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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060
EISSN: 1678-8060
Vol. 95, No. 3, 2000, pp. 339-343
Bioline Code: oc00056
Full paper language: English
Document type: Research Article
Document available free of charge

Memórias do Instituto Oswaldo Cruz, Vol. 95, No. 3, 2000, pp. 339-343

 en A Comparison of Three DNA Extractive Procedures with Leptospira for Polymerase Chain Reaction Analysis
IF Veloso; MTP Lopes; CE Salas & EC Moreira

Abstract

Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 102 leptospira, using the silver stain procedure.

Keywords
polymerase chain reaction, leptospira, DNA extraction

 
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