METHOD FOR ISOLATING OF COCCIDIOIDES IMMITIS DNA FOR POLYMERASE CHAIN REACTION (PCR) Russian patent published in 2007 - IPC C12N15/10 C12R1/645 

Abstract RU 2295569 C1

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

Similar patents RU2295569C1

Title Year Author Number
METHOD OF ISOLATION OF DNA FROM MICROORGANISMS AND ANIMAL CELLS SUITED FOR POLYMERASE CHAIN REACTION CARRYING OUT 1997
  • Shestopalov M.Ju.
  • Balakhonov S.V.
  • Kalinovskij A.I.
  • Golubinskij E.P.
RU2129610C1
OLIGONUCLEOTIDE PRIMERS FOR IDENTIFYING Coccidioides posadasii 2007
  • Tkachenko Galina Aleksandrovna
  • Savchenko Sergej Sergeevich
  • Grishina Marina Anatol'Evna
  • Antonov Valerij Alekseevich
RU2346045C1
OLIGONUCLEOTIDE PRIMERS FOR INDENTIFYING STIMULANTS OF COCCIDIOIDOMYCOSIS Coccidiodes immitis AND Coccidioides posadasii 2007
  • Tkachenko Galina Aleksandrovna
  • Savchenko Sergej Sergeevich
  • Grishina Marina Anatol'Evna
  • Antonov Valerij Alekseevich
RU2346046C1
METHOD FOR RECOVERY OF NUCLEIC ACIDS 2014
  • Khripko Jurij Ivanovich
  • Bateneva Natalja Vladimirovna
  • Smirnov Pavel Nikolaevich
RU2584346C2
METHOD FOR PREDICTING ONCOLOGICAL DISEASES 2004
  • Belokhvostov A.S.
  • Kuznetsova G.V.
RU2245551C1
METHOD FOR EXTRACTION OF NUCLEIC ACIDS FROM NAIL PLATES 2020
  • Domonova Elvira Alekseevna
  • Silveistrova Olga Iurevna
  • Shipulina Olga Iurevna
RU2751244C1
SET FOR DETECTING LEGIONELLOSIS AGENT IN BIOLOGICAL MATERIAL AND ENVIRONMENTAL MEDIUM 2009
  • Portenko Svetlana Anatol'Evna
  • Osina Natal'Ja Aleksandrovna
  • Bugorkova Tat'Jana Vasil'Evna
  • Kutyrev Vladimir Viktorovich
  • Jatsyshina Svetlana Borisovna
  • Shipulin German Aleksandrovich
RU2397251C1
METHOD FOR MYCOPLASMA HOMINIS DNA DETECTION FROM BLOOD SAMPLES 2013
  • Gerasimova Natal'Ja Avenirovna
  • Evstigneeva Natal'Ja Petrovna
RU2533238C1
COMPOSITION FOR REMOVING DNA- AND/OR RNA-CONTAINING BIOLOGICAL MATERIAL (VARIANTS) 2021
  • Kornienko Igor Valerievich
  • Faleeva Tatyana Georgievna
  • Aramova Olga Yurevna
RU2789387C1
METHOD OF DIFFERENTIATION OF TOXIGENIC GENETICALLY MODIFIED STRAINS VIBRIO CHOLERAE OF BIOVAR EL TOR WITH DIFFERENT EPIDEMIC POTENTIAL BY METHOD OF MULTIPLEX POLYMERASE CHAIN REACTION AND TEST SYSTEM FOR ITS IMPLEMENTATION 2014
  • Smirnova Nina Ivanovna
  • Agafonov Dmitrij Anatol'Evich
  • Shashkova Alena Vladimirovna
  • Zadnova Svetlana Petrovna
  • Cheldyshova Nadezhda Borisovna
RU2556127C2

RU 2 295 569 C1

Authors

Tkachenko Galina Aleksandrovna

Grishina Marina Anatol'Evna

Savchenko Sergej Sergeevich

Antonov Valerij Alekseevich

Dates

2007-03-20Published

2005-07-12Filed