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1. WO2021061797 - COMPOSITION ET PROCÉDÉ POUR AMÉLIORER LA DÉTECTION DE BIOMOLÉCULES DANS DES ÉCHANTILLONS DE FLUIDES BIOLOGIQUES

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

WHAT IS CLAIMED IS:

1. A method of detecting a nucleic acid in a biofluid sample, the method comprising adding to the biofluid sample a composition comprising a sufficient amount of dextran sulphate to provide between 50 nM and 5 mM dextran sulphate when the composition is added to the biofluid sample.

2. A method for improving detection of nucleic acid in a biofluid sample, the method comprising adding to the biofluid sample a composition comprising a sufficient amount of dextran sulphate to provide between 50 nM and 5 pM dextran sulphate when the composition is added to the biofluid sample, wherein the detection in increased relative to a reference sample.

3. The method of claim 1 or 2, wherein the composition further comprises:

a. a buffer agent;

b. a chelating agent;

c. a sufficient amount of boric acid to provide between 4 mM and 400 mM boric acid when the composition is added to the biofluid sample;

d. a sufficient amount of sodium citrate to provide between 4 mM and 400 mM sodium citrate when the composition is added to the biofluid sample; and e. a sufficient amount of lithium chloride to provide between 1 mM and 100 mM lithium chloride when the composition is added to the biofluid sample.

4. The method of any one of claims 1-3, wherein the buffer agent comprises a sufficient amount of Tris-HCl (pH 7.0) to provide between 8mM and 800 mM Tris-HCl (pH 7.0) when the composition is added to the biofluid sample.

5. The method of any one of claims 1-4, wherein the chelating agent comprises a sufficient amount of ethyl enedi ami netetraacetic acid (EDTA) to provide between 0.4 mM and 40 mM EDTA when the composition is added to the biofluid sample.

6. The method of claim 5, wherein the composition comprises:

a. a sufficient amount of Tris-HCl (pH 7.0) to provide about 80 mM Tris-HCl (pH 7.0) when the composition is added to the biofluid sample;

b. a sufficient amount of EDTA to provide about 4 mM EDTA when the composition is added to the biofluid sample;

c. a sufficient amount of boric acid to provide about 40 mM boric acid when the composition is added to the biofluid sample;

d. a sufficient amount of sodium citrate to provide about 40 mM sodium citrate when the composition is added to the biofluid sample;

e. a sufficient amount of dextran sulphate to provide about 500 nM dextran sulphate when the composition is added to the biofluid sample; and

f. a sufficient amount of lithium chloride to provide about 10 mM lithium chloride when the composition is added to the biofluid sample.

7. The method of any one of claims 1-6, wherein the composition further comprises:

a. a sufficient amount of aurintricarboxylic acid to provide between ImM and 100 mM aurintricarboxylic acid when the composition is added to the biofluid sample; b. a sufficient amount of polyvinylpyrrolidone to provide between 0.01% and 10% (w/v) polyvinylpyrrolidone when the composition is added to the biofluid sample; and

c. a sufficient amount of Tris (2-carboxyl ethyl) phosphine to provide between 100 nM and 1 mM Tris (2-carboxylethyl) phosphine when the composition is added to the biofluid sample.

8. The method of claim 7, wherein the composition comprises:

a. a sufficient amount of aurintricarboxylic acid to provide about 10 mM aurintricarboxylic acid when the composition is added to the biofluid sample; b. a sufficient amount of polyvinylpyrrolidone to provide about 0.25% (w/v) polyvinylpyrrolidone when the composition is added to the biofluid sample; and c. a sufficient amount of Tris (2-carboxyl ethyl) phosphine to provide about 500 nM Tris (2-carboxylethyl) phosphine when the composition is added to the biofluid sample.

9. The method of any one of claims 1-8, wherein the addition of the composition to the biofluid sample results in improved DNA or RNA detection in the biofluid sample, as measured by % amount of DNA or RNA detected after 3 or 7 days, compared to the amount of DNA or RNA detected at time 0, wherein the % amount of DNA or RNA detected after 3 or 7 days is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 100% of the amount of DNA or RNA that is detected at time 0.

10. The method of any one of claims 1-9, wherein the biofluid sample is urine.

11. The method of any one of claims 1-10, wherein the biofluid sample comprises fecal contaminant.

12. The method of any one of claims 1-11, wherein the composition is powder.

13. The method of any one of claims 1-11, wherein the composition is a tablet.

14. The method of any one of claims 1-11, wherein the composition is a gel.

15. The method of anyone of claims 1-11, wherein the composition is an aqueous solution.

16. The method of any one of claims 1-15, wherein the method further comprises detecting or having detected one or more target DNA or RNA sequence(s) in the biofluid sample after a period of time.

17. The method of claim 16, wherein the period of time is more than 1 hour, 1.5 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, or 5 years.

18. The method of claim 16 or 17, wherein the biofluid sample is stored at a temperature greater than 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, or 40 °C, during the period of time.

19. The method of claim 16, wherein the target DNA or RNA is detected using next- generation sequencing (NGS), real-time PCR, or an isothermal amplification method.

20. The method of any one of claims 16-19, wherein the one or more target DNA or RNA sequence(s) are DNA or RNA sequence(s) of an infectious agent.

21. The method of claim 20, wherein the infectious agent is a virus, a bacteria, or a fungus.

22. The method of claim 21, where the reference sample is not infected by the infectious agent.

23. The method of claim 21 or 22 , wherein the virus is Herpesviridae, Epstein barr virus, Adenovirus, Cytomegalovirus, Human papilloma virus, Enterovirus, Zika virus, Polyomaviruses such as BK virus, Coxsackie A viruses, Hepatitis viruses, Arbovirus, Parvovirus B19, Reovirus, Measles virus, Gastrointestinal viruses, Influenza, Parainfluenza, Mumps, Respiratory syncytial virus, Adenoviruses, Coronaviruses, Enteroviruses, Hemorrhagic fever viruses , Ebola virus, Hantavirus, Dengue, chikungunya, West Nile virus, Nipah virus, Yellow fever virus, Hepatitis A, Hepatitis B, Hepatitis C, Noroviruses, Rabies, Rhinovirus, Birnaviruses (rotaviruses) SARS-CoV-1, SARS-CoV-2, or Middle East Respiratory Syndrome (MERS).

24. The method of claim 23, wherein the virus is SARS-CoV-2.

25. The method of claim 21 or 22, wherein the bacteria is Mycobacteria, Serratia, Staphylococci, Streptococci, Klebsiella, Escherechia, Neisseria, Shigella, Salmonella, Helicobacteria, Listeria, Morganella, Pseudomonas, Acinetobacter, Bacteroides, Burkholderia, Clositridium, Enterobacteriaceae, Enterococcus, Stenotrophomonas. In certain instances, the infectious agent can be a fungus such as Entomophtoromycota, Ascomycota, Basdiomycota, Mucorales, or Histoplasma.

26. The method of claim 21 or 22, wherein the fungus is Entomophtoromycota, Ascomycota, Basdiomycota, Mucorales, or Histoplasma.

27. The method of claim 16, wherein the one or more target DNA sequence(s) are genomic DNA sequences or genomic DNA sequences containing at least one somatic mutation, of a subject from whom the biofluid sample is obtained.

28. The method of any one of claims 1-27, wherein the composition is added to the biofluid sample within one hour after obtaining the biofluid sample from a subject.

29. A method of treating a subject for a condition, the method comprising:

a. obtaining or having obtained a biofluid sample from a subject;

b. performing the method of any one of claims 1-28;

c. determining or having determined the level of a nucleic acid in the biofluid sample, thereby providing a diagnosis for the condition; and

d. treating or having treated the subject for the condition according to the diagnosis.

30. The method of claim 29, wherein the condition is a urinary tract infection, genital tract infection, renal infection, prostate cancer, or cervical cancer.

31. The method of claim 29, wherein the condition is SARS-CoV-2.

32. The method of any one of claims 2-31, where in the reference sample is a sample that is not infected by one or more infectious agents.

33. An aqueous solution comprising:

a. a biofluid sample; and

b. between 50 nM and 5 mM dextran sulphate.

34. The aqueous solution of claim 33, further comprising:

a. a buffer;

b. a chelating agent;

c. between 4 mM and 400 mM boric acid;

d. between 4 mM and 400 mM sodium citrate; and

e. between 1 mM and 100 mM lithium chloride.

35. The aqueous solution of claim 34, wherein the buffer comprises between 8mM and 800 mM Tris-HCl (pH 7.0).

36. The aqueous solution of claim 34 or 35, wherein the chelating agent comprises between 0.4 mM and 40 mM EDTA.

37. The aqueous solution of any one of claims 33-36, wherein the aqueous solution comprises about 500 nM dextran sulphate.

38. The aqueous solution of any one of claims 33-37, wherein the aqueous solution comprises:

a. about 40 mM boric acid;

b. about 40 mM sodium citrate; and

c. about 10 mM lithium chloride.

39. The aqueous solution of any one of claims 33-38, wherein the aqueous solution comprises about 80 mM Tris-HCl (pH 7.0).

40. The aqueous solution of any one of claims 33-39, wherein the aqueous solution comprises about 4 mM EDTA.

41. The aqueous solution of any one of claims 33-40, wherein the aqueous solution further comprises:

a. between ImM and 100 mM aurintricarboxylic acid;

b. between 0.01% and 10% (w/v) polyvinylpyrrolidone; and

c. 100 nM and 1 mM Tris (2-carboxyl ethyl) phosphine.

42. The aqueous solution of claim 41, wherein the aqueous solution comprises:

a. about 10 mM aurintricarboxylic acid;

b. about 0.25% (w/v) polyvinylpyrrolidone; and

c. about 500 nM Tris (2-carboxylethyl) phosphine.

43. The aqueous solution of any one of claims 33-42, wherein the biofluid sample is urine.

44. The aqueous solution of any one of claims 33-42, wherein the biofluid sample comprises fecal contaminant.

45. A composition comprising:

a. a sufficient amount of Tris-HCl (pH 7.0) to provide between 8 mM and 800 mM Tris-HCl (pH 7.0) when the composition is added to a biofluid sample; b. a sufficient amount of EDTA to provide between 0.4 mM and 40 mM EDTA when the composition is added to the biofluid sample;

c. a sufficient amount of boric acid to provide between 4 mM and 400 mM boric acid when the composition is added to the biofluid sample;

d. a sufficient amount of sodium citrate to provide between 4 mM and 400 mM sodium citrate when the composition is added to the biofluid sample; e. a sufficient amount of dextran sulphate to provide between 50 nM and 5 mM dextran sulphate when the composition is added to the biofluid sample; and

f. a sufficient amount of lithium chloride between 1 mM and 100 mM lithium chloride when the composition is added to the biofluid sample,

wherein the addition of the composition to a biofluid sample results in stabilization of a nucleic acid in the biofluid sample.

46. The composition of claim 45, further comprising:

a. a sufficient amount of aurintricarboxylic acid to provide between ImM and 100 mM aurintricarboxylic acid when the composition is added to the biofluid sample; b. a sufficient amount of polyvinylpyrrolidone to provide between 0.01% and 10% (w/v) polyvinylpyrrolidone when the composition is added to the biofluid sample; and

c. a sufficient amount of Tris (2-carboxyl ethyl) phosphine to provide between 100 nM and 1 mM Tris (2-carboxylethyl) phosphine when the composition is added to the biofluid sample.

47. The composition of claim 46, comprising:

a. a sufficient amount of Tris-HCl (pH 7.0) to provide about 80 mM Tris-HCl (pH 7.0) when the composition is added to the biofluid sample;

b. a sufficient amount of EDTA to provide about 4 mM EDTA when the composition is added to the biofluid sample;

c. a sufficient amount of boric acid to provide about 40 mM boric acid when the composition is added to the biofluid sample;

d. a sufficient amount of sodium citrate to provide about 40 mM sodium citrate when the composition is added to the biofluid sample;

e. a sufficient amount of dextran sulphate to provide about 500 nM dextran sulphate when the composition is added to the biofluid sample; and

f. a sufficient amount of lithium chloride to provide about 10 mM lithium chloride when the composition is added to the biofluid sample.

48. The composition of any one of claims 45-47, further comprising:

a. a sufficient amount of aurintricarboxylic to provide about 10 mM aurintricarboxylic acid when the composition is added to the biofluid sample; b. a sufficient amount of polyvinylpyrrolidone to provide about 0.25% (w/v) polyvinylpyrrolidone when the composition is added to the biofluid sample; and c. a sufficient amount of Tris (2-carboxyl ethyl) phosphine to provide about 500 nM Tris (2-carboxylethyl) phosphine when the composition is added to the biofluid sample.

49. The composition of any one of claims 45-48, wherein the composition is powder.

50. The composition of any one of claims 45-48, wherein the composition is a tablet.

51. The composition of any one of claims 45-48, wherein the composition is a gel.

52. The composition of any one of claims 45-48, wherein the composition is an aqueous solution.

53. A kit comprising:

a. a sufficient amount of dextran sulphate to provide between 50 nM and 5 mM dextran sulphate when the dextran sulphate is added to a biofluid sample; and optionally further comprising one or more of:

i. a sufficient amount of Tris-HCl (pH 7.0) to provide between 8mM and 800 mM Tris-HCl (pH 7.0) when the Tris-HCl (pH 7.0) is added to the biofluid sample;

ii. a sufficient amount of EDTA to provide between 0.4 mM and 40 mM EDTA when the EDTA is added to the biofluid sample iii. a sufficient amount of boric acid to provide between 4 mM and 400 mM boric acid when the boric acid is added to the biofluid sample;

iv. a sufficient amount of sodium citrate to provide between 4 mM and 400 mM sodium citrate when the sodium citrate is added to the biofluid sample;

v. a sufficient amount of lithium chloride to provide between 1 mM and 100 mM lithium chloride when the lithium chloride is added to the biofluid sample;

vi. a sufficient amount of aurintricarboxylic acid to provide between ImM and 100 mM aurintricarboxylic acid when the aurintricarboxylic acid is added to the biofluid sample;

vii. a sufficient amount of polyvinylpyrrolidone to provide between 0.01% and 10% (w/v) polyvinylpyrrolidone when the polyvinylpyrrolidone is added to the biofluid sample; and

viii. a sufficient amount of Tris (2-carboxyl ethyl) phosphine to provide between 100 nM and 1 mM Tris (2-carboxyl ethyl) phosphine when the Tris (2-carboxylethyl) phosphine is added to the biofluid sample.

54. The kit of claim 53, comprising:

a. a sufficient amount of dextran sulphate to provide about 500 nM dextran sulphate when the dextran sulphate is added to a biofluid sample; and optionally further comprising one or more of:

i. a sufficient amount of Tris-HCl (pH 7.0) to provide about 80 mM Tris- HC1 (pH 7.0) when the Tris-HCl (pH 7.0) is added to the biofluid sample; ii. a sufficient amount of EDTA to provide about 4 mM EDTA when the EDTA is added to the biofluid sample;

iii. a sufficient amount of boric acid to provide about 40 mM boric acid when the boric acid is added to the biofluid sample;

iv. a sufficient amount of sodium citrate to provide about 40 mM sodium citrate when the sodium citrate is added to the biofluid sample;

v. a sufficient amount of lithium chloride to provide about 10 mM lithium chloride when the lithium chloride is added to the biofluid sample; vi. a sufficient amount of aurintricarboxylic acid to provide about 10 mM aurintricarboxylic acid when the aurintricarboxylic acid is added to the biofluid sample;

vii. a sufficient amount of polyvinylpyrrolidone to provide about 0.25% (w/v) polyvinylpyrrolidone when the polyvinylpyrrolidone is added to the biofluid sample; and

viii. a sufficient amount of Tris (2-carboxyl ethyl) phosphine to provide about 500 nM Tris (2-carboxylethyl) phosphine when the Tris (2-carboxylethyl) phosphine is added to the biofluid sample.

55. A device for detecting a biomolecule in a biofluid sample, comprising a collection container for collecting a biofluid sample, wherein the collection container contains the composition of any one of claims 45-52, and optionally further comprising:

(1) a processing module capable of lysing cells, enriching or separating the biomolecule, and/or amplifying the biomolecule;

(2) a detection module capable of detecting the biomolecule; and/or

(3) a readout module capable of providing information regarding the presence of absence of the biomolecule to a user.

56. The device of claim 55, wherein the biomolecule is a DNA or an RNA molecule having a specific sequence.

57. The device of claim 55, wherein the biomolecule is a DNA molecule having a sequence associated with HPV infection.

58. The device of claim 57, wherein the processing module is capable of amplifying the biomolecule using an isothermic DNA amplification method.

59. The device of claim 58, wherein the isothermic DNA amplification method is LAMP, HDA or RPA.

60. The device of claim 55-59, wherein the detection module is capable of detecting the presence of two or more distinct biomolecules.

61. The device of claim 60, wherein the two or more distinct biomolecules are each associated with two or more different types of HPV.

62. The device of claim 60 or 61, wherein the readout of the presence or the absence of each of the two or more distinct biomolecules are provided in a 2D macro-array or a 3D macro-array, wherein two or more distinct probes that each specifically binds to one of the two or more distinct biomolecules are immobilized on a 2D surface or a 3D structure.

63. The device of claims 55-62, wherein the detection module comprises an RNA probe having a sequence that is complementary to the DNA molecule, and an antibody that is specific for the DNA-RNA hybrid that forms as a result of the RNA probe binding to the DNA molecule, and wherein the antibody is detected using a sandwich ELISA method.

64. The device of claim 55-63, wherein the steps of processing, detecting, and providing a readout are carried out by the device without an input from a healthcare professional.