MICROORGANISM SEPARATION AND DETECTION

Abstract

Methods for separating microorganisms from non-microorganism cells in a non-microorganism cell-containing sample comprise incubating the sample with particles to form particle-microorganism complexes and then separating the particle-microorganism complexes from the non-microorganism cells. These methods are used to detect the absence or presence of a microorganism in a sample that also contains non-microorganism cells. Particular reagents and combinations of reagents enhance the selective capture of microorganisms in mixed samples. Corresponding compositions and kits are also provided.

Claims

1-29. (canceled)

30. A method of separating microorganisms from non-microorganism cells in a non-microorganism cell-containing sample, the method comprising: a) incubating the sample with particles to form particle-microorganism complexes, wherein the step of incubating is performed in the presence of sodium polyanethol sulfonate and a reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample; and b) separating the particle-microorganism complexes from the non-microorganism cells, wherein the sample is a blood containing sample.

31. The method of claim 30, further comprising (c) detecting the microorganisms in the particle-microorganism complex, wherein detecting comprises one or more of: detecting an enzymatic activity of a nucleic acid molecule modifying enzyme associated with the microorganism; (ii) detecting the microorganism directly by cytometry or microscopy; (iii) detecting the microorganism following cell culture; (iv) detecting the microorganism by nucleic acid amplification; and (v) detecting the microorganism by nucleic acid sequencing.

32. The method of claim 31, wherein detecting the microorganisms in the particle-microorganism complex comprises detecting an enzymatic activity of a nucleic acid molecule modifying enzyme associated with the microorganism, the method further comprising: a) lysing the microorganisms in the particle-microorganism complexes; b) incubating the lysate with a nucleic acid molecule which acts as a substrate for nucleic acid modifying activity of the microorganisms; and c) detecting a modified nucleic acid molecule resulting from the action of the nucleic acid modifying enzyme on the substrate nucleic acid molecule to detect the microorganism.

33. The method of claim 32, wherein step (a) comprises adding a lysis reagent containing the substrate nucleic acid molecule.

34. The method of claim 32, wherein the nucleic acid modifying enzyme comprises a DNA or RNA polymerase, optionally wherein the DNA polymerase is DNA polymerase I.

35. The method of claim 30, wherein the method further comprises washing the separated particle-microorganism complexes of step (b) to remove non-microorganism cells or lysate.

36. The method of claim 30, wherein step (b) further comprises removing the non-microorganism cells from the particle-microorganism complexes.

37. The method of claim 30, wherein step (b) is performed using a magnetic field or centrifugation.

38. The method of claim 30, wherein the reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample is a detergent; optionally wherein the detergent is non-ionic.

39. The method of claim 38, wherein the detergent is not conjugated to the particles/beads capable of forming complexes with microorganisms.

40. The method of claim 30, wherein the particles/beads have a diameter of between 0.1 and 2.0 m.

41. The method of claim 30, wherein the particles/beads are magnetic, optionally wherein the particles/beads are superparamagnetic.

42. The method of claim 30, wherein the outer surface of the particles/beads capable of forming complexes with microorganisms comprises a polymer, optionally wherein the polymer is carbon-based.

43. The method of claim 30, wherein the outer surface of the particles/beads capable of forming complexes with microorganisms comprises or is coated with any one or more of: i) carboxylic acid groups; ii) amino groups; iii) hydrophobic groups; and iv) streptavidin.

44. The method of claim 30, wherein the microorganism is a pathogenic microorganism, optionally wherein the pathogenic microorganism is a pathogenic bacterium or fungus.

45. The method of claim 30, wherein the non-microorganism cells comprise red blood cells and/or white blood cells.

46. A method of separating microorganisms from non-microorganism cells in a non-microorganism cell-containing sample, the method comprising: a) incubating the sample with particles to form particle-microorganism complexes; and b) separating the particle-microorganism complexes from the non-microorganism cells, wherein the particles have an outer surface that is not coated with any of (i) an antibody, (ii) an carbohydrate, (iii) a peptide derived from Apolipoprotein H protein, (iv) a Mannose Binding Lectin protein.

47. The method of claim 46, wherein the sample is a blood containing sample.

48. A kit comprising: a) beads capable of forming complexes with microorganisms, wherein the beads have an outer surface; b) sodium polyanethol sulfonate; and c) at least one reagent that selectively lyses non-microorganism cells in the sample whilst retaining intact microorganisms present in the sample.

49. The kit of claim 48, further comprising: detection means for detecting the absence or presence of microorganisms in the bead-microorganism complexes, wherein the detection means comprises a nucleic acid molecule (DNA) which acts as a substrate for nucleic acid modifying activity of the microorganisms, and wherein the nucleic acid molecule (DNA) is at least partially double stranded and comprises uracil residues in the complementary strand.

50. A kit comprising: a) particles capable of forming complexes with microorganisms, wherein the particles have an outer surface that is not coated with any of (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from Apolipoprotein H protein, (iv) a Mannose Binding Lectin protein; and b) detection means for detecting the absence or presence of microorganisms in the particle-microorganism complexes, wherein the detection means comprises a nucleic acid molecule (DNA) which acts as a substrate for nucleic acid modifying activity of the microorganisms, and wherein the nucleic acid molecule (DNA) is at least partially double stranded and comprises uracil residues in the complementary strand.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0315] FIG. 1 is an image of blood samples and shows the extent of blood lysis for each sample-set: E-BUF, UREA, Tris+NaCl, freezing (left to right) (see Example 8).

[0316] FIG. 2 is an image of final sample outputs prior to PCR set-up. The image provides a visual demonstration of the benefit of SPS for sample processing with magnetic beads in blood: SPS appears to enable more thorough removal of blood components as indicated by less red eluates in the presence of SPS. Note, that the BacTec PLUS aerobic broth used for the Blood Broth sample-set also contains SPS (see Example 9).

[0317] The invention will be understood with respect to the following non-limiting examples:

EXPERIMENTAL SECTION

Abbreviations & Definitions

[0318] 5th % Fifth Percentile threshold calculation to determine 5% FPR (formula=PERCENTILE.INC(array,0.05)) [0319] BO Broth Only [0320] BB Blood Broth [0321] Cfu Colony Forming Unit [0322] Confirm A PCR multiplex assay targeting microbial DNA according to gram status (Gram Negative, Gram Positive or Candida) [0323] CPD Citrate Phosphate Dextrose [0324] Ct Cycle Threshold Value [0325] CV Critical Value (cfu): theoretical limit of detection based on cfu value and Ct using formula: sample cfu2.sup.Ct [0326] D1 . . . Dilution point (10-fold series) [0327] E*cfu Extrapolated cfu value using dilution point with highest countable TVC in a dilution series [0328] EC Escherichia coli [0329] ETGA Enzyme Template Generation and Amplification [0330] IPC Internal Process Control: PCR template present in LM to demonstrate correct sample processing and verify PCR amplification in ETGA negative samples [0331] LAWN Confluent microbial growth [0332] LM Microbial Lysis Mix containing a mixture of detergents and microbial lytic enzymes [0333] MM Master Mix [0334] NoCt No amplification above threshold fluorescence after 50 cycles [0335] NSC No Spike Control [0336] O/n Overnight [0337] PC Polymerase-spike Control [0338] PCR Polymerase Chain Reaction [0339] Pt Positivity threshold calculated from NSC/NC results [0340] qPCR Quantitative Polymerase Chain Reaction [0341] RT Room temperature (+19 to +20 C.) [0342] s/n Supernatant [0343] SPS Sodium polyanethol sulfonate [0344] TNTC Too Numerous To Count [0345] TVC Total Viable Count [0346] WB Wash Buffer (containing Tris-HCl+Sodium Chloride+Igepal+Sodium Deoxycholate+Tergitol, unless otherwise stated); or Whole Blood where stated. [0347] Ct Difference between two Ct values (typically NSC Ctpositive sample Ct)

Example 1

[0348] In a manual format, two bead types (Merck Bio-Estapor (streptavidin-conjugated) 300 nm beads (ProductBE-M08/03; Bio-Estapor) and ademtech Bio-Adembeads Streptavidin Plus 200 nm beads (Product number 03222; Bio-Ademtech)) were compared to ApoH Technologies Peps6 beads (ReferenceMP20006; ApoH Peps6).

[0349] In experiment 1A, an aliquot of Bio-Estapor beads (25 L) and an aliquot of ApoH Pep6 beads (10 uL) were compared for binding. The higher volume of Bio-Estapor reflected the lower number of beads per mL in the material provided compared to the ApoH material. Three organisms were tested: E. coli (Gram negative bacterium), S. epidermidis (Gram positive bacterium) and C. albicans (yeast). 0.5 mL of organism suspension was exposed to the beads in 0.5 mL TTGB microbial binding buffer, provided in the ApoH Peps6 kit (Peps6 Captobac, Reference MP10031-50T).

[0350] After allowing the organism to bind for 30 min, the sample of beads was separated from the liquid supernatant by applying a magnetic field to concentrate the beads and removing the supernatant with a pipette. The beads were gently washed with three aliquots of wash buffer (50 mM Tris pH 8, 1% v/v Igepal CA-630, 150 mM NaCl, 0.25% v/v Tergitol 15-S-9) and the retained supernatant and the washed beads were analysed for viable organisms by two methods; colony counts on an Agar Petri dish and detection of microbial DNA by the enzymatic template generation and amplification (ETGA) test (as described in Zweitzig et al., 2012. Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes. Nucleic Acids Research 40, 14, e109, 1-12; and in WO2011/130584, WO2013/103744 and WO2016/005768).

[0351] The plate counts in Table 1A show that with the Bio-Estapor beads and E. coli, the great majority of growth is found from the beads (33 CFU) vs the supernatant (2 CFU) and this is similar to the result from ApoH Peps6. No growth was found with S. epidermidis and this organism did not appear to grow in the original broth. C. albicans showed approximately 10% of the CFU in the supernatant and 90% bound, for both Bio-Estapor and ApoH Peps6. These results indicate that the Bio-Estapor beads appear to bind organisms at an equivalent rate to the commercially available organism-binding beads Peps6 under the conditions of the test. The sensitive ETGA test supports the results but indicates that S. epidermidis may bind to Bio-Estapor better than Peps6 as shown by the lower Cq value.

TABLE-US-00001 TABLE 1A Experiment 1 A Estapor beads at 25 L, ApoH at 10 L (+ve ctl) and no beads (ve ctl) vs E. coli, S. epidermidis & C. albicans dilutions in blood broth (Manual protocol). E. c. ONC dil.sup.n 2 1.01E+04 cfu/mL E. coli S. e. ONC dil.sup.n 2 No growth cfu/ml Staph epidermidis C. a. ONC dil.sup.n 1 3.60E+05 cfu/ml Candida albicans Plate count ETGA Resuts Tube Organism ApoH Estapor CFU Cq Bio-Estapor beads Bio-Estapor in binding buffer (supernatant) E. coli 25 L 2 not tested Bio-Estapor in binding buffer (bound) 33 32.54 Bio-Estapor in binding buffer (supernatant) S. epidermidis 25 L 0 not tested Bio-Estapor in binding buffer (bound) 0 37.63 Bio-Estapor in binding buffer (supernatant) C. albicans 25 L 34 not tested Bio-Estapor in binding buffer (bound) 246 33.34 ApoH Peps6 beads ApoH Peps6 in binding buffer (supernatant) E. coli 10 L 1 not tested ApoH Peps6 in binding buffer (bound) 38 33.18 ApoH Peps6 in binding buffer (supernatant) S. epidermidis 10 L 0 not tested ApoH Peps6 in binding buffer (bound) 0 40.94 ApoH Peps6 in binding buffer (supernatant) C. albicans 10 L 35 not tested ApoH Peps6 in binding buffer (bound) 316 34.01 No beads No beads E. coli 43 not tested No beads s. epidermidis 0 not tested No beads c. albicans 316 not tested No bugs/no beads No beads no microbes 0 not tested

[0352] Experiment 1B demonstrates E. coli binding under similar conditions to Experiment 1A although in 1B the wash steps were omitted. Experiment 1B shows that another bead, Bio-Ademtech, also binds organisms although at a lower level (see Table 1B). Here the plate counts indicate that approximately one third of the viable counts have bound to the bead. The more sensitive ETGA DNA polymerase assay indicates that half of the organisms remain on the beads, as the Cq for the beads and the supernatant are approximately equal.

TABLE-US-00002 TABLE 1B Experiment 1B AdemTech beads and ApoH (+ve ctl) and no beads & no beads/no bugs controls. E. ONC dil.sup.n 2 1.26E+04 cfu/mL Plate count ETGA Resuts Tube AdemTech ApoH Estapor CFU Cq AdemTech beads Ademtech beads supernatant 100 L 78 34.09 Ademtech beads bound 100 L 44 34.02 ApoH Peps6 (5 L in 95 L) ApoH Peps6 supernatant 5 L 0 40.16 ApoH Pep6 bound 5 L 36 32.8 No beads No beads 45 34.22 No bugs/no beads No beads/no organisms 0 Excluded

Example 2

[0353] In Experiment 2, ApoH Peps6, Bio-Estapor and Estapor beads with a carboxylated surface (Product MI-030/40; Estapor COOH) were compared. The number of organisms remaining in the supernatant after binding of E. coli to the beads for 30 min was measured using a fluorescent ATP assay (BacTiter-Glo Microbial Cell Viability Assay; Promega Corporation, G8230). Although this is an indirect test in that it does not directly detect the presence of organisms on the bead, it is a useful comparative test for the ligand-based beads (ApoH Peps6) and the non-ligand beads of the invention (Bio-Estapor and Estapor COOH). After binding of 1 mL of 10.sup.4 CFU/mL E coli for 30 mL from a phosphate saline buffer, an aliquot of the supernatant was assayed for ATP as a measure of organism content using the BacTiter-Glo assay. The results in Table 2 show that the reduction in levels of organisms in the supernatant for Peps6 beads, Bio-Estapor and Estapor COOH were 33%, 27% and 24% respectively when measured using this technique.

TABLE-US-00003 TABLE 2 Sample % Binding (10{circumflex over ()}4 CFU/ Baseline Baseline Based on Signal Bead Supplier ml E. coli) (No E. coli) corrected Depletion of to (Type) Date of assay Time (RFU) (RFU) (RFU) Supernatant noise ApoH (Peps6) Jun. 3, 2018 am 1896 345 1551 32% 5.50 ApoH (Peps6) Jun. 3, 2018 am 1856 345 1511 34% 5.38 ApoH (Peps6) Jun. 3, 2018 pm 2549 378 2171 31% 6.74 ApoH (Peps6) Jun. 3, 2018 pm 2398 378 2020 35% 6.34 Average 33% Std Dev 2.1% % CV 6.4% BioEstapor (Sav) Jun. 3, 2018 am 2067 345 1722 24% 5.99 BioEstapor (Sav) Jun. 3, 2018 am 2138 345 1793 21% 6.20 BioEstapor (Sav) Jun. 3, 2018 pm 2447 378 2069 34% 6.47 BioEstapor (Sav) Jun. 3, 2018 pm 2618 378 2240 28% 6.93 Average 27% Std Dev 5.4% % CV 20.1% Estapor (COOH) Jun. 3, 2018 am 2045 345 1700 25% 5.93 Estapor (COOH) Jun. 3, 2018 am 2500 345 2155 5% 7.25 Estapor (COOH) Jun. 3, 2018 pm 2416 378 2038 35% 6.39 Estapor (COOH) Jun. 3, 2018 pm 2520 378 2142 32% 6.67 Average 24% Std Dev 13.2% % CV 54.3% No Beads Jun. 3, 2018 am 2578 345 2233 N/A 7.47 No Beads Jun. 3, 2018 am 2668 345 2323 N/A 7.73 No Beads Jun. 3, 2018 pm 3594 378 3216 N/A 9.51 No Beads Jun. 3, 2018 pm 3418 378 3040 N/A 9.04

Example 3

[0354] Example 3 shows results from testing E. coli (EC), S. aureus (SA) and C. albicans (CA) in dilution series performed by automating the method for magnetic separation described in Example 1. The assay used Bio-Estapor 300 nm diameter beads as the capture medium with a binding buffer of TTGB containing 0.25% Tergitol. As 10-fold dilutions of each of the three organisms were made, so a continuous change in the Ct was recorded allowing a dose response curve to be constructed.

TABLE-US-00004 TABLE 3 Bugs: EC, SA, CA ETGA threshold 4.338 IPC threshold 0.911 ETGA ETGA GrNeg GrPos Candida Confirm Sample TVCs Ct IPC Ct result Ct (.315) dF Ct (.318) dF Ct (.161) dF Result Confirm EC e-2 640,000 CFU/mL 15.62 32.33 18.89 10.29 No Ct 0.57 No Ct 0.08 GrNeg GrNeg EC e-3 64,000 CFU/mL 20.18 32.20 21.82 9.25 No Ct 0.69 No Ct 0.10 GrNeg GrNeg EC e-4 6,400 CFU/mL 25.39 31.79 24.00 8.11 35.20 1.30 No Ct 0.15 GrNeg GrNeg EC e-5 640 CFU/mL 29.15 31.57 29.56 3.64 32.22 3.23 No Ct 0.13 ND GrNeg EC e-6 64 CFU/mL 34.03 31.59 30.23 2.23 32.07 2.70 No Ct 0.03 ND GrNeg EC e-7 37.32 31.62 35.62 0.79 33.75 2.36 No Ct 0.09 N/A N/A EC e-8 37.77 31.99 34.55 1.05 33.52 2.37 No Ct 0.13 N/A N/A SA e-2 180,000 CFU/mL 17.49 32.40 33.89 0.04 16.83 18.51 No Ct 0.05 GrPos GrPos SA e-3 18,000 CFU/mL 21.34 32.20 No Ct 0.08 20.18 16.59 No Ct 0.02 GrPos GrPos SA e-4 1,800 CFU/mL 24.75 31.38 No Ct 0.73 25.37 16.26 No Ct 0.08 GrPos GrPos SA e-5 180 CFU/mL 28.63 31.45 No Ct 0.76 28.26 12.15 No Ct 0.15 GrPos GrPos SA e-6 18 CFU/mL 35.21 31.95 No Ct 0.54 32.94 2.05 No Ct 0.02 ND N/A SA e-7 37.34 31.99 34.15 0.39 33.45 1.95 No Ct 0.07 N/A N/A SA e-8 37.27 31.91 36.02 0.27 32.84 2.24 No Ct 0.01 N/A N/A CA e-0 293,000 CFU/mL 23.99 32.99 No Ct 0.52 32.31 3.58 21.30 7.66 ND Candida CA e-1 29,300 CFU/mL 27.97 31.73 No Ct 0.75 33.36 1.72 24.87 6.14 Candida Candida CA e-2 2,900 CFU/mL 31.85 31.69 34.44 0.60 31.73 4.44 29.33 2.90 ND Candida CA e-3 290 CFU/mL 35.56 31.65 No Ct 0.32 32.53 3.54 35.14 0.47 ND Candida CA e-4 29 CFU/mL 36.94 31.52 No Ct 0.29 34.12 1.50 No Ct 0.02 N/A N/A CA e-5 3 CFU/mL 35.03 32.03 No Ct 0.11 33.94 1.42 No Ct 0.02 N/A N/A CA e-6 36.96 31.88 36.07 0.33 33.99 1.01 No Ct 0.02 N/A N/A NSC 1 0 CFU/mL 37.35 32.02 No Ct 1.22 32.45 3.07 No Ct 0.10 N/A N/A NSC 2 0 CFU/mL 37.20 32.01 No Ct 0.01 32.11 3.85 No Ct 0.08 GrPos N/A NSC 3 0 CFU/mL 37.10 31.82 No Ct 0.52 31.47 4.24 No Ct 0.02 GrPos N/A NSC Av 37.22 31.90 LOB Result Ct DF ETGA FAM 38.51 ETGA IPC 31.27 Confirm FAM 0.13 Confim HEX 2.13 Confirm Cy5 1.69

[0355] The following examples demonstrate the universal microbial capture of microorganisms by magnetic beads in Momentum's Magnitor test. The Magnitor test consists of two microbial detection read-outs: [0356] ETGA: detection of microbial polymerase from intact microbial cells [0357] Confirm: detection of microbial DNA according to gram status (Gram Negative, Gram Positive, or Candida)

Key Findings:

[0358] Magnetic beads capture bacteria and fungi from simple buffers and a variety of complex biological specimen types [0359] Microbial capture occurs using a variety of different bead sizes (0.2 to 1.5 m diameter beads) and surface coatings (e.g. carboxylated, hydrophobic, aminated etc)

[0360] Certain binding buffer components can improve microbial detection in the Magnitor assay, for example detergent-based lysis of blood.

Example 4: Detection of Microorganisms is Dependent on Capture by Magnetic Beads

Aim:

[0361] Microbial binding performance was assessed for E. coli, S. aureus and C. albicans in a simple Tris+NaCl buffer (pH buffered with physiological salt conc. to prevent microbial osmotic shock that may occur in water only). A no bead control sample-set was also included in this experiment to demonstrate that detection is dependent on the presence of magnetic beads for microbial capture.

Magnetic Bead Preparation:

[0362] Estapor beads (Merck, Cat #M1-30/40) washed 31 mL in 1 Tris+NaCl buffer: 40 L beads resuspended in a final volume of 400 L 1 Tris+NaCl buffer (1% solid content)

Protocol:

[0363] Microorganism overnight liquid cultures (o/n) set-up as standard in BacTec PLUS aerobic broth (inoculation of 3 mL broth from agar plate). The following day (approx. 16 hours later) 1.88 L E. coli and S. aureus liquid culture added to 3 mL broth, and 18.75 L C. albicans liquid culture added to 3 mL broth; and 2-hour outgrowth performed at 37 C., 500 rpm. [0364] Following 2-hour outgrowth, microorganism precultures diluted (DF10) in 1 Tris+NaCl buffer (50 mM Tris-HCl [pH8.0]+150 mM NaCl) to create four dilution points per microorganism. [0365] 100 L TVCs performed for each microbial dilution
Manual simulation of Magnitor performed using DynaMaq-2 magnet and manual liquid transfers: [0366] 1 mL samples added to 2 mL tubes containing 15 L prewashed beadsNote, 112 L 1 Tris+NaCl buffer not added to tube with beads (as per standard protocol), because all microbial samples were diluted in the same 1 buffer. [0367] 30 mins shaking (1000 rpm) @ 37 C. [0368] 5 mins magnetisation on DynaMag-2 [0369] All s/n removed [0370] 1 mL Wash Buffer (WB) added and tubes mixed for 2 mins @ RT (1000 rpm) [0371] 3 mins magnetisation on Dynmag-2 [0372] All s/n removed [0373] 50 L Lysis Mix (LM) added to tubes off magnet (5 L Polymerase Control (PC) added to each PC sample tube) [0374] ETGA reaction performed: 5 mins at 1000 rpm, then 55 mins at 800 rpm @26 C. [0375] Manual qPCR set-up for ETGA and Confirm (10 L reactions)

Results:

TVCs (COL/SAB Agar Plates)

[0376]

TABLE-US-00005 Colonies *E cfu/mL E. coli D1 TNTC 216,000 E. coli D2 TNTC 21,600 E. coli D3 *216 2,160 E. coli D4 26 216 S. aureus D1 *671 6,710 S. aureus D2 121 671 S. aureus D3 33 67 S. aureus D4 0 7 C. albicans D1 *51 510 C. albicans D2 6 51 C. albicans D3 8 5 C. albicans D4 0 1 NSC 0 *E cfu/mL values derived from highest countable TVC plate

ETGA Ct

[0377]

TABLE-US-00006 E. coli S. aureus C. albicans (+) BEADS () BEADS (+) BEADS () BEADS (+) BEADS () BEADS D1 12.64 26.25 18.99 29.29 26.57 30.61 D2 18.09 29.02 24.28 32.40 30.08 33.62 D3 22.75 32.11 28.61 32.70 33.27 33.12 D4 30.52 31.01 30.85 31.56 30.80 31.65 NSC1 35.26 32.22 35.73 31.37 34.69 31.31 NSC2 35.67 33.13 35.61 35.13 35.29 32.40 NSC3 34.92 32.38 35.28 31.54 34.31 31.77 PC 32.10 30.79 31.40 29.64 31.69 29.34 Average NSC 35.28 32.58 35.54 32.68 34.76 31.83

Internal Process Control (IPC) Ct

[0378]

TABLE-US-00007 E. coli S. aureus C. albicans (+) BEADS () BEADS (+) BEADS () BEADS (+) BEADS () BEADS D1 37.36 31.51 32.61 32.20 32.32 31.95 D2 32.85 32.11 32.26 32.38 32.10 32.26 D3 32.44 32.10 31.98 32.19 31.82 33.10 D4 32.15 32.01 32.10 32.15 31.68 32.15 NSC1 32.20 32.15 32.26 31.95 32.12 32.05 NSC2 32.24 32.40 32.13 33.98 32.09 32.20 NSC3 32.17 32.33 32.18 32.39 32.26 32.20 PC 32.30 32.62 32.18 32.51 32.50 32.24
ETGA Ct (averageNSC)

TABLE-US-00008 E. coli S. aureus C. albicans (+) BEADS () BEADS (+) BEADS () BEADS (+) BEADS () BEADS D1 22.64 6.33 16.55 3.39 8.19 1.221 D2 17.19 3.56 11.26 N/A 4.69 N/A D3 12.54 0.47 6.93 N/A 1.49 N/A D4 4.77 1.57 4.69 N/A 3.97 N/A NSC1 N/A N/A N/A N/A N/A N/A NSC2 N/A N/A N/A N/A N/A N/A NSC3 N/A N/A N/A N/A N/A N/A PC 3.18 1.79 4.14 3.04 3.08 2.48
Critical Values Based on Average NSC (cfu/mL)

TABLE-US-00009 E. coli S. aureus C. albicans (+) BEADS () BEADS (+) BEADS () BEADS (+) BEADS () BEADS D1 0.03 2692.80 0.07 641.54 1.74 218.601 D2 0.14 1836.42 0.27 N/A 1.98 N/A D3 0.36 1557.76 0.55 N/A 1.81 N/A D4 7.93 72.82 0.26 N/A 0.03 N/A NSC1 N/A N/A N/A N/A N/A N/A NSC2 N/A N/A N/A N/A N/A N/A NSC3 N/A N/A N/A N/A N/A N/A PC N/A N/A N/A N/A N/A N/A

Confirm Ct

[0379]

TABLE-US-00010 E. coli (GrNeg) S. aureus (GrPos) C. albicans (Candida) (+) BEADS () BEADS (+) BEADS () BEADS (+) BEADS () BEADS D1 24.26 42.44 28.13 NoCt 35.08 NoCt D2 27.83 32.08 31.65 NoCt 39.30 43.21 D3 33.17 NoCt 36.79 NoCt NoCt NoCt D4 NoCt NoCt 36.71 NoCt NoCt NoCt NSC1 NoCt NoCt NoCt 41.51 NoCt 48.84 NSC2 40.57 NoCt NoCt NoCt NoCt NoCt NSC3 NoCt NoCt NoCt 40.49 NoCt NoCt PC NoCt NoCt 43.24 NoCt NoCt NoCt Positivity threshold (Pt) 40 Ct

Analysis:

[0380] Magnitor results for (+) BEADS samples show a very strong cell-density specific ETGA and Confirm signal for all three microorganism species, demonstrating bead-specific binding of a broad range of microorganism groups (GrNeg, GrPos, Candida). [0381] Note, that Candida results could have followed a better cell density trend, but the liquid culture was quite particulate which may have affected the quality of serial dilutions [0382] Some evidence of microbial cell carryover in the () BEADS controls, but this is to be expected with only a single wash step.

Example 5: Microbial Capture from Blood by Magnetic Beads Occurs in Simple and Complex Blood Lysis Buffers, and Allows Microbial Detection Comparable to Capture by Centrifugation

Aim:

[0383] To compare two different blood lysis buffers in two different diluent formats for development of a simple and fast Rapid Magnitor test (no wash step included in protocol).

Test Conditions:

[0384]

TABLE-US-00011 2 EBB 1 mL 2 EBB + 1 mL Specimen 10 EBB 112 L 10 EBB + 1 mL Specimen 2 B-BUF 1 mL 2 B-BUF + 1 mL Specimen 10 B-BUF 112 L 10 B-BUF + 1 mL Specimen 10 EBB: 500 mM Tris-HCl [pH 8.0] + 2.5% Tergitol 10 B-BUF: 500 mM Tris-HCl [pH 8.0] + 1.5M Sodium Chloride + 10% Igepal + 5% Sodium Deoxycholate + 2.5% Tergitol

Sample Set-Up:

[0385] E. coli o/n liquid culture 1E-3 dilution spiked into blood broth (6.25 L o/n per mL: 244 L o/n+39 mL blood broth) and 60 minute out-growth performed in shaking incubator @ 37 C., 500 rpm [0386] Following 2-hour outgrowth, samples produced by adding 1 mL specimen to 2 mL tube containing buffer (and 15 L BioEstapor beads (Merck, Cat #BE-M 08/0.3) for Mag Beads sample-set): triplicate E. coli (EC) and No Spike Control (NSC) samples per test condition [0387] 100 L TVCs performed for NSC and E. coli (including dilutions of specimen to ensure countable plates)

Protocol:

[0388] Samples set up as above, and progressed immediately to Spin or Mag Beads protocol

Spin Protocol

[0389] Samples centrifuged for 3 minutes at 9000g (tube hinges facing outwards for pellet traceability) [0390] Supernatants removed [0391] 50 L LM added to samples (approx. 10 pipette mixes to resuspend pellets) [0392] Samples placed in shaking incubator at 900 rpm for 5 minutes and then 800 rpm for 55 minutes (26 C.) [0393] Centrifuge samples at 17000g for 1 minute before qPCR set-up

Mag Beads Protocol

[0394] Samples placed in shaking incubator at 900 rpm for 30 minutes (37 C.) [0395] Samples placed on DynaMag-2 magnetic rack for 5 minutes and then supernatants removed [0396] 50 L LM added to samples (approx. 10 pipette mixes to resuspend pellets) [0397] Samples placed in shaking incubator at 900 rpm for 5 minutes and then 800 rpm for 55 minutes (26 C.) [0398] Magnetise samples for 3 minutes before qPCR set-up [0399] Manual qPCR set-up (10 L reactions) for ETGA mastermix only

Results:

cfu Calculation

[0400] Sample and dilutions of sample plated on COL plates (100 L)

TABLE-US-00012 TVC *cfu/mL E. coli 1E3 TNTC 68500 E. coli 1E4 685 6850 E. coli 1E5 89 685 NSC 0 0

[0401] Sample source

ETGA Ct

[0402]

TABLE-US-00013 Centrifugation Magnetic Beads cfu/sample 2XEBB 10XEBB 2XB-BUF 10XB-BUF 2XEBB 10XEBB 2XB-BUF 10XB-BUF E. coli 1 68,500 15.89 16.94 21.43 22.83 15.10 17.12 20.78 22.61 E. coli 2 68,500 15.63 16.87 22.72 21.82 15.35 17.50 21.07 23.21 E. coli 3 68,500 16.05 16.85 20.04 22.20 15.69 17.72 21.77 23.20 NSC1 23.72 25.48 47.72 NoCt 29.52 34.45 45.73 44.89 NSC2 24.51 25.01 43.55 46.85 30.02 34.30 46.30 44.26 NSC3 24.16 25.74 NoCt 44.62 30.08 34.21 48.06 45.06

Summary Data

[0403]

TABLE-US-00014 Centrifugation Magnetic Beads cfu/sample 2XEBB 10XEBB 2XB-BUF 10XB-BUF 2XEBB 10XEBB 2XB-BUF 10XB-BUF Ave. E. coli ETGA Ct 68,500 15.86 16.89 21.40 22.28 15.38 17.45 21.21 23.01 Ave.NSC ETGA Ct 24.13 25.41 45.64 45.73 29.88 34.32 46.70 44.74 Ave. ETGA ACt 68,500 8.27 8.52 24.24 23.45 14.49 16.87 25.49 21.73

Analysis:

[0404] Microbial binding by magnetic beads occurs in: [0405] Simple and complex blood lysis buffers (EBB=Tris-HCl+Tergitol; B-BUF=Tris-HCl+Sodium Chloride+Igepal+Sodium Deoxycholate+Tergitol), but blood-derived test signal varies depending on blood lysis buffer components [0406] Diluted (2 buffer: 1-part blood lysis buffer to 1-part specimen) and concentrated (10 buffer: 1-part blood lysis buffer to 9 parts specimen) sample formats
Furthermore, microbial detection signal for microbial capture by magnetic beads is comparable to capture by centrifugation.

Example 6: Microbial Capture from Blood by Magnetic Beads is not Dependent on Blood Lysis, but Downstream Microbial Detection is Improved when Microbial Binding Occurs in Lysed Blood

Aim:

[0407] Given the recent discovery that multiple bead types/sizes produce similar Magnitor results for microbial serial dilutions and NSCs, it was thought that a component within Momentum's binding buffer might be mediating/facilitating this observed universal microbial binding character. To investigate this possibility, a dilution series of E. coli was performed comparing the standard binding buffer (B-BUF) with a detergent-free B-BUF consisting of just Tris-HCl [pH8.0]+NaCl, to test whether the detergents in general are important for microbial binding. Sample-sets were prepared for blood-broth, broth-only and in 1 binding buffer only to compare results for different specimen types.

Preparation:

[0408] 100 mL 10 Binding Buffers prepared fresh: [0409] B-BUF: 500 mM Tris-HCl [pH 8.0]+1.5 M Sodium Chloride+10% Igepal+5% Sodium Deoxycholate+2.5% Tergitol [0410] Tris+NaCl: 500 mM Tris-HCl [pH 8.0]+1.5 M Sodium Chloride

[0411] Estapor beads (Merck, Cat #M1-30/40) washed 31 mL in respective 1 buffer (diluted 10 B-BUF or 10 Tris+NaCl): 40 L beads resuspended in a final volume of 400 L 1 buffer (1% solid content)

Protocol:

[0412] E. coli o/n liquid culture set-up as standard in BacTec PLUS aerobic broth, then following day (approx. 16 hours later) 1.88 L o/n added to 3 mL broth (equivalent to EC 1E-1 dilution added to broth at 6.25 L/mL) and 2-hour outgrowth performed at 37 C., 500 rpm. [0413] Following 2-hour outgrowth, E. coli preculture serially diluted (DF10) down to EC 1E-6 in either prewarmed blood-broth (BB), broth only (BO) or 1 buffer (B-BUF or Tris+NaCl). [0414] 100 L TVCs performed for all E. coli dilutions and NSCs

Manual Simulation of Magnitor Performed Using DyneMag-2 Magnet and Manual Liquid Transfers:

[0415] 1 mL samples added to 2 mL tubes containing 112 L of binding buffer (either B-BUF or Tris+NaCl: 10 for BB and BO sample-sets; and 1 for Buffer sample-sets)+15 L beads (prewashed in respective buffer) [0416] 30 mins shaking (1000 rpm) @ 37 C. [0417] 5 mins magnetisation on DynaMag-2 [0418] All s/n removed [0419] 1 mL WB added and tubes mixed for 2 mins @ RT (1000 rpm) [0420] 5 mins magnetisation on Dynmag-2 [0421] All s/n removed [0422] 50 L LM added to tubes off magnet (5 L Polymerase Control (PC) added to each PC sample tube) [0423] ETGA reaction performed: 5 mins at 1000 rpm, then 55 mins at 800 rpm @26 C. [0424] Manual qPCR set-up for ETGA and Confirm (10 L reactions)

Observations:

[0425] No blood lysis observed for Tris+NaCl, as expected [0426] Beads more grainy/aggregated in absence of detergents

Results:

[0427]

TABLE-US-00015 10X Binding Buffer BB BO 1X B-BUF Tris+NaCl Specimen TVC *E cfu/mL TVC *E cfu/mL TVC *E cfu/mL TVC *E cfu/mL E. coli 1E-3 TNTC 74800 TNTC 52000 TNTC 47600 TNTC 54700 E. coli 1E-4 *748 7480 *520 5200 *476 4760 *547 5470 E. coli 1E-5 88 748 70 520 45 476 57 547 E. coli 1E-6 6 75 5 52 2 48 5 55 NSC 0 0 0 0 0 0 0 0 *Cfu/mL values extrapolated from highest countable TVC

ETGA Ct

[0428]

TABLE-US-00016 10X Binding B-BUF Tris+NaCl Specimen BB BO B-BUF BB BO Tris+NaCl E. coli 1E-3 22.84 21.63 18.93 24.71 17.78 14.91 E. coli 1E-4 27.32 26.16 25.08 29.70 21.58 20.14 E. coli 1E-5 31.40 30.03 29.31 33.58 25.38 24.53 E. coli 1E-6 36.87 32.91 33.62 34.97 29.36 31.24 NSC 1 44.80 35.23 34.71 36.04 38.81 33.73 NSC 2 43.10 34.86 35.26 35.21 38.83 34.89 NSC 3 44.11 34.54 36.72 35.41 38.56 34.19 PC 34.84 28.06 31.37 34.08 32.78 30.55 Average 44.00 34.87 35.56 35.55 38.74 34.27 NSC

IPC Ct

[0429]

TABLE-US-00017 10X Binding B-BUF Tris+NaCl Specimen BB BO B-BUF BB BO Tris+NaCl E. coli 1E-3 33.89 31.43 32.14 33.59 32.15 33.36 E. coli 1E-4 34.41 31.22 31.67 33.63 31.66 31.68 E. coli 1E-5 33.44 31.27 31.41 32.85 31.35 31.27 E. coli 1E-6 33.27 31.34 31.46 33.16 31.11 31.49 NSC 1 34.06 31.29 31.43 33.26 31.34 31.30 NSC 2 35.07 31.53 31.69 33.18 31.50 31.51 NSC 3 33.79 31.29 31.54 33.25 31.11 31.77 PC 34.22 31.29 32.18 33.34 31.37 31.65

ETGA Ct (Average NSC)

[0430]

TABLE-US-00018 10X Binding B-BUF Tris+NaCl Specimen BB BO B-BUF BB BO Tris+NaCl E. coli 1E-3 21.16 13.25 16.63 10.85 20.95 19.35 E. coli 1E-4 16.68 8.71 10.49 5.86 17.15 14.13 E. coli 1E-5 12.61 4.84 6.25 1.97 13.35 9.74 E. coli 1E-6 7.14 1.97 1.94 0.58 9.38 3.03 NSC 1 N/A N/A N/A N/A N/A N/A NSC 2 N/A N/A N/A N/A N/A N/A NSC 3 N/A N/A N/A N/A N/A N/A PC 9.16 6.82 4.20 1.47 5.96 3.72
Critical Values Based on Average NSC (cfu/mL)

TABLE-US-00019 10X Binding Buffer B-BUF Tris+NaCl Specimen BB BO B-BUF BB BO Tris+NaCl E. coli 1E-3 0.03 5.34 0.47 40.63 0.03 0.08 E. coli 1E-4 0.07 12.40 3.32 129.20 0.04 0.31 E. coli 1E-5 0.12 18.10 6.25 190.59 0.05 0.64 E. coli 1E-6 0.53 13.30 12.42 50.03 0.08 6.69 NSC 1 N/A N/A N/A N/A N/A N/A NSC 2 N/A N/A N/A N/A N/A N/A NSC 3 N/A N/A N/A N/A N/A N/A PC N/A N/A N/A N/A N/A N/A

Confirm GrNeg Ct

[0431]

TABLE-US-00020 10X Binding B-BUF Tris+NaCl Specimen BB BO B-BUF BB BO Tris+NaCl E. coli 1E-3 31.36 NoCt 28.13 30.53 31.60 25.94 E. coli 1E-4 36.41 NoCt 30.86 35.72 38.16 26.69 E. coli 1E-5 37.67 NoCt NoCt NoCt 39.26 34.62 E. coli 1E-6 NoCt NoCt NoCt NoCt NoCt NoCt NSC 1 NoCt NoCt NoCt 42.97 NoCt NoCt NSC 2 NoCt NoCt NoCt NoCt 39.64* NoCt NSC 3 NoCt NoCt NoCt 49.52 NoCt NoCt PC NoCt NoCt NoCt NoCt NoCt NoCt Positivity threshold (Pt) 40 Ct; *false positives

Analysis:

[0432] Detergents are important for producing good ETGA results in the presence of blood (as indicated by poorer ETGA results for 10 Tris+NaCl with BB), but microbial capture/detection is still evident in the absence of blood lysis (as demonstrated by results for 10 Tris+NaCl with BB sample-set). [0433] Good ETGA results in the absence of blood, indicate that detergents, as components of the binding buffer, are not necessary for binding of E. coli to beads [0434] Good ETGA results in the 10 Tris+NaCl with 1 Tris+NaCl sample-set demonstrate that biological components in blood and/or broth are not required for microbial binding. [0435] Interestingly, the B-BUF appears to be slightly inhibitory to ETGA signal in 10 B-BUF with BO and 1 B-BUF sample-sets, but recent work elsewhere has shown that Sodium Deoxycholate could be somewhat inhibitory to the assay, so this observation is not unexpected [0436] Confirm performed best in the 10 Tris+NaCl with 1 Tris+NaCl sample-set. All other similar sample-sets produced similar Confirm GrNeg results. [0437] IPC signal was somewhat inhibited by the presence of blood, as can be expected. [0438] These results demonstrate that neither detergents or the biological sample are mediators of microbial binding for E. coli

Example 7: In the Absence of Blood, Microbial Capture by Magnetic Beads Occurs Regardless of any pH Buffering or Osmotic Stabilisation with Salt

Aim:

[0439] To further investigate the importance of Momentum's binding buffer in mediating microbial binding the effect of pH buffering and salt on binding was investigated in a clean system (i.e. in the absence of any blood or broth).

10 Buffer Preparation:

[0440] 25 mL of each buffer made fresh: [0441] BUF-1 500 mM Tris-HCl [pH7.4]+1.5 M NaCl [0442] BUF-2 500 mM Tris-HCl [pH8.0]+1.5 M NaCl [0443] BUF-3 500 mM Tris-HCl [pH8.5]+1.5 M NaCl [0444] BUF-4 500 mM Tris-HCl [pH8.0] ONLY [0445] BUF-5 1.5 M NaCl ONLY [0446] BUF-6 Water ONLY

[0447] Estapor beads (Merck,Cat M1-30/40) washed 31 mL in respective 1 buffer (diluted 10 buffers): 30 L beads resuspended in a final volume of 300 L 1 buffer (1% solid content)

[0448] Protocol: [0449] E. coli o/n liquid cultures set-up as standard in BacTec PLUS aerobic broth (containing SPS) and Nutrient Broth (NB containing no SPS), then the following day (approx. 16 hours later) 1.88 L o/n added to 3 mL broth (equivalent to EC 1E-1 dilution added to broth at 6.25 L/mL) for each broth type (NB in morning and PLUS broth in afternoon), and 2-hour outgrowth incubations performed at 37 C., 500 rpm. [0450] For each experiment (NB and PLUS), 1E-1 E. coli preculture diluted down to E. coli 1E-6 (DF10) in each 1 buffer (BUF-1 to BUF-6) [0451] 100 L TVCs performed using a separate EC dilution set performed in relevant broth (NB or PLUS broth) to prevent plate viability inconsistencies resulting from different 1 buffers

Manual Simulation of Magnitor Performed Using DyneMag-2 Magnet and Manual Liquid Transfers:

[0452] 1 mL samples added to 2 mL tubes containing 112 L of respective 1 buffer+15 L beads (prewashed in respective buffer) [0453] 30 mins shaking (1000 rpm) @ 37 C. [0454] 5 mins magnetisation on DynaMag-2 [0455] All s/n removed [0456] 1 mL WB added and tubes mixed for 2 mins @ RT (1000 rpm) [0457] 5 mins magnetisation on Dynmag-2 [0458] All s/n removed [0459] 50 L LM added to tubes off magnet [0460] ETGA reaction performed: 5 mins at 1000 rpm, then 55 mins at 800 rpm @26 C. [0461] Manual qPCR set-up for ETGA and Confirm (10 L reactions)

Results:

[0462] NB Dataset (i.e. No SPS)Morning Experiment

TABLE-US-00021 Colonies *E cfu/mL E. coli 1e3 TNTC 35000 E. coli 1e4 *350 3500 E. coli 1e5 12 350 E. coli 1e6 5 35

[0463] All buffer (BUF-1 to 6) NC TVCs=0

TABLE-US-00022 Colonies ETGA Ct *E cfu/mL BUF-1 BUF-2 BUF-3 BUF-4 BUF-5 BUF-6 E. coli 1e-3 TNTC 35000 19.00 18.44 17.73 17.45 19.12 16.28 E. coli 1e-4 *350 3500 22.60 21.23 21.53 21.16 22.51 26.22 E. coli 1e-5 12 350 25.57 26.94 28.18 24.77 27.51 28.71 E. coli 1e-6 5 35 30.41 30.47 31.37 29.92 31.36 33.81 NSC 1 0 0 40.37 39.45 39.70 40.18 39.22 38.16 NSC 2 0 0 40.63 36.20 39.74 38.03 40.01 38.64 NSC 3 0 0 39.76 39.36 39.09 39.67 38.47 38.17 PC N/A N/A 30.24 31.51 33.06 32.57 32.90 32.08 NSC Ave. 40.25 38.34 39.51 39.30 39.23 38.32

TABLE-US-00023 Colonies IPC Ct *E cfu/mL BUF-1 BUF-2 BUF-3 BUF-4 BUF-5 BUF-6 E. coli 1e-3 TNTC 35000 32.17 32.36 32.32 32.30 32.05 32.31 E. coli 1e-4 *350 3500 31.58 31.62 31.83 31.67 31.55 31.28 E. coli 1e-5 12 350 31.44 31.65 31.44 31.27 31.67 31.62 E. coli 1e-6 5 35 31.37 31.47 31.48 31.31 31.64 31.50 NSC 1 0 0 31.60 31.25 31.42 31.64 31.48 31.54 NSC 2 0 0 31.53 31.33 31.60 31.40 31.75 31.52 NSC 3 0 0 31.42 31.95 31.37 31.43 31.76 31.67 PC N/A N/A 31.20 31.49 31.65 31.49 31.57 31.97

TABLE-US-00024 Colonies ETGA Ct *E cfu/mL BUF-1 BUF-2 BUF-3 BUF-4 BUF-5 BUF-6 E. coli 1e-3 TNTC 35000 21.26 19.89 21.78 21.85 20.11 22.05 E. coli 1e-4 *350 3500 17.65 17.10 17.98 18.13 16.73 12.10 E. coli 1e-5 12 350 14.68 11.40 11.34 14.53 11.72 9.61 E. coli 1e-6 5 35 9.85 7.86 8.14 9.38 7.87 4.51 NSC 1 0 0 N/A N/A N/A N/A N/A N/A NSC 2 0 0 N/A N/A N/A N/A N/A N/A NSC 3 0 0 N/A N/A N/A N/A N/A N/A PC N/A N/A 10.02 6.83 6.45 6.72 6.33 6.24

TABLE-US-00025 ETGA Critical Values (averageNSC) Colonies *E cfu/mL BUF-1 BUF-2 BUF-3 BUF-4 BUF-5 BUF-6 E. coli 1e-3 TNTC 35000 0.014 0.036 0.010 0.009 0.031 0.008 E. coli 1e-4 *350 3500 0.017 0.025 0.014 0.012 0.032 0.795 E. coli 1e-5 12 350 0.013 0.130 0.135 0.015 0.104 0.448 E. coli 1e-6 5 35 0.038 0.150 0.124 0.053 0.149 1.535 NSC 1 0 0 N/A N/A N/A N/A N/A N/A NSC 2 0 0 N/A N/A N/A N/A N/A N/A NSC 3 0 0 N/A N/A N/A N/A N/A N/A PC N/A N/A N/A N/A N/A N/A N/A N/A

TABLE-US-00026 Confirm GrNeg Ct Colonies *E cfu/mL BUF-1 BUF-2 BUF-3 BUF-4 BUF-5 BUF-6 E. coli 1e-3 TNTC 35000 25.07 25.28 25.98 25.77 25.05 27.70 E. coli 1e-4 *350 3500 28.36 28.58 28.31 30.15 30.62 NoCt E. coli 1e-5 12 350 31.75 31.16 31.82 32.25 38.38 NoCt E. coli 1e-6 5 35 NoCt NoCt 34.94 NoCt NoCt NoCt NSC 1 0 0 NoCt NoCt NoCt NoCt NoCt NoCt NSC 2 0 0 NoCt NoCt NoCt NoCt NoCt NoCt NSC 3 0 0 NoCt NoCt NoCt NoCt NoCt NoCt PC N/A N/A NoCt NoCt NoCt NoCt NoCt NoCt Positivity threshold (Pt) 40 Ct
PLUS Dataset (i.e with SPS)Afternoon Experiment

TABLE-US-00027 Colonies *E cfu/mL E. coli 1e3 TNTC 55600 E. coli 1e4 *556 5560 E. coli 1e5 77 556 E. coli 1e6 7 55.6

[0464] All buffer (BUF-1 to 6) NC TVCs=0

TABLE-US-00028 ETGA Ct Colonies *E cfu/mL BUF-1 BUF-2 BUF-3 BUF-4 BUF-5 BUF-6 E. coli 1e-3 TNTC 55600 17.30 16.32 15.48 18.29 19.15 17.61 E. coli 1e-4 *556 5560 21.89 21.09 20.37 22.37 24.39 26.13 E. coli 1e-5 77 556 26.82 24.63 26.19 26.30 29.27 34.26 E. coli 1e-6 7 55.6 31.76 30.71 32.60 31.57 34.20 34.68 NSC 1 0 0 37.74 39.10 38.95 41.09 41.03 41.46 NSC 2 0 0 38.25 38.48 38.90 39.11 40.58 40.84 NSC 3 0 0 39.46 38.90 38.03 42.26 40.81 41.02 PC N/A N/A 33.25 32.41 32.62 33.85 33.49 33.26 NSC Ave. 38.48 38.83 38.63 40.82 40.81 41.11

TABLE-US-00029 IPC Ct Colonies *E cfu/mL BUF-1 BUF-2 BUF-3 BUF-4 BUF-5 BUF-6 E. coli 1e-3 TNTC 55600 32.46 33.44 32.97 33.31 31.98 33.00 E. coli 1e-4 *556 5560 31.77 32.07 32.00 32.25 31.42 31.64 E. coli 1e-5 77 556 32.01 32.03 31.96 31.70 31.21 31.15 E. coli 1e-6 7 55.6 31.96 31.65 31.75 31.55 31.55 31.63 NSC 1 0 0 31.84 32.11 31.99 31.13 31.58 31.65 NSC 2 0 0 31.90 31.51 32.08 31.25 31.46 31.64 NSC 3 0 0 31.94 31.84 31.68 31.49 31.44 31.59 PC N/A N/A 32.17 32.24 32.34 31.73 31.37 31.87

TABLE-US-00030 ETGA Ct Colonies *E cfu/mL BUF-1 BUF-2 BUF-3 BUF-4 BUF-5 BUF-6 E. coli 1e-3 TNTC 55600 21.18 22.50 23.15 22.53 21.66 23.50 E. coli 1e-4 *556 5560 16.59 17.73 18.26 18.45 16.41 14.98 E. coli 1e-5 77 556 11.66 14.20 12.44 14.52 11.54 6.85 E. coli 1e-6 7 55.6 6.72 8.12 6.03 9.25 6.61 6.43 NSC 1 0 0 N/A N/A N/A N/A N/A N/A NSC 2 0 0 N/A N/A N/A N/A N/A N/A NSC 3 0 0 N/A N/A N/A N/A N/A N/A PC N/A N/A 5.23 6.41 6.01 6.97 7.31 7.85

TABLE-US-00031 ETGA Critical Values (averageNSC) Colonies *E cfu/mL BUF-1 BUF-2 BUF-3 BUF-4 BUF-5 BUF-6 E. coli 1e-3 TNTC 55600 0.023 0.009 0.006 0.009 0.017 0.005 E. coli 1e-4 *556 5560 0.056 0.026 0.018 0.016 0.064 0.172 E. coli 1e-5 77 556 0.172 0.030 0.100 0.024 0.187 4.827 E. coli 1e-6 7 55.6 0.528 0.200 0.849 0.092 0.569 0.644 NSC 1 0 0 N/A N/A N/A N/A N/A N/A NSC 2 0 0 N/A N/A N/A N/A N/A N/A NSC 3 0 0 N/A N/A N/A N/A N/A N/A PC N/A N/A N/A N/A N/A N/A N/A N/A

TABLE-US-00032 Confirm GrNeg Ct Colonies *E cfu/mL BUF-1 BUF-2 BUF-3 BUF-4 BUF-5 BUF-6 E. coli 1e-3 TNTC 55600 28.12 27.49 26.73 28.36 28.13 27.82 E. coli 1e-4 *556 5560 32.91 27.92 27.85 30.15 30.85 NoCt E. coli 1e-5 77 556 34.43 34.83 38.66 43.88 NoCt NoCt E. coli 1e-6 7 55.6 NoCt NoCt NoCt NoCt NoCt NoCt NSC 1 0 0 NoCt NoCt NoCt NoCt NoCt NoCt NSC 2 0 0 NoCt NoCt NoCt NoCt NoCt NoCt NSC 3 0 0 NoCt NoCt NoCt NoCt NoCt NoCt PC N/A N/A NoCt NoCt NoCt NoCt NoCt NoCt Positivity threshold (Pt) 40 Ct

Analysis:

[0465] All buffers, including water only, demonstrated capture of E. coli similarly well (as indicated by similar ETGA and Confirm results)however, there was some indication of osmotic microbial lysis at lower cell densities in water only (BUF-6) [0466] Both PLUS broth and NB grown E. coli produced very similar Magnitor results for all buffers tested, indicating that SPS plays no obvious role in mediating microbial binding of E. coli [0467] These results indicate that no buffer components are essential for binding of E. coli to Estapor (carboxylated) beads

Example 8: Microbial Capture from Blood by Magnetic Beads can be Performed Using a Variety of Different Blood Lysis Methods

Aim:

[0468] To determine whether microbial capture and detection can occur when alternative blood lysis methods are employed.

Preparation:

[0469] Binding Buffers were prepared as follows: [0470] E-BUF=500 mM Tris-HCl [pH 8.0]+1.5 M Sodium Chloride+10% Igepal+2.5% Tergitol [0471] UREA=83 mM Tris-HCl [pH 8.0]+10 M Urea [0472] Tris+NaCl=500 mM Tris-HCl [pH 8.0]+1.5 M Sodium Chloride

[0473] BioEstapor beads (Merck, Cat #BE-M 08/0.3) were re-suspended prior to use.

Protocol:

[0474] S. aureus o/n liquid culture set-up as standard in BacTec PLUS aerobic broth, then following day (approx. 16 hours later) 3.0 L o/n added to 3 mL blood broth (1E-3 dilution) and 4-hour outgrowth performed at 37 C., 500 rpm. [0475] Following the 4-hour outgrowth, S. aureus pre-culture serially diluted (DF10) down to 1E-6 in prewarmed blood broth. [0476] 100 L TVCs performed for all S. aureus dilutions and NSCs

Manual Sample Processing Using DynaMaq-2 Magnet and Manual Liquid Transfers by Pipette:

Initial Set-Up

[0477] For samples using Urea, 0.25 mL specimens added to 2 mL tubes containing 0.75 mL of UREA+15 L beads [0478] For samples to be frozen, 1 mL specimens added to 2 mL tubes then snap frozen on dry ice for 5 minutes. The specimens were thawed at 37 C. for 5 minutes, then 112 L Tris+NaCl+15 L beads were added [0479] For samples using E-BUF or Tris+NaCl, 1 mL specimens added to 2 mL tubes containing 112 L of binding buffer (either E-BUF or Tris+NaCl)+15 L beads

Processing of all Samples

[0480] 30 mins orbital mixing (1000 rpm) @ 37 C. [0481] 5 mins magnetisation on DynaMag-2 [0482] All s/n removed [0483] 1 mL WB added and tubes mixed for 3 mins @ 37 C. (1000 rpm) [0484] 5 mins magnetisation on Dynmag-2 [0485] All s/n removed [0486] 50 L LM added to tubes off magnet [0487] ETGA reaction performed: 5 mins at 1000 rpm, then 55 mins at 800 rpm @26 C. [0488] Manual qPCR set-up for ETGA and Confirm (10 L reactions)

Observations:

[0489] Blood lysis observed for the frozen samples after thawing (see FIG. 1) [0490] Blood lysis observed to be almost instantaneous with UREA [0491] Some beads appeared to be lost during processing for the samples using UREA [0492] No apparent blood lysis observed for Tris+NaCl sample-set (as expected)

Results:

[0493]

TABLE-US-00033 Specimen TVC *E cfu/mL S. aureus 1E4 TNTC 3,360,000 S. aureus 1E5 TNTC 336,000 S. aureus 1E6 *3360 33,600 NSC 0 0 *Cfu/mL values extrapolated from highest countable TVC

ETGA Ct

[0494]

TABLE-US-00034 BLOOD LYSIS METHOD Specimen E-BUF UREA Tris + NaCl FREEZING S. aureus 1E4 14.26 34.98 13.90 14.42 S. aureus 1E5 18.50 30.26 17.79 18.99 S. aureus 1E6 22.59 35.96 22.07 22.96 NSC 1 42.11 37.55 32.56 35.14 NSC 2 41.13 36.52 32.72 34.60 NSC 3 43.83 36.71 32.42 34.58 Average NSC 42.36 36.92 32.57 34.77

ETGA Ct (Average NSC)

[0495]

TABLE-US-00035 BLOOD LYSIS METHOD Specimen E-BUF UREA Tris + NaCl FREEZING S. aureus 1E4 28.10 1.94 18.67 20.35 S. aureus 1E5 23.85 6.66 14.78 15.78 S. aureus 1E6 19.77 0.96 10.49 11.82 NSC 1 N/A N/A N/A N/A NSC 2 N/A N/A N/A N/A NSC 3 N/A N/A N/A N/A
Critical Values Based on Average NSC (cfu/mL)

TABLE-US-00036 BLOOD LYSIS METHOD Specimen E-BUF UREA Tris + NaCl FREEZING S. aureus 1E-4 0.01 875527.09 8.06 2.52 S. aureus 1E-5 0.02 3313.90 11.96 5.96 S. aureus 1E-6 0.04 17241.36 23.34 9.31 NSC 1 N/A N/A N/A N/A NSC 2 N/A N/A N/A N/A NSC 3 N/A N/A N/A N/A

IPC Ct

[0496]

TABLE-US-00037 BLOOD LYSIS METHOD Specimen E-BUF UREA Tris + NaCl FREEZING S. aureus 1E-4 35.68 36.10 37.31 38.12 S. aureus 1E-5 34.49 36.18 35.39 35.54 S. aureus 1E-6 34.22 35.76 35.12 34.50 NSC 1 34.40 36.51 34.39 34.87 NSC 2 34.18 35.86 34.52 34.09 NSC 3 34.85 36.31 34.25 34.04

Confirm GrPos Ct

[0497]

TABLE-US-00038 BLOOD LYSIS METHOD Specimen E-BUF UREA Tris + NaCl FREEZING S. aureus 1E-4 22.34 23.47 18.74 19.55 S. aureus 1E-5 26.03 26.33 21.19 23.03 S. aureus 1E-6 27.67 30.92 24.73 26.37 NSC 1 46.59 35.42* NoCt 48.06 NSC 2 40.33 NoCt NoCt NoCt NSC 3 NoCt NoCt 45.99 41.75 Positivity threshold (Pt) 40 Ct; *false positives

[0498] FIG. 1 shows the extent of blood lysis for each sample-set: E-BUF, UREA, Tris+NaCl, freezing (left to right)

Analysis:

[0499] Microbial capture and detection of S. aureus by magnetic beads is comparable for alternative lysis methods and no blood lysis, as determined by Confirm.

[0500] However, microbial detection by ETGA is improved to differing extents by alternative blood lysis methods, due to effects on the reduction of blood-derived ETGA signal.

Example 9: SPS is Needed for Optimal Bead Performance, Sample Processing and Microbial Detection in Whole Blood

Aim:

[0501] To determine the optimal SPS concentration for the Magnitor Rapid test using 1 mL whole blood samples. The secondary objective was to assess the effect of SPS on microbial viability in whole blood as determined by TVCs.

Test Conditions:

[0502] 25 mL whole blood or BacTec PLUS aerobic blood broth (1:3 ratio) aliquoted for each sample-set (E. coli and NSC sample). Then SPS added as follows:

TABLE-US-00039 Sample-set 10% SPS (L) BB None WB 0% None WB 0.01% 5 WB 0.02% 10 WB 0.04% 20 WB 0.06% 30 WB 0.08% 40 WB 0.10% 50

[0503] Then 5 L of E. coli 1E-2 preculture added to each 5-mL specimen tube to recreate standard 1E-5 dilution sample

Protocol:

[0504] 1.88 L neat o/n in Nutrient Broth (NB) added to 3 mL NB; and incubated for 2 hours at 37 C., 500 rpm [0505] After 2 hours, E. coli preculture diluted 10-fold in warm NB; and then 5 L added to each 5 mL specimen tube (prepared as shown in test conditions) [0506] Magnitor Test initiated immediately, and TVCs performed as detailed below.

Manual Simulation of Magnitor Performed Using DynaMaq-2 Magnet and Manual Liquid Transfers:

[0507] 112 L E-BUF (500 mM Tris-HCl [pH 8.0]+1.5 M Sodium Chloride+10% Igepal+2.5% Tergitol)+15 L Beads (BioEstapor, Merck, Cat #BE-M 08/0.3) preloaded into each sample-tube, then 1 mL specimens added to sample tubes [0508] 30 mins orbital mixing (1000 rpm) @ 37 C. [0509] 5 mins magnetisation on DynaMag-2 [0510] All s/n removed [0511] 1 mL WB added and tubes mixed for 3 mins @ RT (1000 rpm) [0512] 5 mins magnetisation on Dynmag-2 [0513] All s/n removed [0514] 50 L LM added to tubes off magnet [0515] ETGA reaction performed: 5 mins at 1000 rpm, then 55 mins at 800 rpm @26 C. [0516] Manual qPCR set-up for ETGA and Confirm (10 L reactions)

Results:

TVC Analysis

[0517] 100 L on COL plates at Time Zero [0518] Sample bijous, containing approximately 2 mL sample, left at room temperature (20.4 C.) on bench (static) [0519] TVCs performed at time points shown in table: samples mixed thoroughly before plating

TABLE-US-00040 WB SPS % (colonies) BB 0 0.01 0.02 0.04 0.06 0.08 0.1 EC Time ZERO 263 303 147 96 354 128 322 126 EC Time 2 hrs 428 256 498 448 1106 760 491 341 EC Time 4 hrs TNTC 790 TNTC TNTC TNTC TNTC TNTC TNTC EC Time 6 hrs TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC EC Time 21 hrs LAWN LAWN LAWN LAWN LAWN LAWN LAWN LAWN NSC Time ZERO 0 0 N/A N/A N/A N/A N/A N/A NSC Time 2 hrs 0 0 N/A N/A N/A N/A N/A N/A NSC Time 4 hrs 0 0 N/A N/A N/A N/A N/A N/A NSC Time 6 hrs 0 0 N/A N/A N/A N/A N/A N/A NSC Time 21 hrs 0 0 N/A N/A N/A N/A N/A N/A FOLD INCR. 0-2 hr 1.63 0.84 3.39 4.67 3.12 5.94 1.52 2.71

Magnitor Rapid Test Performed at Time ZERO

ETGA Results

[0520]

TABLE-US-00041 WB SPS % BB 0 0.01 0.02 0.04 0.06 0.08 0.1 EC 1 26.94 *28.72 27.90 26.41 29.31 28.70 32.31 33.76 EC 2 27.31 45.22 29.12 25.88 29.43 28.50 31.94 34.37 EC 3 27.34 41.32 28.14 27.24 29.70 27.62 31.40 34.08 NSC 1 39.06 43.71 33.39 33.20 38.93 44.62 50.00 50.00 NSC 2 39.75 44.30 33.06 33.77 39.93 44.11 50.00 50.00 NSC 3 40.28 46.58 32.81 34.41 38.54 43.30 50.00 50.00 Ave. EC 27.20 43.27 28.39 26.51 29.48 28.27 31.88 34.07 Ave. NSC 39.70 44.86 33.09 33.79 39.13 44.01 50.00 50.00 Ave. Ct 12.50 1.59 4.70 7.28 9.65 15.74 18.12 15.93 CV (cfu/mL) 0.45 1003.27 56.59 6.16 4.40 0.02 0.01 0.02 Note: NoCt changed to 50 Ct for analysis; *Outlier excluded due to substantial pellet loss during processing

IPC Results

[0521]

TABLE-US-00042 WB SPS % BB 0 0.01 0.02 0.04 0.06 0.08 0.1 EC 1 34.03 42.50 42.19 40.42 37.87 35.81 38.14 37.06 EC 2 34.83 NoCt 39.34 39.01 38.38 35.70 36.80 37.11 EC 3 34.47 NoCt 40.49 37.85 38.34 36.11 36.80 37.66 NSC 1 34.15 NoCt 39.75 39.26 36.56 36.10 36.80 37.17 NSC 2 34.00 NoCt 39.12 38.40 37.81 35.98 37.87 38.60 NSC 3 34.18 45.91 40.58 37.32 36.90 36.36 38.09 37.46 Ave. NSC 34.11 45.91 39.81 38.33 37.09 36.15 37.59 37.74

Confirm GrNeg Results

[0522]

TABLE-US-00043 WB SPS % BB 0 0.01 0.02 0.04 0.06 0.08 0.1 EC 1 32.33 30.05 27.94 29.15 28.71 31.58 29.20 29.27 EC 2 31.45 34.24 29.18 28.16 28.75 29.77 29.80 28.65 EC 3 31.71 34.97 28.32 29.75 29.82 29.26 28.87 29.61 NSC 1 NoCt NoCt NoCt NoCt NoCt NoCt NoCt 47.82 NSC 2 43.07 NoCt NoCt NoCt NoCt NoCt NoCt NoCt NSC 3 47.16 NoCt NoCt NoCt NoCt NoCt NoCt NoCt Ave. EC 31.83 33.08 28.48 29.02 29.09 30.20 29.29 29.18

Analysis:

[0523] SPS indicates a benefit of providing microbial protection/viability in whole blood based on TVC assay; but no major issues with E. coli viability in whole blood generally [0524] Incorporation of SPS improved sample processing efficacy and microbial detection performance for both ETGA and Confirm readouts [0525] SPS at 0.06% produced the best results for TVC-based viability; ETGA detection (best results taking E. coli and NSC sample Cts into account); and PCR inhibition as indicated by IPC Ct values. Confirm GrNeg results were also improved by SPS addition to whole blood, but the exact concentration of SPS was less critical.

Example 10: Microbial Capture from Blood by Magnetic Beads Occurs Using a Variety of Commercially Available Carboxylated Bead Products of Similar Size (300 nm Diameter

Aim:

[0526] To compare alternative carboxylated magnetic beads of similar size using Momentum's Magnitor assay

Test Conditions:

[0527]

TABLE-US-00044 I.D Bead Size (m) A PS-MAG-COOH (microparticles GmbH #S2003) 0.27 B Sphero Carboxyl magnetic particles 0.1-0.4 (Spherotech #CM-025-10H) C SuperMag Carboxylic Acid Beads 0.2 (Ocean Nanotech #SC0201) D Carboxyl-Adembeads (Ademtech #02120) 0.2 E Carboxyl beads (FG Beads #TAS8848N1140) 0.2 G Bio-Estapor (Merck #BE-M08/03)-streptavidin-conjugated 0.3

Protocol:

[0528] E. coli and S. pyogenes o/n liquid cultures set up as standard in 3 mL broth and blood broth (BacTec PLUS aerobic) respectively, and incubated for 16-20 hours (37 C.)

[0529] The following day: [0530] E. coli liquid culture diluted to 1E-3 in blood broth and then spiked into blood broth (6.25 L per mL blood broth); and pre-incubated for 1 hr 30 mins (37 C.) [0531] S. pyogenes liquid culture diluted to 1E-1 in blood broth and then spiked into blood broth (6.25 L per mL blood broth); and pre-incubated for 2 hr 30 mins (37 C.)
E. coli Experiment Performed in Morning [0532] E. coli 1E-3 pre-culture serially diluted in blood broth to produce 5 dilution points (1E-3 to 1E-7) [0533] Samples set up by adding 1 mL specimen to 2 mL tubes preloaded with 15 l 1% solid beads+112 L Binding Buffer; and Magnitor V4.0 test performed: 5 dilution points+3 NSCs (8 sample-set) per bead type with three bead types tested on each epMotion 5073m
S. pyogenes Experiment Performed in Afternoon [0534] S. pyogenes 1E-3 pre-culture serially diluted in blood broth to produce 5 dilution points (1E-1 to 1E-5) [0535] Samples set up by adding 1 mL specimen to 2 mL tubes preloaded with 15 l 1% solid beads+112 L Binding Buffer; and Magnitor V4.0 test performed: 5 dilution points+3 NSCs (8 sample-set) per bead type with three bead types tested on each epMotion 5073m
Magnitor V4.0 Protocol (Automated Sample Processing on epMotion 5073m) [0536] 30 mins orbital mixing (1000 rpm) @ 37 C. [0537] 15 mins magnetisation [0538] 1 mL s/n removed [0539] 0.82 mL WB added to tubes whilst beads magnetised [0540] 1 mL s/n removed [0541] 50 L LM added to tubes whilst beads magnetised [0542] Magnetisation switched off and ETGA reaction performed: 5 mins at 1000 rpm, then 55 mins at 800 rpm @26 C. [0543] qPCR set-up for ETGA and Confirm (10 L reactions)

Results:

[0544] Internal Positivity Thresholds (Pt) calculated using NSCs (n=6) for each bead type: formula=PERCENTILE.INC(array,0.05)

TABLE-US-00045 A B C D E G NSC1 35.12 37.10 41.11 45.29 43.55 40.33 NSC2 35.00 38.85 42.73 46.18 43.15 40.37 NSC3 34.57 37.02 40.49 50.00 42.32 41.79 NSC4 33.77 38.25 41.20 44.74 43.82 43.80 NSC5 33.72 38.22 40.52 45.25 45.08 44.12 NSC6 35.06 41.19 41.93 44.33 43.32 42.39 Pt (5th %) 33.74 37.04 40.49 44.44 42.53 40.34 Note, that Pt (5th %) method will generate one false positive within n = 6: highlighted in red font within main results table below
E. coli

TABLE-US-00046 ETGA Confirm TVCs *E CFU/ml ETGA Ct Result IPC Ct GrNeg Ct GrPos Ct Cand. Ct I.D. A E. coli 1e-3 TNTC 28,800 27.20 Positive 35.15 30.99 NoCt NoCt GrNeg E. coli 1e-4 *288 2,880 31.30 Positive 34.91 NoCt NoCt NoCt No ID E. coli 1e-5 35 288 33.31 Positive 35.08 NoCt NoCt NoCt No ID E. coli 1e-6 4 29 35.47 Negative 35.37 NoCt NoCt NoCt No ID E. coli 1e-7 0 3 34.63 Negative 35.09 NoCt NoCt NoCt No ID NSC1 0 35.12 Negative 35.67 NoCt NoCt NoCt No ID NSC2 0 35.00 Negative 35.10 NoCt NoCt NoCt No ID NSC3 0 34.57 Negative 35.27 NoCt NoCt NoCt No ID B E. coli 1e-3 TNTC 28,800 25.67 Positive 34.45 36.51 NoCt NoCt GrNeg E. coli 1e-4 *288 2,880 31.31 Positive 34.02 NoCt NoCt NoCt No ID E. coli 1e-5 35 288 32.92 Positive 34.74 NoCt NoCt NoCt No ID E. coli 1e-6 4 29 36.56 Positive 34.26 NoCt NoCt NoCt No ID E. coli 1e-7 0 3 38.68 Negative 34.46 NoCt NoCt NoCt No ID NSC1 0 37.10 Negative 33.86 NoCt NoCt NoCt No ID NSC2 0 38.85 Negative 34.28 NoCt NoCt NoCt No ID NSC3 0 37.02.sup. Positive 33.95 40.26 NoCt NoCt No ID C E. coli 1e-3 TNTC 28,800 23.59 Positive 35.38 29.32 NoCt NoCt GrNeg E. coli 1e-4 *288 2,880 27.50 Positive 35.16 34.29 NoCt NoCt GrNeg E. coli 1e-5 35 288 31.34 Positive 35.16 42.48 NoCt NoCt No ID E. coli 1e-6 4 29 32.91 Positive 35.29 NoCt NoCt NoCt No ID E. coli 1e-7 0 3 40.91 Negative 35.61 NoCt NoCt NoCt No ID NSC1 0 41.11 Negative 34.63 NoCt NoCt NoCt No ID NSC2 0 42.73 Negative 35.09 49.10 NoCt NoCt No ID NSC3 0 40.49.sup. Positive 35.58 NoCt NoCt NoCt No ID D E. coli 1e-3 TNTC 28,800 24.41 Positive 35.51 31.63 NoCt NoCt GrNeg E. coli 1e-4 *288 2,880 28.00 Positive 34.72 NoCt NoCt NoCt No ID E. coli 1e-5 35 288 32.33 Positive 35.39 NoCt NoCt NoCt No ID E. coli 1e-6 4 29 33.46 Positive 34.66 NoCt NoCt NoCt No ID E. coli 1e-7 0 3 48.92 Negative 35.09 NoCt NoCt NoCt No ID NSC1 0 45.29 Negative 35.40 NoCt NoCt NoCt No ID NSC2 0 46.18 Negative 35.11 NoCt NoCt NoCt No ID NSC3 0 50.00 Negative 34.70 NoCt NoCt NoCt No ID E E. coli 1e-3 TNTC 28,800 22.77 Positive 35.70 30.68 NoCt NoCt GrNeg E. coli 1e-4 *288 2,880 27.16 Positive 35.49 39.05 NoCt NoCt GrNeg E. coli 1e-5 35 288 33.84 Positive 34.91 NoCt NoCt NoCt No ID E. coli 1e-6 4 29 42.64 Negative 35.16 NoCt NoCt NoCt No ID E. coli 1e-7 0 3 42.40 Positive 35.16 NoCt NoCt NoCt No ID NSC1 0 43.55 Negative 35.30 NoCt NoCt NoCt No ID NSC2 0 43.15 Negative 34.55 NoCt NoCt NoCt No ID NSC3 0 42.32.sup. Positive 34.19 NoCt NoCt NoCt No ID G E. coli 1e-3 TNTC 28,800 22.91 Positive 36.48 32.84 NoCt NoCt GrNeg E. coli 1e-4 *288 2,880 27.54 Positive 35.61 NoCt NoCt NoCt No ID E. coli 1e-5 35 288 31.36 Positive 35.86 42.94 NoCt NoCt No ID E. coli 1e-6 4 29 41.03 Negative 35.44 NoCt NoCt NoCt No ID E. coli 1e-7 0 3 42.24 Negative 35.45 NoCt NoCt NoCt No ID NSC1 0 40.33.sup. Positive 35.78 NoCt 43.85 NoCt No ID NSC2 0 40.37 Negative 35.31 NoCt NoCt NoCt No ID NSC3 0 41.79 Negative 36.90 NoCt 38.72.sup. NoCt GrPos Confirm Positivity threshold (Pt) 40 Ct; false positivest.sup.
S. pyogenes

TABLE-US-00047 ETGA Confirm TVCs *E CFU/ml ETGA Ct Result IPC Ct GrNeg Ct GrPos Ct Cand. Ct I.D. A S. pyogenes 1e-1 TNTC 2,210,000 26.20 Positive 34.95 NoCt 27.74 NoCt GrPos S. pyogenes 1e-2 TNTC 221,000 30.05 Positive 34.52 NoCt 31.28 NoCt GrPos S. pyogenes 1e-3 TNTC 22,100 32.49 Positive 34.77 NoCt 35.19 NoCt GrPos S. pyogenes 1e-4 *221 2,210 33.67 Positive 35.15 NoCt NoCt NoCt No ID S. pyogenes 1e-5 3 221 33.30 Positive 34.33 NoCt NoCt NoCt No ID NSC1 0 33.77 Negative 35.22 NoCt NoCt NoCt No ID NSC2 0 33.72.sup. Positive 35.09 NoCt NoCt NoCt No ID NSC3 0 35.06 Negative 34.93 NoCt NoCt NoCt No ID B S. pyogenes 1e-1 TNTC 2,210,000 30.57 Positive 33.56 NoCt 32.72 NoCt GrPos S. pyogenes 1e-2 TNTC 221,000 34.19 Positive 33.45 NoCt 38.13 NoCt GrPos S. pyogenes 1e-3 TNTC 22,100 36.31 Positive 34.20 NoCt NoCt NoCt No ID S. pyogenes 1e-4 *221 2,210 37.67 Negative 33.58 NoCt NoCt NoCt No ID S. pyogenes 1e-5 3 221 36.79 Positive 33.44 NoCt NoCt NoCt No ID NSC1 0 38.25 Negative 34.07 43.83 NoCt NoCt No ID NSC2 0 38.22 Negative 33.86 NoCt NoCt NoCt No ID NSC3 0 41.19 Negative 34.23 NoCt NoCt NoCt No ID C S. pyogenes 1e-1 TNTC 2,210,000 26.21 Positive 35.04 NoCt 26.56 NoCt GrPos S. pyogenes 1e-2 TNTC 221,000 30.61 Positive 34.96 NoCt 30.38 NoCt GrPos S. pyogenes 1e-3 TNTC 22,100 34.17 Positive 35.19 NoCt 34.06 NoCt GrPos S. pyogenes 1e-4 *221 2,210 38.99 Positive 34.78 NoCt NoCt NoCt No ID S. pyogenes 1e-5 3 221 41.83 Negative 34.25 NoCt NoCt NoCt No ID NSC1 0 41.20 Negative 34.91 NoCt NoCt NoCt No ID NSC2 0 40.52 Negative 35.07 46.11 NoCt NoCt No ID NSC3 0 41.93 Negative 34.18 43.77 NoCt NoCt No ID D S. pyogenes 1e-1 TNTC 2,210,000 27.51 Positive 35.47 NoCt 28.26 NoCt GrPos S. pyogenes 1e-2 TNTC 221,000 31.66 Positive 35.00 NoCt 30.96 NoCt GrPos S. pyogenes 1e-3 TNTC 22,100 34.50 Positive 35.42 NoCt 34.86 NoCt GrPos S. pyogenes 1e-4 *221 2,210 38.78 Positive 35.53 NoCt 42.04 NoCt No ID S. pyogenes 1e-5 3 221 43.26 Positive 35.40 NoCt NoCt NoCt No ID NSC1 0 44.74 Negative 34.96 NoCt NoCt NoCt No ID NSC2 0 45.25 Negative 34.55 NoCt NoCt NoCt No ID NSC3 0 44.33.sup. Positive 35.20 NoCt NoCt NoCt No ID E S. pyogenes 1e-1 TNTC 2,210,000 26.32 Positive 34.59 NoCt 27.87 NoCt GrPos S. pyogenes 1e-2 TNTC 221,000 30.65 Positive 35.46 NoCt 30.83 NoCt GrPos S. pyogenes 1e-3 TNTC 22,100 34.55 Positive 35.19 NoCt 35.01 NoCt GrPos S. pyogenes 1e-4 *221 2,210 39.11 Positive 35.15 NoCt 41.25 NoCt No ID S. pyogenes 1e-5 3 221 43.96 Negative 35.59 NoCt 46.72 NoCt No ID NSC1 0 43.82 Negative 36.22 NoCt NoCt NoCt No ID NSC2 0 45.08 Negative 35.37 NoCt 42.20 NoCt No ID NSC3 0 43.32 Negative 35.31 NoCt NoCt NoCt No ID G S. pyogenes 1e-1 TNTC 2,210,000 26.36 Positive 36.24 NoCt 28.73 NoCt GrPos S. pyogenes 1e-2 TNTC 221,000 30.74 Positive 36.09 NoCt 32.02 NoCt GrPos S. pyogenes 1e-3 TNTC 22,100 34.60 Positive 35.96 NoCt 35.34 NoCt GrPos S. pyogenes 1e-4 *221 2,210 39.37 Positive 35.53 NoCt 42.28 NoCt No ID S. pyogenes 1e-5 3 221 41.78 Negative 34.66 NoCt 33.29 NoCt GrPos NSC1 0 43.80 Negative 35.29 NoCt 35.27.sup. NoCt GrPos NSC2 0 44.12 Negative 35.37 NoCt 38.56.sup. NoCt GrPos NSC3 0 42.39 Negative 35.96 NoCt NoCt NoCt No ID Confirm Positivity threshold (Pt) 40 Ct; false positives.sup.

Observations:

[0545] Bead B difficult to resuspend before diluting down to 1% solid content; and appeared visually more dilute after dilution to 1% solid [0546] At the end of processing, samples were placed on DynaMag-2 magnetic rack, and all bead types C-G magnetised similarly apart from: Bead A, which appeared to have heavy pellets; and Bead B, which had very small bead pellets

Analysis:

[0547] All carboxylated magnetic beads tested here demonstrate microbial binding as determined by ETGA and Confirm readouts. However, the sensitivity of microbial detection does vary somewhat, depending on the level of blood-derived ETGA signal and/or assay inhibition

Example 11: Microbial Capture from Blood by Magnetic Beads Occurs Using a Variety of Different Bead Sizes and Functional Coatings

Aim:

[0548] To compare microbial capture performance for a variety of commercially-available magnetic beads of different size and functional coating using Momentum's Magnitor test (ETGA and Confirm technologies). Two experiments were performed to demonstrate microbial capture for automated (Protocol 1) and manual (Protocol 2) sample processing. Importantly, Protocol 2 included three bead resuspension washes to more convincingly demonstrate that ETGA/Confirm signal is specific to bead-bound microbial cells, rather than sample carryover (as opposed to Protocol 1, which included a single beads-magnetised wash step).

Test Conditions:

[0549]

TABLE-US-00048 Diameter Heading Description Product (m) Ferrite % Polymer COOH-0.2 Very Small Estapor Merck #M1-020/50 0.160- >50 Polystyrene Carboxylated Nanospheres 0.240 (COOH) COOH-1.0 Original Estapor Merck #M1-070/40 0.700- 35-45 Polystyrene Carboxylated Microspheres 1.300 (COOH) HYDRO-1.0 Original Estapor Merck #MS-070/40 0.700- 35-50 Polystyrene Hydrophobic Microspheres 1.300 NH2-1.5 Original Estapor Aminated Merck #M2-070/40 1.000- 35-45 Polystyrene Microspheres (NH2) 2.000 Peps6 Magnetic beads covered with ApoH Technologies 0.200 Unknown Unknown Peps6 Ltd #MP20006 Speed SpeedBeads magnetic GE Healthcare 1.000 40 Polystyrene carboxylate modified #65152105050250 particles (two layers of magnetite) BioEsta Streptavidin coated Small Merck #BE-M08/03 0.251- 40-60 Polystyrene Estapor Carboxylated 0.400 Nanospheres (COOH)

[0550] All beads washed in 1 mL 1 E-BUF (50 mM Tris-HCl [pH 8.0]+150 mM Sodium Chloride+1% Igepal+0.25% Tergitol) and resuspended to 1% solid in 1 E-BUF

Sample Set-Up (Performed Separately for Protocol 1 and 2 which were Performed on Different Days): [0551] E. coli o/n liquid cultures set up as standard in 3 mL broth (BacTec PLUS aerobic) and incubated for 16-20 hours (37 C.) [0552] The following day, E. coli liquid culture diluted to 1E-3 in blood broth and 2-hour outgrowth incubation performed (37 C. @ 500 rpm) [0553] Following the 2-hour outgrowth incubation, E. coli 1E-3 pre-culture serially diluted in blood broth to produce three dilution points (EC 1E-6 to 1E-8) [0554] 1 mL specimens (three E. coli dilutions and three NSC samples: 6 sample-set) added to 2 mL sample tubes preloaded with 112 L 10 E-BUF (500 mM Tris-HCl [pH 8.0]+1.5 M Sodium Chloride+10% Igepal+2.5% Tergitol)+15 L beads (1% solid), then Magnitor test initiated according to either Protocol 1 or Protocol 2 (see below):
Protocol 1 (Automated Sample Processing on epMotion 5073m): [0555] 30 mins orbital mixing (1000 rpm) @ 37 C. [0556] 15 mins magnetisation [0557] 1 mL s/n removed [0558] 0.82 mL WB added to tubes whilst beads magnetised [0559] 1 mL s/n removed [0560] 50 L LM added to tubes whilst beads magnetised [0561] Magnetisation switched off and ETGA reaction performed: 5 mins at 1000 rpm, then 55 mins at 800 rpm @26 C. [0562] qPCR set-up for ETGA and Confirm (10 L reactions)

Protocol 2 (Manual Sample Processing Using DynaMag-2 Magnet and Manual Liquid Transfers by Pipette):

[0563] 30 mins orbital mixing (1000 rpm) @ 37 C. [0564] 5 mins magnetisation on DynaMag-2 [0565] All s/n removed [0566] 1 mL WB added and tubes mixed for 2 mins @ RT (1000 rpm) [0567] 5 mins magnetisation on Dynmag-2 [0568] All s/n removed [0569] 1 mL WB added and tubes mixed for 2 mins @ RT (1000 rpm) [0570] 5 mins magnetisation on Dynmag-2 [0571] All s/n removed [0572] 1 mL WB added and tubes mixed for 2 mins @ RT (1000 rpm) [0573] 5 mins magnetisation on Dynmag-2 [0574] All s/n removed [0575] 50 L LM added to tubes off magnet [0576] ETGA reaction performed: 5 mins at 1000 rpm, then 55 mins at 800 rpm @26 C. [0577] Manual qPCR set-up for ETGA and Confirm (10 L reactions)

Results:

[0578]

TABLE-US-00049 Protocol 1 (automated sample processing on epMotion 5073m) - performed on 20190221 ETGA Ct Colonies *E cfu/mL COOH-0.2 COOH-1.0 HYDRO-1.0 NH2-1.5 Speed BioEsta Peps6 E. coli 1E-6 TNTC 18500 20.97 20.13 21.01 21.36 21.98 21.01 19.85 E. coli 1E-7 *185 1850 25.28 24.23 26.43 24.61 25.06 25.46 26.27 E. coli 1E-8 12 185 27.56 28.64 30.54 27.24 30.63 30.59 28.63 NSC 1 0 0 38.19 36.32 38.73 37.86 39.18 37.94 40.26 NSC 2 0 0 38.33 36.16 39.41 37.19 38.90 39.22 38.82 NSC 3 0 0 34.34 35.78 37.67 36.76 40.05 39.16 39.42 Confirm GrNeg Ct Colonies *E cfu/mL COOH-0.2 COOH-1.0 HYDRO-1.0 NH2-1.5 Speed BioEsta Peps6 E. coli 1E-6 TNTC 18500 32.56 30.75 33.89 32.42 30.51 32.63 46.19 E. coli 1E-7 *185 1850 40.82 33.67 NoCt 31.99 33.47 NoCt NoCt E. coli 1E-8 12 185 39.21 NoCt NoCt 39.38 NoCt NoCt NoCt NSC 1 0 0 NoCt 42.19 40.84 44.46 40.55 NoCt NoCt NSC 2 0 0 NoCt 40.61 47.17 NoCt NoCt NoCt NoCt NSC 3 0 0 NoCt 37.42.sup. 41.04 NoCt NoCt NoCt NoCt Positivity threshold (Pt) 40 Ct; false positives.sup. IPC Ct Colonies *E cfu/mL COOH-0.2 COOH-1.0 HYDRO-1.0 NH2-1.5 Speed BioEsta Peps6 E. coli 1E-6 TNTC 18500 35.89 35.14 35.72 36.28 36.06 36.07 34.86 E. coli 1E-7 *185 1850 35.98 35.14 34.74 36.15 35.08 35.59 35.63 E. coli 1E-8 12 185 34.56 34.83 34.93 36.15 34.72 35.40 34.82 NSC 1 0 0 34.45 34.83 34.56 35.91 35.55 35.49 34.74 NSC 2 0 0 34.51 34.71 35.13 36.07 34.92 35.04 34.42 NSC 3 0 0 35.04 34.85 34.45 35.62 35.62 35.34 34.85

TABLE-US-00050 Protocol 2 (manual sample processing using DynaMag-2 magnet and manual liquid transfers by pipette)performed on 2019 Feb. 28 ETGA Ct Colonies *E cfu/mL COOH-0.2 COOH-1.0 HYDRO-1.0 NH2-1.5 Speed BioEsta E. coli 1E6 *724 7240 23.97 25.45 24.80 26.29 25.95 24.32 E. coli 1E7 76 724 27.64 30.33 29.14 31.52 30.34 27.35 E. coli 1E8 7 72 32.20 30.81 36.25 32.91 29.64 31.54 NSC 1 0 0 40.47 39.30 40.27 34.36 43.39 41.61 NSC 2 0 0 42.23 37.84 41.03 33.96 43.47 41.70 NSC 3 0 0 42.74 39.66 41.53 34.34 43.27 40.19 Confirm GrNeg Ct Colonies *E cfu/mL COOH-0.2 COOH-1.0 HYDRO-1.0 NH2-1.5 Speed BioEsta E. coli 1E6 *724 7240 27.84 28.54 27.26 27.05 27.72 26.90 E. coli 1E7 76 724 31.29 32.46 37.65 33.26 32.58 29.29 E. coli 1E8 7 72 36.95 NoCt NoCt 36.41 31.61 34.06 NSC 1 0 0 NoCt .sup.33.69.sup. NoCt NoCt NoCt NoCt NSC 2 0 0 NoCt .sup.31.78.sup. NoCt NoCt 42.70 NoCt NSC 3 0 0 NoCt .sup.35.24.sup. NoCt NoCt NoCt NoCt IPC Ct Colonies *E cfu/mL COOH-0.2 COOH-1.0 HYDRO-1.0 NH2-1.5 Speed BioEsta E. coli 1E6 *724 7240 33.22 33.94 33.24 37.43 34.15 33.30 E. coli 1E7 76 724 32.44 32.91 32.78 36.47 33.70 32.53 E. coli 1E8 7 72 33.29 33.07 32.57 35.99 33.80 33.18 NSC 1 0 0 33.32 33.02 33.07 35.78 33.83 32.99 NSC 2 0 0 32.95 33.44 32.95 35.99 33.88 33.54 NSC 3 0 0 33.61 33.66 33.23 35.77 34.33 33.25 Positivity threshold (Pt) 40 Ct; false positives.sup.

Analysis:

[0579] These results demonstrate that a variety of different bead sizes and functional coatings produce comparable levels of microbial binding as determined by ETGA and Confirm readouts.

Example 12: Magnetic Beads of Different Size and Functional Coating can be Used to Capture a Broad Range of Microbial Species (Gram Negative, Gram Positive and Candida) from Blood

Aim:

[0580] To compare microbial capture performance for a variety of commercially-available magnetic beads of different size and functional coating using Momentum's Magnitor test (ETGA and Confirm technologies). E. coli was tested previously (Bead size and coating I: source experiment: 20190221_WP7_Bead-Comparison_Analysis and 20190228_WP7_Bead-Comparison-3-wash_Analysis): to expand on this previous work, three additional microbial species were tested (S. aureus, S. pneumoniae and C. albicans).

Test Conditions:

[0581]

TABLE-US-00051 Heading Description Product Diameter (m) Ferrite % Polymer COOH-0.2 Very Small Estapor Carboxylated Merck #M1-020/50 0.160-0.240 >50 Polystyrene Nanospheres COOH-1.0 Original Estapor Carboxylated Merck #M1-070/40 0.700-1.300 35-45 Polystyrene Microspheres HYDRO-1.0 Original Estapor Hydrophobic Merck #MS-070/40 0.700-1.300 35-50 Polystyrene Microspheres NH2-1.5 Original Estapor Aminated Merck #M2-070/40 1.000-2.000 35-45 Polystyrene Microspheres (NH2)

[0582] All beads washed in 1 mL 1 Tris+NaCl and resuspended to 1% solid in 1 Tris+NaCl

Protocol:

Sample Set-Up:

[0583] Microorganism overnight liquid cultures (o/n) set-up in BacTec PLUS aerobic broth (inoculation of 3 mL broth from agar plate). The following day (approx 16 hours later) 300 L S. pneumoniae and C. albicans liquid culture inoculated in 3 mL blood broth (1E-1 dilution), and 3 L S. aureus liquid culture inoculated in 3 mL blood broth (1E-3 dilution); and 2 hour outgrowth performed at 37 C., 500 rpm. [0584] Following 2-hour outgrowth, microbial pre-cultures diluted (DF10) in blood broth to create three dilution points per microorganism. [0585] 100 L TVCs performed for each microbial dilution

Manual Simulation of Magnitor Performed Using DynaMaq-2 Magnet and Manual Liquid Transfers:

[0586] 1 mL specimens (three dilutions per microorganism species and three NSC samples: 12 sample-set) added to 2 mL sample tubes preloaded 15 L beads (1% solid) and 112 L E-BUF (500 mM Tris-HCl [pH 8.0]+1.5 M Sodium Chloride+10% Igepal+2.5% Tergitol) [0587] 30 mins orbital mixing (1000 rpm) @ 37 C. [0588] 5 mins magnetisation on DynaMag-2 [0589] All s/n removed [0590] 1 mL WB added and tubes mixed for 3 mins @ RT (1000 rpm) [0591] 5 mins magnetisation on Dynmag-2 [0592] All s/n removed [0593] 50 L LM added to tubes off magnet [0594] ETGA reaction performed: 5 mins at 1000 rpm, then 55 mins at 800 rpm @26 C. [0595] Manual qPCR set-up for ETGA and Confirm (10 L reactions)

Results:

[0596]

TABLE-US-00052 ETGA Ct Colonies *E cfu/mL COOH-0.2 COOH-1.0 HYDRO-1.0 NH2-1.5 S. aureus 1E5 TNTC 131,000 21.48 21.37 21.00 22.19 S. aureus 1E6 TNTC 13,100 26.12 26.13 25.73 26.58 S. aureus 1E7 *131 1,310 30.76 30.29 30.25 30.38 C. albicans 1E2 LAWN 956,000 26.68 26.04 26.24 26.64 C. albicans 1E3 TNTC 95,600 28.08 26.46 27.08 26.76 C. albicans 1E4 *956 9,560 31.20 31.09 30.64 31.14 S. pneumoniae 1E2 LAWN 732,000 30.52 31.34 30.61 30.41 S. pneumoniae 1E3 TNTC 73,200 35.07 35.38 34.28 34.01 S. pneumoniae 1E4 *732 7,320 38.73 38.02 37.75 34.93 NSC 1 0 42.44 36.99 41.11 33.84 NSC 2 0 41.52 38.95 40.56 34.52 NSC 3 0 41.64 39.35 40.17 35.27

TABLE-US-00053 Confirm Ct COOH-0.2 COOH-1.0 HYDRO-1.0 NH2-1.5 Colonies *E cfu/mL GrNeg GrPos Candida GrNeg GrPos Candida GrNeg GrPos Candida GrNeg GrPos Candida S. aureus TNTC 131,000 NoCt 29.03 NoCt NoCt 29.05 NoCt NoCt 28.63 NoCt NoCt 26.17 NoCt 1E5 S. aureus TNTC 13,100 NoCt 32.30 NoCt NoCt 33.17 NoCt NoCt 32.67 NoCt NoCt 29.46 NoCt 1E6 S. aureus *131 1,310 NoCt 39.23 NoCt 40.08 NoCt NoCt NoCt 41.21 NoCt NoCt 33.84 NoCt 1E7 C. albicans LAWN 956,000 NoCt NoCt 28.35 NoCt NoCt 27.45 NoCt NoCt 26.37 NoCt NoCt 27.24 1E2 C. albicans TNTC 95,600 NoCt NoCt 30.93 NoCt 36.10* 31.54 NoCt NoCt 30.17 NoCt NoCt 29.05 1E3 C. albicans *956 9,560 NoCt NoCt NoCt NoCt NoCt 38.86 NoCt NoCt 48.57 NoCt 39.91* 44.85 1E4 S. pneumoniae LAWN 732,000 NoCt 25.20 NoCt NoCt 26.26 NoCt NoCt 24.58 NoCt NoCt 26.31 NoCt 1E2 S. pneumoniae TNTC 73,200 NoCt 29.16 NoCt NoCt 29.85 NoCt NoCt 28.22 NoCt NoCt 31.18 NoCt 1E3 S. pneumoniae *732 7,320 NoCt 32.38 NoCt NoCt 33.47 NoCt NoCt 32.33 NoCt NoCt 33.74 NoCt 1E4 NSC 1 0 NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt 41.24 NoCt NSC 2 0 NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NSC 3 0 NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt Positivity threshold (Pt) 40 Ct; *false positives

TABLE-US-00054 IPC Ct Colonies *E cfu/mL COOH-0.2 COOH-1.0 HYDRO-1.0 NH2-1.5 S. aureus 1E5 TNTC 131,000 33.22 33.19 33.14 34.50 S. aureus 1E6 TNTC 13,100 33.19 32.55 33.07 34.55 S. aureus 1E7 *131 1,310 33.61 32.80 33.05 34.20 C. albicans 1E2 LAWN 956,000 34.75 34.71 34.80 35.47 C. albicans 1E3 TNTC 95,600 33.59 33.02 33.49 34.54 C. albicans 1E4 *956 9,560 33.14 33.02 32.81 34.06 S. pneumoniae 1E2 LAWN 732,000 34.02 33.22 33.56 34.17 S. pneumoniae 1E3 TNTC 73,200 33.29 33.05 33.44 35.28 S. pneumoniae 1E4 *732 7,320 33.23 32.84 33.02 33.75 NSC 1 0 33.17 33.21 33.50 33.96 NSC 2 0 33.38 33.17 33.32 34.50 NSC 3 0 33.55 33.59 33.05 34.20

Analysis:

[0597] These results demonstrate that a variety of different bead sizes and functional coatings produce comparable levels of microbial binding as determined by ETGA and Confirm readouts.

Example 13: Magnetic Beads of Different Size and Functional Coating can be Used to Capture a Broad Range of Microbial Species (Gram Negative, Gram Positive and Candida) from a Simple Tris+NaCl Buffer

Aim:

[0598] To compare microbial capture performance for a variety of commercially-available magnetic beads of different size and functional coating using Momentum's Magnitor test (ETGA and Confirm technologies). This experiment was performed using a simple buffer (50 mM Tris-HCl [pH 8.0]+150 mM NaCl) as the specimen and wash buffer i.e. no detergents used until the addition of microbial lysis mix.

Test Conditions:

[0599]

TABLE-US-00055 Heading Description Product Diameter (m) Ferrite % Polymer COOH-0.2 Very Small Estapor Merck #M1-020/50 0.160-0.240 >50 Polystyrene Carboxylated Nanospheres COOH-1.0 Original Estapor Merck #M1-070/40 0.700-1.300 35-45 Polystyrene Carboxylated Microspheres HYDRO-1.0 Original Estapor Merck #MS-070/40 0.700-1.300 35-50 Polystyrene Hydrophobic Microspheres NH2-1.5 Original Estapor Aminated Merck #M2-070/40 1.000-2.000 35-45 Polystyrene Microspheres (NH2)

[0600] All beads washed in 1 mL 1 Tris+NaCl (50 mM Tris-HCl [pH 8.0]+150 mM NaCl) and resuspended to 1% solid in 1 Tris+NaCl

Protocol:

Sample Set-Up:

[0601] Microorganism overnight liquid cultures (o/n) set-up in BacTec PLUS aerobic broth (inoculation of 3 mL broth from agar plate). The following day (approx 16 hours later) 3 L E. coli and S. aureus liquid culture inoculated in 3 mL broth (1E-3 dilution), and 300 L C. albicans liquid culture inoculated in 3 mL broth (1E-1 dilution); and 2-hour outgrowth performed at 37 C., 500 rpm. [0602] Following 2-hour outgrowth, microbial pre-cultures diluted (DF10) in 1 Tris+NaCl buffer to create three dilution points per microorganism. [0603] 100 L TVCs performed for each microbial dilution

Manual Simulation of Magnitor Performed Using DynaMaq-2 Magnet and Manual Liquid Transfers:

[0604] 1 mL specimens (three dilutions per microorganism species and three NSC samples: 12 sample-set) added to 2 mL sample tubes preloaded 15 L beads (1% solid) [0605] 30 mins orbital mixing (1000 rpm) @ 37 C. [0606] 5 mins magnetisation on DynaMag-2 [0607] All s/n removed [0608] 1 mL WB (1 Tris+NaCl) added and tubes mixed for 3 mins @ RT (1000 rpm) [0609] 5 mins magnetisation on Dynmag-2 [0610] All s/n removed [0611] 50 L LM added to tubes off magnet [0612] ETGA reaction performed: 5 mins at 1000 rpm, then 55 mins at 800 rpm @26 C. [0613] Manual qPCR set-up for ETGA and Confirm (10 L reactions)

Results:

[0614]

TABLE-US-00056 ETGA Ct Colonies *E cfu/mL COOH-0.2 COOH-1.0 HYDRO-1.0 NH2-1.5 E. coli 1E6 *629 6,290 18.68 19.24 22.36 30.63 E. coli 1E7 65 629 22.43 22.29 25.24 31.25 E. coli 1E8 21 63 28.70 28.21 29.44 31.35 S. aureus 1E5 *791 7,910 18.32 18.22 18.08 25.72 S. aureus 1E6 102 791 22.26 22.94 22.58 30.12 S. aureus 1E7 5 79 27.65 26.37 26.61 30.26 C. albicans 1E2 LAWN 811,000 25.38 25.51 26.02 29.07 C. albicans 1E3 TNTC 81,100 27.13 27.71 27.50 30.09 C. albicans 1E4 *811 8,110 28.93 28.78 29.03 30.59 NSC 1 0 35.04 32.14 36.86 32.12 NSC 2 0 36.37 31.65 36.47 31.61 NSC 3 0 36.54 32.51 35.46 30.97

TABLE-US-00057 Confirm Ct COOH-0.2 COOH-1.0 HYDRO-1.0 NH2-1.5 Colonies *E cfu/mL GrNeg GrPos Candida GrNeg GrPos Candida GrNeg GrPos Candida GrNeg GrPos Candida E. coli 1E6 *629 6,290 28.24 NoCt NoCt 26.59 NoCt NoCt 27.42 NoCt NoCt 27.39 NoCt NoCt E. coli 1E7 65 629 38.82 NoCt NoCt 29.58 NoCt NoCt 29.43 NoCt NoCt NoCt NoCt NoCt E. coli 1E8 21 63 NoCt NoCt NoCt NoCt NoCt NoCt 34.63 NoCt NoCt NoCt 46.54 NoCt S. aureus 1E5 *791 7,910 NoCt 29.56 NoCt NoCt 28.61 NoCt NoCt 28.79 NoCt NoCt NoCt NoCt S. aureus 1E6 102 791 NoCt 32.82 NoCt NoCt 31.94 NoCt NoCt 31.52 NoCt NoCt NoCt NoCt S. aureus 1E7 5 79 NoCt 36.19 NoCt NoCt 35.89 NoCt NoCt 35.26 NoCt NoCt NoCt NoCt C. albicans 1E2 LAWN 811,000 NoCt 43.68 28.74 NoCt 41.77 27.34 NoCt 32.91 26.74 NoCt NoCt 31.90 C. albicans 1E3 TNTC 81,100 NoCt NoCt 29.59 43.60 NoCt 29.14 NoCt 36.69 29.16 NoCt 38.39 48.53 C. albicans 1E4 *811 8,110 NoCt NoCt 31.92 NoCt NoCt 30.81 NoCt NoCt 30.45 NoCt 32.56 40.26 NSC 1 0 NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt 41.43 NoCt NSC 2 0 NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt NSC 3 0 NoCt 41.20 NoCt NoCt NoCt NoCt NoCt NoCt NoCt NoCt 40.79 NoCt Positivity threshold (Pt) 40 Ct; false positives shown in red font

TABLE-US-00058 IPC Ct Colonies *E cfu/mL COOH-0.2 COOH-1.0 -IYDRO-1.0 NH2-1.5 E. coli 1E6 *629 6,290 31.24 30.86 30.98 36.67 E. coli 1E7 65 629 31.10 30.46 30.27 35.56 E. coli 1E8 21 63 31.01 30.25 30.51 37.00 S. aureus 1E5 *791 7,910 31.43 31.17 31.02 NoCt S. aureus 1E6 102 791 30.89 31.11 30.71 37.43 S. aureus 1E7 5 79 30.84 30.56 30.69 45.23 C. albicans 1E2 LAWN 811,000 35.64 33.75 33.48 NoCt C. albicans 1E3 TNTC 81,100 32.98 31.94 33.61 40.29 C. albicans 1E4 *811 8,110 31.34 31.44 31.74 37.11 NSC 1 0 31.03 30.57 30.62 37.31 NSC 2 0 31.37 30.55 30.51 35.33 NSC 3 0 31.32 30.37 30.75 36.67

Analysis:

[0615] These results demonstrate that in a clean system (i.e. simple Tris+NaCl buffer instead of a biological specimen) a variety of different bead sizes and functional surfaces (carboxylated and hydrophobic) produce comparable levels of microbial binding as determined by ETGA and Confirm readouts. [0616] Interestingly, aminated beads (NH2-1.5) produced very poor Magnitor results in specimen type, indicative of little/no microbial binding. This observation differs to the situation in blood broth specimens, where aminated beads produced comparable levels of microbial capture to the other beads tested.

Example 14: Magnetic Beads of Different Size and Functional Coating can be Used to Capture a Broad Range of Microbial Species (Gram Negative, Gram Positive and Candida) from Non-Lysed Blood

Aim:

[0617] To compare microbial capture performance for a variety of commercially-available magnetic beads of different size and functional coating using Momentum's Magnitor test (ETGA and Confirm technologies) in the absence of blood lysis i.e. no detergents in binding buffer

Test Conditions:

[0618]

TABLE-US-00059 Diameter Heading Description Product (m) Ferrite % Polymer COOH-0.2 Very Small Estapor Merck #M1-020/50 0.160-0.240 >50 Polystyrene Carboxylated Nanospheres COOH-1.0 Original Estapor Merck #M1-070/40 0.700-1.300 35-45 Polystyrene Carboxylated Microspheres HYDRO- Original Estapor Merck #MS-070/40 0.700-1.300 35-50 Polystyrene 1.0 Hydrophobic Microspheres NH2-1.5 Original Estapor Aminated Merck #M2-070/40 1.000-2.000 35-45 Polystyrene Microspheres (NH2)

[0619] All beads washed in 1 mL 1 Tris+NaCl (50 mM Tris-HCl [pH 8.0]+150 mM NaCl) and resuspended to 1% solid in 1 Tris+NaCl

Protocol:

Sample Set-Up:

[0620] Microorganism overnight liquid cultures (o/n) set-up in BacTec PLUS aerobic broth (inoculation of 3 mL broth from agar plate). The following day (approx 16 hours later) 3 L E. coli and S. aureus liquid culture inoculated in 3 mL broth (1E-3 dilution), and 300 L C. albicans liquid culture inoculated in 3 mL blood broth (1E-1 dilution); and 2-hour outgrowth performed at 37 C., 500 rpm. [0621] Following 2-hour outgrowth, microbial pre-cultures diluted (DF10) in blood broth to create three dilution points per microorganism. [0622] 100 L TVCs performed for each microbial dilution

Manual Simulation of Magnitor Performed Using DyneMag-2 Magnet and Manual Liquid Transfers:

[0623] 1 mL specimens (three dilutions per microorganism species and three NSC samples: 12 sample-set) added to 2 mL sample tubes preloaded 15 L beads (1% solid) and 112 L Binding Buffer (Tris-HCl+Sodium Chloride) [0624] 30 mins orbital mixing (1000 rpm) @ 37 C. [0625] 5 mins magnetisation on DynaMag-2 [0626] All s/n removed [0627] 1 mL WB added and tubes mixed for 3 mins @ RT (1000 rpm) [0628] 5 mins magnetisation on Dynmag-2 [0629] All s/n removed [0630] 50 L LM added to tubes off magnet [0631] ETGA reaction performed: 5 mins at 1000 rpm, then 55 mins at 800 rpm @26 C. [0632] Manual qPCR set-up for ETGA and Confirm (10 L reactions)

Results:

[0633]

TABLE-US-00060 ETGA Ct Colonies *E cfu/mL COOH-0.2 COOH-1.0 HYDRO-1.0 NH2-1.5 E. coli 1E6 *671 6,710 28.37 28.22 28.83 28.40 E. coli 1E7 69 671 32.26 32.90 31.21 31.73 E. coli 1E8 24 67 35.02 33.23 34.05 32.73 S. aureus 1E5 TNTC 27,600 22.23 22.86 22.45 22.57 S. aureus 1E6 *276 2,760 26.62 26.64 26.53 26.09 S. aureus 1E7 28 276 30.37 29.44 30.72 31.01 C. albicans 1E2 LAWN 649,000 34.01 34.00 33.52 33.48 C. albicans 1E3 TNTC 64,900 35.00 33.89 34.23 33.66 C. albicans 1E4 *649 6,490 35.47 35.08 34.48 33.73 NSC 1 0 33.95 35.32 32.42 31.15 NSC 2 0 32.27 34.34 32.47 30.76 NSC 3 0 33.89 33.37 32.45 31.24

TABLE-US-00061 Confirm Ct COOH-0.2 COOH-1.0 HYDRO-1.0 NH2-1.5 Colonies *E cfu/mL GrNeg GrPos Candida GrNeg GrPos Candida GrNeg GrPos Candida GrNeg GrPos Candida E. coli 1E6 *671 6,710 30.24 No Ct No Ct 30.51 No Ct No Ct 31.59 No Ct No Ct 30.47 No Ct No Ct E. coli 1E7 69 671 38.27 No Ct No Ct 48.35 No Ct No Ct No Ct No Ct No Ct 35.36 No Ct No Ct E. coli 1E8 24 67 No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct S. aureus 1E5 TNTC 27,600 No Ct 24.94 No Ct No Ct 24.55 No Ct No Ct 24.34 No Ct No Ct 24.24 No Ct S. aureus 1E6 *276 2,760 No Ct 27.34 No Ct No Ct 27.54 48.19 No Ct 28.18 No Ct No Ct 27.37 No Ct S. aureus 1E7 28 276 No Ct 30.39 No Ct No Ct 29.72 No Ct No Ct 31.12 No Ct No Ct 29.65 No Ct C. albicans 1E2 LAWN 649,000 No Ct 40.65 31.51 40.74 No Ct 29.89 No Ct No Ct 30.24 No Ct No Ct 28.70 C. albicans 1E3 TNTC 64,900 No Ct No Ct No Ct 42.94 No Ct 34.41 No Ct No Ct No Ct No Ct No Ct No Ct C. albicans 1E4 *649 6,490 No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct NSC 1 0 No Ct No Ct No Ct No Ct No Ct No Ct No Ct 45.94 No Ct No Ct No Ct No Ct NSC 2 0 No Ct No Ct No Ct 42.39 No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct NSC 3 0 No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct Positivity threshold (Pt) 40 Ct; false positives shown in red font

TABLE-US-00062 IPC Ct Colonies *E cfu/mL COOH-0.2 COOH-1.0 HYDRO-1.0 NH2-1.5 E. coli 1E6 *671 6,710 32.75 32.82 33.21 33.42 E. coli 1E7 69 671 33.02 33.61 32.90 33.81 E. coli 1E8 24 67 34.18 33.17 33.58 33.21 S. aureus 1E5 TNTC 27,600 33.31 33.77 33.86 34.06 S. aureus 1E6 *276 2,760 33.46 33.20 33.74 33.07 S. aureus 1E7 28 276 32.96 33.06 33.93 33.71 C. albicans 1E2 LAWN 649,000 32.94 33.30 33.81 33.95 C. albicans 1E3 TNTC 64,900 33.82 33.25 33.71 34.42 C. albicans 1E4 *649 6,490 33.58 33.53 33.32 34.25 NSC 1 0 34.42 34.62 33.47 33.77 NSC 2 0 33.62 33.76 33.61 33.86 NSC 3 0 33.83 33.81 33.71 33.33

Analysis:

[0634] These results demonstrate that a variety of different bead sizes and functional coatings produce comparable levels of microbial binding from blood in the absence of blood lysis as determined by ETGA and Confirm readouts [0635] However, microbial detection by ETGA is substantially reduced by an increase in NSC ETGA signal in the absence of blood lysis, when compared to previous experiments with blood lysis during microbial binding.

Example 15: Microbial Capture by Magnetic Beads Occurs in a Variety of Complex Biological Specimen Types

Aim:

[0636] To investigate whether microbial capture using magnetic beads is possible for other complex biological fluids, in addition to blood

Test Conditions:

[0637]

TABLE-US-00063 Specimen type Description Tris + NaCl Tris buffer with NaCl as non-biological control sample-set (two identical sample-sets processing in parallel for Magnitor and Regrowth assays) Blood Whole blood with CPD and SPS anticoagulants Saliva Saliva diluted to 50% with distilled H2O Urine Mid-stream urine Milk Semi-skimmed Pasteurised Cow's milk Note: Tris + NaCl: 50 mM Tris-HCl [pH 8.0] + 150 mM Sodium Chloride

Protocol:

[0638] E. coli o/n liquid cultures set-up as standard in BacTec PLUS aerobic broth, then the following day (approx 16 hours later) 3 L o/n added to 3 mL broth (E. coli 1E-3), and 2-hour outgrowth incubation performed at 37 C., 500 rpm. [0639] Following 2-hour outgrowth, E. coli 1E-3 preculture diluted to produce five serial dilution points (E. coli 1E-6 to 1E-9) in each specimen type. 100 L TVCs performed on COL agar plates.

Manual Simulation of Magnitor Performed Using DynaMaq-2 Magnet and Manual Liquid Transfers:

[0640] 1 mL specimens added to 2 mL sample tubes preloaded with 112 L Binding Buffer (500 mM Tris-HCl [pH 8.0]+1.5 M Sodium Chloride+10% Igepal+2.5% Tergitol+0.5% Sodium Deoxycholate)+15 L beads (BioEstapor beads; Merck #BE-M08/03 (1% solid))Note, sample tubes for Tris+NaCl sample-sets not preloaded with 112 L binding buffer (to avoid inclusion of detergents, which might inhibit microbial growth for the Regrowth assay) [0641] 30 mins shaking (1000 rpm) @ 37 C. [0642] 5 mins magnetisation on DynaMag-2 [0643] All s/n removed [0644] 1 mL WB added and tubes mixed for 3 mins @ RT (1000 rpm)note, 1 mL Tris+NaCl buffer added instead of wash buffer for Tris+NaCl sample-sets to avoid inclusion of detergents, which might inhibit microbial growth for the Regrowth assay [0645] 5 mins magnetisation on Dynmag-2 [0646] All s/n removed [0647] 50 L LM added to tubes off magnetnote, beads resuspended in 100 L Tris+NaCl buffer for Regrowth assay [0648] ETGA reaction performed: 5 mins at 1000 rpm, then 55 mins at 800 rpm @26 C. [0649] Manual qPCR set-up for ETGA and Confirm (10 L reactions)

Results:

[0650]

TABLE-US-00064 TVCs (100 L on COL plates) Specimen Colonies Tris + NaCl E. coli D1 TNTC Tris + NaCl E. coli D2 661 Tris + NaCl E. coli D3 121 Tris + NaCl E. coli D4 21 Tris + NaCl E. coli D5 0 Tris + NaCl NSC 0 Blood NSC 0 Saliva NSC LAWN Urine NSC 5 Milk NSC 3

Regrowth Assay on Paired Tris+NaCl Sample-Set

[0651] 1. 100 l of Tris+NaCl specimens plated/inoculatedSpecimen [0652] 2. 100 l of supernatant plated/inoculated after microbial binding stepAfter binding [0653] 3. 100 l of supernatant plated/inoculated after wash stepAfter washing [0654] 4. 50 l of beads resuspended in 100 L Tris+NaCl buffer and plated/inoculated (i.e. 50% material on plate and 50% material inoculated into liquid culture)Beads

[0655] Plates and Liquid Cultures incubated overnight at 37 C.

TABLE-US-00065 TVC (colonies) Nutrient Broth (Growth: YES/NO) Specimen After bind After wash Beads Specimen After bind After wash Beads E. coli D1 TNTC 555 453 LAWN YES YES YES YES E. coli D2 661 104 5 TNTC YES YES YES YES E. coli D3 121 12 1 366 YES YES NO YES E. coli D4 21 12 0 7 YES YES NO YES E. coli D5 0 2 0 0 YES YES NO YES NSC 0 0 0 0 NO NO NO NO Tris + NaCl sample-set

Magnitor Results:

[0656]

TABLE-US-00066 ETGA Ct Colonies *E cfu/mL Tris + NaCl Blood Saliva Urine Milk E. coli D1 TNTC 66100 14.49 21.22 26.45 23.13 20.30 E. coli D2 *661 6610 20.62 25.73 28.50 26.22 24.97 E. coli D3 121 661 24.09 29.41 28.14 29.39 28.22 E. coli D4 21 66 29.84 33.98 28.20 30.83 30.99 E. coli D5 0 7 35.13 36.56 28.33 30.40 30.07 NSC 1 0 0 35.74 38.41 27.32 31.05 30.70 NSC 2 0 0 34.90 39.26 28.06 31.06 30.48 NSC 3 0 0 35.04 39.45 28.73 31.08 30.89 Ave. NSC 35.23 39.04 28.04 31.06 30.69 Pt (5th %) 34.91 38.49 27.39 31.05 30.50 Positivity Thresholds (Pt) calculated using NSCs (n = 3) for each specimen type using formula = PERCENTILE.INC(array, 0.05)

TABLE-US-00067 Confirm GrNeg Confirm GrPos Colonies *E cfu/mL Tris + NaCl Blood Saliva Urine Milk Tris + NaCl Blood Saliva Urine Milk E. coli D1 TNTC 66100 20.56 27.24 NoCt 30.16 21.25 NoCt NoCt 27.22 32.87 28.01 E. coli D2 *661 6610 27.03 31.35 NoCt NoCt 25.38 NoCt NoCt 28.52 33.07 27.43 E. coli D3 121 661 27.64 37.88 NoCt NoCt 29.58 NoCt NoCt 29.99 31.79 26.94 E. coli D4 21 66 NoCt NoCt NoCt NoCt 43.46 NoCt NoCt 28.12 35.25 26.91 E. coli D5 0 7 NoCt NoCt NoCt NoCt NoCt 40.65 NoCt 28.09 35.43 27.38 NSC 1 0 0 NoCt NoCt NoCt NoCt NoCt NoCt 42.73 28.58 35.36 26.83 NSC 2 0 0 NoCt NoCt NoCt NoCt 45.84 NoCt NoCt 28.83 32.96 27.28 NSC 3 0 0 NoCt NoCt NoCt NoCt NoCt 35.55 42.95 26.77 32.95 27.40 Positivity threshold (Pt) 40 Ct

[0657] Note, no observable amplification in Candida channel for Confirm

TABLE-US-00068 IPC Ct Colonies *E cfu/mL Tris + NaCl Blood Saliva Urine Milk E. coli D1 TNTC 66100 38.08 34.81 NoCt 32.46 36.65 E. coli D2 *661 6610 38.79 34.37 NoCt 32.83 37.05 E. coli D3 121 661 46.28 34.24 NoCt 32.76 36.44 E. coli D4 21 66 43.87 34.31 NoCt 32.87 35.68 E. coli D5 0 7 38.34 34.61 NoCt 32.60 35.72 NSC 1 0 0 37.18 34.07 41.24 33.06 36.76 NSC 2 0 0 38.20 33.79 NoCt 32.89 35.60 NSC 3 0 0 36.24 34.14 NoCt 33.10 35.97

Analysis:

[0658] These results demonstrate that magnetic beads can be used to capture microorganisms from a variety of complex biological specimen types as determined by ETGA and Confirm readouts. [0659] The Regrowth assay demonstrates that E. coli can regrow on agar and liquid culture after binding to magnetic beads, as determined by observable growth for Beads sample-set.

Example 16: Microbial Capture and Detection is Possible from Non-Blood Specimens in the Absence of Specimen Lysis

Aim:

[0660] To show microbial capture and detection in non-blood specimens of milk and urine using non-lysing binding buffer and non-lysing wash buffer.

Preparation:

[0661] 10 Tris+NaCl binding buffer=500 mM Tris-HCl [pH 8.0]+1.5 M Sodium Chloride [0662] 1 Tris+NaCl wash buffer=1 in 10 dilution of 10 Tris+NaCl binding buffer [0663] Fresh (Human) urine [0664] Semi-skimmed (pasteurised) cow's milk

[0665] BioEstapor beads (Merck, Cat #BE-M 08/0.3) were re-suspended prior to use.

Protocol:

[0666] E. coli, S. aureus, C. albicans and S. pneumoniae o/n liquid cultures set-up as standard in BacTec PLUS aerobic broth [0667] The following day (approx 16 hours later) 3 L of E. coli and S. aureus o/n used to inoculate fresh 3 mL broth cultures (1E-3 dilutions), and 300 L C. albicans and S. pneumoniae used to inoculate fresh 3 mL broth cultures (1E-1 dilutions); and 2-hour outgrowth performed at 37 C., 500 rpm. [0668] Following 2-hour outgrowth, microbial pre-cultures were serially diluted (DF10) in pre-warmed fresh urine and fresh milk to produce five dilution points: E. coli 1E-5 to 1E-9; S. aureus 1E-5 to 1E-9; C. albicans 1E-2 to 1E-6; and S. pneumoniae 1E-2 to 1E-6. [0669] 100 L TVCs performed for each microbial dilution tested in milk and urine; NSCs for milk and urine were plated on three types of agar plate (SAB, COL and CBA) [0670] 1 mL specimens (five dilutions of each microbial species and four NSC samples: 24 samples per specimen type) added to 2 mL sample tubes preloaded with 112 L Binding Buffer+15 L beads (1% solid), then automated Magnitor test initiated.
Automated Sample Processing on epMotion 5073m: [0671] 30 mins orbital mixing (1000 rpm) @ 37 C. [0672] 15 mins magnetisation [0673] 1 mL s/n removed [0674] 0.82 mL WB (1 Tris+NaCl) added to tubes whilst beads magnetised [0675] 1 mL s/n removed [0676] 50 L LM added to tubes whilst beads magnetised [0677] Magnetisation switched off and ETGA reaction performed: 5 mins at 1000 rpm, then 55 mins at 800 rpm @26 C. [0678] qPCR set-up for ETGA and Confirm (10 L reactions)

Results:

[0679]

TABLE-US-00069 Urine Milk Specimen TVC *E cfu/mL TVC *E cfu/mL E. coli 1E-5 TNTC 141000 TNTC 181000 E. coli 1E-6 TNTC 14100 TNTC 18100 E. coli 1E-7 *141 1410 *181 1810 E. coli 1E-8 32 141 16 181 E. coli 1E-9 3 14 6 18 S. aureus 1E-5 TNTC 19600 *812 8120 S. aureus 1E-6 *196 1960 69 812 S. aureus 1E-7 11 196 15 81 S. aureus 1E-8 2 20 8 8 S. aureus 1E-9 1 2 9 1 C. albicans 1E-2 TNTC 564000 TNTC 597000 C. albicans 1E-3 TNTC 56400 TNTC 59700 C. albicans 1E-4 *564 5640 *597 5970 C. albicans 1E-5 89 564 107 597 C. albicans 1E-6 11 56 6 60 S. pneumoniae 1E-2 TNTC 4080000 TNTC 5740000 S. pneumoniae 1E-3 TNTC 408000 TNTC 574000 S. pneumoniae 1E-4 TNTC 40800 TNTC 57400 S. pneumoniae 1E-5 *408 4080 *574 5740 S. pneumoniae 1E-6 55 408 66 574 NSC_SAB plate 0 20 2 100 NSC_COL plate 0 20 *10 100 NSC_CBA plate *2 20 10 100 *Cfu/mL values extrapolated from highest countable TVC (NB: Urine is a non-sterile solution, therefore, colonies on the NSC plates are not unexpected) (NB: pasteurised milk contains microorganisms, therefore, there should be colonies on the NSC plates)

ETGA Ct

[0680]

TABLE-US-00070 Specimen Urine Milk E. Coli 1E-5 32.36 40.12 E. Coli 1E-6 31.64 37.94 E. Coli 1E-7 32.35 43.03 E. Coli 1E-8 33.06 37.06 E. Coli 1E-9 33.29 41.03 S. aureus 1E-5 32.37 36.09 S. aureus 1E-6 32.41 39.29 S. aureus 1E-7 32.48 38.13 S. aureus 1E-8 32.99 36.77 S. aureus 1E-9 33.11 37.21 C. albicans 1E-2 38.08 46.11 C. albicans 1E-3 33.72 37.14 C. albicans 1E-4 32.20 37.10 C. albicans 1E-5 32.43 36.26 C. albicans 1E-6 32.58 39.70 S. pneumoniae 1E-2 31.78 39.47 S. pneumoniae 1E-3 31.45 41.49 S. pneumoniae 1E-4 31.96 38.17 S. pneumoniae 1E-5 32.71 37.63 S. pneumoniae 1E-6 32.90 48.38 NSC 1 33.42 42.68 NSC 2 33.63 44.35 NSC 3 33.53 38.45 NSC 4 33.51 37.14

IPC Ct

[0681]

TABLE-US-00071 Specimen Urine Milk E. Coli 1E-5 15.80 18.97 E. Coli 1E-6 21.69 21.38 E. Coli 1E-7 26.06 22.19 E. Coli 1E-8 29.54 22.18 E. Coli 1E-9 30.48 22.51 S. aureus 1E-5 19.57 21.47 S. aureus 1E-6 23.73 22.09 S. aureus 1E-7 27.82 22.44 S. aureus 1E-8 28.88 22.15 S. aureus 1E-9 28.95 22.28 C. albicans 1E-2 22.46 22.62 C. albicans 1E-3 24.56 22.51 C. albicans 1E-4 28.56 22.17 C. albicans 1E-5 29.82 22.39 C. albicans 1E-6 29.43 22.00 S. pneumoniae 1E-2 24.12 23.02 S. pneumoniae 1E-3 26.07 22.22 S. pneumoniae 1E-4 29.00 22.24 S. pneumoniae 1E-5 30.78 22.06 S. pneumoniae 1E-6 29.27 22.32 NSC 1 29.48 22.15 NSC 2 29.51 22.17 NSC 3 29.99 22.13 NSC 4 29.99 22.48 Average NSC 29.74 22.23

[0682] NB: Pasteurised milk contains bacteria; these showed as a consistent ETGA Ct-22

Confirm Ct

[0683]

TABLE-US-00072 Urine Milk Specimen GrNeg GrPos Candida GrNeg GrPos Candida E. Coli 1E5 28.86 NoCt NoCt 22.71 22.56 NoCt E. Coli 1E6 29.38 NoCt NoCt 29.95 23.01 NoCt E. Coli 1E7 NoCt NoCt NoCt NoCt 22.61 NoCt E. Coli 1E8 NoCt 45.77 NoCt NoCt 22.73 NoCt E. Coli 1E9 NoCt NoCt NoCt NoCt 22.78 NoCt S. aureus 1E5 NoCt 30.03 NoCt NoCt 21.46 NoCt S. aureus 1E6 NoCt 34.47 NoCt NoCt 22.41 NoCt S. aureus 1E7 NoCt 38.97 NoCt NoCt 22.33 NoCt S. aureus 1E8 NoCt 37.85 NoCt NoCt 22.39 NoCt S. aureus 1E9 NoCt NoCt NoCt NoCt 23.03 NoCt C. albicans 1E2 NoCt 41.75 28.06 NoCt 21.59 31.08 C. albicans 1E3 NoCt NoCt 28.61 NoCt 23.00 33.04 C. albicans 1E4 NoCt NoCt 36.26 NoCt 22.09 39.74 C. albicans 1E5 NoCt NoCt NoCt NoCt 36.30 NoCt C. albicans 1E6 NoCt NoCt NoCt NoCt 22.07 NoCt S. pneumoniae 1E2 NoCt 27.07 NoCt NoCt 20.58 NoCt S. pneumoniae 1E3 NoCt 31.78 NoCt NoCt 22.08 NoCt S. pneumoniae 1E4 NoCt 42.22 NoCt NoCt 22.47 NoCt S. pneumoniae 1E5 NoCt NoCt NoCt NoCt 23.00 NoCt S. pneumoniae 1E6 NoCt 36.55 NoCt NoCt 23.29 NoCt NSC 1 NoCt NoCt 43.91 NoCt 22.18 NoCt NSC 2 NoCt NoCt NoCt NoCt 22.15 NoCt NSC 3 NoCt 40.96 NoCt NoCt 21.87 NoCt NSC 4 NoCt NoCt NoCt NoCt 22.28 NoCt Positivity threshold (Pt) 40 Ct

Analysis:

[0684] These results demonstrate that microbial capture by magnetic beads is possible in alternative specimens to blood (specifically urine and milk) in the absence of specimen lysis, as determined by ETGA and Confirm read-outs. [0685] The presence of commensal microorganisms in these specimen types (particularly milk), does however, effect the level of background signal for ETGA and Confirm readouts.