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 fol lowed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-c hloroform; 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 proteina se K or E6870 afforded positive polymerase chain reaction (PCR) reaction. O ur the other hand from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evalu ated by RAPD-PCR, DNAs from serovars isolated with proteinase K and protein ase 6870 with primers B11/B12. Each of the DNA samples provided PCR profile s in agreement with previous data. Moreover; the results with E6870 showed less background non-specific amplification, suggesting that removal of nucl eases was more efficient with E6870. The limit for detection by PCR using L ep13/Lep14 was determined to be 10(2) leptospira, using the silver stain pr ocedure.