Methods and systems for removing masking agents from test samples, e.g., DNA-containing samples obtained from living subjects, when they are submitted for or subjected to molecular assays. The present invention allows molecular assays of nucleic acids in bodily fluids and excretions, such as urine,
Methods and systems for removing masking agents from test samples, e.g., DNA-containing samples obtained from living subjects, when they are submitted for or subjected to molecular assays. The present invention allows molecular assays of nucleic acids in bodily fluids and excretions, such as urine, blood, blood serum, amniotic fluid, spinal fluid, conjunctival fluid, salivary fluid, vaginal fluid, stool, seminal fluid, and sweat to be carried out with greater sensitivity. The masking agents are suppressed by contacting a test sample with an amount of one or more divalent metal chelators and an amount of one or more chelator enhancing components. The amounts of the divalent metal chelator(s) and the chelator enhancing component(s) are selected such that interference of a masking agent on a molecular assay of a nucleic acid-containing test sample are suppressed, and upon contact with the divalent metal chelator(s)/chelator enhancing component(s), the masking agents are suppressed.
대표청구항▼
What is claimed is: 1. A method of suppressing the interference of a masking agent selected from the group consisting of a leukocyte esterase, a heme protein, a myoglobin analogue, a hemoglobin analogue, a myoglobin derivative, a hemoglobin derivative, a myoglobin oxidation product, a hemoglobin ox
What is claimed is: 1. A method of suppressing the interference of a masking agent selected from the group consisting of a leukocyte esterase, a heme protein, a myoglobin analogue, a hemoglobin analogue, a myoglobin derivative, a hemoglobin derivative, a myoglobin oxidation product, a hemoglobin oxidation product, a myoglobin breakdown product, a hemoglobin breakdown product, a ferritin, methemoglobin, sulfhemoglobin, and bilirubin, on a molecular assay of a nucleic acid-containing bodily fluid, the method comprising: contacting the bodily fluid with a reagent consisting of from about 0.01 M to about 0.1 M of a divalent metal chelator and from about 0.1 M to 1.0 M of a chelator enhancing component selected from the group consisting of lithium chloride, sodium salicyl ate, and combinations thereof; wherein the interference of the masking agent on the molecular assay of the nucleic acid-containing bodily fluid is suppressed. 2. A method according to claim 1, wherein the divalent metal chelator is selected from the group consisting of ethylenediaminetetraacetic acid, imidazole, ethylenebis(oxyethylenenitriol)tetraacetic acid; iminodiacetate; and 1,2-bis(2aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; bis(5-amidino-2-benzimidazolyl)methane and salts thereof. 3. A method of suppressing the interference of a masking agent selected from the group consisting of a leukocyte esterase, a heme protein, a myoglobin analogue, a hemoglobin analogue, a myoglobin derivative, a hemoglobin derivative, a myoglobin oxidation product, a hemoglobin oxidation product, a myoglobin breakdown product, a hemoglobin breakdown product, a ferritin, methemoglobin, sulfhemoglobin, and bilirubin, on a molecular assay of a nucleic acid-containing bodily fluid, the method comprising: contacting the bodily fluid with a reagent having from about 0.01 M to about 0.1 M of a divalent metal chelator and from about 0.1 M to 1.0 M of a chelator enhancing component selected from the group consisting of lithium chloride, sodium salicylate, sodium perchlorate, sodium thiocyanate, and combinations thereof, wherein the interference of the masking agent on the molecular assay of the nucleic acid-containing bodily fluid is suppressed. 4. A method according to claim 3, wherein the divalent metal chelator is selected from the group consisting of ethylenediaminetetraacetic acid, imidazole, ethylenebis(oxyethylenenitriol)tetraacetic acid; iminodiacetate; and 1,2-bis(2aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; bis(5-amidino-2-benzimidazolyl)methane and salts thereof. 5. A method according to claim 3, wherein the divalent metal chelator is selected from the group consisting of ethylenediaminetetraacetic acid and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, and salts thereof. 6. A method decording to claim 3, wherein the masking agent is selected from the group consisting of a leukocyte esterase and a heme protein. 7. A method according to claim 3, wherein the heme protein is selected from the group consisting of a myoglobin analogue, a hemoglobin analogue, a myoglobin oxidation product, a hemoglobin oxidation product, a myoglobin breakdown product, and a hemoglobin breakdown product. 8. A method according to claim 3, wherein the masking agent is selected from the group consisting of a ferritin, methemoglobin, sulthemoglobin and bilirubin. 9. A method according to claim 3, wherein the masking agent is selected from the group consisting of methemoglobin and bilirubin. 10. A method according to claim 3, wherein the nucleic acid is selected from the group consisting of DNA, RNA, mRNA, and cDNA. 11. A method according to claim 3, wherein the nucleic acid is eukaryotic DNA. 12. A method according to claim 3, wherein the molecular assay is selected from the group consisting of a polymerase chain reaction, a ligase chain reaction, nucleic acid sequence-based amplification, strand displacement amplification, and a genetic transformation test. 13. A method according to claim 3, wherein the molecular assay comprises a polymerase chain reaction. 14. A method of improving the signal response of a molecular assay of a nucleic acid-containing bodily fluid the method comprising: contacting the nucleic acid-containing bodily fluid with a reagent consisting of from about 0.01 M to about 0.1 M of a divalent metal chelator and from about 0.1 M to 1.0 M of a chelator enhancing component selected from the group consisting of lithium chloride, sodium salicylate, and combinations thereof to form a preserved test sample, wherein the interference of a masking agent selected from the group consisting of a leukocyte esterase, a heme protein, a myoglobin analogue, a hemoglobin analogue, a myoglobin derivative, a hemoglobin derivative, a myoglobin oxidation product, a hemoglobin oxidation product, a myoglobin breakdown product, a hemoglobin breakdown product, a ferritin, methemoglobin, sulfhemoglobin, and bilirubin on the molecular assay is suppressed; extracting molecular analytes of interest from the preserved test sample; and conducting a molecular assay on the extracted molecular analytes of interest, wherein the signal response of the molecular assay is improved relative to a molecular assay performed without the reagent. 15. A method according to claim 14, wherein the divalent metal chelator is selected from the group consisting of ethylenediaminetetraacetic acid, imidazole, ethylenebis(oxyethylenenitriol)tetraacetic acid; iminodiacetate; and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; bis(5-amidino-2-benzimidazolyl)methane and salts thereof. 16. A method of improving the signal response of a molecular assay of a nucleic acid-containing bodily fluid, the method comprising: contacting the nucleic acid-containing bodily fluid with a reagent having from about 0.01 M to about 0.1 M of a divalent metal chelator and from about 0.1 M to 1.0 M of a chelator enhancing component selected from the group consisting of lithium chloride, sodium salicylate, sodium perchlorate, sodium thiocyanate, and combinations thereof to form a preserved test sample, wherein the interference of a masking agent selected from the group consisting of a leukocyte esterase, a heme protein, a myoglobin analogue, a hemoglobin analogue, a myoglobin derivative, a hemoglobin derivative, a myoglobin oxidation product, a hemoglobin oxidation product, a myoglobin breakdown product, a hemoglobin breakdown product, a ferritin, methemoglobin, sulthemoglobin, and bilirubin on the molecular assay is suppressed; extracting molecular analytes of interest from the preserved test sample; and conducting a molecular assay on the extracted molecular analytes of interest, wherein the signal response of the molecular assay is improved relative to a molecular assay performed without the reagent. 17. A method according to claim 16, wherein the divalent metal chelator is selected from the group consisting of ethylenediaminetetraacetic acid, imidazole, ethylenebis(oxyethylenenitriol)tetraacetic acid; iminodiacetate; and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; bis(5-amidino-2-benzimidazolyl)methane and salts thereof. 18. A method according to claim 16, wherein the divalent metal chelator is selected from the group consisting of ethylenediaminetetraacetic acid and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, and salts thereof. 19. A method according to claim 16, wherein the masking agent is selccted from the group consisting of a leukocyte esterase and a heme protein. 20. A method according to claim 19, wherein the heme protein is selected from the group consisting of a myoglobin analogue, a hemoglobin analogue, a myoglobin oxidation product, a hemoglobin oxidation product, a myoglobin breakdown product, and a hemoglobin breakdown product. 21. A method according to claim 16, wherein the masking agent is selected from the group consisting of a ferritin, methemoglobin, sulihemoglobin and bilirubin. 22. A method according to claim 16, wherein the masking agent is selected from the group consisting of methemoglobin and bilirubin. 23. A method according to claim 16, wherein the bodily fluid is selected from the group consisting of urine, blood, blood serum, amniotic fluid; ccrcbrospinal and spinal fluid; synovial fluid; conjunctival fluid; salivary fluid; vaginal fluid; stool; seminal fluid; lymph; bile; tears, and sweat. 24. A method according to claim 23, wherein the bodily fluid is urine. 25. A method according to claim 16, wherein the nucleic acid is selected from the group consisting of DNA, RNA, mRNA, and cDNA. 26. A method according to claim 16, wherein the nucleic acid is cukaryotic DNA. 27. A method according to claim 16, wherein the molecular assay is selected from the group consisting of a polymerase chain reaction, a ligase chain reaction, nucleic acid sequence-based amplification, strand displacement amplification, and a genetic transformation test. 28. A method according to claim 16, wherein the molecular assay comprises a polymerase chain reaction. 29. A method of suppressing the interference of a masking agent selected from the group consisting of a leukocyte esterase, a heme protein, a myoglobin analogue, a hemoglobin analogue, a myoglobin derivative, a hemoglobin derivative, a myoglobin oxidation product, a hemoglobin oxidation product, a myoglobin breakdown product, a hemoglobin breakdown product, a ferritin, methemoglobin, sulfhemoglobin, and bilirubin, on a molecular assay of a nucleic acid-containing bodily fluid, the method comprising: contacting the bodily fluid with a reagent consisting of from about 0.01 M to about 0.1 M of a chelator selected from the group consisting of ethylenediaminetetraacetic acid, imidazole, ethylenebis(oxyethylenenitriol)tetraacetic acid; iminodiacetate; and 1,2-bis(2 aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; bis(5-amidino-2-benzimidazolyl) methane and salts or combinations thereof, and from about 0.1 M to 1.0 M of a chelator enhancing component selected from the group consisting of lithium chloride, sodium salicylate, and combinations thereof; thereby suppressing the interference of the masking agent on the molecular assay of the nucleic acid-containing bodily fluid. 30. A method of performing a molecular assay on a nucleic acid-containing bodily fluid, the method comprising: suppressing the interference of a masking agent in the sample wherein the masking agent is selected from the group consisting of a leukocyte esterase, a heme protein, a myoglobin analogue, a hemoglobin analogue, a myoglobin derivative, a hemoglobin derivative, a myoglobin oxidation product, a hemoglobin oxidation product, a myoglobin breakdown product, a hemoglobin breakdown product, a ferritin, methemoglobin, sulffiemoglobin, and bilirubin, the suppressing comprising: contacting the bodily fluid with a reagent consisting of from about 0.01 M to about 0.1 M of a chelator selected from the group consisting of ethylenediaminetetraacetic acid, imidazole, ethylenebis(oxyethylenenitriol)tetraacetic acid; iminodiacetate; and 1,2-bis(2 aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; bis(5-amidino-2-benzimidazolyl) methane and salts or combinations thereof, and from about 0.1 M to 1.0 M of a chelator enhancing component selected from the group consisting of lithium chloride, sodium salicylate, and combinations thereof; and performing the molecular assay on the bodily fluid wherein the masking agent is suppressed.
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