Systems and methods for performing amplicon rescue multiplex polymerase chain reaction (PCR)

Abstract

Embodiments of the present disclosure generally relate to systems and methods for performing amplicon rescue multiplex polymerase chain reaction (arm-PCR). In one embodiment, the system includes a processor coupled to a control element and a reader. The control element is configured to control operation of the processor and the reader based on a plurality of settings. The processor is configured to receive a self-contained cartridge for performing PCR amplification of DNA and / or RNA obtained from a bodily sample. The processor engages with the cartridge and manipulates reagents within the cartridge to amplify and detect DNA from the sample. The processor also causes the cartridge to deposit the DNA on a microarray within the cartridge. The reader is configured to receive the cartridge after it has been processed by the processor and capture an image of the microarray for transmission to the control element.

Description

[0001] This application is a divisional application of Chinese patent application No. 201280063646.3, entitled "System and method for performing amplicon rescue multiplex polymerase chain reaction (PCR)," which entered the Chinese national phase of PCT international patent application PCT / US2012 / 064713, filed on November 12, 2012.

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 61 / 558,791, filed November 11, 2011, entitled "Systems and Methods for Performing Amplicon Rescue Multiplex Polymerase Chain Reaction," which is incorporated herein by reference. This application also claims priority to U.S. Provisional Patent Application No. 61 / 592,372, filed January 30, 2012, entitled "Systems and Methods for Performing Amplicon Rescue Multiplex Polymerase Chain Reaction (PCR)," which is incorporated herein by reference. Technical Field

[0004] This application relates to systems and methods for multiplex polymerase chain reactions. More specifically, this application relates to systems and methods for amplicon-rescue multiplex polymerase chain reaction (arm-PCR). Background Technology

[0005] The development of polymerase chain reaction (PCR) has enabled the use of DNA amplification for a variety of applications, including molecular diagnostic assays. However, many challenges exist associated with the use of PCR for molecular differential diagnosis (MDD) assays. PCR utilizes specific primers or primer sets, temperature conditions, and enzymes. PCR reactions can be susceptible to contamination, primer binding may require different conditions for different primers, primers should be specific to the target sequence in order to amplify only that target sequence, and so on. This makes it more difficult to amplify multiple sequences from a single sample.

[0006] In the past, diagnostic testing of clinical samples to detect one or more pathogenic agents required the isolation and culturing of microorganisms. This could take days, while in many cases, a diagnosis must be made within hours to save a patient's life. The goal is to identify one or more pathogenic agents in clinical samples within approximately a few hours, and many methods have been developed to achieve this goal. For example, multiplex PCR and target-enriched multiplex PCR (tem-PCR) techniques have been developed to amplify multiple nucleic acids within a sample to generate sufficient DNA / RNA to enable the detection and identification of multiple organisms. Multiplex PCR and tem-PCR techniques offer the ability to perform multiple assays on a single sample at once, but this is achieved at the cost of high sensitivity, which can be achieved with a single amplification reaction using a single set of target-specific primers. Further improvements to these techniques are needed to provide diagnostic tests with greater sensitivity and shorter diagnostic times. Integration of the amplification and detection steps is also needed to eliminate the open-tube hybridization step to reduce false positives caused by carryover contamination from PCR products. Summary of the Invention

[0007] Embodiments of this disclosure generally relate to systems and methods for performing amplicon rescue multiplex polymerase chain reaction (arm-PCR). In one embodiment, the system includes a processor and a reader coupled to a control element. The control element is configured to control the operation of the processor and the reader based on a variety of settings. The processor is configured to receive a self-contained cassette for PCR amplification of DNA and / or RNA obtained from an in vivo sample. The processor engages with the cassette and manipulates reagents within the cassette to amplify and detect DNA from the sample. The processor also causes the cassette to store the DNA on a microarray within the cassette. The reader is configured to receive the cassette after it has been processed by the processor and to capture an image of the microarray for transmission to the control element. Attached Figure Description

[0008] The present disclosure can be better understood by referring to the accompanying drawings. The elements in the drawings are not necessarily to scale, but the focus is on clearly illustrating the principles of the present disclosure. Furthermore, in all these views, the same reference numerals designate corresponding parts.

[0009] Figure 1 This is a block diagram illustrating an exemplary system for performing PCR amplification according to the present disclosure.

[0010] Figure 2 It is a diagrammatic explanation as follows: Figure 1 A block diagram of an exemplary embodiment of the depicted control element.

[0011] Figure 3 This is a side plan view of an exemplary processor module.

[0012] Figure 4 yes Figure 3 Rear perspective view of the processor module.

[0013] Figure 5 Is it so? Figure 3 A partially exploded view of the heater assembly depicted.

[0014] Figure 6 Is it so? Figure 3 A perspective view of an exemplary heater.

[0015] Figure 7 Is it so? Figure 3 A perspective view of the elevator assembly depicted.

[0016] Figure 8 Is it so? Figure 3 A perspective view of an exemplary camshaft as depicted.

[0017] Figure 9 Is it so? Figure 3 A perspective view of an exemplary plunger depicted.

[0018] Figure 10 Is it so? Figure 3 A perspective view of an exemplary lead screw shaft depicted.

[0019] Figure 11 Depicting engagement with the box Figure 3 The heater assembly of the heater.

[0020] Figure 12 Is it so? Figure 1 A perspective view of an exemplary embodiment of the reader depicted.

[0021] Figure 13 The driver component has been removed. Figure 12 A perspective view of the reader.

[0022] Figure 14 It is the removal of the drive assembly and flywheel assembly. Figure 12 A perspective view of the reader.

[0023] Figure 15 yes Figure 12 Partial exploded view of the driving components.

[0024] Figure 16 yes Figure 12 A perspective view of the flywheel assembly.

[0025] Figure 17 yes Figure 12 Top perspective view of the flywheel.

[0026] Figure 18 yes Figure 17 Bottom perspective view of the flywheel.

[0027] Figure 19 yes Figure 12 A perspective view of the optical components.

[0028] Figure 20 This is a top plan view of an exemplary implementation of the microarray.

[0029] Figure 21 yes Figure 19 Exploded view of the optical cube assembly.

[0030] Figure 22 This is a block diagram depicting an exemplary open platform target solution system according to this disclosure.

[0031] Figure 23 A portion of a microarray image showing exemplary points is depicted.

[0032] Figure 24 A portion of a microarray image depicting exemplary repositioned points is shown.

[0033] Figure 25 It is a side sectional view of the flywheel housing including the counterweight.

[0034] Figure 26 This is a cross-sectional view of the flywheel housing from the other side.

[0035] Figure 27A This is a rear view of the counterweight.

[0036] Figure 27B This is a side view of a counterweight containing two small gears.

[0037] Figure 27C It is a side view of a counterweight containing a small gear.

[0038] Figure 28 An exemplary implementation of the box is described.

[0039] Figure 29 Depicting by Figure 28 An exploded view of the exemplary box depicted. Detailed Implementation

[0040] Embodiments of this disclosure generally relate to systems and methods for performing amplicon rescue multiplex polymerase chain reaction (arm-PCR). In one embodiment, the system includes a processor and a reader coupled to a control element. The control element is configured to control the operation of the processor and reader based on a variety of settings. The processor is configured to receive a self-contained cartridge for PCR amplification of DNA and / or RNA obtained from an in vivo sample. The processor engages with the cartridge and manipulates reagents within the cartridge to amplify and detect DNA from the sample. The processor also causes the cartridge to store DNA on a microarray within the cartridge. The reader is configured to receive the cartridge after it has been processed by the processor and to capture an image of the microarray for transmission as test data to the control element. The control element is further configured to analyze the test data received from the reader and generate output indicating a comparison of the test data with predefined data.

[0041] Figure 1 An exemplary system 10 for PCR amplification of DNA and / or RNA obtained from an organism sample is described. System 10 enables arm-PCR, a technique previously described in U.S. Patent No. 7,999,092 entitled “Amplicon Rescue MultiplexPolymerase Chain Reaction for Amplication of Multiple Targets,” which is incorporated herein by reference. System 10 includes a processor 12 and a reader 14 coupled to a control element 15. In one embodiment, the control element 15 includes a computing device such as a computer, but in other embodiments, other types of control elements 15 are possible. The control element 15 is configured to communicate with the processor 12 and the reader 14 to control the operation of the processor 12 and the reader 14 based on various settings, as discussed in more detail below. The control element 15 is further configured to receive data from the reader 14 indicating amplified DNA of a sample and to generate output indicating a comparison of the amplified DNA with predefined data, as discussed in more detail below. Such a comparison is used to diagnose the sample.

[0042] Processor 12 is configured to receive, engage with, and manipulate cassette 17 containing an organism sample to perform arm-PCR on the sample within cassette 17. An exemplary cassette is disclosed in U.S. Patent Application Serial No. 12 / 780,698, entitled “Apparatus for Performing Amplicon Rescue Multiplex PCR,” which is incorporated herein by reference. In one embodiment, processor 12 includes at least one detection element 19 for detecting cassette 17 within processor 12 and determining various information about cassette 17. Detection element 19 transmits information to control element 15, which manipulates processor 12 based on said information, as will be discussed in more detail below.

[0043] Reader 14 is configured to receive cartridge 17 after it has been processed by processor 12 and capture an image of a microarray (not shown) on cartridge 17. The microarray indicates the detection of DNA generated by PCR amplification. In one embodiment, the image of the microarray comprises a digital image, but in other embodiments, other types of images are possible. Reader 14 is further configured to transmit the image as test data to control element 15 to allow control element 15 to analyze the test data and compare the test data with predefined data.

[0044] Figure 2 Depicting Figure 1 An exemplary embodiment of the control element 15. As stated above, the control element 15 is coupled to at least one processor 12 of the system 10. Figure 1 ) and at least one reader 14 ( Figure 1 The control element 15 is configured to communicate with the processor 12 and the reader 14, and to monitor and control their operation. The control element 15 is further configured to receive test data from the reader 14 and analyze that test data, as will be discussed in more detail below. In one embodiment, the control element 15 is executed via a computer, such as a desktop or laptop computer; however, in other embodiments, other types of devices may be used to execute the control element 15.

[0045] As by Figure 2As shown, the control element 15 includes at least one conventional processing element 20, such as a digital signal processor (DSP) or a central processing unit (CPU), which communicates with and drives other elements of the control element 15 via a local interface 22, which may include at least one bus. The control element 15 further includes a processor interface 24 enabling communication with the processor 12 and a reader interface 25 enabling communication with the reader 14. In one embodiment, the processor interface 24 receives status information from the processor 12 and allows the control element 15 to control the operation of the processor 12 based on various settings, as discussed in more detail below. In one embodiment, the reader interface 25 receives status information from the reader 14 and allows the control element 15 to control the operation of the reader 14. Furthermore, the reader interface 25 receives data from the reader 14 in the form of an image indicating a microarray of amplified DNA from a sample. The control element 15 further includes a user input interface 26 (e.g., a computer keyboard and / or mouse) and a user output interface 28 (e.g., a computer monitor and / or printer). However, in other implementations, different user input interfaces 26 and user output interfaces 28 are possible.

[0046] Control element 15 further includes control logic 31, which is configured to control the operation of processor 12 and reader 14 and manage data and other components within control element 15. In this regard, in one embodiment, control logic 31 manages the application of an appropriate set of predefined settings 32 and data 35 by mapping box 17 to an appropriate set of settings 32 and an appropriate set of data 35 based on ID mapping data 33 and / or information input by the user through user input interface 26. ID mapping data 33 is stored in memory 30 of control element 15 and will be associated with identifiers (not shown) (e.g., barcodes) on and associated with box 17, such as those detected by detection element 19. Figure 1The detected data is mapped to a corresponding set of predefined settings 32 and a set of predefined data 35 for cartridge 17. Each set of predefined settings 32 is stored in the memory 30 of the control element 15 and indicates various operations to be performed by the processor 12 for arm-PCR for the corresponding cartridge 17. Each set of predefined data 35 indicates a set (e.g., one or more sets) of points of the cartridge's microarray that will fluoresce during readout due to DNA from the target pathogen aggregated at the point, as will be discussed in more detail below. The predefined data 35 is compared with test data 34 indicating points of the microarray that have fluoresced during readout to determine the presence of the target pathogen in the sample. The predefined settings 32 may vary depending on the type of test to be run on the sample (e.g., the type of target pathogen designed to be detected in the sample). For example, a set of predefined settings 32 may indicate information such as: the heater ( Figure 2 The duration of arm-PCR of the target pathogen (not shown in the text), the temperature of the corresponding heater, and the sequence of specific operations to be performed by the processor 12 are applied to the cassette 17 to effectively manipulate the cassette 17 to perform arm-PCR of the target pathogen, as discussed in more detail below.

[0047] In one implementation, the predefined settings 32 include multiple sets of operations to be performed by the processor 12. In one exemplary implementation, the predefined settings 32 include three sets of settings. In such an implementation, the three sets of settings 32 are adjusted for high specificity, high sensitivity, and nominal results. In this regard, if a set of settings 32 adjusted for high specificity is selected, then the processor 12 operates on the cassette 17 to isolate a specific target pathogen or DNA sequence in the sample and exclude all other target pathogens or DNA sequences. However, if a set of settings adjusted for high sensitivity is selected, then the processor 12 operates on the cassette 17 to identify a wide range of target pathogens or DNA sequences in the sample. Furthermore, if a nominal set of settings is selected, then the processor 12 operates on the cassette 17 to generate a nominal range of target pathogens or DNA sequences. However, in other implementations, there may be different numbers and types of array settings.

[0048] In one implementation, the array of predefined settings 32 can be used on both closed and open platforms. In this regard, the closed platform allows for specific testing of a particular target pathogen (e.g., a target pathogen regulated by the Food and Drug Administration (FDA),) while the open platform allows for a wide variety of tests on unregulated target pathogens. For example, for a closed platform, a set of predefined settings 32 can be automatically selected by control logic 31 based on the target pathogen as indicated by ID mapping data 33. Therefore, the user can choose not to select or otherwise control the testing of the target pathogen on the closed platform. However, for an open platform, the user can manually select a desired set of predefined settings 32 to obtain the desired results via user input interface 26. In the exemplary implementation described above, the user can select one of three sets of predefined settings 32 to be performed on box 17, where these three sets are tailored for high specificity, high sensitivity, or nominal results. However, in other implementations, different types and numbers of arrays of predefined settings 32 can be selected by the user on the open platform. Furthermore, in one implementation, the user can define a set of customized settings to be performed on box 17 on the open platform. Therefore, on the open platform, users can manipulate the test on the target pathogen by selecting different groups for the test 32 and changing the primers used for the test, as will be discussed in more detail below.

[0049] In one of the above embodiments, the identifier of cartridge 17 is read by detection element 19. In other embodiments, other techniques for determining the identifier are possible. As an example, the identifier may be stored electronically in cartridge 17. Cartridge 17 may be configured to transmit the identifier wirelessly or otherwise to detection element 19. As an example, radio frequency (RF) or infrared communication may be used to communicate with the identifier. In other embodiments, detection element 19 may use other techniques to determine the identifier of the cartridge.

[0050] Control logic 31 is further configured to receive images of the microarray from reader 14 via reader interface 25 and store the images as test data 34 in memory 30 of control element 15. In one embodiment, test data 34 comprises one or more digital images of the microarray of one or more boxes 17, but in other embodiments, different types of test data 34 are possible. Control logic 31 is further configured to compare test data 34 with predefined data 35 mapped to box 17 to determine whether a specific target pathogen is detected in the sample. In this regard, in one embodiment, test data 34 comprises images of the microarray in which DNA corresponding to a specific target pathogen is aggregated at specific points or combinations of points (not shown) in the microarray. The points or combinations of aggregated DNA fluoresce when irradiated by a laser from reader 14. When the microarray is irradiated by a laser, reader 14 captures a digital image of the microarray, and microarray detection logic 36 is configured to analyze the image to determine the points that fluoresce due to the presence of DNA on or in them. As described in more detail herein, each dot is composed of a different material, and the dots are arranged in a predefined pattern. Therefore, the pattern of fluorescent dots in the image indicates the presence of a target pathogen in the tested sample. Furthermore, logic 36 performs digital analysis on the image to determine the fluorescent dots in the image and compares the determined fluorescent pattern with a set of appropriate predefined data 35 for a specific target pathogen. Based on this comparison, logic 36 determines whether the target pathogen is present in the tested sample.

[0051] When analyzing a microarray image, Logic 36 locates and identifies each point within the image. Various techniques are available for locating and identifying points. In one exemplary implementation, Logic 36 identifies regions within the digital image referred to herein as “test regions.” Each test region is an area in the digital image where a particular point is expected to be located based on a predefined pattern of points on the microarray. As an example, Figure 23 A portion of a microarray image showing an exemplary point 101 is depicted. A reference line 102, which is not actually visible in the image, represents a test region 103, in which logic 36 anticipates finding point 101 based on the pixel location within the digital image of test region 103. To determine whether point 101 associated with test region 103 fluoresces in the image, logic 36 calculates the average brightness of all pixels within test region 103 and compares the average brightness to a threshold. If the average brightness exceeds the threshold, then logic 36 determines that point 101 fluoresces in the image. However, if the average brightness does not exceed the threshold, then logic 36 determines that point 101 does not fluoresce in the image.

[0052] As by Figure 23As shown, point 101 may be slightly misaligned with test area 103 for various reasons (including imperfections in the material on which point 101 is placed in the microarray). If the majority of test area 103 is misaligned with point 101, such misalignment can lead to false fluorescence measurements. Therefore, to improve test results, logic 36 automatically shifts the image of point 101 relative to test area 103 so that a larger percentage of points 101 are within test area 103. In this regard, logic 36 performs an alignment algorithm to reposition point 101 within the microarray image before evaluating whether the relevant point 101 fluoresces, as described above.

[0053] According to this algorithm, logic 36 identifies point 101 by comparing pixel color values. In this respect, a group of adjacent pixels with substantially similar color values ​​generally indicates the location of point 101 within the image.

[0054] After identifying point 101, logic 36 is configured to measure the average brightness of test region 103 associated with point 101 (e.g., the closest point 101 within the image). Logic 36 then slightly repositions point 101 relative to test region 103 (e.g., moves point 101 within the image), as by... Figure 24 As shown, the average brightness of the pixels within the test area is calculated again. If the movement causes a large percentage of the test area 101 to be covered by point 101, then the average brightness should increase after the movement. Logic 26 compares the average brightness before the movement with the average brightness after the movement, and determines whether point 101 is more aligned based on this comparison. In this regard, if the average brightness after repositioning is higher, then logic 36 determines that point 101 is now more aligned with the test area 101. However, if the average brightness after repositioning is lower, then logic 36 determines that point 101 is now less aligned with the test area 101.

[0055] Furthermore, logic 36 continues to reposition point 101 and measure the average brightness until a maximum average brightness is found. For such a maximum brightness, the position of point 101 relative to test area 103 is the position that results in the maximum alignment between point 101 and test area 103. This position is selected by logic 103 as the final position within the image to be used for point 101.

[0056] In one exemplary implementation, when performing the repositioning algorithm, logic 36 moves point 101 relative to test region 103 within a widened spiral. In this respect, logic 36 moves point 101 such that its center moves along spiral 104, as described by... Figure 23As shown. Logic 36 continues to reposition point 101 as long as the average brightness of test area 103 continues to increase for a consecutive number of point positions. However, once the average brightness decreases for a consecutive number of point positions, logic 36 stops the repositioning algorithm and selects the position providing the highest average brightness as the final position of the point within the image. In other embodiments, other techniques and / or movement patterns can be used to find the position where point 101 is most aligned with test area 103. In one exemplary embodiment, logic 36 is configured to perform the same repositioning algorithm separately for each point of the microarray image, such that each point is moved individually to better align it with its corresponding test area. Once all corresponding points have been repositioned, the image is stored in memory 30 as test data 34.

[0057] Predefined data 35 is stored in memory 30 and, for a given cartridge 17, indicates one or more points in the microarray corresponding to a specific target pathogen. In this regard, when DNA from a specific target pathogen is present in the sample, predefined data 35 indicates one or more points in the microarray that should fluoresce during reading. Therefore, if test data 34 indicates that the DNA is detected at one or more specific points in the microarray, control logic 31 accesses the predefined data 35 mapped to cartridge 17 to determine the target pathogen corresponding to said point. Control logic 31 compares each point in the microarray to test for one or more target pathogens. Control logic 31 transmits the comparison result between test data 34 and predefined data 35 via user output interface 28. In one embodiment, control logic 31 stores the test result as test result data 37 in memory 30, which can be accessed by the user via user output interface 28. For example, for a closed platform, test result data 37 could indicate "yes" or "no" for each target pathogen tested against it, thus indicating whether such a target pathogen was detected in the sample, but different types of indications are possible. However, for an open platform, test result data 37 could indicate the brightness value of each point, allowing the user to compare multiple sets of test result data 37 for a given target pathogen to determine the preferred solution for the target pathogen, as discussed in more detail below.

[0058] It should be noted that control logic 31 and microarray detection logic 36 can be executed in software, hardware, firmware, or any combination thereof. Figure 2 In the exemplary embodiment illustrated in the diagram, control logic 31 and microarray detection logic 36 are executed in software and stored in the memory 30 of the control element 15.

[0059] Note that when executed in software, control logic 31 and microarray detection logic 36 can be stored and transported on any computer-readable medium for use by or in conjunction with an instruction execution device that can retrieve and execute instructions. In the context of this document, "computer-readable medium" can be any device that can contain or store a computer program for use by or in conjunction with an instruction execution device.

[0060] Figure 3 Depicting Figure 1 An exemplary processor module 40 of the processor 12. In this regard, the processor 12 includes one or more processor modules 40. In one embodiment, the processor 12 includes four processor modules 40 arranged side-by-side within a housing (not shown), but in other embodiments, any number of modules 40 may be utilized. Each processor module 40 is configured to receive and process a single cartridge 17 ( Figure 1 The processor module 40 includes a container 42 for receiving and housing the box 17, when the box 17 is positioned within the processor module 40. When the box 17 is placed within the container 42, the module 40 further includes at least one detection element 19 located adjacent to the container 42 for detecting an identifier (not shown) located on the outer surface of the box 17. In one embodiment, the detection element 19 includes a barcode scanner and the identifier includes a barcode; however, in other embodiments, other types of detection elements 19 and identifiers may be used. The detection element 19 detects the identifier and transmits it to the control element 15. Figure 1 This allows control element 15 to map identifiers to predefined settings 32. Figure 2 As stated above.

[0061] Once the predefined setting 32 is established, control element 15 communicates with onboard control element 48, which controls the operation of processor module 40 based on setting 32. In this regard, onboard control element 48 controls the operation of latch motor 41, cam lever motor 43, pump pin motor 44, lead screw motor 45, heater assembly 46, and elevator assembly 47. Latch motor 41 controls the operation of the latch (not shown), as discussed in more detail below. Cam lever motor 43 is coupled to and controls the rotation of cam lever shaft 50, as discussed in more detail below. In one embodiment, cam lever motor 43 is located behind container 42 within module 40. Cam lever shaft 50 extends horizontally into the rear opening (not shown) of container 42 and engages with cam lever (not shown) of cartridge 17 to control clockwise and counterclockwise rotation of the cam lever and manipulate pipette (not shown) for vertical upward and / or downward movement within cartridge 17. A pump pin motor 44 is coupled to and controls the lateral movement of plunger 52, as discussed in more detail below. The pump pin motor 44 is located behind container 42 within module 40, and plunger 52 extends laterally into container 42 through an opening (not shown) in the container. Plunger 52 engages with a pump pin (or "push rod") (not shown) of cartridge 17 and operates a pipette pump assembly (not shown) within cartridge 17, causing fluid to be drawn into or expelled from the pipette due to the compression of the pump pin by plunger 52.

[0062] Furthermore, the lead screw motor 45 is rotatably coupled to the lead screw shaft 53 and controls the clockwise and counterclockwise rotation of the lead screw shaft 53. In one embodiment, the lead screw motor 45 is located behind the container 42 within the module 40, and the lead screw shaft 53 extends horizontally into the container 42. The lead screw shaft 53 engages with the lead screw (not shown) of the housing 17 to control the lateral movement of the pipette within the housing 17. In this regard, rotating the lead screw shaft 53 clockwise causes the lead screw to rotate clockwise, causing the pipette to travel laterally in one direction within the housing 17, while rotating the lead screw shaft 53 counterclockwise causes the lead screw to rotate counterclockwise, causing the pipette to travel laterally in the opposite direction within the housing 17. The control module 40 of the cam rod, pump pin and lead screw of the cartridge 17 allows the pipette inside the cartridge 17 to manipulate the pipette to remove fluid from the reagent chamber (not shown) or sample chamber (not shown) inside the cartridge 17, or to inject fluid into the reagent chamber or detection chamber (not shown) inside the cartridge 17.

[0063] The heater assembly 46 includes a plurality of heaters 55. In one embodiment, the heater assembly 46 includes three heaters 55, but in other embodiments, other numbers of heaters 55 are possible. In one embodiment, the heater assembly 46 is located directly below the container 42 within the module 40. Each heater 55 is located on an adjustable base 56, which is movable in the vertical direction to adjust the vertical position of the heater 55, as will be described in more detail below. In one embodiment, when the module 40 is in operation, each heater 55 is set to a specific temperature and maintained at that temperature. For example, in one embodiment, one heater 55 is set to 55°C, one heater 55 is set to 72°C, and one heater 55 is set to 95°C, but different temperatures are possible in other embodiments. However, in other embodiments, the temperature of each heater 55 may vary at different times during operation. Each heater 55 has a recess ( Figure 3 (Not shown) is used to receive a sample chamber located at the bottom of the cartridge 17. A sample is inserted into the sample chamber, and the heater 55 engages with the sample chamber at different times to heat the chamber during arm-PCR of the sample. Additionally, in one embodiment, a heater 55 may be raised to contact a microarray (not shown) at the bottom of the detection chamber for hybridization and extraction.

[0064] The heater assembly 46 further includes a base motor 57, a base plate 58, and a track 59. The base plate 58 is coupled to each adjustable base 56, and the base plate 58 is slidably engaged with the track 59 to facilitate horizontal movement of the heater 55 along the track 59. In one embodiment, the motor 57 is rotatably engaged with the base plate 58 to facilitate horizontal movement of the base plate 58 (parallel to the x-direction). Thus, when adjustment of the horizontal position of the heater 55 is required, the motor 57 causes the base plate 58 to slide horizontally along the track 59 a desired distance.

[0065] Module 40 further includes a lift assembly 47 located below the heater assembly 46. The lift assembly 47 includes at least one cam 60 and at least one sensor 61. In one embodiment, assembly 47 includes two cams 60 and two sensors 61, but in other embodiments, other numbers of cams 60 and sensors 61 are possible. Furthermore, the cams 60 are configured to rotate and contact the heater base 56 to lift the heater 55 to contact the sample chamber or detection chamber of the housing 17. In one embodiment, each cam 60 is volute-shaped, such that when the cam 60 is in its home position, the cam 60 does not contact any heater base 56, but when the cam 60 is in the engaged position, the cam 60 contacts and lifts the heater base 56. However, in other embodiments, different cam shapes are possible. Furthermore, in one embodiment, one cam 60 is configured to lift the heater 55 to the sample chamber, and one cam 60 is configured to lift a heater to the microarray on the detection chamber. However, in other embodiments, other configurations are possible. A sensor 61 corresponding to each cam 60 is configured to detect whether the cam 60 is in its original position and transmit such detection results to the onboard control element 48 of component 40.

[0066] Figure 4 Depicting Figure 3 Rear perspective view of processor module 40. (See image) Figure 4 As shown, in one embodiment, a camshaft motor 43 is coupled to a pulley 65 via a belt 66. The pulley 65 is positioned around and coupled to the outer surface of the camshaft 50. The camshaft motor 43 is coupled to an onboard control element 48, which controls the operation of the motor 43 based on a predefined setting 32, as stated above. When the motor 43 rotates, the belt 66 rotates in the direction of rotation of the motor, thereby engaging the pulley 65 and causing the camshaft 50 to rotate in the same direction. As stated above, the rotation of the camshaft 50 causes rotation of the camshaft of the cartridge, which adjusts the vertical position of the pipette within the cartridge 17. In one embodiment, a camshaft sensor 67 is located behind the camshaft 50 and the pulley 65, and detects the camshaft 50 as it extends past the sensor 67. When the camshaft 50 is detected, the sensor 67 transmits a signal to the control element 48. Figure 3 ), to detect the insertion of box 17 and hold box 17 within container 42 for processing.

[0067] Pump pin motor 44 is coupled to plunger 52 ( Figure 3And control the horizontal position of plunger 52. In one embodiment, the pump pin motor 44 comprises a linear motor, but in other embodiments, other types of motors 44 are possible. The motor 44 is coupled to an onboard control element 48, and the control element 48 controls the operation of the motor 44 to manipulate the plunger 52 for performing arm-PCR within cartridge 17, as described above.

[0068] The lead screw motor 45 is coupled to the pulley ( Figure 4 (Not shown in the image), the pulley is coupled around the outer surface of the lead screw shaft 53. In one embodiment, the lead screw motor 45 is coupled to the pulley via a belt 68. When the motor 45 rotates, the belt 68 rotates in the same direction and engages the pulley, causing the pulley and the lead screw shaft 53 to pivot in the same direction. The rotation of the lead screw shaft 53 causes the lead screw of the cartridge 17 to rotate, thereby adjusting the horizontal position of the pipette within the cartridge 17 and facilitating arm-PCR. Module 40 further includes a lead screw shaft sensor 69 located behind the lead screw shaft 53 and the pulley. The sensor 69 is configured to detect the shaft 53 and notify the control element 48 of the position of the shaft to ensure that the shaft 53 is properly engaged with the cartridge 17.

[0069] Figure 5 Depicting Figure 3 The heater assembly 46 includes a heater 55, a base 56, a base motor 57, a base plate 58, and a track 59. In one embodiment, the heater assembly 46 includes three heaters 55 and three bases 56, but in other embodiments, other numbers of heaters 55 and bases 56 are possible. Each heater 55 is located on a base 56, and each base 56 is slidably coupled to the base plate 58. In this respect, each base 56 is coupled to the base plate 58 such that the base 56 can move freely upward and downward (parallel to the y-direction). The cam 60, discussed in more detail below, Figure 3 The heater 55 contacts the base 56 and slides the base 56 perpendicularly (parallel to the y-direction) relative to the substrate 58 so that the heater 55 contacts the sample chamber or detection chamber of the cartridge 17. In addition, each heater 55 has a recess 70 located in the top surface of the heater 55 for receiving the sample chamber of the cartridge 17, as will be discussed in more detail below.

[0070] The substrate 58 is slidably coupled to the track 59, allowing adjustment of the horizontal position of the substrate 58. A motor 57 controls the movement of the substrate 58. Thus, when the setting 32 requires the desired heater 55 to contact the housing 17, the motor 57 adjusts the horizontal position of the substrate 58 such that the desired heater 55 contacts the housing 17 when vertically lifted. In one embodiment, the substrate 58 engages with the track 59 and has a threaded channel (not shown) for receiving a horizontally oriented threaded rod 71. The rod 71 is coupled to the motor 57, and rotation of the motor 57 causes rotation of the rod 71, thereby adjusting the horizontal position of the substrate 58 along the track 59. However, in other embodiments, other devices for adjusting the position of the heater 55 are possible.

[0071] Figure 6 Depicting Figure 3 An exemplary embodiment of heater 55. In one embodiment, heater 55 comprises metal, but in other embodiments, other materials are possible. Heater 55 has a flat top surface 73 having a recess 70 extending downward into the surface 73. The recess 70 is sized to accommodate box 17 ( Figure 1 The sample chamber is configured such that the chamber fits into the recess 70 and the outer surface of the chamber contacts the surface defining the recess 70.

[0072] In one embodiment, heater 55 includes at least one magnet (not shown) positioned adjacent to recess 70. In another embodiment, heater 55 includes a plurality of electromagnets that can be selectively activated, when needed, to magnetically couple to metal beads (not shown) within the sample chamber. In this regard, onboard control element 48 sends control signals to heater 55 to activate the electromagnets in the heater, thereby generating a magnetic flux magnetically coupled to the beads within the chamber. The beads bind DNA from the sample, and the electromagnets magnetically couple the beads through the sample chamber to position the beads in the desired orientation within the chamber. Magnetic coupling to the beads facilitates steps in the arm-PCR process, such as the addition and / or removal of fluids, without unintentionally removing the beads from the chamber. As an example, the electromagnets may be magnetically coupled to the beads to retain them at the bottom of the sample chamber. The magnets also provide sufficient spacing between the beads within the chamber such that all DNA attached to the beads is exposed to the arm-PCR process. In other embodiments, other types of magnets (e.g., permanent magnets) are possible. When heater 50 is raised to the engagement position and receives the sample chamber, heater 55 transfers heat to the chamber, thereby performing the steps in the arm-PCR process.

[0073] In one exemplary embodiment, an electromagnet near the bottom of recess 70 is activated to pull the beads toward the bottom of the sample chamber. To agitate or mix the beads, such an electromagnet is deactivated, and another electromagnet (e.g., one near the side of recess 70) is activated to move the beads from the bottom of the chamber. The activation state of the electromagnets is then reversed to pull the beads toward the bottom of the sample chamber again. However, in other embodiments, the magnet may comprise a permanent magnet that is activated and deactivated by raising and lowering the magnet, respectively.

[0074] The heater 55 further includes an electrical interface 75 located on one side of the heater 55. Interface 75 receives power from a power source (not shown), thereby enabling the heater 55 to achieve a preset setting 32. Figure 2 The desired temperature is stated in the diagram. In this regard, heater 55 has an internal or external resistive element (not shown) that generates heat when current is applied to it. Therefore, control element 48 can control when heat is generated by heater 55 by controlling the electrical signal applied to the resistive element via electrical interface 75. Interface 75 also communicates with control element 48, and control element 48 communicates via a processor interface (…). Figure 2 It communicates with control element 15 and allows control logic 31 to activate the desired magnet within heater 55 based on setting 32.

[0075] Figure 7 Depicting Figure 3 An exemplary embodiment of the lift assembly 47. As stated above, in one embodiment, the lift assembly 47 includes at least one cam 60 and at least one sensor 61. Assembly 47 further includes a base unit 77, the cam 60, and the sensor 61. The cam 60 is rotatably coupled to the base unit 77. In one embodiment, the base unit 77 includes at least one electric motor (not shown) for controlling the rotation of the cam 60, and the base unit 77 is coupled to an onboard control element 48. The onboard control element 48 is connected to control element 15 ( Figure 1 ) communication to allow control logic 31 ( Figure 2 Based on predefined settings 32 ( Figure 2 Control the rotation of cam 60.

[0076] Sensor 61 is also coupled to onboard control element 48, and sensor 61 is configured to detect whether the corresponding cam 60 is in its home position and transmit such detection result to control element 48. In one embodiment, sensor 61 comprises a proximity sensor, but in other embodiments, other types of sensors 61 are possible. Control element 48 controls the rotation of cam 60 based on a comparison of the detection result with the desired orientation of cam 60 as defined in applicable setting 32. For example, if sensor 61 detects that cam 60 is in its home position, but setting 32 requires heater 55 above cam 60 to heat the housing 17, then control element 48 will rotate the motor, thereby rotating cam 60 to an engaged position, which causes cam 60 to contact base 56 and raise heater 55 to the engaged position.

[0077] In one implementation, when box 17 is in container 42 ( Figure 3 When the sample chamber is in the sample compartment, a cam 60 is placed below the housing 17, such that the cam 60 is vertically aligned with the sample chamber. Therefore, when the cam 60 rotates to the engaged position, the heater 55 aligned above the cam 60 is lifted into the sample chamber, causing the recess 70 ( Figure 6 The sample chamber will be received. Furthermore, in one embodiment, another cam 60 is located below the housing 17, such that cam 60 is perpendicularly aligned with the detection chamber, and when cam 60 rotates to the engaged position, the heater 55 aligned above cam 60 will contact the microarray. However, in other embodiments, other orientations of cam 60 are possible.

[0078] Figure 8 Depicting Figure 3 An exemplary embodiment of the camshaft 50. In one embodiment, the camshaft 50 comprises metal, but other materials are possible. The camshaft 50 includes a shaft portion 80 having a slot 81. The shaft portion 80 is connected to the pulley 65 ( Figure 4 ) Extend and enter container 42 ( Figure 3 In. Slot 81 receives the contents of box 17 ( Figure 1 The knob (not shown) is located at the end of the cam rod. Therefore, rotation of the cam rod motor 43 causes rotation of the shaft portion 80, which in turn causes rotation of the cam rod due to the engagement of the slot 81 with the cam rod. Rotation of the cam rod adjusts the vertical position of the pipette within the housing 17, as described above. Although Figure 8 The camshaft 50 has a slot 81, but in other embodiments, other devices for engaging with the camshaft are possible.

[0079] Figure 9 Depicting Figure 3An exemplary embodiment of the plunger 52. In one embodiment, the plunger 52 comprises metal, but other types of materials are possible. The plunger 52 includes a base portion 85 and a tip 86. The base portion 85 is connected to the pump pin motor 44 ( Figure 3 The plunger 52 extends from the base portion 85 to the pump pin of the housing 17. The pump pin motor 44 moves the plunger 52 in a horizontal direction, thereby causing the pipette to release and expel fluid from the housing 17, as described above. Although Figure 9 The plunger 52 is disclosed, but in other embodiments, other devices for engaging the pump pin are possible.

[0080] Figure 10 Depicting Figure 3 An exemplary embodiment of the lead screw shaft 53. In one embodiment, the lead screw shaft 53 comprises metal, but other materials are possible. The lead screw shaft 53 includes a shaft portion 88 having a slot 89. The shaft portion 88 is coupled to the lead screw motor 45 ( Figure 3 ) pulley ( Figure 4 (Not shown in the text) Extends and enters container 42 ( Figure 3 In. Slot 89 receives the contents of box 17 ( Figure 1 The lead screw (not shown) is located at the end of the cam rod. Therefore, rotation of the lead screw motor 45 causes rotation of the shaft portion 88, which in turn causes rotation of the lead screw due to the engagement of the slot 89 with the lead screw. Rotation of the lead screw adjusts the horizontal position of the pipette within the housing 17, as described above. Although Figure 10 The lead screw shaft 53 has a slot 89, but in other embodiments, other devices for engaging with the lead screw are possible.

[0081] Figure 11 A heater 55 is depicted in the sample chamber of receiving box 17. Box 17 is located within container 42. (As shown by...) Figure 11 As shown, the lifting assembly 47 engages with the heater assembly 46. In this regard, the cam 60 of the lifting assembly 47 rotates to the engaged position, causing the cam 60 to contact the base 56 of the heater 55. This contact between the cam 60 and the base 56 raises the base 56 relative to the base plate 58, causing the heater 55 to rise upwards toward the container 42.

[0082] Recess 70 of heater 55 Figure 6 The sample chamber (not shown) extends from the bottom surface of cartridge 17. Heater 55 transfers heat and / or a magnetic field to the sample chamber for steps in the arm-PCR process. Cam 60 remains in the engaged position such that heater 55 contacts cartridge 17 until control logic 32 ( Figure 2Based on the predefined setting 32, cam 60 returns to its original position. When cam 60 returns to its original position, cam 60 rotates to disengage from base 56, causing base 56 to return to a lowered position relative to base plate 58 due to gravity or a return spring (not shown).

[0083] Figure 12 Depicting Figure 1 An exemplary embodiment of the reader 14. In one embodiment, the reader 14 includes a drive assembly 95, a flywheel assembly 96, and an optical assembly 97 located within a housing 98. The drive assembly 95 is located above the flywheel assembly 96 and is configured to engage with and control the rotation of the flywheel assembly 96, as will be discussed in more detail below. The drive assembly 95 includes a large pulley 100, a small pulley 101, and a belt 102. The belt 102 engages tightly around the large pulley 100 and the small pulley 101 such that rotation of the small pulley 101 causes rotation of the large pulley 100 due to the belt 102. The small pulley 101 is connected to a polyflex shaft extending downward from the drive assembly 95. Figure 12 The large pulley 102 is coupled to a motor (not shown) via a flywheel shaft (not shown), and the motor controls the rotation of the small pulley 102. Figure 12 (Not shown) is coupled to flywheel assembly 96. Therefore, the motor controls the rotation of flywheel assembly 96 via drive assembly 95.

[0084] The flywheel assembly 96 includes a flywheel 105 and a plurality of containers 106 located above the top surface of the flywheel 105. In one embodiment, the flywheel 105 is circular and has four containers 106 located above it; however, in other embodiments, other numbers of containers and different shapes of flywheel 105 are possible. Each container 106 is sized to accommodate a box 17 (…). Figure 1 Furthermore, each container 106 faces the edge of the flywheel 105, allowing the reagent kit 17 to be inserted into each container 106, with the container facing the front of the reader 14. The flywheel 105 has multiple openings ( Figure 12 (Not shown in the image) is used to allow the optical component 97 to detect the microarrays of each cartridge 17 located within the flywheel assembly 96, as will be discussed in more detail below. The flywheel assembly 96 is configured to rotate at a high speed, thereby allowing the optical component 97 to rapidly scan the microarrays of each cartridge 17 within the assembly 96.

[0085] Optical component 97 is slidably mounted to flywheel support plate 110 of housing 98. Optical component 97 is configured to detect microarrays located within each housing 17 of flywheel assembly 96 and transmit data indicative of the microarrays to local control element 109. Control element 109 is connected to control element 15 (…). Figure 1The optical component 97 is communicated to control the operation of the components of the reader 14. The optical component 97 slides slowly along the track 108 on the support plate 110 located on the housing 98, so that the component 97 scans the entire microarray of each box 17 within the rotating flywheel component 96, as will be discussed in more detail below.

[0086] Figure 13 Depicting Figure 12 Remove the reader 14 of the driver component 95. (Example) Figure 13 As shown, the flywheel assembly 96 includes a flywheel 105 and a plurality of containers 106 located on the flywheel 105. Each container 106 receives a housing 17. The flywheel 105 is located on a flywheel support plate 110 rigidly mounted to the housing 98. The flywheel 105 is rotatably mounted to the support plate 110 and is rotatable relative to the support plate 110 by 360 degrees. The support plate 110 has an opening (not shown) for allowing light from the optical assembly 97 to pass through to a microarray reaching the housing 17 to allow the optical assembly 97 to capture data indicating a target pathogen detected by the microarray.

[0087] Figure 14 Depicting Figure 12 The reader 14 removes the drive assembly 95 and flywheel assembly 96. The optical assembly 97 includes an optical cube assembly (OCA) 114 and a laser 115 mounted to the mounting plate 116. Figure 14 (Not shown in the image), as discussed in more detail below. The OCA 114 includes a lens 117 for detecting the area located at the flywheel assembly 96 (…). Figure 12 Each box contains 17 microarrays ( Figure 1 Optical component 97 is coupled to control element 109, and optical component 97 is configured to transmit signals indicating the image of the microarray of each box 17 to control element 15. Control element 15 compares the image of each box 17 with predefined data 35 of box 17 to determine whether the target pathogen has been detected in the sample within box 17.

[0088] The reader 14 further includes a power supply 120 configured to supply power to components of the reader 14. The power supply 120 can supply power to components of the reader 14, such as a laser 115, a motor (not shown), and sensors. Figure 14 (Not shown in the image) Powered by electrical components such as [other components].

[0089] Figure 15 Depicting Figure 12 An exemplary embodiment of the drive assembly 95 of the reader 14. As stated above, the drive assembly 95 includes a large pulley 100, a small pulley 101, and a belt 102. The large pulley 100 is generally circular, and the large pulley 100 is connected to the flywheel 105 ( Figure 12The large pulley 100 is vertically aligned. The large pulley 100 is coupled to the flywheel shaft (not shown), and the flywheel shaft extends downward from the center of the large pulley 100 to the center of the flywheel 105 so that the large pulley 100 is coupled to the flywheel 105.

[0090] Small pulley 101 is aligned perpendicularly to a motor (not shown). Small pulley 101 is coupled to a polyflex shaft 123, and shaft 123 extends downward from small pulley 101 to the motor to couple small pulley 101 to the motor. Small pulley 101 is coupled to shaft 123 via coupling mechanism 125. In one embodiment, coupling mechanism 125 includes a locating pin, a screw, a nut, and a washer, wherein the screw engages with a threaded channel within the shaft to couple small pulley 101 to shaft 123. However, in other embodiments, other types of coupling mechanisms 125 are possible. The motor controls the rotation of polyflex shaft 123, and the rotation of polyflex shaft 123 causes rotation of small pulley 101. Belt 102 is positioned around large pulley 100 and small pulley 101 such that the belt is tightly stretched, and rotation of one pulley 100 and 101 causes rotation of the other pulley 100 and 101. Therefore, the rotation of the motor causes the small pulley 101 to rotate, and the rotation of the small pulley 101 causes the large pulley 100 to rotate via the belt 102. The rotation of the large pulley 100 causes the flywheel 105 to rotate. Therefore, the motor controls the rotation of the flywheel 105 via the drive assembly 95.

[0091] Figure 16 Depicting Figure 12 An exemplary embodiment of the flywheel assembly 96. As stated above, the flywheel assembly 96 includes a flywheel 105 and a plurality of containers 106 located on the top surface of the flywheel 105. Each container 106 is sized to accommodate a box 17 ( Figure 1 In one embodiment, the flywheel assembly 96 includes four containers 106 positioned perpendicularly to each other. Therefore, the reader 14 can read four containers 17 simultaneously. Furthermore, in one embodiment, multiple readers 14 can be coupled to each other to read multiple containers 17 simultaneously. For example, 12 readers 14 can be connected to process 48 containers 17 simultaneously. However, in other embodiments, a different number of containers 17 can be read by the readers 14 simultaneously. In one embodiment, when a container 17 is not located within each container, each container 106 includes an automatic balancer (not shown) for maintaining the flywheel's balance during the reading process.

[0092] A flywheel shaft (not shown) extends vertically from the center of flywheel 105 between the back surfaces of container 106 to couple flywheel 105 to large pulley 100, as described above. Flywheel 105 is positioned relative to flywheel support plate 110 ( Figure 13 ) can rotate freely 360 degrees to allow each box 17 to face the reader 14 as needed. Figure 12 The front orientation of the box 17. Note that when the box 17 is located within the container 106, each container 106 is positioned such that the microarray of the box 17 is aligned with the opening in the flywheel 105. Figure 16 (Not shown in the image) Alignment to allow optical component 97 to detect the microarray.

[0093] The flywheel 105 rotates at a high speed, for example, at a horizontal speed exceeding 400 rpm. During this high-speed rotation, the flywheel 105 and other related components experience increased horizontal kinetic energy. Therefore, the load within the flywheel 105 must be carefully balanced. When the flywheel 105 rotates at a high speed, even small differences in mass load can lead to large force imbalances. An unbalanced flywheel 105 may permanently damage the reader 14 or other device components, causing injury to the device user or contamination of the sample. Generally, balancing the flywheel 105 is achieved by using the housing 17 in combination with a balancing device of equal weight, or by using various balancing modes without a balancing device.

[0094] Figure 25 and 26 An exemplary embodiment of the flywheel container 106 is depicted. In one embodiment, the container 106 includes a housing 200. Now turning to Figure 25 The housing 200 includes a first side surface 202 having a beveled recess 204. The recess 204 has evenly spaced and identically sized teeth 206 on its upper side surface 208 and lower side surface 210. The teeth 206 protrude inwardly into the beveled recess 204.

[0095] Figure 26 The diagram illustrates the second side surface 212 of the housing 200. The second side surface 212 has a beveled recess 214, which is located on the lower side surface 218 (not shown, see below). Figure 16 The surface has evenly spaced and identically sized teeth 216. Teeth 218 protrude into the oblique recess. Figure 16 The first side 202 and the second side 212 contain locking tongues 220 that simultaneously have pins 222 and grooves 224. The locking tongues 220 securely link the first side 202 and the second side 212. When engaged, the first side 202 and the second side 212 form the container 106 of the receiving box 17.

[0096] Figure 27AA gear counterweight 240 is depicted for balancing a flywheel 105 containing a non-even number of housings 17 during high-speed rotation. The gear counterweight 240 includes a first side 242 with two rotating pinions 246 and 250. Each of the gears 246 and 250 includes a disc-shaped part with radially projecting interlocking teeth 248 and 252. The edge of each tooth 248 and 252 is straight and aligned parallel to the axis of rotation. The gears 246 and 250 are precisely sized to allow them to be mounted into a slanted recess 204 on the first side 202 of the housing 200. Figure 25 Here, gear teeth 248 and 252 are different from non-rotating tooth 206. Figure 16 Meshing. (Reference) Figure 27A and 27C The counterweight 240 also includes a second side surface 244 having a rotating pinion 254 containing teeth 256. The pinion 254 is precisely sized to fit into a sloping recess 214 on the second side surface 212 of the housing 202. The teeth 256 and non-rotating teeth 216 (… Figure 16 The precise alignment of gears 246, 250, and 254 with the non-rotating gear 206 allows the counterweight 240 to move up and down along the angled recesses 204 and 214. The engagement of gear teeth 248, 252, and 256 with the non-rotating gear 206 maintains the counterweight 240 in an upright position and prevents the counterweight 240 from tilting, rotating, or otherwise becoming misaligned within the recesses 204 and 214.

[0097] Figure 13 An exemplary embodiment of a flywheel container 106 containing a box 17 is depicted. In this embodiment, the box 17 is inserted into the container 106. The box 27 intersects the container 106 such that the rear end 265 of the box 17 contacts the counterweight 240. The box 17 moves into the container, applying force to the counterweight 240 and initiating rotation of gears 246, 250, and 254. This rotation causes gear teeth 248, 252, and 256 to engage and disengage from non-rotating teeth 206, resulting in the counterweight 240 moving upward along the inclined recesses 204, 214. The full insertion of the box 17 stores the counterweight 240 at the end of the container 106 opposite to where the box was inserted. The counterweight 240 is now located near the center of the flywheel 106 and is prevented from moving downward along the container due to the presence of the box 17. Figure 13 Therefore, even during high-speed rotation of the flywheel 106, the counterweight 240 will maintain this central position. Furthermore, the balance center of the flywheel 106 will remain at or near the midpoint of the device, thus preventing any force imbalance during rotation.

[0098] In another embodiment, the box 17 is removed from container 106. Gravity causes the counterweight 240 to move downwards along the recesses 204, 214. These forces cause rotation of gears 246, 250, and 254 and engagement and disengagement of gear teeth 248, 252, and 256 with non-rotating teeth 206. The counterweight 240 moves downwards along the recesses 204, 214 until gears 246, 250, and 254 abut against the bottom edge 260 of the recesses 204 and 214. The bottom edge 260 then prevents further movement of the counterweight 240. As described above, the engagement of gear teeth 248, 252, and 256 with non-rotating teeth 206 maintains the counterweight 240 in an upright position and prevents the counterweight 240 from tilting, rotating, or otherwise becoming misaligned within the recesses 204 and 214. After rotation begins, centrifugal force further pushes the counterweight 240 away from the center of rotation of the flywheel 17. However, the bottom edge 260 prevents the counterweight 240 from moving away from the flywheel 17. The placement of the counterweight 240 near the opening of the container 106 creates a center of gravity similar to that observed when the box 17 is inserted. Therefore, the flywheel 106 can be automatically balanced regardless of which housing 200 is inserted.

[0099] In this regard, if the box 17 is inserted into each of the housings 200, the torque exerted on the flywheel 105 by the housing 200 (including the box 17 and the counterweight 240, all of which are pushed close to the center of the flywheel 105 as described above) is evenly distributed, so that the flywheel 105 is balanced and should rotate smoothly without wobbling or other disturbances. If the box 17 is removed from any of the housings 200, the counterweight 240 for that housing 200 automatically moves from near the center of the flywheel 105 to a position away from this center until the gears 246, 250, and 254 abut against the bottom edge 260 of the recesses 204, 214, as described above. The mass of the counterweight 240 is chosen such that the torque exerted on the flywheel 240 by the housing 200 without the box 17 is substantially equal to the torque exerted on the flywheel 240 by the other housing 200 with the box 17 inserted. In other words, moving the counterweight 240 away from the center of the flywheel increases the torque caused by the counterweight 240, thereby accommodating the removed box 17. Therefore, regardless of which housing 200 actually inserts the box 17, the torque exerted on the flywheel 105 by all housings 200 is substantially equal and evenly distributed around the flywheel 105, so that the flywheel 105 remains balanced during rotation and does not wobble or experience other disturbances.

[0100] Figure 17 Depicting Figure 12The flywheel 105 has a plurality of microarray openings 130 extending vertically through it. The microarray of the housing 17 can be aligned with each opening 130 to allow an optical component 97 located below the flywheel 105 to capture data indicating a target pathogen detected by the microarray of each housing 17. In one embodiment, the flywheel 105 has four microarray openings 130 corresponding to four containers 106. Figure 12 However, in other embodiments, any number of containers 106 and openings 130 are possible.

[0101] The flywheel 105 also has a central opening 131 for receiving a flywheel shaft (not shown). The flywheel 105 is coupled to the flywheel shaft through the opening 131, and the flywheel shaft extends vertically to couple the flywheel 105 to the drive assembly 95. Figure 12 ) large pulley 100 ( Figure 12 The flywheel 105 is rotated by the drive assembly 95 via a shaft.

[0102] Figure 18 Depicting Figure 17 A bottom perspective view of the flywheel 105. In one embodiment, the flywheel 105 includes a rim 135 extending 360 degrees around a central opening 131 on the bottom surface of the flywheel 105. The rim 135 indicates the orientation of the flywheel 105, such that the control element 109 ( Figure 12 ) and control element 15 ( Figure 2 This allows us to determine which box 17 corresponds to which microarray data. As an example, in one embodiment, the rim 135 has three small gaps 137 and one large gap 138, but in other embodiments, other numbers of gaps 137 are possible.

[0103] A sensor (not shown) is coupled to control element 109 and detects gaps 137 and 138 in the rim 135. In one embodiment, the sensor comprises a proximity sensor for detecting the rim 135, but in other embodiments, other types of sensors are possible. When the sensor detects gap 137 or 138 in the rim, the sensor transmits a signal indicating the size of gap 137 or 138 to control element 109. As the flywheel 105 rotates rapidly, the sensor repeatedly transmits signals indicating the size of gaps 137 and 138 in the rim 135 to control element 109, thereby indicating the orientation of each box 17 on the flywheel 105. In this regard, control element 109 identifies each box 17 by its relationship to the large gap 138. This identification is achieved by a detection element (not shown) that detects the identifier of the box 17 in container 106, or by a user via user input interface 26. Figure 2By manually inputting identifiers, control element 109 associates each box 17 with its corresponding container 106. For example, in one embodiment, a large gap 138 is aligned perpendicularly to a particular container 106. Control element 109 associates the large gap 138 with a box 17 in the container 106, and identifies the box 17 as the first box 17, and identifies other boxes 17 sequentially from the first box 17 by the distance of all other boxes 17 on flywheel 105. Thus, when control element 109 receives a signal from a sensor indicating that a large gap 138 has been detected, control element 109 associates the microarray data detected at this time by optical component 97 with the first box 17. Control element 109 further associates subsequent microarray data corresponding to a small gap 137 with its corresponding box 17. Thus, control element 109 can accurately associate microarray data with the appropriate box 17 based on the position of the box relative to the large gap 138.

[0104] Figure 19 Depicting Figure 12 An exemplary embodiment of the optical component 97. The optical component 97 includes an OCA 114 and a laser 115 mounted to a mounting plate 116. The component 97 is slidably mounted to a track 108 via a guide rail 140. Figure 12 ). Figure 20 Depicting Box 17 ( Figure 1 An exemplary microarray 144 is provided. Microarray 144 includes a plurality of dots 145 oriented in multiple rows 146-149. Dots 145 are pre-formed on microarray 144, and each dot is made of a different material. If a specific target pathogen is present in the sample, the DNA of the target pathogen binds to certain dots 145. Figure 20 The exemplary microarray 144 shown comprises four rows 146-149, each with four dots 145; however, in other embodiments, different numbers of rows 146-149, dots 145, and dot patterns are possible. Movement of the optical component 97 along track 108 allows the optical component 97 to detect one dimension of the microarray 144, while the flywheel 105 (… Figure 12 The rotation of the flywheel 105 allows the optical component 97 to detect another dimension of the microarray 144. In this regard, as the flywheel 105 rotates, the optical component 97 performs a raster scan (where the optical component 97 is stationary) and uses a laser to scan the points 145 of a row 146 in the microarray 144. After the flywheel 105 has rotated completely at least once, the optical component 97 slides horizontally to scan the points 145 of the next row 147. The optical component 97 continues this process until the points 145 of each row 146-149 have been scanned. Thus, simultaneously rotating the flywheel 105 and sliding the optical component 97 along the track 108 allows the optical component 97 to capture data indicating each box 17 of the microarray 144 located on the flywheel 105.

[0105] Refer again Figure 19 OCA 114 includes a lens 117 attached to beam splitter cube 142, and OCA 114 is configured to determine which DNA has bound to a point based on which point fluoresces when illuminated with a laser beam, as described in more detail below. In this regard, laser 115 delivers a laser beam (not shown) into OCA 114, and the laser beam travels into beam splitter cube 142. A portion of the beam is reflected and exits OCA 114 through lens 117. In one embodiment, a mirror (not shown) is placed within beam splitter cube 142 to redirect the laser beam away from lens 117, but other methods of reflecting the beam within cube 142 are possible. The laser beam is delivered to microarray 144 on cartridge 17 and causes the amplified DNA attached to point 145 of microarray 144 to fluoresce. Fluorescent point 145 corresponds to a target pathogen designed for detection in an arm-PCR process. In this regard, the arm-PCR process amplifies DNA corresponding to a specific target pathogen. Amplified DNA of a specific target pathogen attaches to a specific spot 145 in the microarray 144, and spot 145 fluoresces when exposed to a laser beam. The fluorescent spot 145 is detected by a photodetector 143 through a lens 117. In one embodiment, the photodetector 143 comprises a photomultiplier tube (PMT), but in other embodiments, different types of photodetectors 143 are possible. For example, in one embodiment, a high-sensitivity camera can be used to capture images of the microarray on each cartridge 17 within the reader 14.

[0106] As the optical component 97 moves along track 108, the fluorescence of each point 145 in the microarray 145 is detected. If the photodetector 143 detects a fluorescent point 145, then control logic 32 ( Figure 2 ) Regarding test data 34 ( Figure 2 The control logic 32 marks each point 145 in the microarray 144. This marking is performed on each point 145 in the test data 34, ensuring that all fluorescent points 145 corresponding to the target pathogen being detected are marked. The control logic 32 compares the test data 34 indicating the fluorescent points 145 with predefined data 35. Figure 2 And through user output interface 28 ( Figure 2 The results of this comparison are then transmitted to the user, as described above. Therefore, the user can make a diagnosis based on the comparison between test data 34 and predefined data 35.

[0107] Figure 21 Depicting Figure 19An exploded view of the OCA 114. The OCA 114 includes a beam splitter cube 142, a lens 117, an input component 150, and a photodetector component 151. The input component 150 is located on one side of the OCA 114 and faces the laser 115. Figure 19 Orientation. Input member 150 receives the laser beam from laser 115 and allows the beam to enter beam splitter cube 142. Beam splitter 152 is located within cube 142 and is configured to redirect a portion of the laser beam toward lens 117. Lens 117 is located on top of OCA 114 and focuses the portion of the laser beam received from beam splitter 152 onto microarray 144. Figure 20 The amplified DNA is attached to the spot 145 in the microarray 144 and fluoresces. The fluorescence from the spot 145 travels back into the lens 117, through the cube 142 and out of the OCA 114 through the photodetector 151.

[0108] The optical detection component 151 is located on the bottom of the OCA 114 and is coupled to the optical detection element 143 via the sleeve 155. Figure 19 The photodetector 151 allows fluorescence from the microarray 144 to be transmitted to the photodetector 143 for transmission to the control element 109. The OCA 114 further includes a beam collecting member 156. The beam collecting member 156 is configured to receive and collect a portion of the laser beam that has not been reflected through the lens 117. The OCA 114 redirects the laser beam from the laser 115 and captures fluorescence from points 145 in the microarray 144 that fluoresce due to exposure to the laser beam. Data indicating which points fluoresce is transmitted to the control element 15 for comparison with predefined data 35.

[0109] Figure 22An exemplary implementation of an open platform target solution system 170 is depicted. System 170 includes at least one communication device 172 coupled to a server 175 via a network 176. In one implementation, server 175 hosts at least one webpage 180 and includes primer selection logic 182, primer data 183, target testing logic 185, and target solution data 186. A user (e.g., a target solution developer) can utilize communication device 172 to access webpage 180 to determine primers for detecting a specific target pathogen. In this regard, the user needs to use an open platform system for performing PCR amplification to detect unmanaged target pathogens. Communication device 172 communicates with server 175 via network 176 (e.g., the Internet) to determine appropriate primers for a given target pathogen. In one implementation, the user inputs the gene sequence of the target pathogen into webpage 180. Based on the input gene sequence, primer selection logic 182 accesses primer data 183 and identifies at least one set of primers for detecting the target pathogen. In this regard, primer data 183 associates various primers with specific gene sequences. Primer selection logic 182 displays suggested primers to the user via webpage 180.

[0110] Based on the primers suggested from server 175, the user can insert the primers into box 17 ( Figure 1 ) and by setting 32 from the predefined settings Figure 2 Users can select a set of settings (e.g., high specificity setting, high sensitivity setting, or nominal setting) to perform open-platform testing on samples, as described above. Users can perform the required number of tests on samples using various primer combinations and different sets of settings 32 suggested by primer selection logic 182 until a successful test is identified. In this regard, different combinations of primers and settings 32 can produce results with varying degrees of reliability in detecting the target pathogen. Once the user has determined a reliable combination of primers and settings for detecting the target pathogen, the user has identified a solution (e.g., the “target solution”) for that target pathogen.

[0111] After determining the target solution, the user can again access server 175 via webpage 180 to submit their target solution to server 175. In this regard, the user can present their combination of primers and settings for the target pathogen as a solution for detecting the target pathogen to server 175, and the user can provide this combination to third-party users interested in obtaining a solution for the target pathogen. For this purpose, the user accesses server 175 via webpage 180 and indicates that they have determined a solution for the target pathogen. The user then manually enters the target pathogen, primers, and the set of settings from predefined settings 32 for the target solution. In one implementation, the server administrator can verify the target solution by performing tests defined by the target solution to ensure its effectiveness. However, in other implementations, other methods for verifying the target solution are possible.

[0112] The target testing logic 185 provides the user with the opportunity to store the target solution in target solution data 186. Target solution data 186 indicates primers and settings 32 for various target pathogens. Therefore, if the user chooses to store the target solution in target solution data 186, then a third-party user can obtain the target solution. In one embodiment, the user can offer the target solution for sale to a third-party user. In this regard, once the target solution is stored in target solution data 186, the third-party user (e.g., an "end user") can access server 175 via another communication device 172. The end user can then browse target solution data 186 via webpage 180 to identify the target solution for the target pathogen they need to detect.

[0113] After identifying the desired target solution, the end user can then purchase or otherwise obtain the target solution from server 175. In one embodiment, the end user can provide a representation of their desired purchase of a specific target solution for a given target pathogen via webpage 180. The server administrator or the user developing the target solution can then ship one or more boxes 17 configured to perform the target solution to the end user. For example, the boxes 17 may contain appropriate primers for detecting the target solution and may have an identifier (e.g., a barcode) on each box 17, and the server administrator can refresh ID mapping data 33 to map the identifier to an appropriate set of predefined settings 32 and predefined data 35 for the desired target solution. Thus, the end user receives one or more boxes 17 configured to detect the target solution without needing to know which specific primers and which set of settings 32 are used in the target solution. In this regard, system 10 can provide an indication of whether the target pathogen is present in the tested sample without the end user needing to know the testing details. In other embodiments, information about the target solution (e.g., the primer and set of settings 32) can be provided directly to the end user if needed.

[0114] Figure 28 An exemplary implementation of box 17 is depicted. (Reference) Figure 28 The cartridge has a pipette 220 operably connected to a rotatable cam rod 216, such that rotation of the rod 216 causes a corresponding upward and / or downward movement of the pipette 220 in the vertical direction. A pipette holder 228 supports the cartridge pipette 220 and guides its vertical movement; the pipette holder 228 is supported by the cartridge 17 and slidably located therein. A guide screw 224 located within the cartridge 17 is operably connected to the pipette holder 228, such that rotation of the guide screw 224 produces a corresponding lateral movement of the pipette holder 228, thereby creating a manner for positioning the pipette 220 above the appropriate fluid orifice 249 at each stage of the amplification / detection process.

[0115] The base 204 of the cartridge 17 includes at least one sample chamber 242 and at least one reagent chamber 249 for containing reagents (not shown). The reagent chambers 249 may have the same, similar, or different sizes, shapes, and depths and may be arranged in multiple locations within the base 204 of the cartridge 17. The required reagents (not shown) are placed in the appropriate reagent chamber 249 such that, while the process is performed within the cartridge 17, the cartridge pipette 220 can collect the reagents required for extraction and two-step dual-primer amplification. The reagent chamber 249 may be preloaded and preferably sealed before shipment with a sealing material comprising a material that will remain in place during shipment and storage but is easily perforated by the downward force of the cartridge pipette to open the reagent chamber 249 to allow retrieval of the contents using the cartridge pipette 220. One such material, either alone or as a whole, suitable for sealing the reagent chambers is a thin aluminum foil sheet (not shown). In various aspects of this disclosure, there are two reagent chambers within the reagent chamber, one for target-specific primers and the other for a common non-target-specific primer. These primers are used in first and second amplification reactions. The first amplification is target-specific to provide amplicons representing a variety of targets that can be found within the sample, and the second amplification is initiated by a common primer to allow semi-quantitative, non-specific amplification of the amplicon from the first amplification. In this two-step process, the first amplification initiated by the target-specific primer provides specificity, while the second amplification initiated by the common primer increases sensitivity.

[0116] A detection chamber 248 containing a microarray 244 is also provided in the base 204 of box 17 for detecting DNA amplified in a two-step ARM-PCR protocol. Microarrays are known in the art, and methods for preparing target-specific microarrays are well known to those skilled in the art.

[0117] A filling port 214 in the top of the box allows the user to insert a pipette (not shown) from the external environment into the sample chamber 242. A transparent plastic window (not shown) may be formed in the box 17 such that when a pipette is inserted into the box 17 to hold a sample (not shown) to be analyzed, the window allows the user to see the tip of the pipette (not shown). In one embodiment, the transparent viewing window is configured to withstand the box's extreme temperatures. Alternatively, the entire housing of the box 17 may be formed of transparent or translucent plastic, allowing the user to see the internal operation of the box 17.

[0118] In one embodiment, the filler cap 212 located on top of the cartridge will be a single-use cap, meaning that once the cap is sealed after sample insertion, it cannot be reopened, thereby maintaining the integrity of the seal and keeping the system closed. In another embodiment, a sliding door 210 can be used, allowing the sliding door 210 to slide and lock into place once a sample (not shown) is introduced into the cartridge 17. The filler cap 212 seals the filler port 214. In one embodiment, the filler port 214 has a minimum inner diameter of 0.3 inches to allow a 20 μl pipette to be inserted through the filler port 214 and into the sample chamber 242. In other embodiments of this disclosure, the filler port 214 can be of other sizes.

[0119] The vertical up-and-down movement of the cartridge pipette 220 is provided by a cam rod 216, which is connected to the processor module 40 by means of a mechanical interface 218 fixedly coupled to the cam rod 216, thereby allowing the movement of the cartridge pipette 220 to be controlled by the processor module 40. In one embodiment, the mechanical interface 18 is a knob; however, other mechanical interfaces may be used in other embodiments.

[0120] The pipette 220 is supported and held in place by a pipette holder 228. The pipette holder 228 is slidably received along the length of the box 17. The pipette holder 228 can be held along the same transverse plane of the box 17 by first and second guide rails (not shown) that can be molded into the side of the box 17. The guide rails can be placed vertically parallel to each other and horizontally between the ends of the box 17. The pipette holder 228 is operatively connected to a lead screw 224. The lead screw 224 is threadedly received into the pipette holder 228 by means of a male-female thread pair between the lead screw 224 and the pipette holder 228. A mechanical interface 240 is fixedly connected to the lead screw 224, thereby allowing clockwise and counterclockwise rotation. Rotation of the mechanical interface 240 causes the lead screw 224 to rotate, and the pipette holder 228 follows the thread of the lead screw 224 and moves laterally along the lead screw 224 along the length of the box 17. Reversing the rotation direction of the lead screw 224 causes a corresponding reversal in the movement of the pipette holder 228. By controlling the number of rotations and the direction of rotation of the lead screw 228, the pipette can be precisely positioned above either the reagent chamber 249 or the sample chamber 242 located in the base 204. In one embodiment, the mechanical interface 240 is a knob; however, in other embodiments, other types of mechanical interfaces may be used.

[0121] It should be emphasized that, Figure 28 and 29 Box 17 is exemplary, and other types of boxes may be used in other embodiments.

Claims

1. A system for performing amplicon-rescue multiplex polymerase chain reaction (arm-PCR), comprising: A self-contained box containing a nucleic acid sample and having a removable pipette, wherein the box has an identifier associated with the box; A processor configured to receive and manipulate the cartridge for performing arm-PCR on the nucleic acid sample, the processor being configured to move the pipette within the cartridge, and the processor having a plurality of movable heaters for heating the nucleic acid sample during the arm-PCR, wherein each of the movable heaters corresponds to a corresponding temperature, wherein the processor is configured to select one of the movable heaters for heating the nucleic acid sample and move the selected heater to a position in contact with the sample chamber of the cartridge for heating the nucleic acid sample in the sample chamber, and wherein the heater is configured to include a plurality of magnets selectively activated to magnetically couple to metal beads within the sample chamber for performing the arm-PCR step; A control element coupled to the processor includes memory for storing control logic and predefined settings groups for performing different types of tests on the nucleic acid sample. The control element also includes at least one conventional processing element that communicates with and drives other elements of the control element via a local interface. The control element includes a computer. The predefined settings groups include a first group for isolating specific target pathogens or DNA in the sample, a second group for identifying a broad range of target pathogens or DNA sequences in the sample, and a third group for generating a nominal range of target pathogens or DNA sequences. Each set of predefined settings indicates what to be performed by the processor. The system includes multiple operations and instructions for the duration of applying a heater to the cartridge for arm-PCR of a target pathogen, the temperature of the corresponding heater, and the sequence of specific operations to be performed by the processor. The control element is configured to receive an identifier from the cartridge and select a set of predefined settings based on the identifier. The control logic of the control element is configured to control the processor to perform arm-PCR on the nucleic acid sample according to specific operational instructions for a test type executed for the selected set of predefined settings. The control logic is also configured to compare test data with predefined data mapped to the cartridge to determine whether a specific target pathogen is detected in the sample. and A reader coupled to the control element includes a drive assembly, a flywheel assembly, and an optical assembly located within a housing. The drive assembly is positioned above the flywheel assembly and configured to engage with and control the rotation of the flywheel assembly. The flywheel assembly is configured to rotate at a high speed, thereby allowing the optical assembly to rapidly scan the microarray of each cell within the flywheel assembly. The optical assembly includes an optical cube assembly and a laser mounted to a mounting plate.

2. The system of claim 1, wherein the cartridge includes a cam rod connected to the pipette, wherein the processor has a cam rod shaft engaging with the cam rod, wherein rotation of the cam rod shaft causes the cam rod to rotate, thereby moving the pipette.

3. The system of claim 1, wherein the processor causes the cartridge to store nucleic acids on a microarray in a detection chamber within the cartridge, and wherein the processor is configured to move one of the plurality of movable heaters to contact the microarray in the detection chamber.

4. The system of claim 1, wherein the control element is configured to automatically select a set of predefined settings based on the target pathogen indicated by the identifier.

5. The system of claim 1, wherein the identifier is displayed on the box, and wherein the processor includes a detection element configured to read the identifier from the box and transmit the identifier to a control element, and wherein the control logic of the control element selects a set of predefined settings from a plurality of predefined settings, the selection mapping the identifier to the selected set of predefined settings using mapping data stored in the control element.

Images