Comparison of techniques for DNA extraction and agarose gel staining of DNA fragments using samples of Cryptosporidium
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 standardis...
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| Format: | Article |
| Language: | English |
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Czech Academy of Agricultural Sciences
2013-10-01
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| Series: | Veterinární Medicína |
| Subjects: | |
| Online Access: | https://vetmed.agriculturejournals.cz/artkey/vet-201310-0004_comparison-of-techniques-for-dna-extraction-and-agarose-gel-staining-of-dna-fragments-using-samples-of-cryptosp.php |
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| author | M.C.M. Couto A.P. Sudre M.F. Lima T.C.B. Bomfim |
| author_facet | M.C.M. Couto A.P. Sudre M.F. Lima T.C.B. Bomfim |
| author_sort | M.C.M. Couto |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-10T08:00:37Z |
| format | Article |
| id | doaj.art-4ca8cc58bfa04aa4aa551600f556e911 |
| institution | Directory Open Access Journal |
| issn | 0375-8427 1805-9392 |
| language | English |
| last_indexed | 2024-04-10T08:00:37Z |
| publishDate | 2013-10-01 |
| publisher | Czech Academy of Agricultural Sciences |
| record_format | Article |
| series | Veterinární Medicína |
| spelling | doaj.art-4ca8cc58bfa04aa4aa551600f556e9112023-02-23T03:50:09ZengCzech Academy of Agricultural SciencesVeterinární Medicína0375-84271805-93922013-10-01581053554210.17221/7085-VETMEDvet-201310-0004Comparison of techniques for DNA extraction and agarose gel staining of DNA fragments using samples of CryptosporidiumM.C.M. Couto0A.P. Sudre1M.F. Lima2T.C.B. Bomfim3Federal Rural University of Rio de Janeiro, Seropedica, Rio de Janeiro, BrazilFluminense Federal University, Niteroi, Rio de Janeiro, BrazilFederal Rural University of Rio de Janeiro, Seropedica, Rio de Janeiro, BrazilFederal Rural University of Rio de Janeiro, Seropedica, Rio de Janeiro, BrazilDifferentiating 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.https://vetmed.agriculturejournals.cz/artkey/vet-201310-0004_comparison-of-techniques-for-dna-extraction-and-agarose-gel-staining-of-dna-fragments-using-samples-of-cryptosp.php18s genegp60 geneethidium bromidegelredtm |
| spellingShingle | M.C.M. Couto A.P. Sudre M.F. Lima T.C.B. Bomfim Comparison of techniques for DNA extraction and agarose gel staining of DNA fragments using samples of Cryptosporidium Veterinární Medicína 18s gene gp60 gene ethidium bromide gelredtm |
| title | Comparison of techniques for DNA extraction and agarose gel staining of DNA fragments using samples of Cryptosporidium |
| title_full | Comparison of techniques for DNA extraction and agarose gel staining of DNA fragments using samples of Cryptosporidium |
| title_fullStr | Comparison of techniques for DNA extraction and agarose gel staining of DNA fragments using samples of Cryptosporidium |
| title_full_unstemmed | Comparison of techniques for DNA extraction and agarose gel staining of DNA fragments using samples of Cryptosporidium |
| title_short | Comparison of techniques for DNA extraction and agarose gel staining of DNA fragments using samples of Cryptosporidium |
| title_sort | comparison of techniques for dna extraction and agarose gel staining of dna fragments using samples of cryptosporidium |
| topic | 18s gene gp60 gene ethidium bromide gelredtm |
| url | https://vetmed.agriculturejournals.cz/artkey/vet-201310-0004_comparison-of-techniques-for-dna-extraction-and-agarose-gel-staining-of-dna-fragments-using-samples-of-cryptosp.php |
| work_keys_str_mv | AT mcmcouto comparisonoftechniquesfordnaextractionandagarosegelstainingofdnafragmentsusingsamplesofcryptosporidium AT apsudre comparisonoftechniquesfordnaextractionandagarosegelstainingofdnafragmentsusingsamplesofcryptosporidium AT mflima comparisonoftechniquesfordnaextractionandagarosegelstainingofdnafragmentsusingsamplesofcryptosporidium AT tcbbomfim comparisonoftechniquesfordnaextractionandagarosegelstainingofdnafragmentsusingsamplesofcryptosporidium |