METHOD FOR ISOLATING OF COCCIDIOIDES IMMITIS DNA FOR POLYMERASE CHAIN REACTION (PCR)

摘要

FIELD: biotechnology, microbiology.;SUBSTANCE: 100 g of dry soil sample is blended with 600 ml of lising buffer containing 300 mul of 6 M guanidine thiocyanate and 300 mum of phenol, buffered with Tris-HCl, pH 8.0 and stirring for 10 s. Mixture is incubated for 30 min at 95°C and centrifuged at 8000 g for 10 s. Then equal volume of chloroform is added, mixture is stirred and centrifuged at 8000 g for 5 min. Aqueous phase is transferred into clear test tube and extracted with equal volume of chloroform. Extraction is twice repeated and further sample is centrifuged at 8000 g for 5 min. Aqueous phase is transferred into test tube and 20 mul of buffer solution containing 10 mM of Tris, 2 mM of EDTA (pH 8.0) and 10 mg of SO₂ sorbent is added. Mixture is incubated at 60°C for 5 min with periodic shaking on vortex. After centrifugation at 8000 g for 20 s supernatant is sampled, and 100 ml of 4 M guanidine thiocyanate solution is added to precipitate. Mixture is stirred to homogenous state for 10-20 s, centrifuged under the same conditions and supernatant is sampled. Further 500 mul of buffer solution containing 10 mM of Tris, 50 mM of NaCl and 70 % of ethanol is added to precipitate. Mixture is stirred and centrifuged at 8000 g for 20 s, and rewashed. Precipitate is dried at 60°C for 10 min. Then 100 mul of deionized water is added, mixture is kept at 60°C for 10 min with periodic shaking on vortex. Slurry is centrifuged at 8000 g for 1-2 min, and supernatant is sampled for PCR.;EFFECT: increased yield of high purity nucleic acid preparation from Coccidioides immitis; increased content of inhibiting admixtures; more effective detection of coccidiomycosis by PCR method.;3 dwg, 3 ex