METHOD OF MINIMALLY INVASIVE ISOLATION OF BACTERIAL DNA FROM TESTICULAR TISSUE BIOPSY IN INFERTILE MEN Russian patent published in 2023 - IPC C12N15/00 G01N33/68 

FIELD: medicine; urology.

SUBSTANCE: invention relates to the section of surgical andrology using the methodology of molecular biology and clinical laboratory diagnostics, and can be used in assisted reproductive technology centers for the treatment of infertility in infertile men with severe spermatogenesis disorders. A sample of testicular testicular tissue is collected intraoperatively by testicular biopsy, followed by sampling from the urethra, and processed in saline under sterile conditions and cryopreserved at a temperature not exceeding -30°C. Purification of intact DNA from testicular tissue is performed using TheReliaPrep kit™ gDNATissueMiniprepSystem, Promega, extracting an intact DNA sample using mini-columns in a benchtop microcentrifuge. About 160 μl of TE buffer and 20 μl of lysozyme with a concentration of 50 mg/ml are added to the testicular tissue sample, mix thoroughly, vortex, incubate at 37°C for 1–2 hours. About 20 µl of Proteinase K solution and 200 µl of Lysis buffer are added and vortexed for 10 seconds. The mixture is incubated again at 56°C for 2 hours, 20 µl of RNase A solution are added, mixed on vortex for 10 seconds, followed by incubation for 10 minutes at 56°C, 250 µl of binding buffer (BBA) are immediately added, vortexed for 1 minute and centrifuged. Mini-columns are placed inside the sample tube and the liquid is transferred to the column, centrifuged for 1 minute twice at 3,000 rpm. for 1 min and 500 μl of column washing solution (CWD) is added, centrifuged for 2 minutes and the liquid is separated. The column is transferred to a microcentrifuge tube, 50 μl of water is added, and centrifuged. The resulting bacterial DNA elution from testicular tissue and a urethral swab sample are cryopreserved for subsequent analysis.

EFFECT: method makes it possible to reduce labor intensity, improve the prediction of diagnostic results of reproductive potential, as well as the validity of the program for choosing a treatment algorithm and increasing medical and social rehabilitation in a shorter period of time.

1 cl, 2 ex