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Kafe 바로가기국가/구분 | United States(US) Patent 등록 |
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국제특허분류(IPC7판) |
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출원번호 | US-0094809 (2011-04-26) |
등록번호 | US-8652782 (2014-02-18) |
발명자 / 주소 |
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출원인 / 주소 |
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대리인 / 주소 |
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인용정보 | 피인용 횟수 : 0 인용 특허 : 139 |
Disclosed are compositions and methods for isolating, detecting, amplifying, and quantitating Mycobacterium-specific nucleic acids in a sample. Also disclosed are compositions and diagnostic kits comprising Mycobacterium IS6110-specific oligonucleotide amplification primers and labeled oligonucleoti
Disclosed are compositions and methods for isolating, detecting, amplifying, and quantitating Mycobacterium-specific nucleic acids in a sample. Also disclosed are compositions and diagnostic kits comprising Mycobacterium IS6110-specific oligonucleotide amplification primers and labeled oligonucleotide detection probes that specifically bind to the amplification products obtained therefrom. Also disclosed are compositions and methods for the isolation and characterization of nucleic acids that are specific to one or more tubercular pathogens, including Mycobacterium tuberculosis, in particular, from a wide variety of samples including those of biological, environmental, clinical and/or veterinary origin.
1. A method for obtaining a population of mycobacterial-specific polynucleotides from a sample suspected of containing one or more pathogenic mycobacterial cells, which comprises: contacting the sample with an effective amount of a composition that comprises: a) one or more chaotropes; b) one or mor
1. A method for obtaining a population of mycobacterial-specific polynucleotides from a sample suspected of containing one or more pathogenic mycobacterial cells, which comprises: contacting the sample with an effective amount of a composition that comprises: a) one or more chaotropes; b) one or more detergents; c) one or more reducing agents; d) one or more chelators; e) one or more buffers; and f) one or more surfactants, for a time sufficient to substantially kill or lyse the one or more pathogenic mycobacterial cells therein, but not degrade the population of mycobacterial-specific polynucleotides, and to denature or inactivate proteins, enzymes, and nucleases present therein; such that, after contact with the composition, the sample is safe for handling and transport; and the integrity of the population of mycobacterial-specific polynucleotides is at least substantially maintained when stored at a temperature from about 10° C. to about 40° C. for a period from about 1 day to about 60 days, wherein the composition further comprises a quantity of a control nucleic acid of about 50 to about 500 nucleotides in length, wherein the control nucleic acid does not substantially hybridize to nucleic acids of the sample, and comprises at least 40 contiguous nucleotides of SEQ ID NO:8, or the complement thereof. 2. The method of claim 1, wherein the (i) the one or more chaotropes comprise guanidine thiocyanate, guanidine isocyanate, guanidine hydrochloride, or any combination thereof; (ii) the one or more detergents comprise sodium dodecyl sulfate, lithium dodecyl sulfate, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium cholate, sodium alkylbenzene sulfonate, N-lauroyl sarcosine, or any combination thereof; (iii) the one or more reducing agents comprise 2-mercaptoethanol, tris(2-carboxyethyl) phosphine, dithiothreitol, dimethylsulfoxide, or any combination thereof; (iv) the one or more chelators comprise ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylene triamine pentaacetic acid, N,N-bis(carboxymethyl)glycine, ethylenediaminetetraacetic acid, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof; or (v) the one or more buffers comprise tris (hydroxymethyl)aminomethane, citrate, 2-(N-morpholino)ethanesulfonic acid, N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 1,3-bis(tris(hydroxymethyl)methyl amino)propane, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 3-(N-morpholino) propanesulfonic acid, bicarbonate, phosphate, or any combination thereof. 3. The method of claim 2, wherein the composition comprises: (a) (i) about 3M guanidine thiocyanate; (ii) about 1 mM TCEP; (iii) about 10 mM sodium citrate; (iv) about 0.5% N-lauroyl sarcosine; (v) about 0.0002% silicone polymer; (vi) about 100 mM 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS); and (vii) about 0.1 mM EDTA; or (b) (i) about 3M guanidine thiocyanate; (ii) about 1 mM TCEP; (iii) about 10 mM sodium citrate; (iv) about 0.5% N-lauroyl sarcosine, sodium salt; (v) about 0.0002% of a silicone polymer; (vi) about 100 mM TRIS; (vii) about 0.1 mM EDTA; and (viii) about 10% to about 25% ethanol (vol./vol.). 4. The method of claim 1, wherein the sample is of a biological, a clinical, or an environmental origin, and comprises one or more of phlegm, sputum, bronchial lavage, pulmonary aspirate, saliva, plasma, whole blood, serum, cells, tissues, bodily fluids, or any combination thereof. 5. The method of claim 1, wherein the sample contains at least a first Mycobacterium-specific nucleic acid segment. 6. The method of claim 5, wherein the first Mycobacterium-specific nucleic acid segment is derived from Mycobacterium tuberculosis, M. bovis, M. africanum, M. microti, M. cannetti, M. caprae, or M. pinnipedi. 7. The method of claim 1, wherein the sample is of human origin. 8. The method of claim 1, wherein the sample is obtained from a human that has, is suspected of having, or is at risk for developing tuberculosis. 9. The method of claim 8, wherein the human has, is suspected of having, or is at risk for developing a secondary bacterial, fungal, or viral infection, or any combination thereof. 10. The method of claim 1, wherein at least a first nucleic acid segment present in the population of mycobacterial-specific polynucleotides is suitable for primer-dependent amplification. 11. The method of claim 1, wherein the population of mycobacterial-specific polynucleotides remains substantially non-degraded upon storage in the composition for a period of about 5 to about 60 days, at an ambient temperature of about 10° C. to about 30° C. 12. The method of claim 1, wherein the population of mycobacterial-specific polynucleotides remains substantially non-degraded upon storage in the composition for a period of about 10 to about 40 days, at an ambient temperature of about 10° C. to about 30° C. 13. The method of claim 1, further comprising detecting within the population of mycobacterial-specific polynucleotides the presence of at least a first Mycobacterium-specific nucleic acid segment by contacting the population with a labeled oligonucleotide detection probe, wherein the presence of a labeled hybridization product is indicative of the presence of one or more Mycobacterium-specific nucleic acid segments in the population of polynucleotides. 14. The method of claim 13, wherein the labeled oligonucleotide detection probe comprises at least a first sequence region that consists of the sequence of SEQ ID NO:4 or SEQ ID NO:7. 15. The method of claim 1, wherein the control nucleic acid is about 70 to about 250 nucleotides in length. 16. The method of claim 1, wherein the control nucleic acid comprises a single-stranded DNA, a double-stranded DNA, a single-stranded RNA, a double-stranded RNA, or a double-stranded DNA:RNA hybrid. 17. The method of claim 16, wherein the control nucleic acid comprises: (a) a first sequence domain that specifically binds to a labeled oligonucleotide detection probe of from about 15 to about 35 nucleotides in length;(b) a second sequence domain that specifically binds to a forward PCR amplification primer of about 15 to about 35 nucleotides in length; and(c) a third sequence domain that specifically binds to a reverse PCR amplification primer of about 15 to about 35 nucleotides in length;wherein the second and third sequence domains are operably positioned upstream, and downstream, respectively, of the first sequence domain to facilitate a PCR-directed amplification of at least a first portion of the control nucleic acid. 18. The method of claim 1, wherein the population of mycobacterial-specific polynucleotides is obtained from the sample using an extraction apparatus. 19. The method of claim 18, wherein the extraction apparatus comprises: (a) a filtration vessel that has at least one receiving end and that comprises a membrane filter adapted to bind the population of polynucleotides thereto, wherein the membrane filter is disposed at least substantially across a width of the filtration vessel and at least partially therein; and(b) a volume-dispensing mechanism adapted to controllably dispense and forcibly inject an amount of liquid operably associated with the filtration vessel to filter the liquid there through. 20. The method of claim 1, further comprising (a) performing at least one thermal cycling step, wherein the cycling comprises at least a first amplifying step and at least a first hybridizing step, wherein the at least a first amplifying step comprises contacting the population of polynucleotides with a composition that comprises at least a pair of Mycobacterium-specific amplification primers, a thermostable polymerase, a first osmolarity agent comprising betaine, at least a first reference dye, and a plurality of deoxynucleoside triphosphates to produce at least a first Mycobacterium-specific amplification product; and(b) detecting the presence of the amplification product so produced by contacting the amplification product with a first labeled Mycobacterium-specific oligonucleotide detection probe, wherein the presence of a labeled hybridization product is indicative of the presence of one or more Mycobacterium-specific nucleic acid segments in the population of polynucleotides. 21. The method of claim 20, wherein the pair of Mycobacterium-specific amplification primers comprises a first oligonucleotide primer of 18 to about 30 nucleotides in length, and a second oligonucleotide primer of 18 to about 30 nucleotides in length, wherein each of the first and second primers specifically hybridize to a first, and a second distinct sequence region, respectively, within the sequence of SEQ ID NO:1 or the complement thereof. 22. The method of claim 20, further comprising performing a primer-dependent amplification of at least a first sequence region of the control nucleic acid in the population of polynucleotides, and quantitating the amount of amplified control nucleic acid. 23. The method of claim 22, further comprising comparing the amount of the control nucleic acid originally present in the composition to the amount of the amplified nucleic acid segment, wherein the ratio is indicative of the quantity of the population of mycobacterial-specific polynucleotides originally present in the sample. 24. The method of claim 22, wherein the primer-dependent amplification of the least a first sequence region of the control nucleic acid is performed as a single amplification reaction subsequent to the first amplifying step. 25. The method of claim 22, wherein the primer-dependent amplification of the least a first sequence region of the control nucleic acid is performed substantially simultaneously with the first amplifying step. 26. The method of claim 25, wherein the amplification product of the control nucleic acid is detected with an oligonucleotide detection probe comprising a first detectable label, and the amplification product of the Mycobacterium-specific nucleic acid segment is detected with an oligonucleotide detection probe comprising a second distinct detectable label. 27. The method of claim 22, wherein the primer-dependent amplification of at least a first sequence region of the control nucleic acid is performed using (a) a forward amplification primer that comprises a sequence region that consists essentially of the sequence of SEQ ID NO:9; (b) a reverse amplification primer that comprises a sequence region that consists essentially of the sequence of SEQ ID NO:10; and (c) a labeled oligonucleotide detection probe that comprises a sequence region that consists essentially of the sequence of SEQ ID NO:11, or the complement thereof. 28. The method of claim 1, further comprising detecting the presence of at least one drug resistance gene within the population of polynucleotides. 29. A method for detecting the presence of a Mycobacterium-specific nucleic acid segment in the population of polynucleotides obtained from the method of claim 1, comprising: (a) performing at least one thermal cycling step, wherein the cycling comprises at least a first amplifying step and at least a first hybridizing step, wherein the at least a first amplifying step comprises contacting the population of polynucleotides with a composition that comprises at least a pair of Mycobacterium-specific amplification primers, a polymerase, a first osmolarity agent comprising betaine, and a plurality of deoxynucleoside triphosphates to produce a Mycobacterium-specific amplification product when a Mycobacterium-specific nucleic acid segment is present in the sample; and(b) detecting the presence of the amplification product by contacting the amplification product with a labeled Mycobacterium-specific oligonucleotide detection probe, wherein the presence of a labeled hybridization product is indicative of the presence of one or more Mycobacterium-specific nucleic acid segments in the population of polynucleotides,wherein the pair of Mycobacterium-specific amplification primers comprises a first oligonucleotide primer of 18 to about 30 nucleotides in length, and a second oligonucleotide primer of 18 to about 30 nucleotides in length, wherein each of the first and second primers specifically hybridizes to a first, and a second sequence region, respectively, within the sequence of SEQ ID NO:1, or the complement thereof. 30. The method of claim 29, wherein the composition used in the at least one thermal cycling step further comprises a first reference dye. 31. The method of claim 30, wherein the first reference dye comprises a passive reference dye. 32. The method of claim 29, wherein (a) at least one of the pair of amplification primers comprises: (i) a first oligonucleotide primer of 18 to about 30 nucleotides in length that comprises at least a first sequence region consisting of a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO 2, or SEQ ID NO 5; or (ii) a second oligonucleotide primer of 18 to about 30 nucleotides in length that comprises at least a second sequence region consisting of a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO 3, or SEQ ID NO 6; and (b) a Mycobacterium-specific oligonucleotide detection probe of 24 to about 35 nucleotides in length that comprises at least a third sequence region consisting of a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO 4, or SEQ ID NO 7, or the complement of either SEQ ID NO 4 or SEQ ID NO 7. 33. The method of claim 29, wherein the method is compatible with at least one high throughput polymerase chain reaction technology. 34. The method of claim 29, wherein the Mycobacterium-specific nucleic acid segment is specific for M. tuberculosis, M. bovis, M. africanum, M. microti, M. cannetti, M. caprae, M. pinnipedi, or any combination thereof. 35. The method of claim 29, further comprising determining the nucleic acid sequence of the detected Mycobacterium-specific nucleic acid segment. 36. A method for obtaining a population of mycobacterial-specific polynucleotides from a sample suspected of containing one or more pathogenic mycobacterial cells, which comprises: adding a control nucleic acid to the sample and contacting the sample with an effective amount of a composition that comprises: a) one or more chaotropes; b) one or more detergents; c) one or more reducing agents; d) one or more chelators; e) one or more buffers; and f) one or more surfactants, which are together exposed to the sample for a time sufficient to substantially kill or lyse the one or more pathogenic mycobacterial cells therein, but not degrade the population of mycobacterial-specific polynucleotides, and to denature or inactivate proteins, enzymes, and nucleases present therein; such that, after contact with the composition, the sample is safe for handling and transport; and the integrity of the population of mycobacterial-specific polynucleotides is at least substantially maintained when stored at a temperature from about 10° C. to about 40° C. for a period from about 1 day to about 60 days, further comprising performing a primer-dependent amplification of at least a first sequence region of the control nucleic acid and quantitating the amount of amplified nucleic acid segment, wherein the primer-dependent amplification of at least a first sequence region of the control nucleic acid is performed using (i) a forward amplification primer that comprises a sequence region that consists essentially of the sequence of SEQ ID NO:9; (ii) a reverse amplification primer that comprises a sequence region that consists essentially of the sequence of SEQ ID NO:10; and (iii) a labeled oligonucleotide detection probe that comprises a sequence region that consists essentially of the sequence of SEQ ID NO:11, or the complement thereof. 37. The method of claim 36, wherein the a) the one or more chaotropes comprise guanidine thiocyanate, guanidine isocyanate, guanidine hydrochloride, or any combination thereof; b) the one or more detergents comprise sodium dodecyl sulfate, lithium dodecyl sulfate, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium cholate, sodium alkylbenzene sulfonate, N-lauroyl sarcosine, or any combination thereof; c) the one or more reducing agents comprise 2-mercaptoethanol, tris(2-carboxyethyl) phosphine, dithiothreitol, dimethylsulfoxide, or any combination thereof; d) the one or more chelators comprise ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylene triamine pentaacetic acid, N,N-bis(carboxymethyl)glycine, ethylenediaminetetraacetic acid, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof; or e) the one or more buffers comprise tris (hydroxymethyl)aminomethane, citrate, 2-(N-morpholino)ethanesulfonic acid, N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 1,3-bis(tris (hydroxymethyl)methyl amino)propane, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 3-(N-morpholino) propanesulfonic acid, bicarbonate, phosphate, or any combination thereof. 38. The method of claim 37, wherein the composition comprises: (a) (i) about 3 M guanidine thiocyanate; (ii) about 1 mM TCEP; (iii) about 10 mM sodium citrate; (iv) about 0.5% N-lauroyl sarcosine; (v) about 0.0002% silicone polymer; (vi) about 100 mM 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS); and (vii) about 0.1 mM EDTA; or (b) (i) about 3M guanidine thiocyanate; (ii) about 1 mM TCEP; (iii) about 10 mM sodium citrate; (iv) about 0.5% N-lauroyl sarcosine, sodium salt; (v) about 0.0002% of a silicone polymer; (vi) about 100 mM TRIS; (vii) about 0.1 mM EDTA; and (viii) about 10% to about 25% ethanol (vol./vol.). 39. The method of claim 36, wherein the sample is of a biological, a clinical, or an environmental origin, and comprises one or more of phlegm, sputum, bronchial lavage, pulmonary aspirate, saliva, plasma, whole blood, serum, cells, tissues, bodily fluids, or any combination thereof. 40. The method of claim 36, wherein the sample contains at least a first Mycobacterium-specific nucleic acid segment. 41. The method of claim 40, wherein the first Mycobacterium-specific nucleic acid segment is derived from Mycobacterium tuberculosis, M. bovis, M. africanum, M. microti, M. cannetti, M. caprae, or M. pinnipedi. 42. The method of claim 36, wherein the sample is of human origin. 43. The method of claim 36, wherein the sample is obtained from a human that has, is suspected of having, or is at risk for developing tuberculosis. 44. The method of claim 43, wherein the human has, is suspected of having, or is at risk for developing a secondary bacterial, fungal, or viral infection, or any combination thereof. 45. The method of claim 36, wherein at least a first nucleic acid segment present in the population of mycobacterial-specific polynucleotides is suitable for primer-dependent amplification. 46. The method of claim 36, wherein the population of mycobacterial-specific polynucleotides remains substantially non-degraded upon storage in the composition for a period of about 5 to about 60 days, at an ambient temperature of about 10° C. to about 30° C. 47. The method of claim 36, wherein the population of mycobacterial-specific polynucleotides remains substantially non-degraded upon storage in the composition for a period of about 10 to about 40 days, at an ambient temperature of about 10° C. to about 30° C. 48. The method of claim 36, further comprising detecting within the population of mycobacterial-specific polynucleotides the presence of at least a first Mycobacterium-specific nucleic acid segment by contacting the population with a labeled oligonucleotide detection probe, wherein the presence of a labeled hybridization product is indicative of the presence of one or more Mycobacterium-specific nucleic acid segments in the population of polynucleotides. 49. The method of claim 48, wherein the labeled oligonucleotide detection probe comprises at least a first sequence region that consists of the sequence of SEQ ID NO:4 or SEQ ID NO:7. 50. The method of claim 36, wherein the control nucleic acid is about 50 to about 500 nucleotides in length and does not substantially hybridize to nucleic acids of the sample. 51. The method of claim 36, wherein the control nucleic acid is of about 70 to about 250 nucleotides in length. 52. The method of claim 36, wherein the control nucleic acid comprises a single-stranded DNA, a double-stranded DNA, a single-stranded RNA, a double-stranded RNA, or a double-stranded DNA:RNA hybrid. 53. The method of claim 36, wherein the control nucleic acid comprises an at least 40 contiguous nucleotide sequence from SEQ ID NO:8, or the complement thereof. 54. The method of claim 36, wherein the control nucleic acid comprises: (a) a first sequence domain that specifically binds to a labeled oligonucleotide detection probe of from about 15 to about 35 nucleotides in length;(b) a second sequence domain that specifically binds to a forward PCR amplification primer of about 15 to about 35 nucleotides in length; and(c) a third sequence domain that specifically binds to a reverse PCR amplification primer of about 15 to about 35 nucleotides in length;wherein the second and third sequence domains are operably positioned upstream, and downstream, respectively, of the first sequence domain to facilitate a PCR-directed amplification of at least a first portion of the control nucleic acid from the forward and reverse primers under conditions effective to amplify the at least a first portion. 55. The method of claim 36, wherein the population of mycobacterial-specific polynucleotides is obtained from the sample using an extraction apparatus. 56. The method of claim 55, wherein the extraction apparatus comprises: (a) a filtration vessel that has at least one receiving end and that comprises a membrane filter adapted to bind the population of polynucleotides thereto, wherein the membrane filter is disposed at least substantially across a width of the filtration vessel and at least partially therein; and(b) a volume-dispensing mechanism adapted to controllably dispense and forcibly inject an amount of liquid operably associated with the filtration vessel to filter the liquid therethrough. 57. The method of claim 36, further comprising (a) performing at least one thermal cycling step, wherein the cycling comprises at least a first amplifying step and at least a first hybridizing step, wherein the at least a first amplifying step comprises contacting the population of polynucleotides with a composition that comprises at least a pair of Mycobacterium-specific amplification primers, a thermostable polymerase, a first osmolarity agent comprising betaine, at least a first reference dye, and a plurality of deoxynucleoside triphosphates to produce at least a first Mycobacterium-specific amplification product; and(b) detecting the presence of the amplification product so produced by contacting it with a first labeled Mycobacterium-specific oligonucleotide detection probe, wherein the presence of a labeled hybridization product is indicative of the presence of one or more Mycobacterium-specific nucleic acid segments in the population of polynucleotides. 58. The method of claim 57, wherein the pair of Mycobacterium-specific amplification primers comprises a first oligonucleotide primer of 18 to about 30 nucleotides in length, and a second oligonucleotide primer of 18 to about 30 nucleotides in length, wherein each of the first and second primers specifically hybridize to a first, and a second distinct sequence region, respectively, within the sequence of SEQ ID NO:1 or the complement thereof. 59. The method of claim 36, further comprising comparing the amount of the control nucleic acid originally present in the composition to the amount of the amplified nucleic acid, wherein the ratio is indicative of the quantity of the population of mycobacterial-specific polynucleotides originally present in the sample. 60. The method of claim 22, wherein the amplification product of the control nucleic acid is detected with an oligonucleotide detection probe comprising a detectable label that is distinct from the first labeled Mycobacterium-specific oligonucleotide detection probe. 61. The method of claim 36, further comprising detecting the presence of at least one drug resistance gene within the population of polynucleotides.
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