METHOD FOR SAMPLE PREPARATION FOR IDENTIFICATION OF MYCOBACTERIA FROM POSITIVE BLOOD CULTURES Russian patent published in 2024 - IPC G01N33/68 G01N33/569 G01N1/28 C12Q1/04 

FIELD: medicine; microbiology.

SUBSTANCE: invention relates to medicine, namely to microbiology, phthisiology, and can be used for sample preparation for identification of mycobacteria from positive haemocultures by matrix laser desorption ionization time-of-flight mass spectrometry (maldi-tof-ms) in patients with tuberculosis and mycobacteriosis. Venous blood is sampled from the patients with suspected bloodstream infection. Obtained blood sample is introduced into flasks with a liquid nutrient medium intended for culturing mycobacteria. Flasks with blood are incubated in an automatic blood culture analyser. Upon receiving a signal on a "positive" flask, Gram and Ziehl-Neelsen staining is performed, and if acid-fast mycobacteria (AFM) are detected in the stained preparations, further sample preparation is carried out. That is ensured by collecting 5-6 ml of haemoculture into a test tube containing a coagulation activator and a separating gel; the test tube is centrifuged at 2,000-3,000 rpm for 15-20 minutes. Obtained precipitate is washed to remove residual medium and lysates of blood corpuscles, and the sample is inactivated by heating, followed by cooling and centrifugation. Obtained precipitate can be washed from residues of the commercial medium with a phosphate-salt buffer with 0.1% sodium dodecyl sulphate. Then 300-600 mcl of deionised water can be added to the precipitate, the mixture is thoroughly mixed on a shaker and the sample is inactivated. To inactivate the sample, it can be heated to temperature of 100 °C and holding at this temperature for at least 30 minutes, then cooling to room temperature and centrifuging until a precipitate is obtained. Obtained precipitate is dried to a creamy consistency. Precipitate can be dried at room temperature for 10-15 minutes. If the precipitate retains a gel-like structure, it can be held at a temperature of -70±2 °C for at least 30 minutes. Dried precipitate is applied on target cells for a mass spectrometer in triplicate, 70% formic acid is applied on top of the precipitate and an HCCA matrix is applied after drying. After complete drying, the sample is placed in a mass spectrometer and an identification procedure is carried out using a library of protein spectra of mycobacteria.

EFFECT: method provides the possibility of direct identification of mycobacteria from haemocultures, reduction of time for species identification of mycobacteria by 3-5 times and simplification of the procedure of extraction of mycobacteria due to introduction of fewer reagents and reduction of time for preparation of positive cultures of mycobacteria.

4 cl, 1 tbl, 2 ex