METHOD simultaneous detection MYCOBACTERIA tuberculosis complex and identification of mutations in mycobacterial DNA, leading to sustainability of microorganisms to rifampicin and isoniazid, microarray, primer set, biochips SET oligonucleotide probes used in the method

摘要

1. method of simultaneous u043eu0431u043du0430u0440u0443u0436u0435u043du0438u00a0 u043cu0438u043au043eu0431u0430u043au0442u0435u0440u0438u0439 tuberculosis complex and the identification of mutations in the dna u043cu0438u043au043eu0431u0430u043au0442u0435u0440u0438u0439, u043fu0440u0438u0432u043eu0434u00a0u0449u0438u0445 to persistence of microorganisms to u0440u0438u0444u0430u043cu043fu0438u0446u0438u043du0443 and isoniazid in clinical samples, including;(a) - u043cu0443u043bu044cu0442u0438u043fu043bu0435u043au0441u043du0443u044e u0430u043cu043fu043bu0438u0444u0438u043au0430u0446u0438u044e fragments of genes rpoB, katG, inhA, ahpC, mobile elements IS6110 using a set of pairs of specific u043fu0440u0430u0439u043cu0435u0440u043eu0432 u0434u043bu00a0 first stage pcr.;(b) u043cu0443u043bu044cu0442u0438u043fu043bu0435u043au0441u043du0443u044e u0430u043cu043fu043bu0438u0444u0438u043au0430u0446u0438u044e fragments of genes rpoB, katG, inhA, ahpC, mobile elements IS6110 using as the matrix product of pcr obtained in the stage (a) and the set of pairs of specific u043fu0440u0430u0439u043cu0435u0440u043eu0432 u0434u043bu00a0 second stage pcr, with one primer in each pair u043fu0440u0430u0439u043cu0435u0440u043eu0432 u00a0u0432u043bu00a0u0435u0442u0441u00a0 fluorescent branded, gaining the advantage u043eu0434u043du043eu0446u0435u043fu043eu0447u0435u0447u043du044bu0445 fluorescent labelled dna fragments;;(b) ensuring the u0431u0438u043eu0447u0438u043fu0430 u0434u043bu00a0 simultaneous u043eu0431u043du0430u0440u0443u0436u0435u043du0438u00a0 u043cu0438u043au043eu0431u0430u043au0442u0435u0440u0438u0439 tuberculosis complex and the identification of mutations u043fu0440u0438u0432u043eu0434u00a0u0449u0438u0445 to sustainability and to u0440u0438u0444u0430u043cu043fu0438u0446u0438u043du0443 isoniazid, u043fu0440u0435u0434u0441u0442u0430u0432u043bu00a0u044eu0449u0435u0433u043e a substrate with u0433u0435u043bu0435u0432u044bu043cu0438 elements, each of which u0438u043cu043cu043eu0431u0438u043bu0438u0437u043eu0432u0430u043d unique u043eu043bu0438u0433u043eu043du0443u043au043bu0435u043eu0442u0438u0434u043du044bu0439 probe has consistently ism. selected from groupincluding the sequence: (a) the sequence of the gene rpoB wild type; b) the sequence of the fragment u043cu0443u0442u0430u043du0442u043du043eu0433u043e variants of the gene rpoB, u043fu0440u0438u0432u043eu0434u00a0u0449u0435u0433u043e to persistence of microorganisms to u0440u0438u0444u0430u043cu043fu0438u0446u0438u043du0443; b) the complementary u043fu043eu0441u043bu0435u0434u043eu0432u0430u0442u0435u043bu044cu043du043eu0441u0442u00a0u043c,described in (a) and (b); (d) the sequence of the gene katG wild type; d) the sequence of the fragment u043cu0443u0442u0430u043du0442u043du043eu0433u043e varus ianthe gene katG, u043fu0440u0438u0432u043eu0434u00a0u0449u0435u0433u043e to persistence of microorganisms to isoniazid; (e) the complementary u043fu043eu0441u043bu0435u0434u043eu0432u0430u0442u0435u043bu044cu043du043eu0441u0442u00a0u043c,described in a) and d); f) the sequence of the gene inhA wild type; 3) the sequence of the fragment u043cu0443u0442u0430u043du0442u043du043eu0433u043e varus ianthe gene inhA, u043fu0440u0438u0432u043eu0434u00a0u0449u0435u0433u043e to persistence of microorganisms to isoniazid and complementary u043fu043eu0441u043bu0435u0434u043eu0432u0430u0442u0435u043bu044cu043du043eu0441u0442u00a0u043c,);described in the right) and 3); to the relevant sequence of gene fragment ahpC wild type; l) the consistency of the u043cu0443u0442u0430u043du0442u043du043eu0433u043e varus ianthe gene ahpC, u043fu0440u0438u0432u043eu0434u00a0u0449u0435u0433u043e to persistence of microorganisms to isoniazid; m) of the complementary u043fu043eu0441u043bu0435u0434u043eu0432u0430u0442u0435u043bu044cu043du043eu0441u0442u00a0u043c,described in) and l); h) the consistency of the mobile element IS6110; (o) the complementary sequence, as described in n).;(g) - u0433u0438u0431u0440u0438u0434u0438u0437u0430u0446u0438u044e u0430u043cu043fu043bu0438u0444u0438u0446u0438u0440u043eu0432u0430u043du043du044bu0445 marked product obtained in stage (b), u0431u0438u043eu0447u0438u043fu0435 in u0443u0441u043bu043eu0432u0438u00a0u0445 to permit one u043du0443u043au043bu0435u043eu0442u0438u0434 between u043eu0431u0440u0430u0437u0443u044eu0449 as a result of hybridization u0438u043cu0438u0441u00a0 perfect and imperfect u0434u0443u043fu043bu0435u043au0441u0430u043cu0438;;(d) - recording and interpretation of the results of hybridization.;2. method for u0445u0430u0440u0430u043au0442u0435u0440u0438u0437u0443u044eu0449u0438u0439u0441u00a0 1, so that at the first stage u043cu0443u043bu044cu0442u0438u043fu043bu0435u043au0441u043du043eu0439 pcr (a) using a set of pairs of sequence specific u043fu0440u0430u0439u043cu0435u0440u043eu0432, which u043fu0440u0435u0434u0441u0442u0430u0432u043b u0435u043du044b SEQ id no: 70, 71, 74, 75, 77, 78, 80, 81, 83, 84.;3. method for u0445u0430u0440u0430u043au0442u0435u0440u0438u0437u0443u044eu0449u0438u0439u0441u00a0 1, so that at the second stage, u043cu0443u043bu044cu0442u0438u043fu043bu0435u043au0441u043du043eu0439 pcr (b) use a set of pairs of sequence specific u043fu0440u0430u0439u043cu0435u0440u043eu0432, which u043fu0440u0435u0434u0441u0442u0430u0432u043b u0435u043du044b SEQ id no: 72, 73, 74, 76, 77, 79, 80, 82, 83, 85.;4. method for u0445u0430u0440u0430u043au0442u0435u0440u0438u0437u0443u044eu0449u0438u0439u0441u00a0 1, so that at the second stage, u043cu0443u043bu044cu0442u0438u043fu043bu0435u043au0441u043du043eu0439 pcr (b), with a view to u043fu043eu043bu0443u0447u0435u043du0438u00a0 mainly u043eu0434u043du043eu0446u0435u043fu043eu0447u0435u0447u043du044bu0445 fluorescent marked u0444u0440u0430u0433u043cu0435u043d in the u0434u043bu00a0 all used pairs of u043fu0440u0430u0439u043cu0435u0440u043eu0432, fluorescent u043cu0435u0447u0435u043du043du044bu0439 primer used in u043cu043eu043bu00a0u0440u043du043eu043c abundance relative to the second u043fu0440u0430u0439u043cu0435u0440u0443, u0441u043eu0441u0442u0430u0432u043bu00a0u044eu0449u0435u043cu0443 the same couple.;5. method for 1, in which u0430u043cu043fu043bu0438u0444u0438u043au0430u0446u0438u044e fragments of genes and the cell element IS6110 u043fu0440u043eu0432u043eu0434u00a0u0442, u0438u0441u043fu043eu043bu044cu0437u0443u00a0 directly material clinical samples (sputum, u044du043au0441u0441u0443 dates, flushing, u0431u0440u043eu043du0445u043e - u0430u043bu044cu0432u0435u043eu043bu00a0u0440u043du044bu0439 lavage) or pre u0432u044bu0440u0430u0449u0435u043du043du0443u044e culture of micro-organisms.;6. method for u0445u0430u0440u0430u043au0442u0435u0440u0438u0437u0443u044eu0449u0438u0439u0441u00a0 1, so that the biochip contains a set of u0438u043cu043cu043eu0431u0438u043bu0438u0437u043eu0432u0430u043du043du044bu0445 u043eu043bu0438u0433u043eu043du0443u043au043bu0435u043eu0442u0438u0434u043eu0432, sequences which are represented SEQ id no: 1 to 69.;7. method for u0445u0430u0440u0430u043au0442u0435u0440u0438u0437u0443u044eu0449u0438u0439u0441u00a0 1, so that, with a view to u043eu0431u0435u0441u043fu0435u0447u0435u043du0438u00a0 u0440u0430u0437u0440u0435u0448u0435u043du0438u00a0 one u043du0443u043au043bu0435u043eu0442u0438u0434 u043eu0431u0440u0430u0437u0443u044eu0449u0438u043cu0438u0441u00a0 resulting from hybridization between committed and u043du0435u0441u043eu0432u0435u0440u0448u0435u043d various u0434u0443u043fu043bu0435u043au0441u0430u043cu0438, use u0433u0438u0431u0440u0438u0434u0438u0437u0430u0446u0438u043eu043du043du044bu0439 buffer u043fu043eu0437u0432u043eu043bu00a0u044eu0449u0438u0439 to u0433u0438u0431u0440u0438u0434u0438u0437u0430u0446u0438u044e in an extended temperature range.;8. method for u0445u0430u0440u0430u043au0442u0435u0440u0438u0437u0443u044eu0449u0438u0439u0441u00a0 1, so that the registration results on the stage (d) u043fu0440u043eu0432u043eu0434u00a0u0442 with portable analyzer and the software is u0444u043bu0443u043eu0440u0435u0441u0446u0435u043du0446u0438u0438 u0438u00a0 that u043fu043eu0437u0432u043eu043bu00a0u0435u0442 using software processing u0438u043du0442u0435u043du0441u0438u0432u043du043eu0441u0442u0435u0439 signals and subsequent interpretation of results.;9. way to 1, where the interpretation of the reported results on the stage (d) u0432u044bu043fu043eu043bu043du00a0u044eu0442 by u0441u0440u0430u0432u043du0435u043du0438u00a0 u0444u043bu0443u043eu0440u0435u0441u0446u0435u043du0446u0438u0438 u00a0u0447u0435u0435u043a intensity signals, in which the u0437u043eu0432u0430u043bu0438u0441u044c perfect and imperfect u0433u0438u0431u0440u0438u0434u0438u0437u0430u0446u0438u043eu043du043du044bu0435 located.;10. method for 1, in which u0438u043du0442u0435u0440u043fu0440u0435u0442u0438u0440u043eu0432u0430u043du043du044bu0435 results can be applied u0434u043bu00a0 u043fu043eu0434u0442u0432u0435u0440u0436u0434u0435u043du0438u00a0 clinical diagnosis tuberculosis epidemiological u0433u0435u043du043eu0442 and goals u0438u043fu0438u0440u043eu0432u0430u043du0438u00a0, u0438u0441u043fu043eu043bu044cu0437u0443u00a0 as a marker of the presence of a mutation.;11. a set of specific pairs of u043fu0440u0430u0439u043cu0435u0440u043eu0432 u0434u043bu00a0 u043eu0441u0443u0449u0435u0441u0442u0432u043bu0435u043du0438u00a0 method simultaneously u043eu0431u043du0430u0440u0443u0436u0435u043du0438u00a0 u043cu0438u043au043eu0431u0430u043au0442u0435u0440u0438u0439 tuberculosis complex and the identification of mutations in the dna u043cu0438u043au043eu0431 u0430u043au0442u0435u0440u0438u0439, u043fu0440u0438u0432u043eu0434u00a0u0449u0438u0445 to u0440u0438u0444u0430u043cu043fu0438u0446u0438u043du0443 and isoniazid resistance in clinical samples and the sequence of u043fu0440u0430u0439u043cu0435u0440u043eu0432 lyrics are brought to SEQ id no: 70 - 85.;12.biochip used in the method of the simultaneous u043eu0431u043du0430u0440u0443u0436u0435u043du0438u00a0 u043cu0438u043au043eu0431u0430u043au0442u0435u0440u0438u0439 tuberculosis complex and the identification of mutations in the dna u043cu0438u043au043eu0431u0430u043au0442u0435u0440u0438u0439, u043fu0440u0438u0432u043eu0434u00a0u0449u0438u0445 to u0443u0441u0442u043eu0439u0447 u0438u0432u043eu0441u0442u0438 to u0440u0438u0444u0430u043cu043fu0438u0446u0438u043du0443 and isoniazid in clinical samples u043fu0440u0435u0434u0441u0442u0430u0432u043bu00a0u044eu0449u0438u0439 a substrate with u0433u0435u043bu0435u0432u044bu043cu0438 elements, each of which u0438u043cu043cu043eu0431u0438u043bu0438u0437u043eu0432u0430u043d unique u043eu043bu0438u0433 u043eu043du0443u043au043bu0435u043eu0442u0438u0434u043du044bu0439 probethe sequence of the probes are SEQ id no: 1 to 69.;13. a set of u043eu043bu0438u0433u043eu043du0443u043au043bu0435u043eu0442u0438u0434u043du044bu0445 probes used u0434u043bu00a0 u043fu043eu043bu0443u0447u0435u043du0438u00a0 u0431u0438u043eu0447u0438u043fu0430 used in the method of simultaneous u043eu0431u043du0430u0440u0443u0436u0435u043du0438u00a0 u043cu0438u043au043eu0431u0430u043au0442u0435u0440u0438u0439 tuberculosis complex and the identification of mutations in the dna u043cu0438u043au043eu0431u0430u043au0442u0435u0440u0438u0439, u043fu0440u0438u0432u043eu0434u00a0u0449u0438u0445 to u0440u0438u0444u0430u043cu043fu0438u0446u0438u043du0443 and isoniazid resistance in clinical samples with the probes are SEQ sequence id no: 1.