NMR METHODS AND SYSTEMS FOR THE RAPID DETECTION OF TICK-BORNE PATHOGENS

20180171388

15/545663

January 21, 2016

The invention features methods, systems, and panels for rapid detection of tick-borne pathogens in a sample (including Borrelia spp. such as B. burgdorferi, B. afzelii, and B. garinii) and for diagnosis and monitoring of tick-transmitted diseases, including Lyme disease, Rocky Mountain spotted fever, Q-fever, babesiosis, ehrlichiosis, tularemia, and anaplasmosis.

1. A method for detecting the presence of at least two different Borrelia burgdorferi sensu lato (Bbsl) species in a biological sample, the method comprising: (a) providing a biological sample and optionally dividing it into portions; (b) amplifying in the biological sample one or more Bbsl target nucleic acids specific to the at least two different Bbsl species; and (c) detecting the amplified nucleic acids to determine whether one or all of the at least two different Bbsl species is present, wherein the method individually detects a concentration of an individual Bbsl species of 10 cells/mL of biological sample or less.

2. The method of claim 1, wherein the at least two different Bbsl species are selected from B. burgdorferi, B. afzelii, and B. garinii.

3. The method of claim 1 or 2, wherein the method comprises detecting the presence of B. burgdorferi, B. afzelii, and B. garinii.

4. The method of any one of claims 1-3, wherein amplifying step (b) further comprises amplifying in the same or different portions of the biological sample a second target nucleic acid specific to a non-Lyme disease pathogen and detecting step (c) further comprises detecting the amplified second target nucleic acid to determine whether the non-Lyme disease pathogen is present.

5. The method of any one of claims 1-4, wherein the method detects a concentration of the non-Lyme disease pathogen of 10 cells/mL of biological sample or less.

6. A method for detecting the presence of a tick-borne pathogen in a biological sample, the method comprising: (a) providing a biological sample and optionally dividing it into portions; (b) amplifying in the same or different portions of the biological sample a target nucleic acid specific to a Bbsl species and a second target nucleic acid specific to a non-Lyme disease pathogen, wherein the non-Lyme disease pathogen is selected from a Rickettsia spp., Coxiella burnetii, a Babesia spp., Anaplasma phagocytophilum, a Francisella tularensis subspp., Streptococcus pneumoniae, or Neisseria meningitidis; and (c) detecting the amplified nucleic acids to determine whether the Bbsl species or the non-Lyme disease pathogen is present in the sample, wherein the method individually detects a concentration of the Bbsl species or the non-Lyme disease pathogen of 10 cells/mL of biological sample or less.

7. The method of any one of claims 1-6, wherein amplifying step (b) further comprises amplifying a pan-Borrelia target nucleic acid in the presence of a forward primer and a reverse primer, each of which is universal to Borrelia species, and detecting step (c) further comprises detecting the amplified pan-Borrelia target nucleic acid to determine whether a Borrelia species is present.

8. The method of any one of claims 1-7, wherein the amplified Bbsl target nucleic acid, non-Lyme disease pathogen target nucleic acid, or pan-Borrelia target nucleic acid is detected by optical, fluorescent, mass, density, magnetic, chromatographic, and/or electrochemical measurement.

9. The method of claim 8, wherein the amplified Bbsl target nucleic acid, non-Lyme disease pathogen target nucleic acid, or pan-Borrelia target nucleic acid is detected by measuring the T2 relaxation response of the biological sample or portion thereof following contacting the biological sample or portion thereof with magnetic particles, wherein the magnetic particles have binding moieties on their surfaces, the binding moieties operative to alter the specific aggregation of the magnetic particles in the presence of the amplified Bbsl target nucleic acid, non-Lyme disease pathogen target nucleic acid, or pan-Borrelia target nucleic acid.

10. A method for detecting the presence of at least two different Borrelia burgdorferi sensu lato (Bbsl) species in a biological sample, the method comprising: (a) providing a biological sample; (b) preparing a first assay sample by contacting a portion of the biological sample with magnetic particles, wherein the magnetic particles have binding moieties on their surfaces, the binding moieties operative to alter the specific aggregation of the magnetic particles in the presence of an analyte associated with a first Bbsl species; (c) preparing a second assay sample by contacting a portion of the biological sample with magnetic particles, wherein the magnetic particles have binding moieties on their surfaces, the binding moieties operative to alter the specific aggregation of the magnetic particles in the presence of an analyte associated with a second Bbsl species; (d) placing each assay sample in a device, the device comprising a support defining a well for holding the assay sample, and having an RF coil configured to detect a signal produced by exposing the assay sample to a bias magnetic field created using one or more magnets and an RF pulse sequence; (e) exposing each assay sample to the bias magnetic field and the RF pulse sequence; (f) following step (e), measuring the signal produced by each assay sample; and (g) using the results of step (f) to determine whether one or all of the at least two different Bbsl species is present in the biological sample.

11. The method of claim 10, wherein the at least two different Bbsl species are selected from B. burgdorferi, B. afzelii, and B. garinii.

12. The method of claim 11, further comprising preparing a third assay sample by contacting a portion of the biological sample with magnetic particles, wherein the magnetic particles have binding moieties on their surfaces, the binding moieties operative to alter the specific aggregation of the magnetic particles in the presence of an analyte associated with a third Bbsl species.

13. The method of any one of claims 10-12, further comprising preparing an additional assay sample by contacting a portion of the biological sample with magnetic particles, wherein the magnetic particles have binding moieties on their surfaces, the binding moieties operative to alter the specific aggregation of the magnetic particles in the presence of an analyte associated with a pathogen selected from an Ehrlichia spp., a Rickettsia spp., Coxiella burnetii, a Babesia spp., Anaplasma phagocytophilum, a Francisella tularensis subspp., Streptococcus pneumoniae, or Neisseria meningitidis.

14. A method for detecting the presence of a tick-borne pathogen in a biological sample, the method comprising: (a) providing a biological sample; (b) preparing a first assay sample by contacting a portion of the biological sample with magnetic particles, wherein the magnetic particles have binding moieties on their surfaces, the binding moieties operative to alter the specific aggregation of the magnetic particles in the presence of an analyte associated with a Bbsl species; (c) preparing a second assay sample by contacting a portion of the biological sample with magnetic particles, wherein the magnetic particles have binding moieties on their surfaces, the binding moieties operative to alter the specific aggregation of the magnetic particles in the presence of an analyte associated with a non-Lyme disease pathogen, wherein the non-Lyme disease pathogen is selected from a Rickettsia spp., Coxiella burnetii, a Babesia spp., Anaplasma phagocytophilum, a Francisella tularensis subspp., Streptococcus pneumoniae, or Neisseria meningitidis; (d) placing each assay sample in a device, the device comprising a support defining a well for holding the assay sample, and having an RF coil configured to detect a signal produced by exposing the assay sample to a bias magnetic field created using one or more magnets and an RF pulse sequence; (e) exposing each assay sample to the bias magnetic field and the RF pulse sequence; (f) following step (e), measuring the signal produced by each assay sample; and (g) using the results of step (f) to determine whether the Bbsl species or the non-Lyme disease pathogen is present in the biological sample.

15. The method of any one of claims 10-14, further comprising preparing a pan-Borrelia assay sample by contacting a portion of the biological sample with magnetic particles, wherein the magnetic particles have binding moieties on their surfaces, the binding moieties operative to alter the specific aggregation of the magnetic particles in the presence of a pan-Borrelia-associated analyte.

16. The method of any one of claims 10-15, wherein the analyte associated with a Bbsl species, the analyte associated with a non-Lyme disease pathogen, or a pan-Borrelia-associated analyte is independently a polypeptide or a nucleic acid.

17. The method of claim 16, wherein the nucleic acid is a target nucleic acid that has been amplified in the biological sample or portion thereof.

18. A method for detecting the presence of at least two different Borrelia burgdorferi sensu lato (Bbsl) species in a biological sample, the method comprising: (a) providing a biological sample; (b) lysing the Borrelia cells in the biological sample; (c) amplifying one or more Bbsl target nucleic acids in the biological sample to form an amplified biological sample; (d) preparing a first assay sample by contacting a portion of the amplified biological sample with a first population of magnetic particles, wherein the magnetic particles of the first population have binding moieties characteristic of a first Bbsl species on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the one or more Bbsl target nucleic acids; (e) preparing a second assay sample by contacting a portion of the amplified biological sample with a second population of magnetic particles, wherein the magnetic particles of the second population have binding moieties characteristic of a second Bbsl species on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the one or more Bbsl target nucleic acids; (f) providing each assay sample in a detection tube within a device, the device comprising a support defining a well for holding the detection tube comprising the assay sample, and having an RF coil configured to detect a signal produced by exposing the mixture to a bias magnetic field created using one or more magnets and an RF pulse sequence; (g) exposing each assay sample to a bias magnetic field and an RF pulse sequence; (h) following step (g), measuring the signal produced by each assay sample; and (i) on the basis of the result of step (h), determining whether one or all of the at least two different Bbsl species is present in the biological sample.

19. The method of claim 18, wherein the at least two different Bbsl species are selected from B. burgdorferi, B. afzelii, and B. garinii.

20. The method of claim 18 or 19, wherein the method further comprises preparing a third sample assay by contacting a portion of the amplified biological sample with a third population of magnetic particles, wherein the magnetic particles of the third population have binding moieties characteristic of a third Bbsl species on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the Bbsl target nucleic acid.

21. The method of any one of claims 18-20, wherein the amplifying of step (c) comprises amplifying a Bbsl target nucleic acid to be detected in the presence of a forward primer and a reverse primer, each of which is universal to Bbsl species, thereby forming a pan-Bbsl amplicon.

22. The method of any one of claims 18-21, wherein the magnetic particles of each population comprise two subpopulations, a first subpopulation bearing a first probe on its surface, and a second subpopulation bearing a second probe on its surface.

23. The method of any one of claims 18-22, wherein the amplifying of step (c) comprises amplifying two target nucleic acids characteristic of a single Bbsl species, thereby forming a first amplicon characteristic of a single Bbsl species and a second amplicon characteristic of a single Bbsl species, for at least one of the Bbsl species to be detected.

24. The method of claim 23, wherein the amplifying of step (c) comprises amplifying two target nucleic acids characteristic of a single Bbsl species for at least two of the Bbsl species to be detected.

25. The method of claim 24, wherein the amplifying of step (c) comprises amplifying two target nucleic acids characteristic of B. burgdorferi and two target nucleic acids characteristic of B. afzelii.

26. The method of any one of claims 23-25, wherein the magnetic particles of step (d) or (e) comprise a first subpopulation conjugated to a first probe and a second probe, and a second subpopulation conjugated to a third probe and a fourth probe, wherein the first probe and third probe are operative to bind a first segment and a second segment, respectively, of the first amplicon; and the second probe and fourth probe are operative to bind a first segment and a second segment, respectively, of the second amplicon, wherein the magnetic particles form aggregates in the presence of the first amplicon and form aggregates in the presence of the second amplicon.

27. The method of any one of claims 18-26, further comprising detecting the presence of a non-Lyme disease pathogen in the biological sample or a portion thereof.

28. The method of claim 27, wherein the non-Lyme disease pathogen is detected by: (j) lysing the non-Lyme disease pathogen cells in the biological sample or portion thereof; (k) amplifying a non-Lyme disease pathogen target nucleic acid in the biological sample to form an amplified biological sample; (l) preparing an assay sample by contacting the amplified biological sample with magnetic particles, wherein the magnetic particles have binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the non-Lyme disease pathogen target nucleic acid; (m) providing the assay sample in a detection tube within a device, the device comprising a support defining a well for holding the detection tube comprising the assay sample, and having an RF coil configured to detect a signal produced by exposing the assay sample to a bias magnetic field created using one or more magnets and an RF pulse sequence; (n) exposing the assay sample to a bias magnetic field and an RF pulse sequence; (o) following step (m), measuring the signal; and (p) on the basis of the result of step (o), determining whether the non-Lyme disease pathogen is present in the biological sample.

29. The method of claim 28, wherein steps (b) through (i) and (j) through (p) are performed simultaneously.

30. A method for detecting the presence of a Borrelia burgdorferi (B. burgdorferi) cell in a biological sample, the method comprising: (a) lysing the B. burgdorferi cells in the biological sample to form a lysate; (b) amplifying a B. burgdorferi target nucleic acid in the lysate in the presence of a first primer pair or a second primer pair to form an amplified lysate comprising a B. burgdorferi amplicon, wherein the first primer pair comprises a forward primer comprising the oligonucleotide sequence: 5′-AGA GGA CTT TTA ATA CTG GGC ATT GCT G-3′ (SEQ ID NO: 1) and a reverse primer comprising the oligonucleotide sequence: 5′-GGC CAT TAT GTA GGA ATC TCT AAT GGT GC-3′ (SEQ ID NO: 2), and the second primer pair comprises a forward primer comprising the oligonucleotide sequence: 5′-ACA TAA TGG CCT TAG AAA ATG AGC TTG ATG-3′ (SEQ ID NO: 5) and a reverse primer comprising the oligonucleotide sequence 5′-CCC GCT TGT AAC CAT GTT TTC TGA GC-3′ (SEQ ID NO: 6); (c) following step (b), contacting the amplified lysate with magnetic particles to form an assay sample, wherein the magnetic particles comprise binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the B. burgdorferi amplicon; (d) providing the assay sample in a detection tube within a device, the device comprising a support defining a well for holding the detection tube comprising the assay sample, and having an RF coil configured to detect a signal produced by exposing the assay sample to a bias magnetic field created using one or more magnets and an RF pulse sequence; (e) exposing the assay sample to a bias magnetic field and an RF pulse sequence; (f) following step (e), measuring the signal from the detection tube; and (g) on the basis of the result of step (f), determining whether a B. burgdorferi cell was present in the biological sample.

31. The method of claim 30, wherein the magnetic particles comprise a first population of magnetic particles conjugated to a first probe, and a second population of magnetic particles conjugated to a second probe, the first probe operative to bind to a first segment of the B. burgdorferi amplicon and the second probe operative to bind to a second segment of the B. burgdorferi amplicon, wherein the magnetic particles form aggregates in the presence of the B. burgdorferi amplicon.

32. The method of claim 31, wherein step (b) comprises amplifying a B. burgdorferi target nucleic acid in the presence of the first primer pair, and the first probe comprises the oligonucleotide sequence: 5′-CTA AAC CAA AAG ATG ATA TTG TCT TTG GTG-3′ (SEQ ID NO: 3), and the second probe comprises the oligonucleotide sequence: 5′-GGA CAT TTC TTA CGA CAA CAC CTG CT-3′ (SEQ ID NO: 4).

33. The method of claim 31, wherein step (b) comprises amplifying the B. burgdorferi target nucleic acid in the presence of the second primer pair, and the first probe comprises the oligonucleotide sequence: 5′-AAC CTA TTA ACA TCA AAG ATA AAA AAT GC-3′ (SEQ ID NO: 7), and the second probe comprises the oligonucleotide sequence: 5′-GCT TAC ACA CCC AAT ATT TAT ACC C-3′ (SEQ ID NO: 8).

34. The method of claim 30, wherein step (b) comprises amplifying the first B. burgdorferi target nucleic acid in the presence of the first primer pair to form a first B. burgdorferi amplicon and amplifying the second B. burgdorferi target nucleic acid in the presence of the second primer pair to form a second B. burgdorferi amplicon, and step (g) comprises detecting the first B. burgdorferi amplicon and the second B. burgdorferi amplicon.

35. The method of claim 34, wherein the magnetic particles comprise a first population of magnetic particles conjugated to a first probe and a second probe, and a second population of magnetic particles conjugated to a third probe and a fourth probe, wherein the first probe and third probe are operative to bind a first segment and a second segment, respectively, of the first B. burgdorferi amplicon; and the second probe and fourth probe are operative to bind a first segment and a second segment, respectively, of the second B. burgdorferi amplicon, wherein the magnetic particles form aggregates in the presence of the first B. burgdorferi amplicon and form aggregates in the presence of the second B. burgdorferi amplicon.

36. The method of claim 35, wherein the first probe comprises an oligonucleotide sequence of SEQ ID NO: 3, the second probe comprises an oligonucleotide sequence of SEQ ID NO: 7, the third probe comprises an oligonucleotide sequence of SEQ ID NO: 4, and the fourth probe comprises an oligonucleotide sequence of SEQ ID NO: 8.

37. A method for detecting the presence of a Borrelia garinii (B. garinii) cell in a biological sample, the method comprising: (a) lysing the B. garinii cells in a biological sample to form a lysate; (b) amplifying a B. garinii target nucleic acid in the lysate in the presence of a forward primer comprising the oligonucleotide sequence: 5′-GGA TAT TCA AAT CCT GAG GTT GAC GAA CTA-3′ (SEQ ID NO: 9) and a reverse primer comprising the oligonucleotide sequence: 5′-CTG ATA GGG CAA ATC TTT CTG AAG CA-3′ (SEQ ID NO: 10) to form an amplified lysate comprising a B. garinii amplicon; (c) following step (b), contacting the amplified lysate with magnetic particles to form an assay sample, wherein the magnetic particles comprise binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the B. garinii amplicon; (d) providing the assay sample in a detection tube within a device, the device comprising a support defining a well for holding the detection tube comprising the assay sample, and having an RF coil configured to detect a signal produced by exposing the assay sample to a bias magnetic field created using one or more magnets and an RF pulse sequence; (e) exposing the assay sample to a bias magnetic field and an RF pulse sequence; (f) following step (e), measuring the signal from the detection tube; and (g) on the basis of the result of step (f), determining whether an B. garinii cell was present in the biological sample.

38. The method of claim 37, wherein the magnetic particles comprise a first population of magnetic particles conjugated to a first probe, and a second population of magnetic particles conjugated to a second probe, the first probe operative to bind to a first segment of the B. garinii amplicon and the second probe operative to bind to a second segment of the B. garinii amplicon, wherein the magnetic particles form aggregates in the presence of the B. garinii amplicon.

39. The method of claim 38, wherein the first probe comprises the oligonucleotide sequence: 5′-CTG AGA TTG AAG TTG ACG AAA AAA TCA G-3′ (SEQ ID NO: 11), and the second probe comprises the oligonucleotide sequence: 5′-GAT CAT CCA ATA ATT CCA ATC TAC AGC G-3′ (SEQ ID NO: 12).

40. A method for detecting the presence of a Borrelia afzelii (B. afzelii) cell in a biological sample, the method comprising: (a) lysing the B. afzelii cells in the biological sample to form a lysate; (b) amplifying a B. afzelii target nucleic acid in the lysate in the presence of a first primer pair or a second primer pair to form an amplified lysate comprising a B. afzelii amplicon, wherein the first primer pair comprises a forward primer comprising the oligonucleotide sequence: 5′-CCG TGG GCA GAG TCT ATG ACA ATC AG-3′ (SEQ ID NO: 13) and a reverse primer comprising the oligonucleotide sequence: 5′-GCC CAA AAA ACC ATC AAC ACT AAT AAG G-3′ (SEQ ID NO: 14), and the second primer pair comprises a forward primer comprising the oligonucleotide sequence: 5′-CAA GGT GCA ATG ACT TTG TTT GGG CA-3′ (SEQ ID NO: 17) and a reverse primer comprising the oligonucleotide sequence 5′-GCA ACT TCA AAG TGT ACA GTA TTG GTA TCC C-3′ (SEQ ID NO: 18); (c) following step (b), contacting the amplified lysate with magnetic particles to form an assay sample, wherein the magnetic particles comprise binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the B. afzelii amplicon; (d) providing the assay sample in a detection tube within a device, the device comprising a support defining a well for holding the detection tube comprising the assay sample, and having an RF coil configured to detect a signal produced by exposing the assay sample to a bias magnetic field created using one or more magnets and an RF pulse sequence; (e) exposing the assay sample to a bias magnetic field and an RF pulse sequence; (f) following step (e), measuring the signal from the detection tube; and (g) on the basis of the result of step (f), determining whether a B. afzelii cell was present in the biological sample.

41. The method of claim 40, wherein the magnetic particles comprise a first population of magnetic particles conjugated to a first probe, and a second population of magnetic particles conjugated to a second probe, the first probe operative to bind to a first segment of the B. afzelii amplicon and the second probe operative to bind to a second segment of the B. afzelii amplicon, wherein the magnetic particles form aggregates in the presence of the B. afzelii amplicon.

42. The method of claim 41, wherein step (b) comprises amplifying a B. afzelii target nucleic acid in the presence of the first primer pair, and the first probe comprises the oligonucleotide sequence: 5′-TAG CAG CTC CTA CTC TTA GCT TGC-3′ (SEQ ID NO: 15), and the second probe comprises the oligonucleotide sequence: 5′-AAT ATT GCT TTG TAA GCA TTT TGG TTT-3′ (SEQ ID NO: 16).

43. The method of claim 41, wherein step (b) comprises amplifying the B. afzelii target nucleic acid in the presence of the second primer pair, and the first probe comprises the oligonucleotide sequence: 5′-AAC CTA TTA ACA TCA AAG ATA AAA AAT GC-3′ (SEQ ID NO: 7), and the second probe comprises the oligonucleotide sequence: 5′-GCT TAC ACA CCC AAT ATT TAT ACC C-3′ (SEQ ID NO: 8).

44. The method of claim 40, wherein step (b) comprises amplifying the first B. afzelii target nucleic acid in the presence of the first primer pair to form a first B. afzelii amplicon and amplifying the second B. afzelii target nucleic acid in the presence of the second primer pair to form a second B. afzelii amplicon, and step (g) comprises detecting the first B. afzelii amplicon and the second B. afzelii amplicon.

45. The method of claim 44, wherein the magnetic particles comprise a first population of magnetic particles conjugated to a first probe and a second probe, and a second population of magnetic particles conjugated to a third probe and a fourth probe, wherein the first probe and third probe are operative to bind a first segment and a second segment, respectively, of the first B. afzelii amplicon; and the second probe and fourth probe are operative to bind a first segment and a second segment, respectively, of the second B. afzelii amplicon, wherein the magnetic particles form aggregates in the presence of the first B. afzelii amplicon and form aggregates in the presence of the second B. afzelii amplicon.

46. The method of claim 45, wherein the first probe comprises an oligonucleotide sequence of SEQ ID NO: 3, the second probe comprises an oligonucleotide sequence of SEQ ID NO: 7, the third probe comprises an oligonucleotide sequence of SEQ ID NO: 4, and the fourth probe comprises an oligonucleotide sequence of SEQ ID NO: 8.

47. A method for detecting the presence of a tick-borne pathogen in a biological sample, the method comprising: (a) providing a biological sample; (b) lysing the tick-borne pathogen cells in the biological sample; (c) amplifying a Bbsl target nucleic acid and a non-Lyme disease pathogen target nucleic acid in the biological sample to form an amplified biological sample, wherein the non-Lyme disease pathogen is selected from Rickettsia spp., Coxiella burnetii, Babesia spp., Anaplasma phagocytophilum, a Francisella tularensis subspp., Streptococcus pneumoniae, or Neisseria meningitidis; (d) preparing a first assay sample by contacting a portion of the amplified biological sample with a first population of magnetic particles, wherein the magnetic particles of the first population have binding moieties characteristic of a Bbsl species on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the Bbsl target nucleic acid; (e) preparing a second assay sample by contacting a portion of the amplified biological sample with a second population of magnetic particles, wherein the magnetic particles of the second population have binding moieties characteristic of the non-Lyme disease pathogen on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the non-Lyme disease pathogen target nucleic acid; (f) providing each assay sample in a detection tube within a device, the device comprising a support defining a well for holding the detection tube comprising the assay sample, and having an RF coil configured to detect a signal produced by exposing the mixture to a bias magnetic field created using one or more magnets and an RF pulse sequence; (g) exposing each assay sample to a bias magnetic field and an RF pulse sequence; (h) following step (g), measuring the signal produced by each assay sample; and (i) on the basis of the result of step (h), determining whether a tick-borne pathogen is present in the biological sample.

48. The method of claim 6, 27, or 47, wherein the non-Lyme disease pathogen is selected from an Ehrlichia spp., a Rickettsia spp., Coxiella burnetii, a Babesia spp., Anaplasma phagocytophilum, a Francisella tularensis subspp., Streptococcus pneumoniae, or Neisseria meningitidis.

49. The method of claim 48, wherein the Ehrlichia spp. is Ehrlichia chaffeensis, Ehrlichia ewingii or Ehrlichia muris-like; the Rickettsia spp. is Rickettsia rickettsii or Rickettsia parkeri; the Babesia spp. is Babesia microti or Babesia divergens, and the Francisella tularensis subspp. is Francisella tularensis subspp. tularensis, holarctica, mediasiatica, or novicida.

50. The method of any one of claim 18-29 or 47-49, wherein the Bbsl target nucleic acid is derived from a single-copy chromosomal locus, a single-copy plasmid, a multi-copy chromosomal locus, or a multi-copy plasmid.

51. The method of claim 50, wherein the multi-copy plasmid encodes an outer surface protein opsA and/or opsB.

52. The method of any one of claim 18-29 or 47-49, wherein the Bbsl target nucleic acid is a B. burgdorferi target nucleic acid, and the B. burgdorferi target nucleic acid is derived from the oppA gene encoded on linear plasmid 54 (lp54), an outer membrane protein (OMP), the crasp1 gene encoded on lp54, the lipoprotein ORF 01110, lipoprotein S2, acetyl CoA acetyltransferase encoded on the chromosome, or lipoprotein encoded on lp54.

53. The method of any one of claim 18-29 or 47-49, wherein the Bbsl target nucleic acid is a B. garinii target nucleic acid, and the B. garinii target nucleic acid is derived from the oppA gene, protein 24 (p24) encoded on lp54, chitibiose transporter protein encoded on lp54, chitibiose transporter protein (chbC) encoded on circular plasmid 26 (cp26), lipoprotein encoded on lp54 (lp54-69226), or dbpB gene encoded on lp54.

54. The method of any one of claim 18-29 or 47-49, wherein the Bbsl target nucleic acid is a B. afzelii target nucleic acid, and the B. afzelii target nucleic acid is derived from conserved hypothetical protein bb0242 encoded on the chromosome, lipoprotein S2. chitibiose transporter protein encoded on lp54, inosine-5′-monophosphate dehydrogenase (guaB) encoded on cp26, or phosphotransferase (PTS) glucose transporter subunit encoded on cp26.

55. The method of any one of claim 28, 29, or 47-54, wherein the non-Lyme disease pathogen target nucleic acid is derived from an essential housekeeping gene, a virulence factor, or a ribosomal DNA (rDNA) operon.

56. The method of claim 55, wherein the non-Lyme disease pathogen target nucleic acid is derived from a single-copy locus.

57. The method of any one of claim 18-29 or 47-56, wherein amplifying step (c) further comprises amplifying a pan-Borrelia target nucleic acid in the biological sample.

58. The method of claim 57, further comprising preparing a pan-Borrelia assay sample by contacting a portion of the amplified biological sample with a population of magnetic particles, wherein the magnetic particles have binding moieties operative to bind the pan-Borrelia target nucleic acid on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the pan-Borrelia target nucleic acid, and determining whether a Borrelia species is present in the biological sample on the basis of the result of step (h).

59. The method of any one of claims 30-46, wherein step (b) further comprises amplifying a pan-Borrelia target nucleic acid in the biological sample, and wherein the method further comprises preparing a pan-Borrelia assay sample by contacting a portion of the amplified lysate with a population of magnetic particles, wherein the magnetic particles have binding moieties operative to bind the pan-Borrelia target nucleic acid on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the pan-Borrelia target nucleic acid; exposing the pan-Borrelia assay sample to the bias magnetic field and the RF pulse sequence; and measuring the signal from the detection tube, thereby determining whether a Borrelia cell was present in the biological sample.

60. The method of any one of claims 1-59, wherein the biological sample or portion thereof is between 0.1 and 4 mL.

61. The method of claim 60, wherein the biological sample is between 1.25 and 2 mL.

62. The method of any one of claims 1-61, wherein the biological sample is blood, cerebrospinal fluid (CSF), urine, or synovial fluid.

63. The method of claim 62, wherein the biological sample is blood.

64. The method of claim 63, wherein the blood is whole blood or platelet-rich plasma (PRP).

65. The method of claim 63 or 64, wherein amplifying is in the presence of whole blood proteins and non-target nucleic acids.

66. The method of claim 63 or 64, wherein the biological sample or portion thereof is a fraction of blood enriched for white blood cells.

67. The method of any one of claims 18-66, wherein lysing comprises mechanical lysis or heat lysis.

68. The method of claim 67, wherein the mechanical lysis is beadbeating or sonicating.

69. The method of any one of claims 1-68, wherein the steps of the method are completed within 5 hours.

70. The method of claim 69, wherein the steps of the method are completed within 4 hours.

71. The method of claim 70, wherein the steps of the method are completed within 3 hours.

72. The method of any one of claims 9-71, wherein an assay sample is contacted with 1×106 to 1×1013 magnetic particles per milliliter of the biological sample.

73. The method of any one of claim 18-29 or 47-72, wherein step (h) or (o) comprises measuring the T2 relaxation response of the assay sample, and wherein increasing agglomeration in the assay sample produces an increase in the observed T2 relaxation time of the assay sample.

74. The method of any one of claims 30-46, wherein step (f) comprises measuring the T2 relaxation response of the assay sample, and wherein increasing agglomeration in the assay sample produces an increase in the observed T2 relaxation time of the assay sample.

75. The method of any one of claims 9-74, wherein the magnetic particles have a mean diameter of from 150 nm to 699 nm or from 700 nm to 1200 nm.

76. The method of claim 75, wherein the magnetic particles have a mean diameter of from 700 nm to 950 nm.

77. The method of claim 76, wherein the magnetic particles have a mean diameter of from 700 nm to 850 nm.

78. The method of any one of claims 9-77, wherein the magnetic particles have a T2 relaxivity per particle of from 1×109 to 1×1012 mM−1s−1.

79. The method of any one of claims 9-78, wherein the magnetic particles are substantially monodisperse.

80. A method for detecting the presence of a tick-borne pathogen in a sample of whole blood, the method comprising: (a) providing an sample produced by lysing the red blood cells in a whole blood sample from a subject, centrifuging the sample to form a supernatant and a pellet, discarding some or all of the supernatant, and optionally washing the pellet; (b) lysing the tick-borne pathogen cells and subject cells in the pellet to form a lysate; (c) amplifying a Bbsl target nucleic acid and a non-Lyme disease pathogen target nucleic acid in the lysate to form an amplified lysate, wherein the non-Lyme disease pathogen is selected from Rickettsia spp., Coxiella burnetii, Babesia spp., Anaplasma phagocytophilum, a Francisella tularensis subspp., Streptococcus pneumoniae, or Neisseria meningitidis; (d) following step (c), preparing a first assay sample by adding to a portion of the amplified lysate from 1×106 to 1×1013 magnetic particles per milliliter of the amplified lysate, wherein the magnetic particles have a mean diameter of from 700 nm to 950 nm and binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the Bbsl target nucleic acid, wherein said magnetic particles have a T2 relaxivity per particle of from 1×109 to 1×1012 mM−1s−1; (e) following step (c), preparing a second assay sample by adding to a portion of the amplified lysate from 1×106 to 1×1013 magnetic particles per milliliter of the amplified lysate, wherein the magnetic particles have a mean diameter of from 700 nm to 950 nm and binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the non-Lyme disease pathogen target nucleic acid, wherein said magnetic particles have a T2 relaxivity per particle of from 1×109 to 1×1012 mM−1s−1 (f) providing each assay sample in a detection tube within a device, the device comprising a support defining a well for holding the detection tube comprising the assay sample, and having an RF coil configured to detect a signal produced by exposing the mixture to a bias magnetic field created using one or more magnets and an RF pulse sequence; (g) exposing each assay sample to a bias magnetic field and an RF pulse sequence; (h) following step (i), measuring the signal produced by each assay sample; and (i) on the basis of the result of step (h), determining whether a tick-borne pathogen is present in the sample of whole blood.

81. The method of claim 80, wherein the non-Lyme disease pathogen is selected from an Ehrlichia spp., a Rickettsia spp., Coxiella burnetii, a Babesia spp., Anaplasma phagocytophilum, a Francisella tularensis subspp., Streptococcus pneumoniae, or Neisseria meningitidis.

82. The method of claim 81, wherein the Ehrlichia spp. is Ehrlichia chaffeensis, Ehrlichia or Ehrlichia muris-like; the Rickettsia spp. is Rickettsia rickettsii or Rickettsia parkeri; the Babesia spp. is Babesia microti or Babesia divergens, and the Francisella tularensis subspp. is Francisella tularensis subspp. tularensis, holarctica, mediasiatica, or novicida.

83. The method of any one of claims 80-82, wherein amplifying step (c) further comprises amplifying a pan-Borrelia target nucleic acid in the lysate.

84. The method of claim 83, further comprising preparing a pan-Borrelia assay sample by contacting a portion of the amplified biological sample with a population of magnetic particles, wherein the magnetic particles have binding moieties characteristic of all Borrelia species on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the pan-Borrelia target nucleic acid, and determining whether a Borrelia species is present in the biological sample on the basis of the result of step (h).

85. The method of any one of claims 80-84, wherein the whole blood sample is from 0.05 to 4 mL.

86. The method of any one of claims 80-85, wherein the method is capable of detecting a tick-borne pathogen concentration of 1 cells/ml in the whole blood sample.

87. The method of any one of claims 80-86, wherein step (h) comprises measuring the T2 relaxation response of the mixture, and wherein increasing agglomeration in the mixture produces an increase in the observed T2 relaxation time of the mixture.

88. The method of any one of claims 80-87, wherein steps (a) through (i) are completed within 3 hours.

89. The method of any one of claims 80-88, wherein the amplified lysate of step (c) comprises whole blood proteins and non-target DNA from subject cells.

90. A method for detecting the presence of a B. burgdorferi cell in a sample of whole blood, the method comprising: (a) providing an sample produced by lysing the red blood cells in a whole blood sample from a subject, centrifuging the sample to form a supernatant and a pellet, discarding some or all of the supernatant, and optionally washing the pellet; (b) lysing the B. burgdorferi cells and subject cells in the pellet to form a lysate; (c) amplifying a B. burgdorferi target nucleic acid in the lysate in the presence of a first primer pair or a second primer pair to form an amplified lysate comprising a B. burgdorferi amplicon, wherein the first primer pair comprises a forward primer comprising the oligonucleotide sequence: 5′-AGA GGA CTT TTA ATA CTG GGC ATT GCT G-3′ (SEQ ID NO: 1) and a reverse primer comprising the oligonucleotide sequence: 5′-GGC CAT TAT GTA GGA ATC TCT AAT GGT GC-3′ (SEQ ID NO: 2), and the second primer pair comprises a forward primer comprising the oligonucleotide sequence: 5′-ACA TAA TGG CCT TAG AAA ATG AGC TTG ATG-3′ (SEQ ID NO: 5) and a reverse primer comprising the oligonucleotide sequence 5′-CCC GCT TGT AAC CAT GTT TTC TGA GC-3′ (SEQ ID NO: 6); (d) following step (c), preparing at least a first assay sample by adding to a portion of the amplified lysate from 1×106 to 1×1013 magnetic particles per milliliter of the amplified lysate, wherein the magnetic particles have a mean diameter of from 700 nm to 950 nm and binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the B. burgdorferi target nucleic acid, wherein said magnetic particles have a T2 relaxivity per particle of from 1×109 to 1×1012 mM−1s−1; (e) providing each assay sample in a detection tube within a device, the device comprising a support defining a well for holding the detection tube comprising the assay sample, and having an RF coil configured to detect a signal produced by exposing the mixture to a bias magnetic field created using one or more magnets and an RF pulse sequence; (f) exposing each assay sample to a bias magnetic field and an RF pulse sequence; (g) following step (f), measuring the signal produced by each assay sample; and (h) on the basis of the result of step (g), determining whether a tick-borne pathogen is present in the sample of whole blood.

91. The method of claim 90, wherein step (c) comprises amplifying the first B. burgdorferi target nucleic acid in the presence of the first primer pair to form a first B. burgdorferi amplicon and amplifying the second B. burgdorferi target nucleic acid in the presence of the second primer pair to form a second B. burgdorferi amplicon, and step (g) comprises detecting the first B. burgdorferi amplicon and the second B. burgdorferi amplicon.

92. The method of claim 91, wherein the magnetic particles comprise a first population of magnetic particles conjugated to a first probe and a second probe, and a second population of magnetic particles conjugated to a third probe and a fourth probe, wherein the first probe and third probe are operative to bind a first segment and a second segment, respectively, of the first B. burgdorferi amplicon; and the second probe and fourth probe are operative to bind a first segment and a second segment, respectively, of the second B. burgdorferi amplicon, wherein the magnetic particles form aggregates in the presence of the first B. burgdorferi amplicon and form aggregates in the presence of the second B. burgdorferi amplicon.

93. The method of claim 92, wherein the first probe comprises an oligonucleotide sequence of SEQ ID NO: 3, the second probe comprises an oligonucleotide sequence of SEQ ID NO: 7, the third probe comprises an oligonucleotide sequence of SEQ ID NO: 4, and the fourth probe comprises an oligonucleotide sequence of SEQ ID NO: 8.

94. The method of any one of claims 90-93, wherein step (c) further comprises amplifying a B. garinii target nucleic acid in the lysate in the presence of a forward primer comprising the oligonucleotide sequence: 5′-GGA TAT TCA AAT CCT GAG GTT GAC GAA CTA-3′ (SEQ ID NO: 9) and a reverse primer comprising the oligonucleotide sequence: 5′-CTG ATA GGG CAA ATC TTT CTG AAG CA-3′ (SEQ ID NO: 10) to form an amplified lysate comprising a B. garinii amplicon.

95. The method of claim 94, wherein the method further comprises detecting the presence of the B. garinii amplicon.

96. The method of any one of claims 90-95, wherein step (c) further comprises amplifying a B. afzelii target nucleic acid in the lysate in the presence of a first primer pair or a second primer pair to form an amplified lysate comprising a B. afzelii amplicon, wherein the first primer pair comprises a forward primer comprising the oligonucleotide sequence: 5′-CCG TGG GCA GAG TCT ATG ACA ATC AG-3′ (SEQ ID NO: 13) and a reverse primer comprising the oligonucleotide sequence: 5′-GCC CAA AAA ACC ATC AAC ACT AAT AAG G-3′ (SEQ ID NO: 14), and the second primer pair comprises a forward primer comprising the oligonucleotide sequence: 5′-CAA GGT GCA ATG ACT TTG TTT GGG CA-3′ (SEQ ID NO: 17) and a reverse primer comprising the oligonucleotide sequence 5′-GCA ACT TCA AAG TGT ACA GTA TTG GTA TCC C-3′ (SEQ ID NO: 18).

97. The method of claim 96, wherein step (c) comprises amplifying the first B. afzelii target nucleic acid in the presence of the first primer pair to form a first B. afzelii amplicon and amplifying the second B. afzelii target nucleic acid in the presence of the second primer pair to form a second B. afzelii amplicon, and step (g) comprises detecting the first B. afzelii amplicon and the second B. afzelii amplicon.

98. The method of claim 97, wherein the method further comprises detecting the presence of the first B. afzelii amplicon and the second B. afzelii amplicon.

99. A composition comprising: (a) a liquid sample, wherein the liquid sample (i) is suspected of containing at least one B. burgdorferi target nucleic acid, or (ii) contains at least one B. burgdorferi target nucleic acid amplicon generated from an amplification reaction; and (b) within the liquid sample, from 1×106 to 1×1013 magnetic particles per milliliter of the liquid sample, the magnetic particles having a mean diameter of from 700 nm to 950 nm, a T2 relaxivity per particle of from 1×104 to 1×1012 mM−1s−1, the magnetic particles comprising a first population and a second population, the first population having a first nucleic acid probe and a second nucleic acid probe conjugated to their surface and the second population having a third nucleic acid probe and a fourth nucleic acid probe conjugated to their surface, wherein the first probe comprises an oligonucleotide sequence of SEQ ID NO: 3, the second probe comprises an oligonucleotide sequence of SEQ ID NO: 7, the third probe comprises an oligonucleotide sequence of SEQ ID NO: 4, and the fourth probe comprises an oligonucleotide sequence of SEQ ID NO: 8.

100. A composition comprising: (a) a liquid sample, wherein the liquid sample (i) is suspected of containing a B. garinii target nucleic acid, or (ii) contains a B. garinii amplicon generated by amplifying the B. garinii target nucleic acid; and (b) within the liquid sample, from 1×106 to 1×1013 magnetic particles per milliliter of the liquid sample, the magnetic particles having a mean diameter of from 700 nm to 950 nm, a T2 relaxivity per particle of from 1×104 to 1×1012 mM−1s−1, wherein the magnetic particles comprise a first population of magnetic particles conjugated to a first nucleic acid probe comprising the oligonucleotide sequence: 5′-CTG AGA TTG AAG TTG ACG AAA AAA TCA G-3′ (SEQ ID NO: 11), and a second population of magnetic particles conjugated to a second nucleic acid probe comprising the oligonucleotide sequence: 5′-GAT CAT CCA ATA ATT CCA ATC TAC AGC G-3′ (SEQ ID NO: 12).

101. A composition comprising: (a) a liquid sample, wherein the liquid sample (i) is suspected of containing at least one B. afzelii target nucleic acid, or (ii) contains at least one B. afzelii target nucleic acid amplicon generated from an amplification reaction; and (b) within the liquid sample, from 1×106 to 1×1013 magnetic particles per milliliter of the liquid sample, the magnetic particles having a mean diameter of from 700 nm to 950 nm, a T2 relaxivity per particle of from 1×104 to 1×1012 mM−1s−1, the magnetic particles comprising a first population and a second population, the first population having a first nucleic acid probe and a second nucleic acid probe conjugated to their surface and the second population having a third nucleic acid probe and a fourth nucleic acid probe conjugated to their surface, wherein the first probe comprises an oligonucleotide sequence of SEQ ID NO: 3, the second probe comprises an oligonucleotide sequence of SEQ ID NO: 7, the third probe comprises an oligonucleotide sequence of SEQ ID NO: 4, and the fourth probe comprises an oligonucleotide sequence of SEQ ID NO: 8.

102. A removable cartridge comprising a plurality of wells, wherein the removable cartridge comprises one or more of the following: (a) a first well comprising the composition of claim 99; (b) a second well comprising the composition of claim 100; and (c) a third well comprising the composition of claim 101.

103. The removable cartridge of claim 102, wherein the removable cartridge comprises (a) through (c).

104. The removable cartridge of claim 102 or 103, further comprising one or more chambers for holding a plurality of reagent modules for holding one or more assay reagents.

105. The removable cartridge of any one of claims 102-104, further comprising a chamber comprising beads for lysing cells.

106. The removable cartridge of any one of claims 102-105, further comprising a chamber comprising a polymerase.

107. The removable cartridge of any one of claims 102-106, further comprising a chamber comprising one or more primers.

108. The removable cartridge of claim 107, wherein the one or more primers comprise oligonucleotide sequences selected from SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, and 18.

109. A method for diagnosing Lyme disease in a subject, the method comprising: (a) providing a biological sample obtained from the subject; and (b) detecting the presence of at least two different Bbsl species in the biological sample according to the method of any one of claim 1-5, 7-13, 15-29, or 48-79, wherein the presence of a Bbsl species in the biological sample obtained from the subject identifies the subject as one who may have Lyme disease.

110. The method of claim 109, wherein the presence of two different Bbsl species selected from B. burgdorferi, B. afzelii, and B. garinii is detected.

111. The method of claim 109, wherein the presence of B. burgdorferi, B. afzelii, and B. garinii is detected.

112. A method for diagnosing a tick-transmitted disease in a subject, the method comprising: (a) providing a biological sample obtained from the subject; (b) detecting the presence of a tick-borne pathogen according to the method of any one of claim 6-9, 14-17, or 47-89, wherein the presence of a tick-borne pathogen identifies the subject as one who may have a tick-transmitted disease.

113. A method for diagnosing Lyme disease in a subject, the method comprising: (a) providing a biological sample obtained from the subject; and (b) detecting the presence of a B. burgdorferi cell, a B. afzelii cell, or a B. garinii cell in the sample according to the method of any one of claim 30-46, 58-79, or 90-98, wherein the presence of a B. burgdorferi cell, a B. afzelii cell, or a B. garinii cell in the biological sample obtained from the subject identifies the subject as one who may have Lyme disease.

114. The method of claim 113, wherein the presence of at least two of a B. burgdorferi cell, a B. afzelii cell, and a B. garinii cell is detected.

115. The method of claim 113, wherein the presence of a B. burgdorferi cell, a B. afzelii cell, and a B. garinii cell is detected.

116. A method for treating Lyme disease in a subject, the method comprising: (a) providing a biological sample obtained from the subject; (b) detecting the presence of at least two different Bbsl species in the biological sample according to any one of claim 1-5, 7-13, 15-29, or 48-79, wherein the presence of a Bbsl species in the biological sample obtained from the subject identifies the subject as one who may have Lyme disease, and (c) administering a Lyme disease therapy to the subject identified as one who may have Lyme disease.

117. The method of claim 116, wherein the presence of two different Bbsl species selected from B. burgdorferi, B. afzelii, and B. garinii is detected.

118. The method of claim 116, wherein the presence of B. burgdorferi, B. afzelii, and B. garinii is detected.

119. A method for treating a tick-transmitted disease in a subject, the method comprising: (a) providing a biological sample obtained from the subject; (b) detecting the presence of a tick-borne pathogen according to any one of 6-9, 14-17, or 47-89, wherein the presence of a tick-borne pathogen identifies the subject as one who may have a tick-transmitted disease; and (c) administering a tick-transmitted disease therapy to the subject identified as one who may have a tick-transmitted disease.

120. A method for treating Lyme disease in a subject, the method comprising: (a) providing a biological sample obtained from the subject; and (b) detecting the presence of a B. burgdorferi cell, a B. afzelii cell, or a B. garinii cell in the sample according to the method of any one of claim 30-46, 58-79, or 90-98, wherein the presence of a B. burgdorferi cell, a B. afzelii cell, or a B. garinii cell in the biological sample obtained from the subject identifies the subject as one who may have Lyme disease; and (c) administering a Lyme disease therapy to the subject identified as one who may have Lyme disease.

121. The method of claim 120, wherein the presence of at least two of a B. burgdorferi cell, a B. afzelii cell, and a B. garinii cell is detected.

122. The method of claim 120, wherein the presence of a B. burgdorferi cell, a B. afzelii cell, and a B. garinii cell is detected.

123. The method of any one of claim 1-29 or 47-98, wherein the method detects a concentration of an individual Bbsl species of between 1-10 cells/mL of biological sample.

124. The method of claim 123, wherein the method individually detects a concentration of an individual Bbsl species of 8 cells/mL of biological sample.

125. The method of claim 123, wherein the method individually detects a concentration of an individual Bbsl species of 5 cells/mL of biological sample.

126. The method of claim 123, wherein the method individually detects a concentration of an individual Bbsl species of 3 cells/mL of biological sample.

127. The method of claim 123, wherein the method individually detects a concentration of an individual Bbsl species of 1 cells/mL of biological sample.

128. The method of any one of claim 1-29 or 47-98, wherein the method results in 95% correct detection when the individual Bbsl species is present in the biological sample at a frequency of less than or equal to 10 cells/mL.

129. The method of claim 128, wherein the method results in 95% correct detection when the individual Bbsl species is present in the biological sample at a frequency of less than or equal to 8 cells/mL.

130. The method of claim 128, wherein the method results in 95% correct detection when the individual Bbsl species is present in the biological sample at a frequency of less than or equal to 5 cells/mL.

131. The method of any one of claims 123-130, wherein the individual Bbsl species is B. burgdorferi, B. afzelii cell, or B. garinii.

132. The method of any one of claim 30-36, 59-72, 74-79, or 90-98, wherein the method detects B. burgdorferi at a concentration of 10 cells/mL of biological sample or less.

133. The method of claim 132, wherein the method detects B. burgdorferi at a concentration of 1-10 CFU/mL.

134. The method of claim 133, wherein the method detects B. burgdorferi at a concentration of 8 CFU/mL.

135. The method of any one of claims 132-134, wherein the method results in 95% correct detection when the B. burgdorferi is present in the biological sample at a frequency of less than or equal to 10 cells/mL.

136. The method of any one of claim 37-39, 59-72, or 74-79, wherein the method detects B. garinii at a concentration of 10 cells/mL of biological sample or less.

137. The method of claim 136, wherein the method detects B. garinii at a concentration of 1-10 CFU/mL.

138. The method of claim 137, wherein the method detects B. garinii at a concentration of 8 CFU/mL.

139. The method of any one of claims 136-138, wherein the method results in 95% correct when the B. garinii is present in the biological sample at a frequency of less than or equal to 10 cells/mL.

140. The method of any one of claim 40-46, 59-72, or 74-79, wherein the method detects B. afzelii at a concentration of 10 cells/mL of biological sample or less.

141. The method of claim 140, wherein the method detects B. afzelii at a concentration of 1-10 CFU/mL.

142. The method of claim 141, wherein the method detects B. afzelii at a concentration of 5 CFU/mL.

143. The method of any one of claims 140-142, wherein the method results in 95% correct when the B. afzelii is present in the biological sample at a frequency of less than or equal to 10 cells/mL.

12; 01; 12

edspap.20180171388