System and method for testing for infectious diseases

Abstract

A method and apparatus for detecting one or more target analytes in a sample, the apparatus comprising: (i) a sample container for encapsulating a sample mixture comprising the sample and at least one lysis reagent; (ii) at least one vacuum blood collection tube sealed under negative pressure using a sealing element and containing a stabilizing reagent corresponding to at least one of the target analytes being detected; and (iii) at least one fluid communication channel oriented to enable the sample mixture to be fluidly connectable from the sample container to one or more of the at least one vacuum blood collection tube via the sealing element of each such fluidly connected vacuum blood collection tube, wherein establishing an fluidly connectable connection with the at least one vacuum blood collection tube sealed under negative pressure causes at least some of the sample mixture to communicate from the sample container to the at least one such fluidly connected vacuum blood collection tube.

Description

[0001] Cross-references to related applications The application claims priority to U.S. Provisional Application No. 63 / 609,013, filed on December 12, 2023, the disclosure of which is hereby incorporated by reference in its entirety. Background Technology

[0002] Unless appropriate responses are established and maintained, people remain vulnerable now and in the future to health threats from existing, evolving, and yet-to-emerge infectious pathogens. For example, the recent convergence of Covid-19 (SARS-CoV-2) variants, influenza (InfA / InfB), and respiratory syncytial virus (RSV) demonstrates that patterns of co-transmission and co-infection are dynamic. Relatively low vaccination rates, new infections, co-infections with multiple viruses, reinfection in immunocompromised populations, antigenic drift of the virus itself, antigenic shifts involving more than one virus, and other factors contribute to a highly insecure and uncertain potential health threat. Significant technical and logistical gaps exist in preparedness.

[0003] Therefore, what is needed is timely delivery of rapid, cost-effective, and accurate testing that meets the needs of ordinary users and differentiates between known infections. This can be linked to targeted allocation, integrated surveillance, contact tracing, real-time digital recording and reporting, and modeling.

[0004] Existing technologies do not meet this need, nor can they be satisfied by centralized testing, small clinics, or current over-the-counter testing. Each of these testing methods is less than ideal for addressing the challenges of a pandemic, from a zero-point event to a global outbreak. Turnaround times, queues, single-pathogen testing, and poor sensitivity during the asymptomatic phase are some of the corresponding key drawbacks of each of these approaches.

[0005] The device design, which will enable a wide range of applications for self-testing, tracking, tracing, and clinical monitoring of a variety of target infectious organisms (including microorganisms and viruses), will have significant practical applications in residential, commercial, and clinical settings. Summary of the Invention

[0006] Disclosed is an apparatus for detecting the presence of one or more target analytes in a sample, the apparatus comprising: (i) a sample container for encapsulating a sample mixture comprising a sample and at least one lysis reagent; (ii) at least one vacuum blood collection tube sealed under negative pressure using a sealing element and containing a stabilizing reagent corresponding to at least one of the target analytes; (iii) at least one engageable fluid communication channel oriented to allow the sample mixture to be fluidly connected from the sample container to one or more of the at least one vacuum blood collection tube via the sealing element of each such fluidly connected vacuum blood collection tube, wherein establishing a fluid connection with the at least one vacuum blood collection tube sealed under negative pressure causes at least some of the sample mixture to be connected from the sample container to the at least one such fluidly connected vacuum blood collection tube; and (iv) a detection module in communication with the at least one vacuum blood collection tube and capable of detecting the presence of the target analyte. In an embodiment, the at least one target analyte is a control.

[0007] In embodiments, the stabilizing reagent includes at least one of: (i) a stabilizing amplification reagent; and (ii) a labeled probe associated with at least one of the target analytes; and the detection module may be capable of detecting the labeled probe associated with one or more target analytes. Therefore, it is understood that vacuum blood collection tubes may encapsulate a “master mixture” or reagent to allow for the identification, amplification, and detection of target analytes based on methods known to those skilled in the art. In the currently disclosed systems, the amount of sample mixture communicating from the sample container to each fluidly connected at least one vacuum blood collection tube is based on one or more of: (i) the volume of each such fluidly connected vacuum blood collection tube; and / or (ii) the magnitude of the negative pressure in each such fluidly connected vacuum blood collection tube when the fluid connection is established. In some embodiments, the engageable fluid communication channel is a non-drilled hollow needle. In at least one such embodiment, the fluid connection is manually established by pressing the sample container onto the engageable fluid communication channel / needle, and the engageable fluid communication channel is disengaged by removing the manual pressure from the sample container when the sample container is positioned on the needle, thus deactivating the fluid connection. In embodiments, a connectable fluid communication channel includes a first end for communication with a sample container and at least one second end shaped for communication with one or more vacuum blood collection tubes. For example, a connectable fluid communication channel (e.g., a needle) may include a first end for communication with a sample container and a second inverted Y-shaped end for communication with two vacuum blood collection tubes. In embodiments, a connectable fluid communication channel may include a first end and a second end, the first end for communication with a sample container, and the second end having two or more sub-channels or sub-ends for establishing a fluid connection with negative pressure in two or more vacuum blood collection tubes, wherein the first end is in fluid communication with each of the two or more second ends and / or sub-channels. The fluid communication channel may be arranged or configured to engage the first end of the fluid communication channel with the sample container prior to engagement of at least one second end of the fluid communication channel with negative pressure in either of at least one vacuum blood collection tube. At least one second end of the fluid communication channel may be arranged or configured such that when the first of such at least one second end is engaged with a vacuum blood collection tube, each of the at least one second end is either pressure-sealed away from or engaged with a vacuum blood collection tube.

[0008] A person skilled in the art will understand that when a connectable fluid communication channel includes two or more second ends that are fluidly in communication with two or more vacuum blood collection tubes, if any one of the two or more second ends is connected to the negative pressure in the two or more vacuum blood collection tubes, then each of the other two or more second ends must be connected to the negative pressure in the other of the two or more vacuum blood collection tubes, or sealed (i.e., not under atmospheric pressure).

[0009] In some embodiments, the stable amplification reagent is an isothermal amplification reagent. For example, the stable amplification reagent can be used for loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), chain invasion-based amplification (SIBA), and multi-enzyme isothermal rapid amplification (MIRA). The stable amplification reagent may include materials required for target-specific amplification, including but not limited to enzymes, probes, labeled probes, intercalation dyes, and buffers.

[0010] In at least some embodiments, the sealing element is a diaphragm or diaphragm, and in some embodiments, the sealing element is permeable. In some cases, the sealing element comprises bromobutyl rubber. For example, the sealing element may be in the form of a plug comprising or made entirely of bromobutyl rubber. Those skilled in the art will understand that the sealing element can be of many different shapes and materials and needs to be able to maintain negative pressure within the vacuum blood collection tube until at least the moment when the sample mixture is transferred from the sample container to the vacuum blood collection tube. For example, the sealing element may include a mechanical valve, a diaphragm valve, a ball valve, a piston valve, and / or a clamp valve.

[0011] Therefore, it should be understood that at least one of (i) the volume of the vacuum blood collection tube and / or (ii) the magnitude of the negative pressure in each vacuum blood collection tube is selected and / or determined and / or established based on the amount of sample mixture expected to be fluidly connected to such vacuum blood collection tubes when establishing a fluid connection between the sample container and such vacuum blood collection tubes. In embodiments, the amount of sample mixture to be connected to the fluidly connected vacuum blood collection tubes may be at least 25 μl, at least 50 μl, at least 100 μl, at least 200 μl, or at least 300 μl.

[0012] In some embodiments of the disclosed device, the detection module further includes a heating component, for example, for facilitating the amplification of a labeled target analyte in a sample mixture within one or more vacuum blood collection tubes containing a combination of a sample mixture, a stable amplification reagent, and a labeled probe. In one exemplary embodiment, the heating component may be configured to heat the vacuum blood collection tube and / or its contents to a predetermined temperature for isothermal amplification. In one embodiment, the heating component may be configured to surround the vacuum blood collection tube and may be, for example, an aluminum heating block. After amplification, the detection module may detect the amount of amplified labeled antigen in each vacuum blood collection tube. For example, the detection module may include at least one optical sensor for optically detecting fluorescent labels. In some embodiments, each vacuum blood collection tube in the device is configured with a labeled probe different from other vacuum blood collection tubes in the device, such that each vacuum blood collection tube in the device can be associated with a different target analyte. In at least one embodiment, an optical detector may be associated with each vacuum blood collection tube to detect the labeled target analyte in such vacuum blood collection tubes, while in other embodiments, an optical detector may be associated with more than one vacuum blood collection tube to detect the labeled target analyte in more than one vacuum blood collection tube.

[0013] In embodiments, the optical sensor may include an optical sensor system comprising at least one LED for illuminating one or more vacuum blood collection tubes, and at least one photodiode detector for optically detecting emission from the vacuum blood collection tubes when the tubes are illuminated. In embodiments having multiple vacuum blood collection tubes and multiple target analytes, a labeling probe positioned in each vacuum blood collection tube within the device may be selected to emit at a different wavelength upon illumination, and the optical detector may be selected to selectively detect at each of the different wavelengths. The labeling probe may include a fluorescent dye.

[0014] The detection module may include a microcontroller and a graphical user interface. The microcontroller may be configured with programmable instructions to transmit user instructions to the graphical user interface, allowing the user to use the device to detect target analytes. In some embodiments, the microcontroller includes programmable instructions for communicating via an application or, for example, via email or text with a remote device such as a telephone.

[0015] Methods for using the device are also disclosed. Therefore, a method for detecting at least one target analyte in a fluid sample is disclosed. In one embodiment, the method includes: (i) acquiring a fluid sample; (ii) combining the fluid sample in a sample container with at least one lysis reagent to produce a sample mixture; (iii) providing at least one vacuum blood collection tube, each vacuum blood collection tube being sealed under negative pressure using a sealing element and containing a stabilizing reagent corresponding to the detection of at least one of the target analytes; (iv) engaging a fluid communication channel to create a fluid connection between the sample container and the at least one vacuum blood collection tube via the sealing element of each fluidly connected vacuum blood collection tube, wherein the engagement of the fluid communication channel causes at least some of the sample mixture to communicate from the sample container to such fluidly connected at least one vacuum blood collection tube; and (v) determining the presence or absence of at least one target analyte in the at least one vacuum blood collection tube.

[0016] The lysis reagent may be lyophilized and / or may be included in a sub-container within a given vacuum blood collection tube.

[0017] The disclosed method includes engaging a first end of a fluid communication channel with a sample container prior to negative pressure engagement of at least one second end of the fluid communication channel with any of at least one vacuum blood collection tube. The method includes providing a fluid communication channel arranged or configured such that the first end of the fluid communication channel is engaged with the sample container prior to negative pressure engagement of at least one second end of the fluid communication channel with any of at least one vacuum blood collection tube. In an embodiment, the method includes providing a fluid communication channel wherein each of the at least one second end is pressure-sealed away from or engaged with an atmospheric pressure-sealed vacuum blood collection tube when the first of such at least one second end is engaged with the vacuum blood collection tube.

[0018] In some embodiments, the method includes disengaging at least a first or second end of a fluid communication channel before determining the presence or absence of at least one target analyte.

[0019] In some embodiments, it is determined that providing an optical detection system is included to detect the fluorescently labeled target analyte.

[0020] In an embodiment, the stabilizing agent comprises a master mixture of reagents capable of identifying, amplifying, and facilitating and / or allowing the detection of a target analyte. Therefore, the stabilizing agent may include an amplification reagent (e.g., a probe) and a fluorescently labeled probe, and the method includes loading the amplification reagent and the fluorescently labeled probe into at least one vacuum blood collection tube before sealing the at least one vacuum blood collection tube under negative pressure.

[0021] In some embodiments, the method includes, prior to sealing such vacuum blood collection tubes with a sealing element at a determined magnitude or amount of negative pressure, determining, selecting, and / or establishing at least one of the following: (i) the volume of the vacuum blood collection tube; and / or (ii) the magnitude or amount of negative pressure stored in each vacuum blood collection tube to allow the sample mixture to be dispensed between fluidly connected vacuum blood collection tubes. The sealing element may be a permeable sealing element, and / or a self-healing permeable sealing element.

[0022] The method further includes, upon determining the presence or absence of a target analyte, heating a vacuum blood collection tube sample mixture (which includes a stabilizing reagent for identifying, amplifying, and detecting the target analyte) to induce a reaction between the sample mixture and the stabilizing reagent. In some embodiments, the reaction includes amplifying the target analyte, and in some of such embodiments, the reaction includes LAMP. The target analyte may be a target nucleic acid. In some embodiments, the detection of the presence or absence of the target analyte may not begin until after heating the vacuum blood collection tube.

[0023] As provided herein, the disclosed methods may include determining at least one detection threshold for each of at least one vacuum blood collection tube containing a sample mixture and reagents and subjected to heating. In embodiments where multiple target analytes are detected in a single vacuum blood collection tube, determining the presence or absence of a target analyte includes determining at least one detection threshold for each vacuum blood collection tube. In one embodiment, determining at least one detection threshold includes determining a detection threshold for each target analyte in each vacuum blood collection tube.

[0024] In some embodiments, determining at least one detection threshold includes performing automated determination of at least one detection threshold. For example, performing automated determination may include performing measurements on a vacuum blood collection tube before amplifying the target analyte to a detectable level, and determining at least one detection threshold based on the performed measurements. The method may also include determining at least one detection threshold based on at least one statistic, which is further based on the performed measurements. For example, at least one statistic may be a combination of the mean, median, and / or standard deviation derived from and / or based on the performed measurements.

[0025] In embodiments where optical detection can be used, the measurements performed may include performing optical measurements on at least one vacuum blood collection tube. For example, if multiple target analytes are present in a single vacuum blood collection tube, and each target analyte is fluorescently labeled for emission at different wavelengths, determining the detection threshold may include performing optical measurements at one or more of the different wavelengths and determining the detection threshold at one or more of the different wavelengths.

[0026] Measurements performed to determine detection thresholds may include: performing optical measurements before amplifying the target analyte to a detectable level / quantity, determining at least one of a mean and a standard deviation based on the performed optical measurements, and determining at least one detection threshold based on the mean and standard deviation. As provided herein, the measurements are performed at one or more different wavelengths associated with the fluorescent label of the target analyte.

[0027] The method may further include: reporting a result once the amount of the target analyte detected exceeds a detection threshold; and stopping the detection of a given target analyte once the amount of the target analyte detected exceeds the detection threshold. In some embodiments, the method includes: stopping the detection of all target analytes when the amounts of all detected target analytes exceed one or more detection thresholds associated with the target analyte. In embodiments, the method includes determining that the target analyte is absent if the amount of such target analyte detected within at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, or at least 30 minutes after heating one or more vacuum blood collection tubes does not exceed the detection threshold.

[0028] In some methods, engaging the fluid communication channel includes manually pressing the sample container to engage the fluid communication channel and / or contacting the fluid communication channel to establish a fluid connection between the sample container and at least one vacuum blood collection tube. In some embodiments, the method includes providing instructions to a user via a graphical user interface. Providing instructions may include instructing the user to: take a sample, insert the sample into the sample container, manually press the sample container to engage the fluid communication channel, release the sample container, and / or update the results to the user in a timely manner. Providing instructions may include providing results to the user. Providing results to the user may include displaying results related to the presence or absence of the target analyte on a graphical user interface, transferring results to an application, transferring results to email, and / or transferring results to a networked device, wherein such a network may be wired or wireless, secure or insecure.

[0029] Therefore, those skilled in the art will understand that this disclosure includes methods, systems, and apparatus for performing homogeneous analysis to detect target analytes from fluid samples suspected of containing such target analytes.

[0030] Other objects and advantages will become apparent from the description and accompanying drawings. Attached Figure Description

[0031] Figure 1 It is a perspective view of the disclosed methods, apparatus and systems according to some embodiments.

[0032] Figure 2 yes Figure 1Cross-sectional view of the cylinder and sample container.

[0033] Figures 3A-3D These are cross-sectional views of different inner cylinders and sample containers according to some embodiments.

[0034] Figures 4A-4E Various exemplary vacuum blood collection tube and fluid communication channel configurations according to some embodiments are shown.

[0035] Figure 5 It includes Figure 1 and Figure 2 A perspective view of the assembly of a disposable vacuum blood collection tube.

[0036] Figures 6A-6C This is a schematic diagram of a base with a microcontroller according to some embodiments, the microcontroller being configured to communicate with additional devices and / or networks.

[0037] Figure 7 Various aspects of a sample container, according to some embodiments, are shown, which is engaged in a disposable tube (and further engaged in a reusable base).

[0038] Figure 8 This is a cross-sectional view of an example detection unit for a single vacuum blood collection tube according to some embodiments.

[0039] Figure 9 This is a perspective view of an exemplary sample container according to some embodiments, the container being engaged with a fluid communication channel, for example, a hollow needle.

[0040] Figure 10A The diagram is a schematic of an exemplary circuit system according to some embodiments, which may be included on a printed circuit board (“PCB”) and configured as a disclosed method, system and apparatus that allows for multiple detection of up to eight different target analytes.

[0041] Figure 10B This is a graph showing the wavelength (nm) and spectral response of a device according to some embodiments. Detailed Implementation

[0042] To provide a general understanding, certain exemplary embodiments will now be described; however, it will be understood by those skilled in the art that the systems and methods described herein can be adapted and modified to provide systems and methods suitable for other applications, and other additions and modifications can be made without departing from the scope of the systems and methods described herein.

[0043] Unless otherwise specified, the illustrated embodiments are to be understood as providing exemplary features of different details of certain embodiments, and therefore, unless otherwise specified, the illustrated features, components, modules and / or aspects may be otherwise combined, separated, interchanged and / or rearranged without departing from the disclosed system or method. Furthermore, the shape and size of the components are also exemplary only and may be changed unless otherwise specified without affecting the scope of the disclosed and exemplary system or method.

[0044] The disclosed methods and systems generally relate to the testing of samples for infectious diseases, and more particularly to the testing of samples for infectious diseases that can be performed by the individual providing the sample within minutes of its presentation (“home testing”). Home testing can also be performed with the assistance of another person (e.g., a guardian, parent, spouse, clinician, etc.) and is not limited to use in a residential setting. Existing technologies employ motors, pumps, compressors, capillaries, valves, and other means to flow fluid through a microlaboratory workflow. Motors and pumps are expensive, have large error ranges, provide poor mixing, and can trap problematic gas plugs in the fluid path. Therefore, nucleic acid testing has traditionally been limited due to the complexity of the workflow required for capturing, lysing, amplifying, and detecting infectious diseases. As used herein, vacuum blood collection tubes are sample containers. Vacuum blood collection tubes are typically sterile and formed of glass or plastic. Vacuum blood collection tubes may have stoppers (such as rubber stoppers) to create a vacuum seal within the tube and may include a needle for drawing samples (such as blood).

[0045] Figure 1 It is a perspective view of the disclosed methods, devices and systems. Figure 2 yes Figure 1 A cross-sectional view of the tube and sample container in the image. The device 100 includes a sample container, depicted as a sample container or tube 200, and as shown, a sample mixture encapsulated therein. Figure 1 In the illustration, the sample is a fluid sample. Device 100 includes a disposable cartridge 300 for receiving the sample container, and the disposable cartridge 300 further includes vacuum blood collection tubes 340A and 340B, which are sealed under negative pressure, wherein a stabilizing reagent is contained within the vacuum blood collection tubes 340A and 340B. The reusable base 400 shown includes an opening 410 for receiving the disposable cartridge 300 and includes a detection module, which includes, for example, a heating system and an optical detection system. However, it should be understood that the detection module can be incorporated into any suitable component or provided separately. For example, the detection module can be disposed in the base 400 or the disposable cartridge 300.

[0046] Figure 2 Disposable cartridges (such as those according to...) are shown. Figure 1The image shows an internal view of a disposable cartridge (shown in the image), in which a sample container 200 is engaged within a disposable cartridge 300. Figure 2 The connectable fluid communication channels 320A, 320B, and 320C shown are non-drilled hollow needles capable of transferring sample mixtures from a sample container to two different vacuum blood collection tubes 340A and 340B, as illustrated. Figure 2 As shown, the two vacuum blood collection tubes are placed parallel to each other, but such an arrangement of vacuum blood collection tubes 340A and 340B is for illustration and not a limitation. As shown, the needle is oriented in a "sample splitter" orientation, having a first end and two second ends, so as to transfer at least some of the sample mixture from the sample container to each of the two vacuum blood collection tubes shown.

[0047] Figures 3A-3D These are cross-sectional views of different inner cylinders and sample containers according to some embodiments. Figures 3A-3D This illustrates the various stages of fluid connection achieved by the "sample splitter" needle configuration. For example... Figures 3A-3D As shown, when sample container 200 is pressed into disposable cylinder 300 ( Figure 3C In this process, the first tip of the needle pierces the bottom of the sample container, and the two tips or portions of the second tip of the needle pierce the sealing element of each vacuum blood collection tube. As disclosed, at least one of (i) the volume of each (fluidly connected) vacuum blood collection tube 340A, 340B; and / or (ii) the magnitude of the negative pressure in each (fluidly connected) vacuum blood collection tube 340A, 340B is related to the amount of sample mixture transferred from the sample container to each such fluidly connected vacuum blood collection tube 340A, 340B. The sample mixture may be mixed with a stabilizing reagent contained in the vacuum blood collection tube before the vacuum blood collection tubes 340A, 340B are sealed with negative pressure. As shown in Figure 3, the sealing element 342 is a stopper comprising bromobutyl rubber; however, it should be understood that any suitable sealing element may be used.

[0048] Figures 4A-4E Various exemplary vacuum blood collection tube and fluid communication channel configurations according to some embodiments are shown. Figures 4A-4E A vacuum blood collection tube / fluid connection channel is shown, which allows the detection of multiple target analytes in a single sample mixture leading to multiple vacuum blood collection tubes (“sample splitter” configurations), wherein each vacuum blood collection tube may be configured with a stabilizing reagent to allow the detection of different target analytes. As shown, the fluid connection channel may have one or more second ends, which may take the form of multiple segments, sub-channels, forks, tips, etc., each segment, sub-channel, fork, tip, etc., fluidly connected to a first end of the connection channel capable of engaging with a sample container. Therefore, any suitable number of vacuum blood collection tubes and fluid connection channels can be used.

[0049] Figure 5 It includes Figure 1 and Figure 2 A perspective view of the engagement of a disposable vacuum blood collection tube 300. The reusable base 400 includes a detection module that also includes a heater, for example, as follows: Figure 8 As shown. Figure 5 As shown, the base 400 includes a tension loading docking mechanism 420 for engaging the disposable tube 300 and the vacuum blood collection tube with the reusable base 400.

[0050] Figures 6A-6C This is a schematic diagram of a base with a microcontroller according to some embodiments, the microcontroller being configured to communicate with additional devices and / or networks. For example... Figures 6A-6C As shown, the reusable dock 400 is equipped with a microcontroller to allow the transmission of results via wired and / or wireless network connections, such as intranets, the Internet, applications, or another wired or wireless network, which can transmit results from dock 400 to another computer, such as a mobile phone P.

[0051] Figure 7 Various aspects of a sample container 200 are shown, which is engaged in a disposable cartridge 300, which is further engaged in a reusable base 400. The base 400 also includes a user interface or display D for communicating results or other operations to the user.

[0052] Figure 8 This is a cross-sectional view of an example detection module for a single vacuum blood collection tube 340 according to some embodiments. As shown, a printed circuit board (“PCB”) 360 may include a microprocessor having instructions for causing the microprocessor to control heating units as disclosed herein, control optical detection systems as disclosed herein, and transmit detection results of one or more target analytes, for example, as shown in FIG. 6. As shown, a heating element or wire 370 may be positioned around the vacuum blood collection tube 340, and an LED 350 may be positioned to illuminate a sample in the vacuum blood collection tube 340. An insulating layer 352 may surround the vacuum blood collection tube. An optical detector 380 may be positioned to detect light signals from the vacuum blood collection tube 340. Although the optical detector 380 may be positioned on the side of the vacuum blood collection tube 340, it should be understood that any suitable location may be used, such as below the vacuum blood collection tube 340.

[0053] although Figure 8Heating wire 370 is shown, but heating assemblies, units, or elements can be configured to surround a vacuum blood collection tube, and may be, for example, an aluminum heating block in the form of a tube. However, the disclosed apparatus, systems, and methods should not be limited to heating elements of a particular shape and / or type, and it is understood that heating elements can be of various forms and types, as long as they are capable of heating the contents of the vacuum blood collection tube to induce a reaction between the sample mixture and the reagent master mixture encapsulated within the vacuum blood collection tube. In one version of the illustrated embodiment, the heating assembly includes a heating block heated using a spring-loaded heat transfer mating plate in a reusable base, which in some embodiments optionally includes a flexible heater element and a thermocouple. Closed-loop temperature control is implemented using a microprocessor, which may be connected to or additionally communicate with the heater and thermocouple. Similarly, the microprocessor may have instructions for communicating user commands to a GUI, which may be, for example, on the reusable base, an application, or another wired or wirelessly connected device (e.g., a smartphone). Figure 8 The image also shows two LEDs configured to illuminate the vacuum blood collection tube. The LEDs can be controlled by a microcontroller.

[0054] Figure 9 This is a perspective view of an exemplary sample container that is engaged with a fluid communication channel, for example, a hollow needle.

[0055] The disclosed systems and methods include devices and methods for detecting and / or determining the presence or absence of infectious diseases, providing a highly sensitive and largely automated workflow. Therefore, this disclosure relies on stored negative pressure to move a sample mixture comprising a fluid sample. This approach significantly reduces or eliminates costs, moves fluid without generating interfering bubbles, and utilizes, in part, at least one vacuum blood collection tube to forcefully mix reagents and samples. This vacuum blood collection tube stores negative pressure and stable (e.g., lyophilized) reagents (e.g., amplification reagents, fluorescently labeled probes) to move the sample mixture from a sample vessel to a vacuum blood collection tube / reaction chamber while establishing or engaging a fluid connection between the vacuum blood collection tube and the sample container. The vacuum blood collection tube / reaction chamber can then be heated and its contents irradiated to provide a signal to at least one optical detector.

[0056] This disclosure includes a testing apparatus comprising at least one enclosed bottom vacuum blood collection tube / reaction chamber with a known void volume. The vacuum blood collection tube / reaction chamber may be made of any suitable material, but in one embodiment is glass. The vacuum blood collection tube may also comprise plastic or another suitable material for sealing under negative pressure and for heating and / or optical observation as provided herein.

[0057] The vacuum blood collection tube / reaction chamber described herein can be configured to operate under negative pressure, and in some embodiments, the lyophilized reagent is encapsulated prior to sealing using a sealing element. The sealing element can take many different forms, as long as it seals the vacuum blood collection tube / reaction chamber. In some embodiments, it is self-healing.

[0058] Self-healing sealing elements (e.g., sealing element 342 in Figure 3) provide a convenient way to establish negative pressure in a vacuum blood collection tube during manufacturing. For example, a vacuum blood collection tube may be filled with a stabilizing agent at ambient pressure and sealed using a self-healing sealing element. The sealing element can then be punctured, for example, with a needle to extract pressure and create the desired amount of negative pressure within the vacuum blood collection tube before the needle is removed. In such embodiments, the self-healing aspect of the sealing element allows for maintaining substantially the same negative pressure over extended periods; however, other manufacturing processes that do not require a self-healing sealing element may also be used, and such other processes may include filling the vacuum blood collection tube with a stabilizing agent and applying a seal in a (micro)environment maintained at the desired negative pressure.

[0059] It is understood that the self-healing sealing element also ensures that during the use of the disclosed systems, apparatus and methods, such as after the fluid communication channel is engaged (and optionally disengaged), no reagent, sample mixture, amplicon and / or any combination thereof may leave the vacuum blood collection tube; however, the disclosed systems, methods and apparatus do not require the sealing element to be airtight, or otherwise completely or perfectly seal the vacuum blood collection tube, especially after the fluid communication channel is engaged.

[0060] Stabilizing agents may include isothermal amplification enzymes and oligonucleotide sequences, and may include fluorescently labeled probes. In some embodiments, the reagents may be included as part of a sealing element. The sealing element may be a membrane, and may include, for example, bromobutyl rubber, but other materials (e.g., neoprene rubber) may also be used to seal negative pressure in a vacuum blood collection tube, and such materials may be based, for example, on a fluid communication channel to ensure that the fluid communication channel is permeable to the sealing material. Those skilled in the art will understand that suitable sealing elements may include polymers or elastomers, metals, ceramics, and / or cementitious materials.

[0061] The disclosed apparatus and methods involve acquiring a known volume of liquid sample, such as saliva, in quantities of at least 10 μl, at least 50 μl, at least 100 μl, at least 250 μl, at least 500 μl, and up to 1000 μl, which is combined with an aqueous medium (such as lysis buffer) in a sample vessel to produce a sample mixture. Those skilled in the art will understand that the lysis buffer (which may be lyophilized) may be in an amount equal to, for example, one to four times the sample volume. The sample may include other fluid samples containing a target analyte for detection, and may include nasal secretions / swabs, urine, blood, blood components (e.g., serum), vaginal or penile secretions, etc. The sample vessel may be maintained at atmospheric pressure. The disclosed methods and apparatus are intended to detect at least one target analyte in a sample by processing a sample mixture as disclosed herein. Therefore, it will be understood that the target analyte mentioned herein includes target nucleic acids.

[0062] This disclosure also includes fluid communication channels oriented to enable fluid communication between a sample mixture in a sample container and one or more vacuum blood collection tubes / reaction chambers, wherein in some embodiments (e.g., permeable sealing elements), such a connection is established via a sealing element of each such fluidly connected vacuum blood collection tube / reaction chamber. Therefore, it is understood that in such embodiments, the fluid communication channel must be oriented or arranged to penetrate the sealing element of the vacuum blood collection tube. As described herein, the fluid communication channel may also be arranged to penetrate or additionally puncture the sample container to contact the sample mixture, allowing fluid communication from the sample container to the vacuum blood collection tube. Upon establishment of a fluid connection, at least one of the volume of each fluidly connected vacuum blood collection tube and / or the negative pressure within such vacuum blood collection tube causes at least some of the sample mixture to communicate from the sample container to such fluidly connected vacuum blood collection tube.

[0063] In one embodiment, the engageable fluid communication channel can be engaged using manual pressure. In one embodiment where the engageable fluid communication channel is a non-drilled hollow needle, the needle may be in a fixed position, and the user of the device and method can engage the communication channel by manually pressing a sample container onto a first end of the needle. Such manual force can similarly (and simultaneously in some embodiments) cause a second end of the needle to penetrate the sealing element of one or more vacuum blood collection tubes, thereby allowing a sample mixture to flow from the sample container through the needle into the vacuum blood collection tube / reaction chamber, wherein such sample mixture flow is based on and / or related to the negative pressure in the vacuum blood collection tube. Releasing the manual pressure can cause the engageable fluid communication channel to disengage from both the first and second ends. In some embodiments, detection of the target analyte may not require disengagement of the fluid communication channel.

[0064] Those skilled in the art will understand that in the disclosed methods and systems employing a sample shunt configuration, for example, a fluid communication channel having a first end and two or more second ends, the sample container must be engaged with the first end before any of the two or more second ends is engaged with the negative pressure of two or more vacuum blood collection tubes; and when one of the second ends is engaged with the negative pressure of two or more vacuum blood collection tubes, the other of the two or more second ends must be sealed (i.e., not under atmospheric pressure) or in negative pressure communication with one of the other two or more vacuum blood collection tubes.

[0065] In one embodiment, a spring or other mechanism providing resistance may be used to ensure engagement of the sample container with the first end of the engageable fluid communication channel before any of the second ends of the fluid communication channel engages with negative pressure in any of two or more vacuum blood collection tubes. Therefore, the disclosed method comprises engaging the first end of the fluid communication channel with the sample container before at least one second end of the fluid communication channel engages with negative pressure in any of at least one vacuum blood collection tube. In the illustrated embodiment, sealing of the two or more second ends of the fluid communication channel is achieved by providing a sufficiently long sealing element (e.g., a butyl rubber stopper or plug) in the vacuum blood collection tube such that any minor differences in the second ends / shunts of the fluid communication channel are compensated for by sealing such ends with a sufficiently long sealing element before engagement with negative pressure in two or more vacuum blood collection tubes. As provided herein, by selecting a sealing element of sufficient length to seal the contents of the vacuum blood collection tube and the second end of the engageable fluid communication channel, a sufficient volume of sample mixture can be delivered to each of the vacuum blood collection tubes via negative pressure to allow reaction and detection of the presence or absence of a target analyte (or control). Therefore, in some of the illustrated embodiments, applying a first amount of manual pressure allows the sample container to engage with a first end of the fluid communication channel, and thereafter, applying a second amount of pressure allows the second end of the fluid communication channel to engage with the negative pressure in the vacuum blood collection tube.

[0066] In some embodiments, a graphical user interface (GUI) (e.g., an application or other wired or wireless connection to the device) may be used to inform the user when to begin applying manual force and when to end applying manual force, respectively, to engage and disengage the engageable fluid communication channel.

[0067] The introduction of a sample mixture into the vacuum blood collection tube / reaction chamber can be detected via a sensor, which can be, for example, an optical sensor capable of monitoring the presence of liquid within the vacuum blood collection tube / chamber. Some embodiments may use electromechanical or optical sensors that monitor the presence of an engageable fluid communication channel (e.g., a needle) with a valve and / or the vacuum blood collection tube / reaction chamber.

[0068] In embodiments, the disclosed device includes a detection module, and a vacuum blood collection tube / reaction chamber may be pre-installed in the detection module and positioned to allow engagement with a second end of an engageable fluid communication channel. In some cases, the detection module includes a heating module / assembly, and the vacuum blood collection tube may be thermally connected to the heating module / assembly. The heating assembly may include a heat sink made of aluminum or another material with sufficient thermal conductivity for the vacuum blood collection tube to allow a reaction between the sample mixture and stabilizing reagents (e.g., probes and labeled probes) according to the disclosed method and system. Stabilizing reagents may include, for example, BST polymerase (16-32 units), 6-10 LAMP probes per target (concentration range of 0.2 μM to 2.0 μM), labeled probes ranging from 0.8 μM to 2 μM, probe quenchers ranging from 0.8 μM to 2 μM, etc., and their concentrations may vary depending on whether a single or multiple reaction is occurring in a given vacuum tube. In some cases, stabilizing agents may include, for example, BST polymerase (16-32 units), 6-10 LAMP probes per target (concentration range of 0.1 μM to 20 μM), labeled probes ranging from 0.4 μM to 2 μM, probe quenchers ranging from 0.4 μM to 2 μM, etc., and their concentrations may vary depending on whether a single or multiple reaction is occurring in a given vacuum tube. In one embodiment, the heat sink may be surrounded by, for example, an electrically heated resistance heater. In some embodiments, the vacuum blood collection tube / reaction chamber may be pushed into the heating module / assembly by the same action of engaging a fluid connection channel (e.g., a needle) to the vacuum blood collection tube.

[0069] The heating assembly may also include a temperature sensor arranged in a closed-loop configuration with the heat sink to regulate the temperature of the heat sink and / or vacuum blood collection tube to a target temperature or one of a plurality of selectable target temperatures. In embodiments, a microcontroller and / or analog control loop are provided to control the temperature feedback loop. Power may be provided by a main power adapter or a battery. The battery may include, for example, an alkaline battery and may include a rechargeable battery. Those skilled in the art will understand that in embodiments including a microcontroller, the microcontroller will include instructions for establishing and regulating the heating assembly.

[0070] In one embodiment, when a sensor (e.g., a fluid sensor, an optical sensor) detects the introduction of a sample from the sample mixture into the vacuum blood collection tube / reaction chamber, the heating assembly can be activated to establish the vacuum blood collection tube at a preselected temperature suitable for isothermal amplification of nucleic acids. In other embodiments, the heating assembly can be activated / turned on as long as the device is powered on to establish the vacuum blood collection tube at a temperature suitable for isothermal amplification of nucleic acids. In some embodiments, the heating assembly can be activated to preheat the vacuum blood collection tube to a temperature suitable for isothermal amplification of nucleic acids upon detection of sample introduction into the vacuum blood collection tube / reaction chamber.

[0071] As described herein, when the sample mixture is introduced into the vacuum blood collection tube / reaction chamber (e.g., via an engageable fluid communication channel), the sample mixture is combined or mixed with a stable (e.g., lyophilized) reagent in the vacuum blood collection tube / reaction chamber. Depending on the embodiment, the heating assembly may then maintain or achieve an operating temperature suitable for isothermal amplification of nucleic acids for at least 30 minutes, or other times sufficient to yield a reliable positive or negative result based on the abundance / presence or absence (e.g., fluorescence) of the amplified nucleic acid sequence in the reaction chamber / vacuum blood collection tube.

[0072] The abundance / presence or absence of amplified nucleic acid sequences in a reaction chamber / vacuum blood collection tube can be detected in a variety of different ways known to those skilled in the art, and current systems and methods are not limited to such detection techniques. With the aid of illustrations and without limitation, fluorescent markers can be detected (e.g., in real-time) in a vacuum blood collection tube / reaction chamber, for example, by providing suitable illuminators, spectral filters, and detectors with appropriate arrangement of the reaction chamber. Heating components can provide transparent openings for one or more light sources and detectors. For example, light-emitting diodes (“LEDs”) can be used as light sources, and photodiodes can be used as detectors. Exemplary multispectral detectors may include an AS734x detector from AMS OSRAM. Fluorescent markers can be disposed in a reagent mixture pre-loaded in the vacuum blood collection tube / reaction chamber such that the reagents are present in the vacuum blood collection tube / reaction chamber at a substantially constant concentration throughout the isothermal amplification process. Fluorescent markers can be designed in one of a variety of ways known in the art to alter or change their fluorescence emission levels based on whether the marker binds (directly or indirectly) to a specific nucleic acid sequence. For example, embedded dyes or dye / quencher pairs coupled to specific complementary nucleic acid sequences can be used. Once the abundance of the target analyte / nucleic acid is detected to determine its presence, amplification and detection can be stopped, and positive results can be communicated. In an embodiment, the abundance of the target nucleic acid equivalent to the presence of the target analyte in a given vacuum blood collection tube can be determined automatically by performing measurements on the given vacuum blood collection tube to obtain a “baseline” or “control” level. Such measurements can be performed before any substantial amplification of the target analyte in the vacuum blood collection tube. For example, these measurements can be performed before or after the sample mixture is introduced into the vacuum blood collection tube. If the measurements are performed after the sample mixture is introduced into the vacuum blood collection tube, they can be performed before heating the vacuum blood collection tube and / or before heating the vacuum blood collection tube to a time known to those skilled in the art as inducing detectable amplification of the target analyte.

[0073] Multiple measurements can be performed on each vacuum blood collection tube. For example, measurements can be performed at different emission wavelengths (e.g., fluorescent labels / probes associated with different target analytes) associated with a given vacuum blood collection tube. Therefore, more than one detection threshold can be determined for each vacuum blood collection tube. For example, in embodiments where multiple target analytes are present in the vacuum blood collection tube, multiple detection thresholds can be determined based on measurements performed on that vacuum blood collection tube. In some embodiments, a single detection threshold can be used even if multiple target analytes are to be detected.

[0074] From the performed measurement, at least one statistic can be obtained or determined. For example, the mean, median, and / or standard deviation can be determined based on the performed measurement. One or more detection thresholds can be determined using a combination of one or more statistics.

[0075] In exemplary embodiments using an optical detection system, fluorescence emission measurements can be performed on vacuum blood collection tubes prior to detectable amplification of the target analyte. In such examples, statistics, such as the mean and / or median fluorescence intensity at each relevant wavelength in each vacuum blood collection tube, and the corresponding standard deviation, can be calculated based on measurements performed (e.g., optical) prior to detectable amplification of the target analyte. A detection (“abundance”) threshold can then be determined based on the statistics (e.g., a multiple of the mean plus the standard deviation) to indicate the presence of the target analyte (e.g., at various applicable emission wavelengths, such as at various wavelengths within the applicable emission wavelength range). Once the detection level is reached, a result can be determined as long as there is only one target analyte in a given vacuum blood collection tube. Therefore, in embodiments, the method and system are designed to provide notification of a positive result as early as possible / once detection is determined. As provided herein, such results can be communicated to a GUI located on the device and / or another remote device (e.g., via an application, email, etc.).

[0076] Therefore, those skilled in the art will understand that the disclosed methods and systems may have embodiments for multiplex testing, wherein multiple target nucleic acids (or target analytes) can be detected. It will be understood that multiplex testing can be performed at least in the form of samples and controls to allow for validation of system performance via control analytes. As is known, control nucleic acid sequences (e.g., actin) can be used in multiplexed configurations with samples to confirm whether the samples were correctly taken, introduced, and whether amplification and other reactions were performed correctly.

[0077] In multiple embodiments using fluorescent markers, different types of fluorescent markers, excited at different wavelengths and emitting at different wavelengths, can be provided in the same vacuum blood collection tube / reaction chamber, each type of fluorescent marker being coupled to a different (amplified) target analyte-specific nucleic acid sequence. In some embodiments, these different fluorescent markers can be individually quantified by alternating irradiation at corresponding different fluorescence / emission wavelengths and detecting fluorescence at corresponding different emission wavelengths (e.g., optical detection). In some embodiments employing optical detection, the emission wavelength and detection wavelength can be in the visible light range (e.g., 450 nm to 700 nm).

[0078] In one exemplary embodiment, fluorescence detection can be achieved using an LED with a full-spectrum bandwidth of 20 to 50 nm. An LED on the order of 10 nm can also be used. -2 Out-of-band suppression is used for the light source without additional spectral filtering. The photodiode detector can be used in conjunction with a suitable spectral filter to define the emission band. One embodiment may employ an integrated multi-band detector (Osram AS7341) that includes a full-spectral bandwidth of 30 to 60 nm and an order of magnitude of 10. -2 The filter employs out-of-band suppression. In such embodiments, a dye concentration of 1 μM can be detected. By more carefully correcting for the background signal, a dye concentration of 0.1 μM can be detected. Therefore, the sensitivity is limited by the background signal from the LED light, which reaches the detector via scattering from the sample and / or reflection from the vacuum blood collection tube / reaction chamber, and is not substantially suppressed by the spectral filter. Therefore, those skilled in the art will understand that if it is desired to detect lower dye concentrations, a narrower optical filter with higher out-of-band suppression can be used in the excitation and / or emission paths.

[0079] In some embodiments, nucleic acids may be labeled and detected after an amplification time using one of many lateral flow analysis methods known to those skilled in the art. In such embodiments, a sealed, negative-pressure detection chamber may be provided to the testing site and fluidly connected to a vacuum blood collection tube / reaction chamber using a second engageable fluid communication channel. After the sample mixture is introduced into the vacuum blood collection tube / reaction chamber and mixed with lyophilized reagents at an operating temperature suitable for isothermal amplification of nucleic acids for a predetermined amplification time, the second engageable fluid communication channel may be engaged to transfer the amplified mixture to a lateral flow strip. In some embodiments, as known in the art, the lateral flow strip provides optical markings and one or more binding regions for the target analyte. After a predetermined incubation time, the lateral flow strip may be read using one or more of several detection techniques known to those skilled in the art, including but not limited to visual, photographic, image analysis, or photoelectric sensors.

[0080] Those skilled in the art will also understand that, as is known in the art, lateral flow analysis can achieve multiplexing of different target analyte-specific nucleic acid sequences with downstream lateral flow detection by configuring lateral flow strips to have different target analyte-specific binding regions corresponding to different target analyte-specific nucleic acid sequences.

[0081] In some embodiments, the disclosed systems and methods can be multiplied by dispensing a single sample into multiple vacuum blood collection tubes / reaction chambers upon introduction into the testing apparatus. In such embodiments, the engageable fluid communication channel may be configured as a fluid splitter (e.g., an inverted Y-shaped or other multi-branched fluid channel) to allow connection from one sample container to two or more vacuum blood collection tubes / reaction chambers (each under negative pressure but each pre-filled with a different reagent mixture). In another embodiment, a single vacuum blood collection tube / reaction chamber may be pre-filled and / or pre-loaded with multiple different reagent mixtures for the detection of more than one target analyte. In still other embodiments, one vacuum blood collection tube may be pre-filled with a reagent mixture for the detection of one target analyte, and another vacuum blood collection tube may be pre-filled for the detection of two or more target analytes. Those skilled in the art will understand that many of the above combinations are possible. In any of such embodiments, any suitable detection technique may be used on the amplified nucleic acid sequences generated individually or collectively in the vacuum blood collection tubes / reaction chambers.

[0082] Therefore, those skilled in the art will understand that the disclosed methods and systems can be used, for example, to determine whether a sample contains one or more target analytes associated with respiratory viruses, such as InfA, InfB, SARS-CoV-2, RSV, while also including human controls (actin, RNaseP). In one exemplary embodiment, reagents associated with these target analytes may be pre-filled into three vacuum blood collection tubes, each containing reagents for two target analytes and two different colors associated with each target analyte in a given vacuum blood collection tube. As another example, InfA and InfB may be in different vacuum blood collection tubes. In such embodiments, each vacuum blood collection tube of the same or similar volume may be initialized to have the same negative pressure, such that when fluidly connected via an engageable fluid communication configured as a three-way sample splitter, the sample mixture will be divided into three portions approximately equally among the three vacuum blood collection tubes. Human controls can be used to demonstrate that sufficient sample mixture has been added to each vacuum blood collection tube, particularly in cases where no target analyte is detected.

[0083] In another embodiment, the target analyte may include other respiratory viruses, including HPIV, HRV, ADV, HMPV, HBoV, and AIV H5N1. In yet another embodiment, infectious pathogens, including tuberculosis, Ebola virus, streptococci, methicillin-resistant Staphylococcus aureus, monkeypox virus, etc., may be used as target analytes.

[0084] Therefore, the disclosed methods and systems allow for modular and integrated systems with disposable components. For example, the sample container and the cylinder containing the vacuum blood collection tube can be disposable. The cylinder can be filled with vacuum blood collection tubes containing different reagent mixtures for detecting different target analytes. In some embodiments, the reusable base may include a microcontroller, a heating assembly, a heater control system, and a detection (e.g., optical, lateral flow) system (and optional heat sink). The cylinder containing the vacuum blood collection tube can be coupled to the reusable base to allow for the detection and transfer of target analyte detection results.

[0085] Therefore, the disclosed methods and systems allow and envision automatic notification of results, for example, using network communication with various devices, such as... Figures 6A-6C As shown in the illustration. For example, in embodiments with optical inspection, the inspection system may be integrated into an intranet and / or the Internet, and may be configured to transmit and / or deliver inspection results via electronic messaging (e.g., text, email, SMS, etc.). The results of such transmission may be direct or transmitted via a remote server, and may be in various formats, including QR codes and other information formats known in the art.

[0086] Figure 10A This is a schematic diagram of an exemplary circuit system that may be included on a printed circuit board (“PCB”) and is configured to allow for multiple detection of up to eight different target analytes.

[0087] Figure 10B This is a graph showing the wavelength (nm) and spectral response of a device according to some embodiments. Figure 10B The measured spectral response is shown to be normalized for a specific channel, indicating the different emission wavelength windows detected by the optical sensor.

[0088] Therefore, the described content is a method and system for detecting one or more target analytes in a sample, the apparatus comprising: (i) a sample container for encapsulating a sample mixture comprising a sample and at least one lysis reagent; (ii) at least one vacuum blood collection tube sealed under negative pressure using a sealing element and containing a stable reagent corresponding to at least one of the target analytes being detected; and (iii) at least one engageable fluid communication channel oriented to allow the sample mixture to be fluidly connected from the sample container to one or more of the at least one vacuum blood collection tube via the sealing element of each such fluidly connected vacuum blood collection tube, wherein establishing a fluid connection with the at least one vacuum blood collection tube sealed under negative pressure causes at least some of the sample mixture to be connected from the sample container to the at least one such fluidly connected vacuum blood collection tube.

[0089] The methods and systems described herein are not limited to specific hardware or software configurations and are applicable to many computing or processing environments. The methods and systems can be implemented in hardware or software, or a combination of both. The methods and systems can be implemented in one or more computer programs, wherein the computer program is understood to include one or more processor-executable instructions. The computer program can execute on one or more programmable processors and can be stored on one or more storage media (including volatile and non-volatile memory and / or storage elements), one or more input devices, and / or one or more output devices that are readable by the processor. The processor can therefore access one or more input devices to obtain input data and access one or more output devices to transmit output data. The input devices and / or output devices can include one or more of the following: random access memory (RAM), redundant array of independent disks (RAID), floppy disk drives, CDs, DVDs, disks, internal hard disk drives, external hard disk drives, memory sticks, or other storage devices accessible by a processor as provided herein, wherein such foregoing examples are not exhaustive and are for illustrative purposes only and are not limiting.

[0090] Computer programs can be implemented using one or more high-level procedural or object-oriented programming languages ​​to communicate with computer systems; however, programs can also be implemented using assembly language or machine language if desired. The language can be assembled or interpreted.

[0091] As provided herein, the processor can therefore be embedded in one or more devices that can operate independently or together in a networked environment, wherein the network may include, for example, a local area network (LAN), a wide area network (WAN), and / or may include an intranet and / or the Internet and / or another network. The network may be a wired network or a wireless network or a combination thereof, and one or more communication protocols may be used to facilitate communication between different processors. The processor can be configured for distributed processing, and in some embodiments, a client-server model may be utilized as needed. Therefore, methods and systems can utilize multiple processors and / or processor devices, and processor instructions can be partitioned among such single or multiple processors / devices.

[0092] Devices or computer systems integrated with processors may include, for example, personal computers, workstations (e.g., Sun, HP), personal digital assistants (PDAs), handheld devices (such as mobile phones, laptops, and handheld devices), or other devices capable of being integrated with processors that can operate as described herein. Therefore, the devices provided herein are not exhaustive and are provided for illustration purposes only and are not limiting.

[0093] References to “microprocessor” and “processor,” or “the microprocessor” and “the processor”, are understood to include one or more microprocessors capable of communicating in independent and / or distributed environments, and thus configurable to communicate with other processors via wired or wireless communication, wherein such one or more processors may be configured to operate on a device controlled by one or more processors (which may be similar or different devices). Therefore, the use of such terms “microprocessor” or “processor” is also understood to include central processing units, arithmetic logic units, application-specific integrated circuits (ICs), and / or task engines, wherein such examples are provided for illustrative purposes and are not limiting.

[0094] Furthermore, unless otherwise specified, references to memory may include one or more processor-readable and accessible memory elements and / or components that may be internal to the processor control unit, external to the processor control unit, and / or accessible via wired or wireless networks using various communication protocols, and unless otherwise specified, may be arranged to include a combination of external and internal memory devices, wherein such memory may be contiguous and / or partitioned on an application-based basis. Therefore, references to databases can be understood to include one or more memory associations, wherein such references may include commercially available database products (e.g., SQL, Informix, Oracle) as well as proprietary databases, and may also include other structures for associated memory, such as links, queues, graphs, trees, wherein such structures are provided for illustration and are not limited thereto.

[0095] Unless otherwise provided, references to networks may include one or more intranets and / or the Internet. Based on the foregoing, references to microprocessor instructions or microprocessor executable instructions herein may be understood to include programmable hardware.

[0096] Unless otherwise stated, the use of the word "substantially" can be interpreted to include precise relationships, conditions, arrangements, orientations and / or other characteristics as understood by one of ordinary skill in the art, as well as deviations thereof (within the scope that such deviations do not materially affect the disclosed methods and systems).

[0097] Throughout this disclosure, unless otherwise specifically stated, the use of the article “a” or “a kind” to modify a noun can be understood as a convenience and includes one or more of the nouns being modified.

[0098] The use of “include” or “includes” should be understood as meaning to include or encompass, and should not be limited to such included components, parts, elements, actions, etc.

[0099] Unless otherwise specified herein, elements, components, modules and / or portions thereof described and / or otherwise depicted by means of figures as communicating, relating to and / or based on other things shall be understood as communicating, relating to and / or based on them directly and / or indirectly.

[0100] Although the methods and systems are described with respect to their particular embodiments, they are not so limiting. In light of the above teachings, many modifications and variations may become apparent.

[0101] Many additional changes to the details, materials, and arrangements of the portions described and shown herein can be made by those skilled in the art. Therefore, it will be understood that the following claims are not limited to the embodiments disclosed herein, but may include practices other than those specifically described, and should be interpreted as per the broadest permissible law.

Claims

1. An apparatus for detecting the presence of one or more target analytes in a sample, the apparatus comprising: (i) A sample container for encapsulating a sample mixture comprising the sample and at least one lysis reagent; (ii) At least one vacuum blood collection tube, said at least one vacuum blood collection tube being sealed under negative pressure using a sealing element, and containing a stable reagent corresponding to the detection of at least one of the target analytes; (iii) At least one engageable fluid communication channel, said at least one engageable fluid communication channel being oriented to enable the sample mixture to be fluidly connected from the sample container to one or more of the at least one vacuum blood collection tubes via the sealing element of each such fluidly connected vacuum blood collection tube, wherein establishing the fluid connection with the at least one vacuum blood collection tube sealed under negative pressure causes at least some of the sample mixture to be connected from the sample container to the at least one such fluidly connected vacuum blood collection tube; as well as (iv) A detection module, which is connected to the at least one vacuum blood collection tube and is capable of detecting the presence of the target analyte.

2. The device according to any of the preceding claims, wherein, At least one target analyte serves as a control, and the stabilizing agent comprises at least one of the following: (i) a stabilizing amplification reagent; and (ii) a labeled probe associated with at least one of the target analytes; and the detection module is configured to detect the labeled probe associated with one or more target analytes.

3. The device according to any of the preceding claims, wherein, The at least one vacuum blood collection tube is configured to encapsulate one or more reagents for the identification, amplification, and / or detection of the target analyte.

4. The device according to any of the preceding claims, wherein, The amount of sample mixture fluidly connected from the sample container to the at least one vacuum blood collection tube is based on one or more of the following: (i) the volume of each such fluidly connected vacuum blood collection tube; and / or (ii) the magnitude of the negative pressure in each such fluidly connected vacuum blood collection tube when the fluid connection is established.

5. The device according to any of the preceding claims, wherein, The connectable fluid communication channel is a non-drilled hollow needle.

6. The device according to any of the preceding claims, wherein, The engageable fluid communication is manually established by pressing the sample container onto the engageable fluid communication channel / needle, and the engageable fluid communication channel is configured to disengage and deactivate the fluid connection when the sample container is positioned on the needle by removing the manual pressure from the sample container.

7. The device according to any of the preceding claims, wherein, The connectable fluid communication channel includes a first end for communication with the sample container and at least one second end shaped for communication with one or more vacuum blood collection tubes.

8. The device according to any of the preceding claims, wherein, The connectable fluid communication channel includes a first end configured to communicate with the sample container and a second inverted Y-shaped end configured to communicate with two vacuum blood collection tubes.

9. The device according to any of the preceding claims, wherein, The connectable fluid communication channel includes a first end and a second end, the first end being configured to communicate with the sample container, and the second end having two or more sub-channels or sub-ends, the sub-channels or sub-ends being configured to establish a fluid connection with negative pressure in two or more of the vacuum blood collection tubes, wherein the first end is in fluid communication with each of the two or more second ends and / or sub-channels.

10. The device according to claim 9, wherein, The fluid communication channel is configured to engage a first end of the fluid communication channel with the sample container before engaging a negative pressure at at least one second end of the fluid communication channel with either of the at least one vacuum blood collection tube.

11. The device according to claim 10, wherein, At least one second end of the fluid communication channel is configured such that when the first of such at least one second end is engaged with the vacuum blood collection tube, each of the at least one second end is either sealed away from atmospheric pressure or engaged with the vacuum blood collection tube.

12. The device according to any of the preceding claims, wherein, The connectable fluid communication channel includes two or more second ends configured to be in fluid communication with two or more vacuum blood collection tubes, and if any one of the two or more second ends is connected to negative pressure in the two or more vacuum blood collection tubes, then each of the other two or more second ends must be connected to negative pressure in another of the two or more vacuum blood collection tubes, or sealed.

13. The device according to any of the preceding claims, wherein, The stable amplification reagent is an isothermal amplification reagent.

14. The device according to claim 13, wherein, The stable amplification reagent is configured for loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), chain invasion-based amplification (SIBA), and multi-enzyme isothermal rapid amplification (MIRA).

15. The device according to claim 13, wherein, The stable amplification reagent includes materials required for target-specific amplification, including enzymes, probes, labeled probes, intercalation dyes, and buffer solutions.

16. The device according to any of the preceding claims, wherein, The sealing element includes a diaphragm or a diaphragm.

17. The device according to claim 16, wherein, The sealing element is permeable.

18. The device according to claim 16, wherein, The sealing element comprises bromobutyl rubber.

19. The device according to claim 16, wherein, The sealing element includes a plug, which is made of bromobutyl rubber or is made entirely of bromobutyl rubber.

20. The device according to any of the preceding claims, wherein, The sealing elements include mechanical valves, diaphragm valves, ball valves, piston valves, and / or pinch valves.

21. The device according to any of the preceding claims, wherein, (i) the volume of the vacuum blood collection tube and / or (ii) the magnitude of the negative pressure in each vacuum blood collection tube are selected and / or determined and / or established based on the amount of sample mixture expected to be fluidly connected to such vacuum blood collection tubes when establishing a fluid connection between the sample container and such vacuum blood collection tubes.

22. The device according to any of the preceding claims, wherein, The amount of sample mixture to be connected to the vacuum blood collection tube in a fluid connection may be at least 25 μl, at least 50 μl, at least 100 μl, at least 200 μl, or at least 300 μl.

23. The device according to any of the preceding claims, wherein, The detection module further includes a heating component configured to facilitate the amplification of the labeled target analyte in the sample mixture within one or more vacuum blood collection tubes containing a combination of a sample mixture, a stable amplification reagent, and a labeled probe.

24. The device according to claim 23, wherein, The heating assembly is configured to heat the vacuum blood collection tube and / or its contents to a predetermined temperature to facilitate isothermal amplification.

25. The device according to claim 23, wherein, The heating assembly is configured to surround the vacuum blood collection tube and includes an aluminum heating block.

26. The device according to claim 25, wherein, After amplification, the detection module is configured to detect the number of amplified labeled antigens in each vacuum blood collection tube.

27. The device according to claim 26, wherein, The detection module includes at least one optical sensor configured to optically detect fluorescent markers.

28. The device according to any of the preceding claims, wherein, The at least one vacuum blood collection tube includes a plurality of vacuum blood collection tubes, and each of the plurality of vacuum blood collection tubes in the device is configured with a labeled probe that is different from that of the other vacuum blood collection tubes in the device, such that each of the plurality of vacuum blood collection tubes in the device is associated with a different target analyte.

29. The device according to claim 28, wherein, At least one optical detector is associated with each of the plurality of vacuum blood collection tubes and configured to detect the labeled target analyte in the corresponding vacuum blood collection tube among the plurality of vacuum blood collection tubes.

30. The device according to claim 28, wherein, At least one optical detector is associated with more than one of the plurality of vacuum blood collection tubes and configured to detect the labeled target analyte in more than one vacuum blood collection tube.

31. The device according to claim 29 or 30, wherein, The at least one optical sensor may include an optical sensor system, wherein the optical sensor system includes at least one LED configured to illuminate one or more vacuum blood collection tubes, and at least one photodiode detector configured to optically detect emissions from one or more vacuum blood collection tubes when the vacuum blood collection tubes are illuminated.

32. The device according to claim 31, wherein, The one or more vacuum blood collection tubes include a plurality of vacuum blood collection tubes, and the target analytes include a plurality of target analytes, wherein a marker probe in each of the plurality of vacuum blood collection tubes is positioned in the device and configured to selectively emit at different wavelengths upon irradiation, and an optional optical detector is configured to selectively detect at each of the different wavelengths.

33. The device according to claim 32, wherein, The labeled probe includes a fluorescent dye.

34. The device according to any of the preceding claims, wherein, The detection module includes a microcontroller and a graphical user interface, wherein the microcontroller is configured with programmable instructions to communicate user instructions to the graphical user interface to allow the user to use the device to detect the target analyte, and the microcontroller includes programmable instructions configured to communicate with a remote device such as a telephone via an application, through email or text.

35. A method for detecting at least one target analyte in a fluid sample, the method comprising: (i) Obtain the fluid sample; (ii) Combining the fluid sample in the sample container with at least one lysis reagent to produce a sample mixture; (iii) Provide at least one vacuum blood collection tube, each vacuum blood collection tube being sealed under negative pressure using a sealing element, and containing a stable reagent corresponding to the detection of at least one of the target analytes; (iv) Engaging a fluid communication channel to create a fluid connection between the sample container and the at least one vacuum blood collection tube via the sealing element of each fluidly connected vacuum blood collection tube, wherein engagement of the fluid communication channel causes at least some of the sample mixture to communicate from the sample container to such fluidly connected at least one vacuum blood collection tube. as well as (v) Determine the presence or absence of the at least one target analyte in the at least one vacuum blood collection tube.

36. The method according to claim 35, wherein, The lysis reagent is lyophilized and / or included in a sub-container within the at least one vacuum blood collection tube.

37. The method according to claim 35 or 36, wherein, Engaging the fluid communication channel in step (iv) includes connecting a first end of the fluid communication channel to the sample container before engaging at least one second end of the fluid communication channel with negative pressure in either of the at least one vacuum blood collection tube.

38. The method of claim 37, further comprising providing a fluid communication channel configured to engage a first end of the fluid communication channel with the sample container prior to negative pressure engagement of at least one second end of the fluid communication channel with either of the at least one vacuum blood collection tube.

39. The method of claim 38, further comprising providing a fluid communication channel, wherein when the first of such at least one second end is engaged with a vacuum blood collection tube, each of the at least one second end is pressure-sealed away from or engaged with a vacuum blood collection tube.

40. The method of claim 39, further comprising disengaging at least a first or second end of the fluid communication channel before determining the presence or absence of the at least one target analyte.

41. The method according to any one of claims 35-40, wherein, The determination includes providing an optical detection system to detect fluorescently labeled target analytes.

42. The method according to any one of claims 35-41, wherein, The stabilizing reagents include those capable of identifying, amplifying, and facilitating and / or allowing the detection of the target analyte.

43. The method according to any one of claims 35-42, wherein, The stabilizing reagent includes an amplification reagent, a probe, and a fluorescently labeled probe, and the method includes loading the amplification reagent and the fluorescently labeled probe into at least one vacuum blood collection tube before sealing the at least one vacuum blood collection tube under negative pressure.

44. The method according to any one of claims 35-42, further comprising, prior to sealing such vacuum blood collection tubes with a determined amount or magnitude of negative pressure using a sealing element, determining, selecting and / or establishing at least one of the following: (i) the volume of the vacuum blood collection tubes; and / or (ii) the amount or magnitude of negative pressure stored in each vacuum blood collection tube to allow the sample mixture to be distributed between the fluidly connected vacuum blood collection tubes.

45. The method according to any one of claims 35-44, wherein, The sealing element includes a permeable sealing element and / or a self-healing permeable sealing element.

46. ​​The method according to any one of claims 35-45, further comprising, upon determining the presence or absence of the target analyte, heating the vacuum blood collection tube sample mixture comprising a stabilizing reagent for identifying, amplifying, and detecting the target analyte to induce a reaction between the sample mixture and the stabilizing reagent.

47. The method according to any one of claims 35-46, wherein, The reaction involves amplifying the target analyte or LAMP.

48. The method according to any one of claims 35-47, wherein, The target analyte includes the target nucleic acid.

49. The method according to any one of claims 35-48, wherein, The detection of the presence or absence of the target analyte begins only after the vacuum blood collection tube is heated.

50. The method according to any one of claims 35-49, further comprising determining at least one detection threshold for each of the at least one vacuum blood collection tube that encapsulates the sample mixture and reagents and is heated.

51. The method according to any one of claims 35-50, further comprising detecting a target analyte in a single vacuum blood collection tube, and determining the presence or absence of the target analyte comprising determining at least one detection threshold for each vacuum blood collection tube.

52. The method according to any one of claims 35-51, wherein, Determining at least one detection threshold involves determining a detection threshold for each target analyte in each vacuum blood collection tube.

53. The method according to any one of claims 50-52, wherein, Determining at least one detection threshold includes performing the automatic determination of the at least one detection threshold.

54. The method according to claim 53, wherein, The automatic determination includes performing measurements on the vacuum blood collection tube before amplifying the target analyte to a detectable level, and determining the at least one detection threshold based on the performed measurements.

55. The method according to any one of claims 50-54, wherein, The at least one detection threshold is determined based on at least one statistic, which is further based on the measurement performed.

56. The method according to claim 55, wherein, The at least one statistic is a combination of the mean, median, and / or standard deviation derived from and / or based on the performed measurement.

57. The method according to any one of claims 35-56, wherein, Determining the presence or absence of the at least one target analyte to perform a measurement includes performing an optical measurement on the at least one vacuum blood collection tube.

58. The method according to claim 57, wherein, If a single vacuum blood collection tube contains multiple target analytes, and each target analyte is fluorescently labeled for emission at different wavelengths, then determining the detection threshold may include performing optical measurements at one or more of the different wavelengths and determining the detection threshold at one or more of the different wavelengths.

59. The method according to claim 58, wherein, Performing measurements to determine a detection threshold may include: performing an optical measurement before amplifying the target analyte to a detectable level / amount; determining at least one of a mean and a standard deviation based on the performed optical measurement; and determining the detection threshold based on at least one of the mean and the standard deviation.

60. The method according to any one of claims 35-59, further comprising: Once the amount of the target analyte is detected to exceed the detection threshold, the result is reported; And once the amount of the detected target analyte exceeds the detection threshold, the detection of the given target analyte is stopped.

61. The method according to any one of claims 35-60, further comprising: When the amount of all detected target analytes exceeds one or more detection thresholds associated with the target analytes, the detection of all target analytes is stopped.

62. The method according to any one of claims 35-61, further comprising determining that the target analyte is absent if the amount of such target analyte detected within at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, or at least 30 minutes from the time the one or more vacuum blood collection tubes are heated does not exceed a detection threshold.

63. The method according to any one of claims 35-62, wherein, Engaging the fluid communication channel includes manually pressing the sample container to engage the fluid communication channel and / or making contact with the fluid communication channel to induce a fluid connection between the sample container and the at least one vacuum blood collection tube.

64. The method according to any one of claims 35-63, further comprising providing instructions to a user via a graphical user interface, the instructions including providing instructions to the user to: take a sample, insert a sample into a sample container, manually press the sample container to engage the fluid communication channel, release the sample container, and / or update the results to the user in a timely manner, and optionally, providing the results to the user includes displaying results related to the presence or absence of the target analyte on a graphical user interface, transmitting the results to an application, transmitting the results to an email address, and / or transmitting the results to a networked device, wherein such network may be wired or wireless, secure or insecure.

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