Resumen
FIELD: medicine. ^ SUBSTANCE: bacteria freely immobilised on a slide are processed with a lysis solution containing an ionogenic protein-denaturing detergent and EDTA. Further, DNA-nucleoid is dry heating and microwave incubation stabilised. DNA is stained by high-sensitivity DNA-specific fluorochrome, in conclusion DNA integrity is evaluated. Offered is a kit for evaluating the DNA bacteria integrity which comprises all the components required. ^ EFFECT: invention can be used for determining the bacterial DNA fragmentation levels by a precise express technique. ^ 16 cl, 11 dwg, 5 tbl, 9 ex
Reivindicaciones
CLAIMS 1. 1.- A process for evaluating the DNA integrity of microorganisms comprising the steps of: a) immobilizing the microorganism on a slide, without fixing, by means of including it in an inert medium, b) treating with a lysis solution to extract cell walls, membranes and proteins; c) stabilizing the DNA nucleoid of the microorganism on the slide; and d) staining and evaluating the DNA integrity. 2. 2.- A process according to claim 1, wherein step a) is optional 3. 3.- A process according to any of claims 1 and 2, wherein the lysis solution comprises an ionic protein denaturing detergent. 4. 4.- A process according to any of claims 1 to 3, wherein the ionic detergent is a detergent selected from the group of sodium dodecyl sulfate (SDS), alkylbenzene sulfate, lauryl sarcosine (sarkosyl), hydrated salt of glycolic acid, and mixtures thereof. 5. 5.- A process according to claim 4, wherein the ionic detergent is preferably sodium dodecyl sulfate (SDS). 6. 6.- A process according to any of claims 1 to 5, wherein the lysis solution comprises between 0.001 and 2M dithiothreitol (DTT); between 0.001 and 2M 2- amino- 2(hydroxymethyl)-1,3-propanediol (Tris); between 0.001 and 2M ethylenediaminetetraacetic acid (EDTA), and between 0.1 and 3% sodium dodecyl sulfate (SDS). 7. 7.- A process according to claim 6, wherein the lysis solution is adjusted to a pH between 6.5 and 10.5. 8. 8.- A process according to any of claims 6 and 7, wherein the lysis solution preferably comprises 0.1 M dithiothreitol (DTT); 0.01 M (hydroxymethyl)-1,3- propanediol (Tris); 0.05M ethylenediaminetetraacetic acid (EDTA) and 2% sodium dodecyl sulfate (SDS). 9. 9.- A process according to claim 8, wherein the lysis solution is adjusted to a pH of about 10 with NaOH. 10. 10.- A process according to claim 1, wherein the staining of step d) is carried out with a fluorochrome solution. 11. 11.- A process according to claim 1, wherein the sample containing the microorganisms is included in an inert microgel 12. 12.- A process according to claim 11, wherein the sample containing the microorganisms is preferably included in an agarose microgel. 13. 13.- A process for evaluating the DNA integrity of microorganisms according to claim 1, wherein the stabilization and adherence of the DNA of the microorganism is carried out quickly by means of dry heat, incubating the slide with the lysed sample in a microwave oven. 14. 14.- A process for evaluating the DNA integrity of microorganisms according to claim 1, wherein the evaluation is carried out by means of a direct visual analysis. 15. 15.- A process for evaluating the DNA integrity of microorganisms according to claim 1, wherein the evaluation is carried out in an automated manner by means of applying software for analyzing digitized images, obtained by means of cameras coupled to microscopy platforms. 16. 16.- A computer program executing the evaluation of the DNA integrity of microorganisms in the previously claimed process. 17. 17.- A kit for evaluating the DNA integrity of microorganisms comprising. a) pretreated slides; b) agarose solution, c) lysis solution; and d) fluorochrome. 18. 18.- A kit according to claim 16, wherein the lysis solution comprises between 0.001 and 2M dithiothreitol (DTT); between 0.001 and 2M 2-amino- 2(hydroxymethyl)- 1,3-propanediol (Tris); between 0.001 and 2M ethylenediaminetetraacetic acid (EDTA), and between 0.1 and 3% sodium dodecyl sulfate (SDS). 19. 19.- A kit according to any of claims 16 and 17, wherein the lysis solution is adjusted to a pH between 6 5 and 10.5. 20. 20.- A kit according to any of claims 16 to 18, wherein the lysis solution preferably comprises 0.1 M dithiothreitol (DTT); 0.01 M (hydroxymethyl)-1,3- propanediol (Tris), 0.05M ethylenediaminetetraacetic acid (EDTA), and 2% sodium dodecyl sulfate (SDS). 21. 21.- A kit according to any of claims 16 to 19, wherein the lysis solution is adjusted to a pH of about 10 with NaOH.
