bos taurus. J. Eukaryot. Microbiol. 47, 91-95.
6 Fayer, R., Trout, J. M., Xiao, L., Morgan, U. M., Lai, A. A., and Dubey, J. P. (2001) Cryptosporidium canis n. sp. from domestic dogs. J. Parasitol. 87, 1415-1422.
7 Awad-El-Kariem, F.M., Robinson, H.A., Petry, F., McDonald, V., Evans, D., and Casemore, D. (1998) Differentiation between human and animal isolates of Cryptosporidium parvum using molecular and biological markers. Parasitol. Res. 84, 297-301.
8 Peng, M. M., Xiao, L., Freeman, A. R., et al. (1997) Genetic polymorphism among Cryptosporidium parvum isolates: evidence of two distinct human transmission cycles. Emerg. Infect. Dis. 3, 567-573.
9. Widmer, G. (1998) Genetic heterogeneity and PCR detection of Cryptosporidium parvum. Adv. Parasitol. 40, 223-239.
10 LeChevallier, M. W., Norton, W. D., and Lee, R. G. (1991) Occurrence of Giardia and Cryptosporidium spp. in surface water supplies. Appl. Environ. Microbiol. 57, 2610-2616.
11 Smith, H. V. and Rose J. B. (1998) Waterborne cryptosporidiosis: current status. Parasitol. Today 14, 14-22.
12. Caccio, S., Homan, W., Camilli, R., Traldi, G., Kortbeek, T., and Pozio, E. (2000) A microsatellite marker reveals population heterogeneity within human and animal genotypes of Cryptosporidium parvum. Parasitology 120, 237-244.
13 Feng, X., Rich, S. M., Akiyoshi, D., et al. (2000) Extensive polymorphism in Cryptosporidium parvum identified by multilocus microsatellite analysis. Appl. Environ. Microbiol. 66, 3344-3349.
14 Strong, W. B., Gut, J., and Nelson, R. G. (2000) Cloning and sequence analysis of a highly polymorphic Cryptosporidium parvum gene encoding a 60-kilodalton glycoprotein and characterization of its 15- and 45-kilodalton zoite surface antigen products. Infect. Immun. 68, 4117-4134.
15 Leav, B. A., Mackay, M. R., Anyanwu, A., et al. (2002) Analysis of sequence diversity at the highly polymorphic Cpgp40/15 locus among Cryptosporidium isolates from human immunodeficiency virus-infected children in South Africa. Infect. Immun. 70,3881-3890.
16. Trevors, J. T. and van Elsas, J. D. (1995) Introduction to nucleic acids in the environment: methods and applications. In: Nucleic Acids in the Environment. Methods and Applications (Trevors, J. T. and van Elsas, J. D., eds.). Springer-Verlag, Berlin, pp. 1-7.
17 Sluter, S. D., Tzipori, S., and Widmer, G. (1997) Parameters affecting polymerase chain reaction detection of waterborne Cryptosporidium parvum oocysts. Appl. Microbiol. Biotechnol. 48, 325-330.
18. Rickwood, D. (1987) Centrifugation. A Practical Approach. IRL, Washington, DC, p. 29.
19 Anderson, B. C. (1981) Patterns of shedding of cryptosporidial oocysts in Idaho calves. J. Am. Vet. Med. Assoc. 178, 982-984.
20. Current, W. L. (1990) Techniques and laboratory maintenance of Cryptosporidium. In: Cryptosporidiosis of Man and Animals (Dubey, J. P., Speer, C. A., and Fayer, R., eds.). CRC, Boca Raton, FL, pp. 31-49.
21 Widmer, G., Tzipori, S., Fichtenbaum, C. J., and Griffiths, J. K. (1998) Genotypic and phenotypic characterization of Cryptosporidium parvum isolates from people with AIDS. J. Infect. Dis. 178, 834-840.
22 Xiao, L., Alderisio, K., Limor, J., Royer, M., and Lal, A. A. (2000) Identification of species and sources of Cryptosporidium oocysts in storm waters with a small-subunit rRNA-based diagnostic and genotyping tool. Appl. Environ. Microbiol. 66, 5492-5498.
23 Sulaiman, I. M., Xiao, L., and Lal, A. A. (1999) Evaluation of Cryptosporidium parvum genotyping techniques. Appl. Environ. Microbiol. 65, 4431-4435.
24 Feng, X., Rich S. M., Tzipori S., and Widmer G. (2002) Experimental evidence for genetic recombination in the opportunistic pathogen Cryptosporidium parvum. Mol. Biochem. Parasitol. 119, 55-62.
25 Strong W. B. and Nelson, R. G. (2000) Preliminary profile of the Cryptosporidium parvum genome: an expressed sequence tag and genome survey sequence analysis. Mol. Biochem. Parasitol. 107, 1-32.
26 Widmer, G., Akiyoshi, D., Buckholt, M. A., et al. (2000) Animal propagation and genomic survey of a genotype 1 isolate of Cryptosporidium parvum. Mol. Biochem. Parasitol. 108, 187-197.
27 Tzipori, S., Rand, W., Griffiths, J., Widmer, G., and Crabb, J. (1994) Evaluation of an animal model system for cryptosporidiosis: therapeutic efficacy of paromomycin and hyperimmune bovine colostrum-immunoglobulin. Clin. Diagn. Lab. Immunol. 1,450-463.
28 Okhuysen, P. C., Rich, S. M., Chappell, C. L., et al. (2002) Infectivity of a Cryptosporidium parvum isolate of cervine origin for healthy adults and interferon-gamma knockout mice. J. Infect. Dis. 185, 1320-1325.
29 Carraway, M., Tzipori, S., and Widmer, G. (1996) Identification of genetic heterogeneity in the Cryptosporidium parvum ribosomal repeat. Appl. Environ. Microbiol. 62,712-716.
30 Limor, J. R., Lal, A. A., and Xiao, L. (2002) Detection and differentiation of Cryptosporidium parasites that are pathogenic for humans by real-time PCR. J. Clin. Microbiol. 40, 2335-2338.
31. Tanriverdi, S., Tanyeli, A., Baslamisli, F., et al. (2002) Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis. J. Clin. Microbiol. 40, 3237-3244.
Was this article helpful?