Comparison of techniques for DNA extraction and agarose gel staining of DNA fragments using samples of Cryptosporidium

M.C.M. Couto, A.P. Sudre, M.F. Lima and T.C.B. Bomfim
Additional contact information
M.C.M. Couto: Federal Rural University of Rio de Janeiro, Seropedica, Rio de Janeiro, Brazil
A.P. Sudre: Fluminense Federal University, Niteroi, Rio de Janeiro, Brazil
M.F. Lima: Federal Rural University of Rio de Janeiro, Seropedica, Rio de Janeiro, Brazil
T.C.B. Bomfim: Federal Rural University of Rio de Janeiro, Seropedica, Rio de Janeiro, Brazil

Veterinární medicína, 2013, vol. 58, issue 10, 535-542

Abstract: Differentiating between the Cryptosporidium species and their subtypes using only microscopy is impossible. Therefore, molecular tools are indispensable for accurate species and subtype diagnosis. However, if these tools are to be used correctly and accurately, the techniques used must be standardised. In the present study, two molecular techniques for diagnosing Cryptosporidium infection in cows were compared to determine the optimal methods. For each technique, we tested two DNA extraction methods, several annealing temperatures for nested PCR reactions targeting the 18S, SSU rRNA (small subunit ribosomal RNA), and the GP60 (60 kDa glycoprotein) genes, and two types of DNA staining reagents, ethidium bromide and GelRedTM. We determined that one of the tested protocols yields a higher purity of extracted DNA. Additionally, optimised temperatures for the nested PCR of the 18S and GP60 genes were established. Finally, we determined that the GelRedTM dye was more sensitive than ethidium bromide, and its low toxicity facilitates handling and disposal and reduces environmental contamination.

Keywords: 18S gene; GP60 gene; ethidium bromide; GelRedTM (search for similar items in EconPapers)
Date: 2013
References: View complete reference list from CitEc
Citations:

Downloads: (external link)
http://vetmed.agriculturejournals.cz/doi/10.17221/7085-VETMED.html (text/html)
http://vetmed.agriculturejournals.cz/doi/10.17221/7085-VETMED.pdf (application/pdf)
free of charge

Related works:
This item may be available elsewhere in EconPapers: Search for items with the same title.

Export reference: BibTeX RIS (EndNote, ProCite, RefMan) HTML/Text

Persistent link: https://EconPapers.repec.org/RePEc:caa:jnlvet:v:58:y:2013:i:10:id:7085-vetmed

DOI: 10.17221/7085-VETMED

Access Statistics for this article

Veterinární medicína is currently edited by Ing. Helena Smolová Ph.D.

More articles in Veterinární medicína from Czech Academy of Agricultural Sciences
Bibliographic data for series maintained by Ivo Andrle ().