Target detection

Abstract

Methods and cartridges for detecting a target are provided. A biological sample is introduced into the cartridge. The target in the sample is photon labeled with fluorescent particles in a first liquid layer in a cartridge. Photon-labeled targets are separated from the sample into a second liquid layer within the cartridge, detected and counted to show the presence of the targets in the subject. The cartridge includes a containing reservoir, a mixing aperture for introducing the sample into the photonic marker and the magnetic particles, and an imaging aperture for detecting and counting targets from the sample. The sample may be a human fecal sample. A filter may be used to filter out particulates from the sample.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 660,075, filed on April 19, 2018, and U.S. Provisional Application No. 62 / 711,784, filed on July 30, 2018, the contents of each of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates generally to the analysis and detection of microorganisms and molecules. Background Art

[0004] Detection of microorganisms and molecules is the basis for important applications in human medicine, veterinary medicine, agriculture, industrial microbiology and scientific research. Infectious disease diagnosis is an important field where microbial testing plays a central role.

[0005] Infectious diseases caused by a range of pathogens are the leading cause of death. For example, Clostridium difficile (C. difficile) tops the CDC's emergency threat level microbial category, causing more deadly hospital infections than any other pathogen. In the U.S., Clostridium difficile infection (CDI) causes an estimated 450,000 infections, 29,000 deaths, and $1-6 billion in health care costs each year. Clostridium difficile infection causes severe diarrhea that may lead to pseudomembranous colitis, toxic megacolon, multiple organ failure, and death. Unfortunately, conventional Clostridium difficile detection methods lack sensitivity and specificity.

[0006] C. difficile is a common commensal Gram-positive, spore-forming anaerobic intestinal microorganism with a colonization rate of up to 50% in hospital patients. Most infected patients carry C. difficile in its dormant and benign spore form. Patients taking antibiotics are at a higher risk of C. difficile infection due to changes in intestinal metabolites that induce microbial spores to germinate and become virulent when other intestinal microorganisms are cleared by antimicrobials. Toxigenic strains of C. difficile secrete cytopathic toxins including toxin B and toxin A. Colonized patients without infection can be distinguished from patients with C. difficile infection based on toxin levels in the stool. Patients with C. difficile infection have higher levels of toxin B than colonized, non-infected patients.

[0007] Rapid commercial immunoassays are available to detect C. difficile toxins in stool samples, but are not clinically sensitive enough to detect all patients with C. difficile infection because they have analytical sensitivities of several ng / ml that are significantly above the clinical threshold. More sensitive tests based on the gold standard cytotoxicity neutralization assay (CCNA) are commercially available but are slow (requiring 1-3 days to obtain results), can be subjective, and are too expensive for routine clinical use.

[0008] Compared with current rapid immunoassay tests, nucleic acid amplification tests (NAAT) have high clinical sensitivity. However, these tests detect the presence of the Clostridium difficile genome, but cannot distinguish between patients with benign colonization of Clostridium difficile spores and patients with Clostridium difficile infection. Although only patients with severe diarrhea should be tested for Clostridium difficile infection, many patients in hospitals experience diarrhea due to other reasons (such as laxatives, drug reactions and viral infections). A large part of these patients are colonized with Clostridium difficile in the form of benign spores and have no active infection. Therefore, samples from such patients produce false positive results. Therefore, although clinically sensitive, nucleic acid-based assays lack clinical specificity and have relatively poor positive predictive values.

[0009] Hospitals that have switched from toxin immunoassays to nucleic acid-based assays have seen a 50-100% increase in C. difficile infection diagnoses, in part due to an increase in false positives from patients who are colonized but not infected. False positives are problematic because patients may receive unnecessary antibiotic therapy that actually increases the risk of C. difficile infection in uninfected patients. False positives also increase the financial burden on hospitals.

[0010] The relatively low sensitivity of available rapid toxin immunoassays and the lack of clinical specificity of nucleic acid-based tests make it impossible to find a single CDI diagnostic test on the market that has high clinical sensitivity and specificity to distinguish colonized patients from those with active C. difficile infection. Summary of the invention

[0011] The present invention provides methods and devices for detecting microorganisms and biomarkers in samples that require minimal sample preparation. The clinician obtains the sample and places it directly into the cartridge to detect the desired clinical analyte (or target).

[0012] Certain embodiments of the present invention relate to methods, devices and kits for detecting target microorganisms, which can be used to quickly, sensitively and accurately count target microorganisms in samples. In one aspect of the present invention, a method for determining the presence of a target microorganism in a sample is provided. For example, the target microorganism can be Clostridium difficile or a component thereof, and the detection determines whether the subject has a Clostridium difficile infection. The method comprises testing a stool sample from a subject for the presence of Clostridium difficile or a component thereof, such as toxin A or toxin B. For example, certain embodiments of the methods of the present invention have an analytical sensitivity of 45 pg / ml for a 30-minute Clostridium difficile toxin B test using a stool sample, and an analytical sensitivity of 365 pg / ml for a 30-minute Clostridium difficile toxin A test using a stool sample. Therefore, the present invention provides rapid and sensitive detection of target microorganisms.

[0013] The methods and devices of the present invention require minimal sample preparation, thereby saving time and costs associated with microbial detection and analysis. For example, when using stool samples to test toxin A and toxin B of Clostridium difficile, sample preparation only requires diluting the stool in an assay buffer and then passing it through a nylon mesh filter to remove large particles before adding the assay reagent. Compared to other test methods, simple sample preparation without washing steps and eliminating the step-by-step addition of reagents reduces a lot of hands-on time, reduces costs, and simplifies instrumentation.

[0014] Methods of the present invention relate to detecting and counting targets. Targets are entities to be detected. For example, methods of the present invention can be used to detect cells, proteins, nucleic acids and carbohydrates. Examples in this application demonstrate detection of secreted molecules. However, the present invention can be used to detect a wide range of molecules and cells, including biomarkers, hormones, cell surface proteins, intracellular proteins, nucleic acids and intact cells.

[0015] The present invention can detect and count these targets by using target binding photons and magnetic markers to mark targets. As an example, the liquid sample obtained by the subject is analyzed after being introduced into a box or a barrel, and the box or barrel comprises an imaging hole with a detection surface at the bottom. The bottom surface of the imaging hole is coated with a dye pad reagent, and these dye pad reagents include a density agent and a light-absorbing dye. The barrel also contains magnetic and fluorescent particles coated with a binding agent, such as an antibody specifically combined with a target. When adding a liquid sample, the dye pad dissolves, thereby forming an opaque water layer with a lower density. These magnetic and fluorescent particles and sample form an upper determination layer. The target is combined with the fluorescence and magnetic marker in the determination layer. The barrel is placed above a magnet, and the magnet attracts all magnetic particles through the dye pad layer, thereby depositing these magnetic particles on the detection surface of the imaging hole. The target combined with magnetic particles and fluorescent particles also can be deposited on the imaging surface, and imaging and counting can be performed. The dye pad is used to optically isolate the sample and uncombined fluorescent markers from the detection surface. This can greatly improve the signal-to-noise levels when imaging the sample, thereby minimizing or eliminating the need for sample preparation and washing steps by the user.

[0016] In some instances, the sample is a human stool sample or is derived from human stool, and the targets are pathogens, such as Clostridium difficile. Exemplary targets include toxin A and toxin B secreted by Clostridium difficile. In another example, the targets include lethal factor, a subunit of a lethal toxin secreted from Bacillus anthracis. Those skilled in the art are aware of many additional targets that will be apparent upon consideration of this disclosure.

[0017] In some embodiments, the detection step further includes counting the targets. The targets of interest are detected and counted on the detection surface by observing the fluorescence of the fluorescent particles from the fluorescently labeled targets. The detection step includes digital imaging. Digital imaging includes irradiating the fluorescent particles and detecting the signals emitted from the fluorescent particles on a photoelectric array detector. Any suitable digital imaging device can be used. In certain embodiments, the detection does not employ an optical magnification greater than 5× or does not employ amplification. In some embodiments, the box or cartridge is used to perform the steps of the method. The cartridge is preloaded with fluorescent particles and magnetic particles. The cartridge comprises: a storage reservoir into which the sample is introduced by the user; a dye pad and a detection surface, the dye pad and the detection surface being disposed in an imaging well in fluid communication with a mixing well; and a plurality of paired imaging wells and a mixing well group parallel to each other. The cartridge may also include a filter that filters particles from the sample, such as large particles that may block the flow of fluid in the cartridge.

[0018] Certain aspects of the present invention relate to performing assays in parallel in multiple channels in the box. The first channel includes sample detection. The second channel includes a positive control. The positive control includes detecting and counting targets in a positive control sample that introduces a known amount of the target of interest. The third channel includes a neutralization control. The neutralization control includes detecting and counting targets in a neutralization control sample that introduces a neutralizing binder that isolates the target of interest, thereby preventing fluorescent labeling of these targets. The method further includes calculating the ratio of the detection signal from the sample to the signal detected from the neutralization control. The method further includes determining whether the ratio exceeds a threshold value.

[0019] In certain embodiments, the photonic markers may include fluorescent particles, fluorophores, chemiluminescent agents, bioluminescent agents, resonance light scattering particles, light absorbing or chromogenic signaling agents, quantum dots, or up-converting phosphors.

[0020] In certain embodiments, the particle reagents are conjugated to binding molecules that bind to the target of interest or a component thereof. Examples of binding molecules include antibodies or antigen-binding fragments thereof or aptamers. In certain embodiments, the target of interest is a toxin secreted from a pathogen, such as toxin A and toxin B secreted from Clostridium difficile.

[0021] Certain aspects of the present invention relate to a box or barrel. The barrel includes a storage reservoir into which a user introduces a sample. The barrel may also include a mixing hole for introducing the sample into fluorescent particles and magnetic particles and an imaging hole for detecting and counting targets from the sample. The imaging hole is in fluid communication with the mixing hole. In some embodiments of the present invention, the barrel further includes a plurality of paired imaging holes and sample hole groups parallel to each other. The barrel further includes a filter for filtering particles from the sample before mixing and detection.

[0022] The imaging hole further comprises a dye pad and a detection surface. The dye pad comprises a density medium and a dye, and the density medium makes the unbound sample and unbound fluorescent particles in the sample away from the detection surface, and the dye interference light is transmitted from the unbound fluorescent particles in the sample. When applying a magnetic field through the dye pad, the magnetic field draws these magnetic particles through the dye pad to the detection surface. In certain embodiments, the dye pad is provided in a dry or lyophilized state in the imaging hole in the barrel, until being moistened by the sample. For example, in certain embodiments, the dye pad is in a dry form in the barrel, and provides a sample that makes the dye pad hydration in a dry form.

[0023] In some embodiments of the invention, the cartridge further comprises a positive control sample having a known amount of target. In some instances, the cartridge comprises a neutralization control sample comprising the sample and free binding molecules that bind to the targets to reduce complex formation.

[0024] In some aspects, the method of the present invention further includes detecting and counting the target in the positive control sample, and the positive control includes a known amount of target. For example, in certain embodiments, the method further includes repeating these steps with a positive control sample, and the positive control sample includes the sample and a known amount of target. The method can also include repeating these steps with a neutralization control sample, and the neutralization control sample includes the sample and is combined with these targets to reduce the free binding molecules formed by the complex. The method can also include performing both positive control and neutralization control. The method can include determining whether the target counted from the sample exceeds the threshold value of the target counted from the neutralization control sample. The method can include determining whether the ratio of the target counted from the sample and the target counted from the neutralization control sample exceeds the threshold value. The method can also include determining whether the target counted from the positive control sample exceeds the threshold value.

[0025] In some embodiments, the methods of the invention are performed in a competitive format that can enable detection of small molecule targets. In such examples, the magnetic particles and fluorescent particles bind to each other to form a complex, and the targets bind to one of the magnetic particles or fluorescent particles to reduce the number of complexes formed.

[0026] In some aspects, the invention provides a test kit comprising fluorescent particles, magnetic particles and liquid density reagents or dry density reagents. These fluorescent particles and magnetic particles are combined with the target of interest in the sample to form a complex of these targets. The test kit can also be formulated for competitive determination, wherein these fluorescent particles and magnetic particles are combined with each other to form a complex, and these targets are combined with these fluorescent particles or magnetic particles to reduce the quantity of the complex of the formed particles. The test kit may further include free binding agents and / or known amounts of targets. The test kit may further include a barrel with a detection surface, a plurality of barrels or a barrel with a plurality of holes to allow the execution of a control test. In certain embodiments, the liquid density reagent or dry density reagent is stored in the barrel. The test kit may further include a dye or dye pad placed in the liquid density reagent or dry density reagent. In certain embodiments, the dye interferes with the transmission of light to or from these fluorescent particles. In certain embodiments, these photon markers include fluorescent particles, fluorophores, chemiluminescent agents, bioluminescent agents, resonance light scattering particles, light absorption or color development signal transducers, quantum dots or up-conversion phosphors. In other embodiments, the fluorescent particles and magnetic particles are conjugated to binding molecules that independently bind to the target of interest or a component thereof. Examples of binding molecules include antibodies or antigen binding fragments thereof or aptamers. Exemplary components of the target are secretory components, such as Clostridium difficile toxin A or toxin B. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a flow chart of an exemplary embodiment of the method of the present invention.

[0028] Figure 2 An exemplary embodiment of the method of the present invention is shown.

[0029] Figure 3 An exemplary embodiment of a cartridge according to the invention is shown.

[0030] Figure 4 An exemplary embodiment of an apparatus for use with the present invention is shown.

[0031] Figure 5 A schematic representation of the present invention is shown.

[0032] Figure 6 A non-magnified image of a Clostridium difficile toxin B test is shown showing individual microscopic fluorescent particles that have been tethered to the toxin B molecules to the magnetic particles, which are attracted by the dye pad and deposited on the detection surface.

[0033] Figure 7 is a diagram depicting how the dye and pad in the assay vessel eliminates the need for a wash step.

[0034] Figure 8 Images of wells containing unbound particles covered with a pad with or without a dye are shown, demonstrating the effectiveness of the dye and pad in eliminating background from unbound fluorescent particles.

[0035] Fig. 9 The analytical sensitivity of the C. difficile toxin B test is depicted.

[0036] Fig.10 The assay precision curve for the C. difficile toxin B test is depicted.

[0037] Fig.11 The dose response and dynamic range of the C. difficile toxin B test are depicted.

[0038] Fig.12 is a graphical representation of the threshold value called positive in the C. difficile toxin B test using clinical specimens.

[0039] Fig.13 Depicted are the results of Clostridium difficile toxin B testing using clinical samples.

[0040] Fig.14 Toxin A performance (FoD) is shown.

[0041] Fig.15 Toxin A clinical sample testing of antibody pairs against magnetic and fluorescent particles is shown.

[0042] Fig.16 Toxin A clinical sample testing of antibody pairs against magnetic and fluorescent particles is shown.

[0043] Fig.17 Graphs showing data from the use of the plates on fresh clinical samples using Toxin A.

[0044] Fig.18 Graphs showing data from the use of the plates for fresh clinical samples using toxin B.

[0045] Figure 19-21 A technique for detecting toxin B of Clostridium difficile is shown.

[0046] Fig. 22 The platform workflow for detecting Clostridium difficile toxin B is shown.

[0047] Figure 23-25 Results of an assay for detection of toxin B of C. difficile are shown.

[0048] Fig.26 An overview of an assay for detecting the lethal factor of Bacillus anthracis toxin is shown.

[0049] Fig. 27 A workflow for detecting the lethal factor of Bacillus anthracis toxin is shown.

[0050] Fig.28 Results of detecting the lethal factor of Bacillus anthracis toxin are shown.

[0051] Fig.29 Signal distributions of populations of unspiked and spiked samples for detection of Bacillus anthracis toxin lethal factor are shown.

[0052] Fig.30 The dynamic range results for the detection of the lethal factor of Bacillus anthracis toxin are shown.

[0053] Fig.31 Detection of the target without sample preparation or washing steps is shown.

[0054] Fig.32 Analytical performance of the anthrax test is shown.

[0055] Fig.33 Example 1 of anthrax testing is shown.

[0056] Fig.34 Example 2 of anthrax testing is shown.

[0057] Fig.35 Testing of the NHP inhalation anthrax samples of Example 2 is shown.

[0058] Fig.36 The AST workflow in a cartridge is shown.

[0059] Fig.37 A comparison of conventional FISH and multipath FISH is shown.

[0060] Fig.38 Shown is the UTI ID test: microtiter plate study.

[0061] Fig.39 High analytical sensitivity for UTI pathogens was demonstrated.

[0062] Fig.40 Shown is the UTI AST test: microtiter plate study.

[0063] Fig.41 UTI AST accuracy is shown.

[0064] Fig.42 The robustness of multipath rapid AST for different inoculum levels is shown.

[0065] Fig.43 The effect of polymicrobial samples on rapid AST results is shown.

[0066] Fig.44 Results from UTI ID / AST are shown.

[0067] Fig.45 Option 1 UTI ID / AST Summary is shown.

[0068] Fig.46 FISH probe inclusion is shown.

[0069] Fig.47 FISH probe exclusivity is shown.

[0070] Fig.48 An example platform cartridge is shown. DETAILED DESCRIPTION

[0071] The methods and apparatus of the present invention enumerate individual targets with simple optical equipment, minimal sample preparation, and rapid turnaround time. The present invention can ultrasensitively detect informative targets in complex samples. For example, it can detect low levels of pathogenic Clostridium difficile toxins directly in minimally processed fecal samples. As another example, the methods and apparatus of the present invention can be used to detect low concentrations of toxins that cause anthrax.

[0072] The assay of C. difficile toxin B using the methods of the present invention demonstrated comparable performance to the CCNA reference method, which is highly sensitive to toxin B and is used for regulatory approval of C. difficile toxin testing. The analytical sensitivity of the 30-minute C. difficile toxin B test in stool samples was found to be high (e.g., 45 pg / ml), and the analytical sensitivity of the 30-minute C. difficile toxin A test in stool samples was found to be 365 pg / ml. Moreover, the accuracy of the clinical samples tested was comparable to that of the cytotoxicity assay, a sensitive reference method.

[0073] The method and apparatus of the present invention require minimal sample preparation, thereby saving time and cost associated with microbial detection and analysis. The use of dye pads allows detection with minimal sample handling, because appropriate pads optically isolate the sample and unbound fluorescent particles from the detection surface. Specifically, the preparation of the method of the present invention only involves diluting feces in assay buffer and passing it through a nylon mesh filter to remove large particles before adding assay reagents. Compared with other test methods, simple sample preparation without washing steps and eliminating the stepwise addition of reagents reduces a large amount of practical time, reduces costs and simplifies instruments.

[0074] Figure 1The method 101 for detecting a target is illustrated. The method 101 preferably includes obtaining a sample suspected of containing a target of interest, such as a stool sample from a patient suffering from an infection. The sample can be delivered 107 to a collection tube, a hole, a reservoir or a cartridge to be processed according to the steps herein. For example, a patient can use a collection cup to collect a stool sample and deliver it to a clinician. The sample can be frozen. The clinician can use a disposable pipette (such as a 1mL plastic graduated pipette) to transfer 107 a portion of the sample to a storage reservoir of a test device or cartridge. The method may further include filtering 109 samples because stool samples have a high potential for microparticles. In order to identify or detect the presence of a target in a sample, the method 101 includes mixing 113 the sample with fluorescent particles and magnetic particles that are only bound to a specific target. For example, fluorescent particles and magnetic particles can be conjugated to molecules that are independently bound to a target selected from cells, proteins, nucleic acids, carbohydrates and sugars. In one example, the target includes at least one of toxin A and toxin B of Clostridium difficile. In one example, the targets include lethal factor, a subunit of a lethal toxin secreted from Bacillus anthracis. The method 101 further comprises separating 129 bound and unbound magnetic particles from the sample. Magnetic particles bound to the target are separated from the remainder of the sample. Targets bound to the magnetic particles will also be bound to the fluorescent particles. Thus, if the target is present, the target will be bound to the magnetic particles and the fluorescent particles and separated from the remainder of the sample. The method further comprises detecting 135 and counting the target by observing fluorescence from the fluorescent particles bound to the target.

[0075] Figure 2 The method according to the present invention is shown. The method detects and counts these targets by using target binding photons and magnetic markers to mark the targets. As an example, the liquid sample obtained by the subject is analyzed after being introduced into a box or a barrel, and the box or barrel comprises an imaging hole with a detection surface at the bottom. The bottom surface of the imaging hole is coated with a dye pad reagent, which includes a density agent and a dye that absorbs light. The barrel also contains magnetic and fluorescent particles coated with a binding agent, such as an antibody that specifically binds to the target. When the liquid sample is added, the dye pad dissolves, thereby forming an opaque water layer with a lower density. These magnetic and fluorescent particles and the sample form an upper determination layer. The target is combined with the fluorescence and magnetic markers in the determination layer. The barrel is placed above a magnet, which attracts all magnetic particles through the dye pad layer, thereby depositing these magnetic particles on the detection surface of the imaging hole. The target combined with magnetic particles and fluorescent particles will also be deposited on the imaging surface, and imaging and counting can be performed. The dye pad is used to optically isolate the sample and unbound fluorescent markers from the detection surface. This can greatly improve the signal-to-noise levels when imaging the sample, thereby minimizing or eliminating the need for sample preparation and washing steps by the user.

[0076] Figure 2 Shown is the separation of magnetic particle binding target from unbound fluorescent particles and residual sample by passing through density gradient medium using applied magnetic field traction target.Density medium can be supplied in tube or hole as shown (and can include dye to provide "dye pad"), so that separation can include magnetic particle binding target distribution above dye pad and use magnetic field traction combined target to pass through dye pad and arrive on imaging surface, so that unbound probe is left on the surface of dye pad.Then, detection step can include using digital imaging to image imaging surface.Therefore, as shown, mixing step includes sample exposure to magnetic particle combined with target of interest, and separation step includes using magnetic field to pull combined target away from unbound label.Preferably, separation step includes magnetic particle binding target distribution above the surface of dye pad, and use magnetic field traction combined target to pass through dye pad and arrive on imaging surface, so that unbound label is left on the surface of dye pad.

[0077] As discussed, the embodiment of separation utilizes density gradient medium dye pad, and this density gradient medium dye pad can comprise the dye for providing dye pad and density agent.Dye pad 803 can comprise density agent (such as iodixanol) and light absorbing dye.Dye pad can be optionally dried or lyophilized before being exposed to sample, and this sample further comprises the dye of absorbing light.Dye pad forms the water layer of density higher than the determination layer containing sample and determination reagent.Dye pad can comprise various density agents individually or in combination (and under various concentrations), comprise for example sucrose, pantotrium salt, iodixanol (akaOptiPrep), NaCl, CsCl, Percoll or albumin.Embodiment can also be incorporated with other density agents, comprise other common density agents, such as pantotrium sodium, other sugars, oligosaccharides, synthetic polymers (such as, Ficoll) and such as various salts such as potassium bromide etc.Embodiment can use dye to match the corresponding photon signal conduction characteristics of various different excitation and emission schemes for photon detection and the photon marker used. For example, the dye toluidine blue O can be used with the fluorescent marker Texas Red (sulforhodamine). One embodiment uses a 65 μL aliquot of a dye pad reagent, i.e., 2 mg / mL of chromotropin R2 and 10% v / v of OptiPrep (60% w / v of iodixanol solution) plus 5% w / v of trehalose, which is pipetted into the assay well. The dye pad can be 15% OptiPrep and 5 mg / mL of chromotropin R2 pre-aliquoted in a 96-well half-area diameter bottom transparent black plate or aliquoted into the imaging well of the cartridge. Referring to well 915, dye pad 903 can be formed by preparing a solution of iodixanol or polyvinyl pyrrolidone (including any optional dye) and drying or lyophilizing the solution in well 915 to form dye pad 915. Dye pad 915 will then be substantially solid (e.g., dry, e.g., well 915 can be stored in any orientation (including inversion) until use). When the liquid sample is delivered into the hole 915, the liquid rehydrates the dye pad 803. In fact, the reagents used in the present method disclosed and discussed throughout this article can be provided in a dried or lyophilized form for later use. This enables the preparation of the reagents and their dry loading onto the cylinder, which can then be transported or stored and subsequently used in the method of the present disclosure.

[0078] Certain embodiments of methods and devices for detecting microorganisms and molecules use magnetic particles conjugated to target-specific antibodies.

[0079] Figure 3An embodiment of a box or cartridge 901 that can be used to perform the method is shown. Cartridge 901 includes a mixing well 911. A sample that may include a target 201 of interest is delivered to the mixing well 911. The sample can pass through a filter 955 before being delivered to the mixing well 911. The sample can be a fecal sample that includes particles. Passing the sample through the filter will reduce the risk of cartridge clogging. Any suitable filter can be used, such as a 0.45 micron membrane filter, a 0.45 μm nitrocellulose filter, a 0.65 μm nitrocellulose filter, or a 0.6 μm polycarbonate filter.

[0080] The cartridge 901 also includes magnetic particles 605 that bind to a target of interest; and a dye pad adjacent to a detection surface 805. When a magnetic field is applied across the dye pad 803, the magnetic field pulls the magnetic particles 605 through the dye pad to the transparent wall. The dye pad 803 includes a solution of a density gradient medium 801 that further includes a dye that absorbs light from unbound fluorescent particles 200. In the depicted embodiment, the dye pad 803 and the detection surface 805 (e.g., the transparent wall) are disposed in an imaging well 915 that is fluidly connected to a mixing well 911. The dye pad 803 is provided in a dry or lyophilized state in the imaging well within the cartridge until it is wetted by the sample.

[0081] As shown, the cartridge may contain multiple pairs of imaging apertures / mixing aperture groups parallel to each other. Here, cartridge 901 is shown as containing 8 parallel "channels", where each channel contains a separation aperture 901, a mixing aperture 911, and an imaging aperture 915. An embodiment of the cartridge may contain 2 sets of 8 channels, such that Figure 4 The pictures in will look about the same, because the additional 8 channels will be behind the eight visible channels (the cylinder is a 3D object). The cylinder can be described in terms of dimensions such as height h, length l, and width w (where width w is the length perpendicular to the Figure 3 ). The height h can be between about 3 cm and 10 cm. The length l can be between about 5 cm and 12 cm. The width w can be between about 0.5 cm and 3 cm. For example, in one embodiment, h is about 6 cm, l is about 8 cm, and w is about 2 cm.

[0082] The barrel 901 preferably includes a storage reservoir 925 in which a user can pipette a sample into the barrel. In certain embodiments, the barrel 901 includes a slidable door 931 including a gasket through which a channel passes. When the door 931 is positioned at the first position, the storage reservoir 925 is at least in fluid communication with the first partition hole 907. When the door 931 is in the second position, the storage reservoir 925, the first partition hole 907, and the first mixing hole 911 are all sealed to each other. When the door 931 is in the third position, the first partition hole 907 and the first mixing hole 911 are in fluid communication with each other.

[0083] The cartridge 901 may include a fitting 935 for connecting to an external instrument to receive pneumatic pressure therefrom, thereby dividing (hereinafter "partitioning") the sample from the receiving reservoir 925 into the partitioning holes 927 and subsequently transferring the liquid from the partitioning holes 907 to the corresponding mixing holes 911 .

[0084] The dye pad 803 includes a density agent 801 and a light absorbing dye. The dye pad 803 can be provided in a dry or lyophilized state in the imaging well in the cartridge until wetted by the sample.

[0085] Figure 4 An exemplary instrument 1001 (e.g., an analyzer) for performing target identification and analysis on the sample in the barrel 901 is shown. The instrument 1001 can be used to interact with the barrel 901 to perform the process involved in the method 101 and the present invention. The instrument 1001 can include a user interface 1003 (e.g., a touch screen) for displaying prompts, results, reports 129 and receiving commands. The instrument 1001 can include multiple workstations. The instrument can include a conveyor belt 1005 for transporting the barrel, an upper compartment 1007 for accommodating processing and optional incubation equipment, and a lower compartment 1009 for accommodating electronic equipment, imaging and pneumatic equipment. The instrument 1001 can include an input mechanism 1013 (e.g., a loading rack or a tray) for accommodating multiple analysis barrels and classifying them. The instrument 1001 can also include a conveyor belt 1005 and a propeller mechanism for moving the barrel in the instrument. The instrument 1001 can also include a task scheduler. The instrument 1001 is preferably controlled by a computer to automatically manipulate the analysis barrel, perform microbial identification and analysis, and generate results. The apparatus 1001 may include a plurality of subsystems for performing the methods of the present invention.

[0086] The subsystems of the instrument 1001 may include a pneumatic subsystem, a magnetic subsystem, a clamshell heater, and an imaging subsystem 1023. The magnetic subsystem may include, for example, a permanent magnet or an electromagnet for providing a magnetic field B to pull the magnetic particles and targets on the detection surface of the analysis tube for imaging. The imaging subsystem may be a subsystem for capturing target images as described in U.S. Patents Nos. 9,643,180 and 8,021,848, all of which are incorporated herein by reference; and a workbench for manipulating the detection surface of the tube relative to the imaging module of the instrument 1001. The imaging subsystem 1023 may be operably associated with a computer to provide image processing, analysis, and display capabilities. The pneumatic subsystem may be operable to drive the movement of samples and reagents within the tube.

[0087] In some embodiments, a pusher mechanism (e.g., a mechanical conveyor arm) is operable to move the tube 901 between each subsystem in the instrument 1001. In some embodiments of the present invention, a pusher mechanism transfers the tube between the conveyor belt 1005 and each subsystem of the instrument. The pusher mechanism pushes the tube up or pulls down the conveyor belt 1005. The conveyor belt 1005 rotates so that the tube is positioned near another subsystem in the subsystem, and then the pusher can apply force to slide the tube onto the subsystem. In some embodiments, the instrument includes a task scheduler for managing the analysis tube in the instrument 1001. The task scheduler is operable to control movement, such as transportation and transfer between multiple subsystems for each of the analysis tubes. In some embodiments, the time each tube spends in the subsystem can also be managed by the task scheduler. The task scheduler can reserve time on each subsystem as needed to analyze each of the analysis tubes. In some embodiments of the present invention, the task scheduler can manage the movement of the tube (i.e., the step / parameter of the analysis to be performed) by identifying the contents of the tube.

[0088] In certain embodiments, instrument 1001 can also include reader, and this reader is operable to analyze the unique identifier (for example, bar code) on the cylinder.The content of cylinder and required processing can be associated with the bar code on the cylinder.Instrument 1001 can read the unique bar code by reader, and the unique bar code is associated with the specific instruction set for the task scheduler to execute.Instrument preferably includes the computer (for example, in interface 1003 or be connected to this interface) for controlling operation as described herein.Computer preferably includes the processor that is coupled to non-transitory memory device.Memory preferably stores the instruction that can be executed by processor so that the system manipulates the analysis cylinder in instrument 1001 and obtains and processes the image of labeling microorganism.

[0089] Processor refers to any device or device system that performs processing operations. The processor will generally include a chip for providing a central processing unit (CPU), such as a single-core or multi-core chip. Processing can be provided by a chip from Intel Corporation (Intel) or AMD. The processor can be any suitable processor, such as a microprocessor sold by Intel Corporation (Santa Clara, California) with the XEON E7 trademark or a microprocessor sold by AMD Corporation (Sunnyvale, California) with the OPTERON 10200 trademark.

[0090] Memory refers to a device or device system that stores data or instructions in a machine-readable format. The memory may include one or more instruction sets (e.g., software) that, when executed by one or more of the processors of the computer, can implement some or all of the methods or functions described herein. Preferably, the computer includes non-transitory memory, such as a solid-state drive, a flash drive, a disk drive, a hard disk drive, a subscriber identity module (SIM) card, a secure digital card (SD card), a micro SD card or a solid-state drive (SSD), optical and magnetic media, etc., or a combination thereof.

[0091] An input / output device is a mechanism or system for transferring data to or from a computer to an instrument. Exemplary input / output devices include a video display unit (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)), an alphanumeric input device (e.g., a keyboard), a cursor control device (e.g., a mouse), a barcode scanner, a reader, a disk drive unit, a signal generating device (e.g., a speaker), a touch screen, an accelerometer, a microphone, a cellular radio frequency antenna, and a network interface device, which may be, for example, a network interface card (NIC), a Wi-Fi card, or a cellular modem. The input / output device may be used to enable a user to control the instrument and display results, and to generate a report obtained from the analysis of the cartridge.

[0092] Thus, the instrument can be used with the cartridge to perform the method of the present invention for detecting microorganisms by mixing a sample with magnetic particles and fluorescent particles that are specific for binding to a target of interest; separating bound and unbound magnetic particles in the sample from the sample, wherein the magnetic particles bound to the target are separated from the sample; and detecting and counting the target by observing fluorescence from a fluorescent marker that binds to the target to show the presence of the target in the sample. The mixing step preferably comprises exposing the sample to the magnetic particles and fluorescent particles that bind to the target of interest. The detecting step preferably comprises imaging the fluorescently labeled target using digital imaging.

[0093] The target of interest can be any type of cell or molecule. In certain embodiments, the target includes at least one of toxin A and toxin B of Clostridium difficile. In other embodiments, the target includes lethal factor, a subunit of a lethal toxin secreted from Bacillus anthracis. In some cases, the target is a pathogen.

[0094] The present invention provides methods and kits for detecting targets such as Clostridium difficile toxins that are more sensitive than commercial immunoassays, faster than cytotoxicity neutralization assays (CCNA), and more specific than nucleic acid tests. The technology can enumerate individual targets with simple optical equipment, minimal sample preparation, and a fast turnaround time for sample-response formats. It has also been demonstrated to be comparable in performance to the highly sensitive toxin B cytotoxicity assay reference method for regulatory approval of Clostridium difficile toxin testing.

[0095] The present invention provides a novel method for rapid and sensitive detection of diagnostic markers directly in complex patient samples. As illustrated below, it is found that the analytical sensitivity of 30-minute Clostridium difficile toxin B test in fecal samples is 45pg / ml, which is more than 15 times more sensitive than the main enzyme immunoassay (EIA). Moreover, the accuracy of the tested clinical samples is comparable to that of CCNA. The unique dye pad form allows detection with minimal sample processing because it optically isolates the sample and unbound marker from the detection surface. Specifically, feces can be diluted only in the assay buffer and passed through a nylon mesh filter to remove large particles before adding the assay reagent. When compared with other EIAs, simple sample preparation, no washing steps and the cancellation of the stepwise addition of reagents provide the possibility of reducing a large amount of practical time, reducing costs and simplifying instruments. In general, the present invention is characterized by a promising technology for accurately and sensitively detecting Clostridium difficile toxins and other targets (e.g., microorganisms and components thereof).

[0096] The uniqueness of the present invention lies in its ability to quickly and sensitively detect a wide range of analytes. In addition to detecting targets such as molecules (including but not limited to toxins, nucleic acids and biomarkers), these methods can also detect and count cell pathogens (bacteria, fungi and parasites), viruses and diagnostically important human cells. The methods described herein can be used to simultaneously test a variety of analytes of a single sample, including diagnostic informative human cells (e.g., neutrophils), toxins (e.g., Clostridium difficile toxin B), viruses (e.g., norovirus) and biomarkers (e.g., cytokines).

[0097] General methods, kits and analysers for carrying out the invention are described in WO 03 / 036290, WO 03 / 073817, WO 2010 / 036808, WO 2010 / 036827 and WO 2010 / 036829, the contents of each of which are incorporated herein by reference.

[0098] The methods of the invention employ labeled particles and magnetic particles that bind to a target of interest, such as a microorganism (eg, C. difficile), a cell, or a component thereof, such as a secreted substance (eg, C. difficile toxin A or toxin B), to form a complex.

[0099] The complex is placed or formed in a liquid layer in the vessel. The method uses at least two liquid layers, and the complex is initially present in a covering layer, which is separated from the detection surface of the vessel by a cushion layer. The cushion layer is denser than the covering layer. Magnetic particles - whether unbound or complex - can be moved through the covering layer and the cushion layer by magnetic force to deposit the magnetic particles and anything bound to them in a detection zone near the detection area of ​​the vessel. Unbound labeled particles, targets and other sample components are retained in the covering layer. The amount of target in the sample can then be detected by counting the number of labeled particles in the detection zone. In certain embodiments, the pad may further include a dye that blocks the signal from being generated or emitted from the unbound labeled particles in the covering layer.

[0100] The method can also use one or more controls. For example, the method can use a positive control, wherein an aliquot of the sample is spiked with a known amount of a target, such as a secretory substance (e.g., Clostridium difficile toxin A or toxin B), to form a complex. The assay is then completed with an unspiked sample. The positive control can be used to determine whether a component in the sample is inhibiting any part of the assay. Since the positive control sample contains a known amount of a target, the amount of the target detected should be proportional to the amount spiked. If the amount detected is significantly lower than the expected amount, it can be determined that the sample is interfering with the assay, and the assay of the sample is invalid. For example, if the amount detected in the positive control is less than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 50% or some other fraction of the expected amount, the assay of the sample may be invalid. Of course, if there are more targets in the sample than spiked, the positive control will produce a higher target amount than expected. So this sample will not be invalid.

[0101] The method may alternatively or additionally include neutralization controls, wherein the sample is mixed with binding molecules, which interfere with the combination of label particles and / or magnetic particles or both. The added binding molecules may be identical or different with the binding molecules on the label particles and the magnetic particles. Neutralization controls may be used to determine background counts that are not specific to the target. The background can be deducted from the amount detected in the sample assay. Alternatively, the amount from the neutralization sample may be used to set a threshold value for determining a positive result. For example, for a sample that is considered to be positive, the amount of the target detected may need to be greater than a threshold value. Alternatively or additionally, for a sample that is considered to be positive, the ratio of the signal from the sample to the signal from the neutralization control may need to be greater than a threshold value (or conversely, the ratio may need to be lower than a threshold value). In an example, for a positive sample, the ratio of the signal from the neutralization control to the signal from the sample assay is less than 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45 or 0.4, for example, less than 0.5.

[0102] In addition, the assay does not require or requires only minimal sample preparation. The present invention simplifies the test operation by adopting the detection and enumeration of the target of individual labeling without the need for washing steps to transmit high sensitivity. The scope of sample source can be very wide. Human samples can include, for example, urine, feces, blood, serum, blood plasma, saliva, nasal secretions, cerebrospinal fluid, skin, wounds and many other materials. Industrial samples can include food, beverages and medicines, and environmental samples can include water, air or surface samples. In one embodiment, for example, for Clostridium difficile or other intestinal microorganisms, the sample is a fecal sample (for example, formed, semi-formed or unformed) or a sample derived from feces. For example, a fecal sample can be diluted and filtered to remove large particles.

[0103] The labeled particle can be any suitable particle known in the art, for example, the labeled particle can be polystyrene, glass or latex beads or quantum dots. The particle can be labeled by any detectable portion, typically an optically detectable portion, such as a fluorophore, a chemiluminescent agent, a bioluminescent agent, a resonance light scattering particle, a light absorbing or color developing signal transducing agent, a quantum dot or an up-conversion phosphor. Alternatively, the particle can be naturally detectable, for example optically detectable. For example, the particle can be detected by fluorescence, absorption, light scattering, phosphorescence or luminescence. Magnetic particles are known in the art and include paramagnetic and superparamagnetic particles.

[0104] The labeled particles and magnetic particles can be provided in an amount that is conducive to forming a complex of a target (e.g., a biomarker or molecule). This amount can be determined by providing an excess of particles in view of the expected or maximum amount of the target found in the sample. In this way, the present invention allows counting of individual targets present in a sample by determining the number of labeled particles in the detection zone.

[0105] Binding molecules are known in the art and include antibodies or antigen binding fragments thereof or aptamers. Depending on the target (eg, microorganism), the binding molecule may also include a ligand or other compound that binds to a cell surface receptor or marker.

[0106] The dye pad may contain various density agents, either alone or in combination (and at various concentrations), including, for example, sucrose, diatrizoate, iodixanol (trade name )、NaCl、CsCl、 The density of the cushion layer can be any suitable value, for example at least 1.01 g / ml, at least 1.05 g / ml, at least 1.1 g / ml, at least 1.2 g / ml, at least 1.3 g / ml or more. The density of the layer can also be uniform or non-uniform, for example, a gradient. When a non-uniform layer is present, its average density can be at least 1.01 g / ml, at least 1.05 g / ml, at least 1.1 g / ml, at least 1.2 g / ml, at least 1.3 g / ml or more. For example, hydration of the cushion by liquid from the cover layer can result in a density gradient in the cushion.

[0107] When the reaction medium is substantially transparent to the excitation light or other illumination light and the reflected light or emitted light that produces the imaging signal, the unbound labeled particles outside the detection zone may provide a large non-specific optical signal for the image. The addition of dyes to the pad can be used to eliminate or reduce the signal generated by the unbound labeled particles present outside the detection zone. For example, the dye at an appropriate concentration enables the detection of fluorescence in the detection zone at or near the detection surface while shielding the signal from the unbound labeled particles in the remaining part of the vessel. When the signal transduction portion has fluorescence, the dye used can have an absorbance that overlaps with the excitation or emission wavelength of the fluorescent signal transduction portion, or can absorb the excitation and emission light. For example, when the fluorescent signal transduction portion is yellow-green, the dyes that can be used in the present invention include chromotropin 2R and acid red 1. Many other dyes suitable for this spectral region and other spectral regions are known to those familiar with the art, such as India ink or Direct Black, such as 19 or 168.

[0108] The combination of a dense cushion layer and a dye provides an efficient method for imaging labeled targets without washing. The method can eliminate background signals generated by unbound labeled particles and labeled entities other than the target. The cushion can ensure that only targets that pass through the dense layer by association with magnetic particles can reach the detection zone. The dye prevents the detection of signals caused by free labeled particles in the covered bulk reaction mixture, thereby separating the signal of the labeled target complexed with the magnetic particles deposited in the detection zone.

[0109] In the method, a pad and dye in liquid or dry form can be used. In one embodiment, the pad with or without dye is in liquid form in the vessel, and the sample (or control) is added above the pad. In another embodiment, the pad with or without dye is in dry form in the vessel, and the addition of liquid hydrates the pad. Advantageously, the dry pad with or without dye can be hydrated by the liquid from the sample (or control). When adding the sample (or control) to the vessel, the liquid from the sample (or control) hydrates the dry reagent to form a cushion layer, which then separates the remainder of the sample from the detection surface.

[0110] These methods employ one or more vessels having a detection surface or detection area on which a labeled target is selectively deposited for subsequent detection. The vessel typically has properties and features that support optical detection of the labeled target. These properties and features may include optically suitable materials, geometric shapes, and fiducial features for focusing.

[0111] Typically, the face of the vessel containing the detection region is optically transparent, with properties that are well suited for detecting the labeling particles used to label the target. For example, if fluorescence is to be detected, the optical window should be non-fluorescent at wavelengths in the corresponding spectral regime of the target. The vessel will also have a low reflectivity for incident light at specific wavelengths, which may also interfere with imaging by increasing background signal.

[0112] The imaging surface may be protected from dust, scratches, and contamination. This may be useful in limiting non-specific background or artifacts that may complicate imaging. Some means of protecting the surface include incorporating physical supports, feet, or barriers, or covering the optical surface with a foil or plastic cover. Alternatively, a hinged or sliding door may be used to protect the surface. These protective features may be removed before imaging occurs, or may be automatically removed during the imaging process. Alternatively, these features may not be moving features, as would be the case with a protective or scratch-resistant coating.

[0113] The vessel or detection surface can be plastic, such as cycloolefin copolymer, acrylic acid, polystyrene and other transparent materials. It can also be made of glass (such as borosilicate glass, fused quartz, quartz, etc.). Other materials include but are not limited to PDMS, RTV, optical adhesives and laminates. The vessel or detection surface can have a built-in optical filtering function, which can include a coating or structural composition, such as a laminate or additional physical layer that blocks or absorbs certain energy wavelengths.

[0114] Detection can use an array photodetector, such as a CMOS or CCD. The size of the detector can be adjusted to detect the entire detection area in a single image. Detection can also be performed without optical magnification or with an optical magnification of 5× or less. Typically, the method uses large area detection. For example, the detector will typically detect at least one area with a cross-sectional size of 1 mm (e.g., at least 1 cm).

[0115] Washing means the process of physically removing the liquid containing the undesired components from the target from the container, as opposed to the undesired components, either of which remain in the container.

[0116] A test that does not require washing refers to a test that detects a target without using a washing step.

[0117] Pad, density pad, liquid pad, pad layer or liquid density pad means a basic liquid layer that is denser than the cover layer. In the present invention, before selection, the pad is present in a vessel between the detection surface and the liquid layer, which contains the sample and the test reagent. The pad provides physical isolation between the test reagent and the detection surface. Using selection, the labeled particles and the target complexed with the magnetic particles move through the pad and are deposited in the detection zone. The labeled particles that are not complexed with the magnetic particles are excluded from the detection zone by the dense liquid layer of the pad.

[0118] Dye means a substance or mixture added to the reaction that interferes with the generation or transmission of light to or from the labeled particles. The dye reduces or eliminates signals originating from outside the detection zone while allowing detection of signals originating from the labeled particles in the detection zone. For fluorescently labeled particles, the dye can absorb light with a fluorescence excitation frequency, a fluorescence emission frequency, or both. Various dye properties can be used for this purpose, including light scattering and absorbance. In various embodiments, the dye reduces the signal by at least 50%, 75%, 85%, 90%, 95%, 99%, or even greater than 99%.

[0119] A dye pad means a pad comprising a dye. The dye pad simultaneously provides for physical exclusion of the bulk reaction from the detection zone (as a function of the density of the dye pad) while preventing or reducing the transmission of signals from the overlying reaction to the detector (as a function of the dye contained in the dense layer).

[0120] In some embodiments, target refers to a microorganism (e.g., Clostridium difficile) or a component thereof, such as a secreted product (e.g., Clostridium difficile toxin A or toxin B), which may be present in a sample and tested for its presence by the present invention. The term also encompasses cells from multicellular organisms (e.g., mammals such as humans) and components thereof.

[0121] A binding molecule means a molecule or a molecular complex that specifically binds to a target. Examples of binding molecules are antibodies, antigen-binding fragments thereof, and aptamers.

[0122] Particle means a matrix with a size less than 50 microns. The size of a particle population or a particle batch is defined as the average measurement of the longest orthogonal dimension of a particle sample. Many particles have some characteristics of solids. However, molecular scaffolds or complexes (which may not be rigid) are also defined as particles. For example, dendritic macromolecules or other branched molecular structures are considered to be particles. Similarly, liposomes are another type of particle. Particles can be associated or conjugated with signal elements. Particles are generally referred to as terms reflecting their size or geometry. For example, the terms nanosphere, nanoparticle or nanobead are used to refer to particles less than 1 micron along any given axis. Similarly, the terms microsphere, microparticle or microbead are used to refer to particles less than 1 millimeter along any given axis. Examples of particles include latex particles, polyacrylamide particles, magnetite microparticles, ferrofluids (magnetic nanoparticles), quantum dots, etc.

[0123] Fluorescent particles or labeled particles refer to particles that can specifically bind to a target and generate a signal.

[0124] A substantially planar surface or substrate means a surface that can be aligned parallel to an imaginary plane such that when measuring the distance from a point in any 1 mm x 1 mm square on the surface to the closest point on the imaginary plane the absolute value of the average distance is less than 50 microns.

[0125] The detection surface means the surface of the substantially planar substrate. The detection surface is transparent to the signal of the label particles.

[0126] The detection area means the area of ​​the vessel that is simultaneously analyzed by the present invention. The longest linear dimension of the detection area is usually greater than 1 mm, such as greater than 5 mm, 10 mm or 15 mm. For example, the portion of the slide that is simultaneously imaged by the optical device comprising the collection lens and the CCD chip may measure 0.8 cm x 0.5 cm. The detection area is then 0.4 cm 2 .

[0127] The detection zone refers to the volume in which the target can be detected. The cross-sectional dimensions of the detection zone are the same as those of the detection area, but the depth corresponds to the depth of the labeled particles that can be detected and identified. Therefore, the depth of the detection zone depends on the threshold criteria used to score a positive signal. When optical detection is used, the depth of the detection zone depends on the optical depth of field.

[0128] Simultaneously detecting a target in a portion of the detection area means detecting the signal from a portion of the substantially planar detection surface in one step.

[0129] Sample refers to the material scanned by the present invention for the presence of a target.

[0130] Photodetector means a man-made device or instrument that converts a photon signal into an electrical signal. Examples of photodetectors include CCD detectors, CMOS detectors, photomultiplier tube detectors, and photodiode detectors, such as avalanche photodiodes.

[0131] "Irradiation" means irradiating with electromagnetic radiation. Irradiation with electromagnetic radiation of various wavelengths can be used. This includes, for example, radiation with wavelengths in the X-ray, UV, visible or infrared spectral regions. Note that the irradiation radiation is not necessarily in the visible range. Preferably, irradiation is in the range of 190 to 1100 nm.

[0132] Microorganism means a single-celled organism (eg, bacteria, protists, archaea, or fungi) or a virus.

[0133] In one embodiment, the method enables single molecule counting and counts individual targets without amplification. Figure 5 The authors show how these methods can detect targets labeled with fluorescent nanoparticles without the use of amplification. Illuminating a target labeled with a fluorescent particle causes the labeled target to emit photons. The photons impinge on a CMOS chip in a digital camera (such as the one in a cell phone), which contains an array of individual light-sensitive pixel elements. As a result, the pixel elements directly above the individual targets "light up" as white dots in the resulting image ( Figure 6 The present invention features a dye cushion that allows the method to rapidly and specifically enumerate targets, such as microorganisms (e.g., Clostridium difficile toxin molecules) or components thereof, in complex stool samples with minimal sample preparation. Figure 7). A liquid sample that may contain a target is added to a bottom transparent vessel containing two types of dry reagents: a dye (e.g., direct black) and a density agent (OptiPrep). The dry dye pad reagent at the bottom surface of the vessel forms a dense layer when hydrated. Target-specific fluorescent and magnetic nanoparticles are stabilized in small freeze-dried balls (about 1 mm in diameter). The magnetic nanoparticles are coated with antibodies that are specific to an antigenic site on a target such as a microorganism (e.g., Clostridium difficile toxin molecule) or a component thereof, and the fluorescent nanoparticles are coated with complementary antibodies that bind to different antigenic sites on the same target. After the dry reagent is hydrated by the sample, two layers are formed: a dense dye pad layer and a determination layer. In the determination layer, the target molecule is combined with magnetic and fluorescent nanoparticles, thereby tethering them together. High concentrations (about 109 / ml) and small sizes (200-500nm) of particles can drive fast binding kinetics only by diffusion mixing, which simplifies the instrument by eliminating the need for mechanical mixing functions. Placing the vessel or cartridge over a permanent magnet for 3 minutes draws the magnetic particles—and any fluorescent particles tethered to them by target molecules—through the dye pad, depositing them in the detection zone. The captured fluorescent particles are instantly imaged and counted by non-amplified digital imaging. A computer instantly enumerates the number of luminescent pixels indicating the number of targets present. At low analyte concentrations, digital counting of individually labeled targets produces a better signal-to-noise ratio than the more common method of integrating signals across the detection area. Non-amplified imaging allows large fields of view to be imaged instantaneously—thus allowing small amounts of targets to be quickly detected in large sample volumes. The key technical advantage of the method's innovative non-amplified digital imaging approach is that the technology can rapidly detect extremely low levels of target and has very low component costs. Because a single molecule can tether a fluorescent particle to a magnetic particle at low target concentrations, counting the number of magnetically deposited labeled particles corresponds to the number of captured target molecules. The dye pad eliminates or reduces sample preparation and washing steps. Figure 8 The dye pad is shown to completely block the intense fluorescence of tens of millions of highly fluorescent unbound particles.

[0134] The dye pad passively forms a dense colored layer that absorbs both the excitation and emission wavelengths of light. This layer prevents light from reaching the unbound fluorescent particles in the assay layer (there are millions of them, which would otherwise be very bright). Similarly, the dye pad optically and physically isolates the sample from the detection surface, making the assay robust to even the most difficult sample matrices without requiring extensive sample preparation by the user.

[0135] In addition to the methods discussed above, the method can also be used as a competitive assay in which magnetic particles are conjugated to target competitors. At least two types of complexes are formed, one complex is formed between the magnetic particles and the labeled particles, and another complex is formed between the target and the magnetic particles or labeled particles. When present, the target reduces the number of complexes formed between the magnetic particles or labeled particles by binding to one of the particles and preventing it from binding to another particle. The target is detected indirectly by reducing the number of complexes of the magnetic particles and labeled particles formed in the absence of the target. As will be appreciated, the threshold for determining a positive result or an effective assay from a control will be reversed in a competitive assay.

[0136] The present invention also provides a test kit for performing the method described herein. These test kits are included in label particles, magnetic particles and liquid pad reagents or dry reagents that form pads when hydrated. These test kits may further include a vessel or a cylinder with a detection surface, wherein the liquid or dry pad reagent is optionally stored in the vessel. The test kit may further include a dye such as mixed with the liquid or dry pad reagent or stored separately. The test kit may further include a positive control (e.g., a predetermined amount of a usually purified target) and / or a neutralization control (e.g., a free binding molecule) required reagent.

[0137] References

[0138] Other documents, such as patents, patent applications, patent publications, magazines, books, papers, web page content, have been referenced and cited throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0139] Equivalent

[0140] Various modifications of the present invention, in addition to those shown and described herein, as well as many additional embodiments thereof, will become apparent to those skilled in the art based on the entire content of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, illustrations, and guidance that can be adapted to practice the present invention in its various embodiments and its equivalents.

[0141] Examples

[0142] The present invention is described with respect to the following non-limiting examples. Specifically, although the examples illustrate the detection of Clostridium difficile toxin B, the methods can also be used for other targets. Unless otherwise specified, any element of the device specifically described in the examples can generally be used with the device or kit of the present invention.

[0143] Reagents. Fluorescent microparticles (500 nm) were purchased from Thermo Fisher Scientific (Waltham, MA). Polystyrene carboxylate magnetic particles (292 nm) were purchased from Ademtech (Pessac, France). Magnets used to capture magnetic particles were from Dexter Magnetic Technologies (Elk Grove, IL). Microtiter plates (96-well clear bottom, half-area black plates) were from Greiner Bio-One (Monroe, NC). Native toxin B standards purified from C. difficile (RNA type 087) were purchased from List Laboratories (Campbell, CA). Mouse monoclonal antibodies raised against C. difficile toxin B were from BBi Solutions (Cardiff, UK) and Fitzgerald (Acton, MA). Heterophile blocking agent (HBR-11) was from Scantibodies (Santibodies, CA). Bovine serum albumin (BSA), casein, acid hydrolyzed casein (Hy-casein SF), Alkali, -HCl, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), Triton X-100, and OptiPrep were from Sigma-Aldrich (St. Louis, MO). Direct Black 19 was from Orient Corporation (Cranford, NJ). Protease inhibitor cocktail was from Takara Biotech (Mountain View, CA).

[0144] Clinical samples. De-identified discarded stool samples were obtained from Beth Israel Deaconess Medical Center (Beth Israel Deaconess Medical Center, Boston, Massachusetts) and Discovery Life Sciences (Discovery Life Sciences, Los Osos, California). These samples were collected over a period of 12 months. Samples of molded stool samples (semi-solid or solid) and samples from children under 2 years old were excluded. The samples were stored in the clinical microbiology laboratory at 4°C for 3-7 days, and the samples were transferred to a refrigerator in the laboratory with ice or ice packs to maintain>4°C. Upon receipt of the sample, it was diluted with water to 40% and made into disposable aliquots, and then stored at -80°C until use. The pooled negative stool samples were made from 14 individual stool samples, which were scored as negative for Clostridium difficile by real-time PCR. Prior to testing, fecal diluent was added to the sample, so that the final composition in the assay mixture was 8% feces, 4 mg / ml casein, 8 mg / ml Hy-casein SF, 50 mM Tris-HCl, pH 7.3, supplemented with 1 mg / ml HBR-11 and protease inhibitor cocktail, at a dilution ratio of 1: 150. The diluted fecal samples were filtered through a 10 micron nylon mesh filter (PluriSelect, San Diego, USA) to remove particulates prior to testing.

[0145] Imaging System. The imaging system is a custom instrument and software that automatically captures image data from selected wells of a microtiter plate using a high-precision linear stage from Prior Technologies (Rockland, MA) to position each well on a fluorescence-based image acquisition subsystem. The instrument can image in 4 independent color channels and uses an objective lens, illumination LED, fluorescence filter set, and camera. The field of view of the objective lens is designed to capture an image of the entire microtiter plate well. The illumination module light source consists of 2 high-power LEDs per color channel. A series of fluorescence image frames are captured with a camera using a 3.1MP Sony IMX265 monochrome sensor with 12 bits per pixel quantization. The final image of each well is then formed by merging multiple frames. For the C. difficile toxin B test, a 470 / 40nm excitation filter and a 515 / 30nm emission filter were used, and 2 frames were captured at 20 milliseconds exposure.

[0146] Preparation of microtiter plates containing dye pads. Dye pads were prepared by adding 50 μL of a solution containing 0.25 mg of Direct Black 19, 10% (v / v) OptiPrep in 50 mM Tris-HCl, pH 7.5, to each well of a surface-plasma treated 96-well microtiter plate and drying for 3 hours at 60° C. The dried plates were stored dry for up to 1 month.

[0147] Preparation of Antibody-Conjugated Magnetic and Fluorescent Particles. Anti-toxin B monoclonal antibodies were conjugated to magnetic and fluorescent particles by carboxyl linkage (EDC / NHS chemistry) using standard conjugation methods recommended by the particle manufacturers (Adtech, Pessac, France and Thermo Fisher Scientific, Waltham, Mass.) The conjugated magnetic particles were quantified by visible light absorption, and the conjugated fluorescent particles were quantified using flow cytometry for subsequent assay formulations.

[0148] C. difficile toxin B test. To prepare the assay mixture, stool was diluted to 8% with a mixture of stool diluent, 7e8 particles / ml of antibody-conjugated magnetic particles, 1.1e7 particles / ml of antibody-conjugated fluorescent particles, and the indicated amount of toxin B (diluted in 50mM Tris-HCl buffer, pH 7.8, containing 2mg / ml BSA, 0.05% w / v Tween-20, and 0.05% v / v Proclln-300) or blank buffer alone. 100 μL of the assay mixture was pipetted into each well containing a dried dye pad. After incubation at 35°C for 30 minutes, the magnetic particles were pulled down by placing the assay plate on a Dexter magnet for 3 minutes. The plate was then imaged using an imaging system, and the signal was quantified as described below.

[0149] Image Analysis. The number of fluorescent particles is quantified for each acquired image as follows. The images are masked with a fixed pixel threshold, creating a binary image where all pixels with intensities above the threshold are set to 1. Connectivity analysis is used to group pixels from the images so that each active pixel is grouped with all active pixels immediately adjacent in the x or y image direction. The groups of pixels, or blobs, are then processed to determine a set of parameters such as area (number of pixels), blob intensity (total intensity of all pixels in the blob), and compactness. The spot list is then filtered to remove non-specific signals that may be generated by the sample matrix. This is done by removing spots based on size, intensity and / or irregular shape. Once the spot list is filtered, the total spot intensity is calculated by summing the intensity of each spot. The number of fluorescent particles detected is then calculated by dividing the total spot intensity by the reference intensity of a single fluorescent particle.

[0150] Limit of Detection, Blank Limit, Dynamic Range, and Precision Curves. These measurements were performed using pooled negative stool samples. The limit of detection for the C. difficile toxin B test was determined by running 24 replicates of the sample without analyte and 12 replicates each with seven toxin B concentrations. The blank limit, detection limit, and precision curve were determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.

[0151] Testing of C. difficile clinical samples. As described above, 320 clinical samples were tested using a 3-well assay (test, positive control, and neutralization control for each sample). Each sample examined was tested independently by two operators. For the positive control, 100 pg / ml of C. difficile toxin B was spiked into the assay mixture containing the patient sample to detect matrix inhibition. For the neutralization control, the assay was spiked with 2.5 μg / ml of C. difficile anti-toxin B antibody to confirm that any signal in the unneutralized sample was the result of toxin B detection.

[0152] Cytotoxicity Neutralization Assay (CCNA). Aliquots of samples that have been used for testing were sent frozen on dry ice to Microbiology Specialists Inc., Houston, Texas, for CCNA. Upon receipt, the integrity of the samples was checked and CCNA was performed using MRC-5 fibroblasts and Quidel cytotoxicity reagent (Quidel, Inc., catalog number: 03-05000). The samples were diluted 5 times with sample diluent and centrifuged at 2000 to 6000 × g for 10 minutes to precipitate solid materials. The supernatant was filtered through a sterile 0.45 micron membrane filter, the supernatant was used to inoculate tissue culture plates with appropriate controls, and the plates were incubated at 35° C. for 24-48 hours. The development of specific cytopathic effects in positive samples was observed within 24 hours, while negative samples were kept for up to 48 hours.

[0153] Data analysis. Data were analyzed using JMP and Graph Pad Prism software. Confidence intervals were determined using Clopper-Pearson analysis.

[0154] Example 1: Detection of Clostridium difficile toxin B in samples

[0155] The method uses digital imaging without magnification to detect molecules labeled with fluorescently dyed nanoparticles. The fluorescent nanoparticle markers are illuminated causing them to emit photons, which are collected using a 1:1 f / 4 relay lens. The light emitted by the particles impinges on a small cluster of pixels on the CMOS chip of the digital camera, resulting in white spots in the final image. At low analyte concentrations, digitally counting individually labeled targets results in a better signal-to-noise ratio than simply integrating the signal from the entire detection area. Non-magnified imaging allows large fields of view to be imaged, enabling the detection of small numbers of target molecules in large sample volumes in milliseconds.

[0156] The sample is first mixed with a diluent and a target-specific immunoreagent consisting of fluorescent and magnetic particles coated with complementary antibodies specific for C. difficile toxin B. The assay mixture is then added to a clear-bottomed microtiter well that has been coated with a dry dye pad reagent. The dye pad reagent is a dye that absorbs visible light (Direct Black 19 in these experiments) and a density agent, iodixanol (OptiPrep TM ). The dried dye pad recovers after adding the assay mixture and forms a dense, opaque layer of water below the assay layer. Light cannot penetrate the dye pad layer to reach the assay layer. This feature optically isolates unbound fluorescent markers and sample matrix from the detection surface. Therefore, the dye pad eliminates the need for laborious sample preparation and washing steps required in other immunoassay formats to eliminate background signals from unbound markers and sample matrix components.

[0157] Example 2: Evaluating analytical performance.

[0158] To evaluate the analytical sensitivity of the C. difficile toxin B test in a stool matrix, a pooled stool sample containing 14 randomly selected clinical samples that gave negative results when tested by the real-time PCR C. difficile test was used. The pooled samples spiked with C. difficile toxin B were tested in a series of two-fold dilutions. The method yielded a limit of detection of 45 pg / ml for C. difficile toxin B ( Fig. 9 ). Similar results were observed when a different pool of PCR-negative stool samples was used. At a toxin B concentration of 45 pg / ml, the reaction contained approximately a 100-fold excess of magnetic particles compared to the number of toxin B molecules. At this analyte concentration, the magnetic and fluorescent particles must be tethered together by, on average, a single toxin B molecule, confirming that the method detects single molecules by imaging without the need for amplification. In addition, Fig.10 The accuracy curve in Fig. 9 The coefficient of variation (CV) of the data shown in FIG. 5 was less than 10%, demonstrating the potential of this method to produce reproducible results at low toxin B concentrations.

[0159] Fig.11 The dose response of the C. difficile toxin B test in pooled stool samples containing a range of exogenously added purified C. difficile toxin B is shown. The data are essentially linear over a 4-5 order of magnitude concentration range up to approximately 1 μg / ml, beyond which the response plateaus. Notably, coverage of this range exceeds the highest level of C. difficile toxin B reported clinically, approximately 100 ng / ml.

[0160] Example 3: Detection and Mitigation of Matrix Effects by Assay Controls

[0161] Positive and neutralization assay controls are designed to facilitate the detection and subsequent mitigation of sample matrix effects. Assay controls and toxin B tests are performed in parallel on equal aliquots of a mixture containing clinical samples and assay reagents. The positive control contains a limited amount of spiked toxin (100 pg). Deviations from the positive control signal below the expected result indicate negative assay interference (assay inhibition). The neutralization control contains toxin B neutralizing antibodies that isolate toxin B in clinical samples, making it undetectable in the assay. In this way, the neutralization control distinguishes specific signals from nonspecific signals of toxin B in the sample. Nonspecific signals may be caused by analyte-independent deposition of fluorescent particles or autofluorescent sample components on the detection surface. In this work, a training set of clinical samples is used to establish signal, neutralization and interference thresholds empirically to optimize the diagnostic accuracy relative to the cytotoxicity assay reference method.

[0162] Fig.12 A graphical representation of the decision matrix for positive and negative calls made by a C. difficile test. The decision matrix contains 2 thresholds, one is the signal threshold (on the x-axis) and the other is the neutralization threshold (on the y-axis). Only samples that exceed both the signal threshold and the neutralization threshold are called positive. This is shown in Fig.12 and 13 In the lower right quadrant of the , a positive call is visualized and in the other three quadrants a negative call. In addition, if interference is detected in the positive control (change > 75% compared to the expected signal), the sample is declared invalid.

[0163] Example 4: Accuracy of Clostridium difficile Toxin B Test on Clinical Samples

[0164] The C. difficile toxin B test was used to analyze 320 clinical stool samples from patients suspected of having C. difficile infection. Samples were tested in duplicate. The results from the sample training set were compared with the results of the toxin B cytotoxicity assay reference method. Receiver operating characteristic curve analysis was used to empirically develop assay thresholds to optimize accuracy. Of the 320 clinical samples, only one sample (two replicates) was excluded from the analysis because it showed an inhibition rate of more than 98% to the positive control.

[0165] Fig.13The training set results are plotted. The data show that the selected threshold effectively distinguishes positive samples from negative samples. Samples (red dots) scored as positive using the cytotoxicity assay fall almost entirely in the lower right quadrant, indicating samples with significant neutralizable signals. In contrast, samples (blue dots) scored as negative by the reference test fall almost entirely in one of the other three quadrants, which represent results with low signals, non-neutralizable signals, or both. Table 1 compares the results of the C. difficile toxin B test with the reference cytotoxicity assay.

[0166] Table 1: Comparison of C. difficile Toxin B Test and Cytotoxicity Assay (Microplate)

[0167]

[0168] Using the selected thresholds, the novel method presented herein achieved 97.0% sensitivity (95% CI, 91.4%-99.4%); 98.3% specificity (95% CI, 96.8%-99.2%); and 98.2% accuracy (95% CI, 96.7%-99.0%) when compared to a cytotoxicity assay reference method.

[0169] Example 5: Improving the performance of the C. difficile assay and adding C. difficile toxin A

[0170] Table 1 shown above shows the microplate assay performance. Table 2 shows the cartridge / analyzer performance.

[0171] Table 2: Comparison of C. difficile Toxin B Test and Cytotoxicity Assay (Cartridge / Analyzer)

[0172]

[0173] To select toxin A antibody pairs, a total of 144 toxin A antibody pairs were screened. Fig.14 Toxin A LoD (antibody vs. J / L) (1 / 2LoD) is shown. Fig.15 ) and magnetic particles J with fluorescent particles L ( Fig.16 ) were tested on clinical samples of toxin A using antibody pairs. Antibodies to toxin A were selected based on the LoD and performance testing of clinical samples. The LoDs of toxin A and toxin B met product requirements. The LoD of toxin B was 45 pg / mL. The LoD of toxin A was 365 pg / mL. In addition, toxin A ( Fig.17 ) and toxin B ( Fig.18 ) showed excellent performance when tested on fresh clinical samples.

[0174] Table 3 shows the cartridge / analyzer performance.

[0175] Table 3: Comparison of C. difficile Toxin A+B Test and Cytotoxicity Assay (Cartridge / Analyzer)

[0176]

[0177] Example 6: Rapid single-molecule counting method for sensitive detection of Clostridium difficile toxin B directly in fecal samples

[0178] Background. An ultrasensitive multipathway Clostridium difficile toxin B test based on novel digital imaging technology has been developed that can enumerate individual target molecules in stool samples with little or no sample preparation. Current tests for Clostridium difficile gastrointestinal infection can be inaccurate. Clostridium difficile toxin immunoassays generally lack clinical sensitivity. Nucleic acid amplification tests have excellent clinical sensitivity, but their clinical specificity is diminished by their inability to distinguish patients with Clostridium difficile infection from patients who are carriers of the Clostridium difficile organism. Because toxin production is a hallmark of Clostridium difficile infection, an ultrasensitive Clostridium difficile toxin test (such as the test presented in this report) can address problems associated with current tests and provide improved accuracy in detecting patients with this devastating infection.

[0179] Technical Methods. The multipathway Clostridium difficile toxin B test uses non-amplified digital imaging to count target-specific magnetic and fluorescent particles that have been tethered by toxin molecules. The method incorporates the use of a novel dye pad that eliminates the need for sample preparation and washing steps. Clinical stool samples were tested to estimate detection limits, imprecision, and dynamic range. The potential to achieve good clinical accuracy was assessed by comparing the results of the new toxin test with those of a sensitive cytotoxicity reference method for toxin detection.

[0180] Technology. The technology used contains a dye pad, which eliminates sample preparation and washing. The technology has a 30-minute test turnaround time. The technology used contains positive and neutralizing internal controls. All steps are performed in a cartridge on an automated analyzer. All reagents are stabilized in the cartridge. Figure 19-21 The technique used is shown in .

[0181] Platform workflow. Fig. 22 A sample cartridge, designated by reference numeral 1 , and a sample analyzer, designated by reference numeral 2 , are shown in FIG.

[0182] Analyze the results. Figure 23-25 The analysis results are shown in .

[0183] Interferences. Twenty potential interfering substances commonly associated with diarrheal stool samples were found to have no effect on the results of samples spiked with toxin B.

[0184] Inclusivity. Analysis of toxins from strains expressing common ribotypes (027, 106, 014, 002, 017, 001, 078, 036, 087) showed similar dose / response when spiked into pooled fecal samples.

[0185] Exclusion / Cross-Reactivity. Toxin assay performance in fecal samples was evaluated in the presence of 23 common off-target species at >1e8 CFU / mL. None of these species inhibited detection of spiked toxin B nor produced false positive results.

[0186] Clinical feasibility results.

[0187] Semi-manual analysis. A training set of 320 clinical unformed stool samples from patients suspected of having C. difficile infection was used to select parameters to produce the best accuracy relative to a cytotoxicity neutralization assay (CCNA) reference test. The assay was performed using microtiter plates and manual pipetting steps. Results from commercial enzyme immunoassays and PCR tests were compared to CCNA results.

[0188] Table 4: Comparison of Multipath 30-minute Test and CCNA

[0189]

[0190] Table 5: Comparison of Nucleic Acid Amplification Test and CCNA

[0191]

[0192] Table 6: Comparison of enzyme immunoassay testing and CCNA

[0193]

[0194]

[0195] Fully Automated Analysis. A random subset of samples was tested on the automated multipath analyzer prototype and multipath consumable cartridges and the results were compared to the CCNA results.

[0196] Table 7: Comparison of Multipath Analyzer Test and CCNA

[0197]

[0198] Limitations. This test detects only C. difficile toxin B, not toxin A or binary toxin. This study was not blinded, and the samples were considered as a training set for optimizing parameters. Only unformed stool samples were tested, which did not have associated patient information for subanalysis, and which were not fresh but frozen at -80°C.

[0199] Conclusion. The data presented demonstrate the potential of ultrasensitive multiplex technology to produce a rapid, accurate, easy-to-use test for C. difficile toxin B. This technology should also be valuable for a variety of other important infectious disease applications.

[0200] Example 7: Rapid and sensitive detection of anthrax toxin lethal factor directly from blood samples

[0201] Abstract. Secreted lethal factor (LF), a subunit of the lethal toxin, is the earliest known biomarker of Bacillus anthracis infection, making it a logical diagnostic target for detecting exposure to this potentially deadly pathogen. Currently there is no commercial method for anthrax LF detection that is rapid (time to result less than 30 minutes), simple enough to use in a physician's office laboratory, and sensitive enough to detect low concentrations of LF (<100 pg / mL) early in infection. Multipath anthrax testing can be performed on small volumes (<60 mL) of venous or finger-stick whole blood added to a disposable cartridge without sample preparation. Once loaded into the analyzer, the test is run automatically without further user input. The time from sample loading to diagnostic result is less than 20 minutes, and up to 20 samples can be processed simultaneously on the platform. The limit of detection (LoD) determined using whole blood samples spiked with pure LF protein is <60 pg / mL. The dynamic range of the assay covers 5 logs of LF concentrations, an important performance indicator given the wide range of LF concentrations observed during anthrax infection. With ease of use, rapid time to result, and high sensitivity, the Multipath Anthrax Test potentially fills an important gap in the anthrax diagnostic toolkit.

[0202] Determination overview. Fig.26 An overview of the assay is shown in .

[0203] Workflow. The multipath platform currently under development requires no sample preparation. Add venous whole blood or finger stick blood to the sample diluent stored in the cartridge. Load the cartridge onto the multipath analyzer. The analyzer provides a diagnostic readout in <20 minutes. Fig. 27 The workflow is shown in .

[0204] Analytical Sensitivity. Lethal factor was serially diluted into venous whole blood samples and then run through the multiplex platform. The blank limit was determined to be 3 standard deviations above the mean of 24 independently prepared blank samples. Analytical sensitivity was determined by determining the lowest interpolated concentration of lethal factor at which 95% of the data points were expected to lie above the blank limit. Comparable performance was seen across a panel of blood samples. Fig.28 The results are shown in .

[0205] Detection of lethal factor spiked into whole blood samples. 48 negative individual patient samples of whole venous blood were run in duplicate on the multiplex platform. Lethal factor was then spiked into the same 48 samples at 150 pg / mL and run in duplicate on the multiplex platform. Fig.29 The signal distributions for the populations of unspiked and spiked samples are shown in Tables 8 and 9 below, as is the assumed diagnostic performance using a signal cutoff point of 4000 fluorescence units.

[0206] Table 8: Comparison of spiked and unspiked

[0207]

[0208] Table 9: Diagnostic performance

[0209]

[0210] Robustness to microbial interference. The ability of the multiplex system to correctly identify the presence or absence of lethal factor was tested in the presence of various common microorganisms spiked into whole blood at 1E7 cfu / mL according to CLSI guidelines. The multiplex system was able to correctly detect the presence or absence of lethal factor in all cases.

[0211] Table 10: Presence of lethal factors for potential microbial disruptors

[0212]

[0213]

[0214] Robustness to Chemical Interferences. The ability of the multipathway platform to correctly identify the presence or absence of lethal factor was tested in the presence of a variety of potential interfering substances. Common potential interfering endogenous and exogenous substances were tested at recommended concentrations according to CLSI guidelines. The multipathway platform was able to correctly detect the presence or absence of lethal factor in all cases.

[0215] Table 11: Presence of lethal factors for potential chemical interferents

[0216]

[0217]

[0218] Dynamic Range. The dynamic range of lethal factor for the multiplex platform is over 1E5 pg / mL. Lethal factor was serially diluted from 1 μg / mL to 100 pg / mL and spiked into pooled human plasma. Samples were then run in triplicate on the multiplex platform for each spike level. Fig.30 The results are shown in .

[0219] Overview. The MultiPath Anthrax Test, run on a MultiPath platform, requires minimal sample preparation, returns results in <20 minutes, and can detect lethal factor over a large dynamic range as low as <60 pg / mL in whole blood while demonstrating robustness to common interfering substances and organisms, thereby providing healthcare providers with a rapid point-of-care testing solution in the event of public exposure to B. anthracis.

[0220] Example 8: Rapid and sensitive anthrax and AST testing on an automated platform

[0221] Testing using the methods described in this article saves lives, reduces costs, and reduces drug resistance. Patients receive targeted narrow-spectrum therapies at the onset of infection. These approaches reduce morbidity, mortality, and length of hospital stay, and reduce the spread of drug resistance.

[0222] The technology used addresses a uniquely broad range of key market applications. Proof-of-concept data were obtained from clinical samples and a working platform prototype was used. There is pressure to reduce inappropriate use of antibiotics (POC, hospital) and there is significant financial pressure on hospitals to reduce infection rates.

[0223] The platform provides high-performance, rapid, affordable testing for hospitals, clinics, and physician office laboratories. The dual-use platform is suitable for critical clinical and public health applications and provides rapid AST for all major syndromic infections and rapid ultrasensitive testing for toxins and pathogens.

[0224] The technology detects infection within 30 minutes and counts molecules, cells, viruses and toxins, and detects the phenotypic AST of pathogens within 4 hours. The technology uses samples (blood, nasal swabs, feces, urine) directly without sample preparation. The technology is highly sensitive and specific and counts individual targets (such as low pg / mL LoD of biomarkers / toxins), where bacterial detection is comparable to qPCR (-10CFU / mL). The technology determines antibiotic susceptibility within 1 to 3 times the time. All reagents are included in the cartridge. The test being developed addresses important medical applications, such as biodefense testing, like anthrax testing and commercial testing for Clostridium difficile, UTI ID / AST and CAUTI ID / AST. Fig.31 Detection of the target without sample preparation or washing steps is shown. The dye pad eliminates user sample preparation and washing steps.

[0225] The analyzer includes a housing and thermal controls required for assay robustness, reduced test time, and bacterial growth. The cartridge rack loading interface is safety related. The analyzer includes a built-in computer and integrated results analysis and database as well as a graphical user interface and touch screen. The analyzer has an industrial design and a small footprint. The dimensions of an exemplary analyzer are H18"×W20"×25"D. The dimensions of another exemplary analyzer are H15"×W 15"×24"D.

[0226] The software includes the analyzer user interface, software development quality program, automated image and result analysis, a results database, software to expand the test menu without software updates, cartridge loading and trash can monitoring software, and automated result verification testing.

[0227] The cartridges contain universal modular cartridges designed for all tests in the pipeline. The cartridges increase the number of holes from 6 to 16, improve fluidics (i.e., minimize blistering), reduce parts count while increasing the number of holes, reducing assembly steps, and reducing pneumatic ports from 5 to 1.

[0228] Anthrax test

[0229] Tests included to detect anthrax following a biothreat event. The test detects lethal factor (LF) in a blood sample. LF is a toxin subunit secreted by Bacillus anthracis that is present early in inhalation anthrax infection and is present free or complexed with protective antigen (lethal toxin). The test consists of a finger stick or venous whole blood sample (70uL). The test result is positive if >150pg / ml blood. Internal controls improve accuracy.

[0230] Fig.32 The analytical performance is shown in . Fig.33 Example 1 is shown, and Fig.34 Example 2 is shown. The test was performed together on 4 different days. Venous blood samples from 4 patients were tested. The replicates were n 空白 =24,n 经过掺加 =12. Fig.35 NHP inhalation anthrax samples tested in Example 2 are shown in FIG. The samples covered the infection time course of 2 animals. The multipath results (pos / neg) were compared to the reference method results.

[0231] There is value in rapid ID / AST testing. Current ID / AST testing takes days to determine the best therapy. Unnecessary or ineffective antibiotics may be prescribed. Empirical broad-spectrum antibiotic therapy increases resistance. The goal of rapid AST is to prescribe narrow-spectrum therapy initially and treat only infected patients. The value to clinicians and patients includes improved patient outcomes, reduced antibiotic resistance, and improved antibiotic stewardship. Fig.36The AST workflow in the cartridge is shown in FIG.

[0232] A streamlined FISH method for multipathway ID / AST has been developed. It provides key competitive advantages over other rapid AST methods. The test detects specific pathogens after differential growth and performs rapid AST of non-sterile and polymicrobial infections. The result is a streamlined classical fluorescence in situ hybridization (FISH). Fig.37 A comparison of conventional FISH and multipath FISH is shown in FIG.

[0233] The UTI ID / AST test determines if the patient does not have a UTI (<10K total CFU / mL urine), if the patient is infected with one of the 4 most common UTI pathogens, and which of the 4 major antibiotics is effective. The workflow involves adding urine directly to a cartridge containing growth medium. If positive for one of the UTI pathogens, it reflexes to AST. Time to target: ID within 30 minutes, AST within 4 hours.

[0234] UTI ID Test: Fig.38 A microtiter plate study is shown in . Fig.39 High analytical sensitivity for UTI pathogens was shown. The test time was 30 minutes, 30% of the urine was spiked with pathogens, and 4 different urine samples were tested. The method detected <10K·CFU / ml urine, which is a common threshold for UTI.

[0235] UTI AST Test: Fig.40 A microtiter plate study is shown in . Fig.41 UTI AST accuracy is shown. The method compares multipath AST (4 hours) to a culture broth microdilution reference test (18 hours). Four species (7-10 strains each), 3-4 Abx were included with 1 small error in 200 observations and no very large or large errors. Multipath AST showed higher accuracy compared to the reference method.

[0236] Fig.42 The robustness of multipath rapid AST for different inoculum levels is shown. The effect on AST of inocula covering 4 orders of magnitude was tested for 3 species and 4 antibiotics, and all samples were 100% essentially consistent. No inoculum effect was observed when the inoculum covered 4 orders of magnitude.

[0237] Fig.43The effect of various microbial samples on rapid AST results is shown. The added non-target bacteria include Staphylococcus epidermidis, Micrococcus luteus, Corynebacterium minutissimum, Staphylococcus aureus, Acinetobacter baumannii, Citrobacter freundii and Klebsiella pneumoniae NDM1. In the presence of high levels of other bacteria, multipath AST is run on Escherichia coli. The Escherichia coli MIC is compared with the reference test of 5 antibiotics. Compared with the reference method, the basic consistency of 84 samples is> 98%. In the presence of 1E7 CFU / mL of Klebsiella pneumoniae NDM1, the Escherichia coli MIC of imipenem is not affected. Therefore, rapid AST results are not affected by various microbial samples.

[0238] Fig.44 Results from UTI ID / AST are shown. Resistant and sensitive E. coli strains were tested on the platform using ID / AST. The method involves measuring growth fold after 4 hours of incubation in 32ug / ml clofazimine. The potential for automated rapid multipath AST on the platform is shown. Fig.45 Option 1 UTI ID / AST Summary is shown.

[0239] The performance of the LF test across a range of blood samples was explored. Whole blood samples from 54 patients. In the LF assay, each sample was tested without and with 150 pg / ml of LF. With a single exception, all samples were correctly identified as spiked or unspiked. The results are shown in Tables 12 and 13 below. Fig.46 FISH probe inclusion is shown. Fig.47 FISH probe exclusivity is shown. 55 strains were tested for cross-reactivity and microbial interference specifications.

[0240] Table 12: Results

[0241]

[0242]

[0243] Table 13: Conditions

[0244]

[0245] Platform and Workflow

[0246] A multi-path platform is available. The platform is a benchtop, sample-and-respond laboratory instrument (analyzer). No sample preparation or culturing / isolation is required. One sample is tested in one cartridge for either organism identification (ID) or antibiotic susceptibility testing (AST) or a combination / variation thereof. Approximately 40 patient samples are run on a rotating basis every 8 hours. Tests provide test results in approximately 1 / 2 hour (ID) and 4 hours (AST). Test Menu: Expandable cartridge barcode contains test and sample identification. Location: Physician's office, emergency department, or hospital laboratory. Size: Approximately 15" (38mm) wide, 15" (38mm) high, 24" (61mm) deep. Power: 120-240VAC, 50-60Hz. Computer: Internal. Communications: Via customer network / LIS or manually via USB port.

[0247] Workflow steps include: 1) load 1-5 cartridges into the removable rack; 2) dispense the sample specified by the test (e.g., blood, urine, feces) into the cartridge, where all reagents are contained; 3) load the rack into the analyzer; 4) start the run and report the results using the touch screen; 5) empty the removable trash bin (up to 20 cartridges).

[0248] The analyzer has a rotating carousel and pusher to transport and incubate up to 20 cartridges. The analyzer has an upper compartment for handling and incubating the cartridges (eg at 35°C) and a lower compartment containing electronics, optics and pneumatics. Fig.48 An example sample cartridge is shown in . The cartridge contains up to 16 wells, each of which can perform up to 4 assays.

[0249] The platform user interface (UI) can report results. The touch screen U1 is used to start / monitor runs and display, print and output test results. After the final imaging of the tube, the software generates results and stores all test data in a database. U1 also provides a means to query, sort and perform other data management functions on the database. The software has a laboratory information system (LIS) API that users can access to automatically upload data to their LIS. Users can configure local or network printers. The software has user identification and permission levels that control access.

Claims

1. A method for detecting a target, comprising: The biological sample is introduced directly into the cartridge for analysis; labeling a target in the sample with a photonic marker in a first liquid layer in the cartridge; separating photon-labeled targets from the sample into a second liquid layer within the cartridge; as well as The photon-labeled targets in the second layer are detected.

2. The method of claim 1, wherein the target is selected from the group consisting of a cell, a virus, and a molecule.

3. The method of claim 2, wherein the molecule is selected from the group consisting of proteins, nucleic acids, carbohydrates and sugars. The method of claim 1 , wherein the photon signal is fluorescence. The method of claim 1 , wherein the photonic marker is a fluorescent particle or a fluorophore.

6. The method of claim 1, wherein the target comprises at least one of toxin A and toxin B of Clostridium difficile.

7. The method of claim 1, wherein the target comprises a biomarker secreted by Bacillus anthracis cells.

8. The method of claim 7, wherein the biomarker is a lethal factor.

9. The method of claim 1, wherein the target is labeled with magnetic particles.

10. The method of claim 2, wherein the target is labeled with magnetic particles and photonic labels.

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