A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis.
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 100 degrees C, 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 10(2) leptospira, using the silver stain procedure.
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Laboratório de Zoonoses, Departamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, MG, 31270-901, Brasil. profisio@brhs.co.br
Lopes
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Moreira
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D002725:Chloroform; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D010450:Endopeptidases; D007919:Leptospira / Q000235:genetics / Q000302:isolation & purification*; D019800:Phenol; D010944:Plants / Q000201:enzymology; D016133:Polymerase Chain Reaction / Q000379:methods*
D004269:DNA, Bacterial; D019800:Phenol; D002725:Chloroform; D010450:Endopeptidases; D019286:Endopeptidase K