Systems, methods, and devices for pathogen identification

Abstract

Systems, methods, and devices for pathogen identification are described herein. A system includes a housing configured to receive a sample comprising one or more pathogens, a pipette system disposed within the housing, one or more centrifuges disposed within the housing, a mechanical agitator disposed within the housing, and a controller. The controller is configured to transfer the sample to a treatment tube using the pipettor system, centrifuge the treatment tube using the one or more centrifuges to concentrate the one or more pathogens in the sample, remove fluid from the treatment tube using the pipettor system, and remove the one or more pathogens from the treatment tube using the pipettor system. Thereby leaving a concentrated pathogen in the treatment tube; adding a cleavage buffer into the treatment tube using the pipettor system; moving the treatment tube to the mechanical agitator using the pipette system; and agitating the treatment tube using the mechanical agitator for cleavage of the concentrated pathogen. The system is further configured for PCR using nucleic acids extracted from the sample.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 299,611, filed January 14, 2022, which is incorporated herein by reference in its entirety. This application is related to co-pending PCT Patent Application No. _____________, filed January 13, 2023, by Bru Gibert et al., entitled "Systems, Methods, and Devices for Antimicrobial Susceptibility Testing" (Attorney Docket No. 4518.002PC01) and PCT Application No. PCT / US2022 / 039290, filed August 3, 2022, the disclosures of which are incorporated herein by reference in their entirety. Background Art Technical Field

[0002] Embodiments of the present disclosure relate to systems, methods, and devices for performing pathogen identification and / or identification of drug resistance genes from whole blood or other samples to help determine treatment for a patient. Background Art

[0003] Sepsis is defined as a life-threatening organ dysfunction caused by a host dysregulation of infection. Patients who experience or suffer from sepsis may begin with a local infection, such as pneumonia, that results in inflammation of the body caused by the patient's immune system going into overdrive. If untreated, inflammation may ultimately lead to organ failure and death of the patient. Sepsis causes 11 million deaths each year, and many of these cases can be prevented through early diagnosis, appropriate clinical management, and treatment.

[0004] Sepsis diagnosis can be made by running laboratory tests to culture a blood sample for identification of infection. Current testing times can take several days to get results from the laboratory because time is required to obtain and process blood cultures of the bacteria that cause sepsis to ultimately determine their sensitivity to effective antimicrobial agents for treatment. However, sepsis testing can be time-sensitive for patients in the hospital because sepsis can lead to septic shock and death within hours if not properly identified and treated promptly. In addition, blood culture tests are slow and may not consistently provide reliable bacterial or fungal test results in patients who are clinically suspected of having sepsis, especially for patients who have been subjected to antibiotic therapy. The yield of obtaining positive blood cultures for patients is generally low, and even if a positive blood culture is not identified, the patient may be experiencing sepsis.

[0005] Without improved solutions, patients may continue to suffer and often get worse as physicians treat with empirical antibiotics while awaiting more actionable information from the laboratory regarding the causative agent or agents (e.g., pathogens) of the infection and which antimicrobial agents to use to treat the highly resistant pathogens in patients experiencing sepsis. Summary of the invention

[0006] Embodiments of the present disclosure provide cost-effective solutions for improved diagnostic methods, systems, and devices for isolating and identifying pathogens so that appropriate treatments can be provided to patients for better patient outcomes.

[0007] Described herein is a system, method and device for directly carrying out pathogen identification from blood samples or other samples (such as urine, sterile body fluids, etc.). In the embodiment proposed herein, a pathogen identification system, an analyzer device, a polymerase chain reaction (PCR) box, a sample preparation box and a processing tube are provided, for the identification of pathogens and drug resistance genes determined by whole blood samples without a culture step and by using a multiplex PCR method, and the analysis time is significantly reduced. Pathogen identification includes using a liquid transfer system to transfer samples to consumables for further processing (e.g., nucleic acid extraction, purification and amplification), concentrating pathogens, lysing pathogens (e.g., by mechanical crushing), purifying nucleic acids from pathogens, and amplifying and identifying pathogens by nested PCR. In some embodiments, the system, method and device for pathogen identification described herein can be used to treat patients with sepsis and / or other potential diseases.

[0008] In the embodiments, an exemplary sample preparation box is described. The sample preparation box includes a housing, a removable treatment tube arranged in the housing, a first removable needle arranged in the housing, and one or more reservoirs coupled to the housing. The removable treatment tube includes a septum and is configured to hold the sample. The first removable needle is configured to transfer the sample to the removable treatment tube and / or transfer the sample from the removable treatment tube by inserting the first removable needle through the septum. The one or more reservoirs are configured to store materials for sample concentration, cracking and nucleic acid amplification.

[0009] In another embodiment, an exemplary system for analyzing a sample is described. The system includes: a housing configured to receive a sample tube containing a sample containing one or more pathogens; a pipette system disposed within the housing; one or more centrifuges disposed within the housing; a mechanical agitator disposed within the housing; and a controller. The controller is configured to transfer the sample from the sample tube to a processing tube using the pipette system, centrifuge the processing tube using the one or more centrifuges to concentrate the one or more pathogens in the sample; remove fluid from the processing tube using the pipette system, thereby leaving concentrated pathogens in the processing tube; add a lysis buffer to the processing tube using the pipette system; move the processing tube to the mechanical agitator using the pipette system; and agitate the processing tube using the mechanical agitator to perform cell lysis of the concentrated pathogens.

[0010] In another embodiment, an exemplary method is described. The method includes: receiving a sample preparation box and a sample tube by an analyzer device, the sample tube containing a sample containing one or more pathogens; installing a first needle from the sample preparation box in a pipette system in the analyzer device; inserting the first needle into the sample tube using the pipette system; transferring the sample from the sample tube to a processing tube in the sample preparation box through the first needle; adding one or more lysis reagents to the processing tube using the pipette system; and mixing the one or more lysis reagents with the sample in the processing tube to lyse blood cells in the sample. The method further includes moving the processing tube to a centrifuge in the analyzer device; centrifuging the processing tube in the centrifuge to concentrate one or more pathogens in the sample; removing fluid from the processing tube using the pipette system, thereby leaving concentrated pathogens in the processing tube; adding a lysis buffer to the processing tube using the pipette system; moving the processing tube to a device in the analyzer device using the pipette system; and agitating the processing tube using the device to perform cell lysis of the concentrated pathogens.

[0011] In another embodiment, an exemplary method is described. The method includes transferring a nucleic acid to at least one primary reaction chamber in a polymerase chain reaction (PCR) box inserted into the analyzer device by a pipette system in the analyzer device; performing a first amplification of the nucleic acid in the at least one primary reaction chamber to produce a first amplification product; and inserting a needle of the analyzer device through the septum of the at least one primary reaction chamber to remove the first amplification product. The method further includes distributing a plurality of aliquots of the first amplification product to the plurality of secondary reaction chambers in the PCR box through the respective septa of the plurality of secondary reaction chambers, wherein each aliquot corresponds to a respective secondary reaction chamber, and wherein each secondary reaction chamber includes a set of reagents for reacting with the respective aliquots of the first amplification product; and performing a second amplification of the aliquots of the first amplification product in the plurality of secondary reaction chambers.

[0012] In another embodiment, an exemplary polymerase chain reaction (PCR) box is described. The PCR box includes at least one primary reaction chamber and a plurality of secondary reaction chambers, wherein the at least one primary reaction chamber is configured to perform a first amplification of nucleic acid, thereby producing a first amplification product, and the plurality of secondary reaction chambers are configured to perform a second amplification of product nucleic acid. The at least one primary reaction chamber and the plurality of secondary reaction chambers are each sealed by a diaphragm. The diaphragm is configured to receive a needle, and each of the plurality of secondary reaction chambers is configured to receive a corresponding aliquot of the first amplification product from the needle. Each secondary reaction chamber includes a set of reagents for reacting with a corresponding aliquot of the first amplification product.

[0013] The further features and advantages and structure and operation of various embodiments are described in detail below with reference to the accompanying drawings. It should be noted that the specific embodiments described herein are not intended to be restrictive. Such embodiments are presented herein for illustrative purposes only. Based on the teachings contained herein, additional embodiments will be clear to one or more technicians in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate embodiments of the present disclosure and, together with the description, further serve to explain the principles of the present disclosure and to enable those skilled in the relevant art to make and use the present disclosure.

[0015] Figure 1 A diagram of a system for performing pathogen identification according to an embodiment of the present disclosure is shown.

[0016] Figure 2 A diagram of an analyzer apparatus according to an embodiment of the present disclosure is shown.

[0017] Figure 3A A diagram showing a front view of an analyzer device according to an embodiment of the present disclosure.

[0018] Figure 3B A diagram showing a top view of an analyzer device according to an embodiment of the present disclosure is shown.

[0019] Figure 4 A diagram of an analyzer device having three open compartments according to an embodiment of the present disclosure is shown.

[0020] Figure 5 A diagram of a sample preparation kit according to an embodiment of the present disclosure is shown.

[0021] Fig. 6A A diagram showing processing tubes and other components within a sample preparation cartridge according to an embodiment of the present disclosure is shown.

[0022] Figure 6B A diagram showing processing tubes and other components within a linear sample preparation cartridge according to an embodiment of the present disclosure is shown.

[0023] Fig. 7A and Figure 7B A diagram of a processing tube according to an embodiment of the present disclosure is shown.

[0024] Figure 7C and Fig.7D A diagram showing an exemplary reagent tube according to an embodiment of the present disclosure is shown.

[0025] Fig. 8A , Figure 8B and Figure 8C A diagram showing a needle configured for insertion into a processing tube and / or a reagent tube according to an embodiment of the present disclosure is shown.

[0026] Fig.9A and Fig. 9B Diagrams of a high volume needle and a low volume needle are shown, respectively, according to embodiments of the present disclosure.

[0027] Fig.10 A diagram showing an example of a high capacity needle according to an embodiment of the present disclosure is shown.

[0028] Fig.11A and Fig. 11B Shown is a diagram of a spin column and spin column basket according to an embodiment of the present disclosure.

[0029] Fig. 12A A diagram of a low volume needle docked with a sample preparation cartridge is shown according to an embodiment of the present disclosure.

[0030] Fig. 12BA low volume needle docked with a spin column basket is shown according to an embodiment of the present disclosure.

[0031] Fig.13A and Fig. 13B A diagram of a PCR cassette according to an embodiment of the present disclosure is shown.

[0032] Fig.14 A diagram showing a low volume needle inserted into a PCR cassette according to an embodiment of the present disclosure is shown.

[0033] Fig.15A and Fig. 15B A diagram showing an exemplary centrifuge for use in an analyzer according to an embodiment of the present disclosure is shown.

[0034] Fig.16A , Fig. 16B , Fig. 16C and Fig.16D A diagram of an exemplary homogenization subsystem for use in an analyzer according to an embodiment of the present disclosure is shown.

[0035] Fig.17A , Fig. 17B and Fig. 17C A diagram showing an exemplary mechanical device for use in an analyzer according to an embodiment of the present disclosure is shown.

[0036] Figures 18A-18C A PCR cartridge is shown docked with a fluorescent sensor in a PCR subsystem in an analyzer according to an embodiment of the present disclosure.

[0037] Figure 19A-19B Further shown is a PCR cartridge interfaced with a fluorescent sensor in a PCR subsystem in an analyzer according to an embodiment of the present disclosure.

[0038] Fig. 20 A flow chart of a method for sample processing prior to pathogen identification, including concentration and lysis of the sample, is shown according to an embodiment of the present disclosure.

[0039] Fig.21 A flow chart of a method for performing PCR for pathogen identification of a sample according to an embodiment of the present disclosure is shown.

[0040] Fig. 22 A block diagram is shown of exemplary components of a computer system according to an embodiment of the present disclosure.

[0041] Figure 23-Figure 29 Experimental results from testing based on embodiments of the present disclosure are presented.

[0042] Embodiments of the present disclosure will be described with reference to the accompanying drawings. DETAILED DESCRIPTION

[0043] Although specific configurations and arrangements are discussed, it should be understood that this is done for illustrative purposes only. Those skilled in the relevant art will recognize that other configurations and arrangements may be used without departing from the spirit and scope of the present disclosure. It will be clear to those skilled in the relevant art that the present disclosure may also be used in various other applications.

[0044] Units, prefixes and symbols are all expressed in the form approved by the International System of Units (SI). Numerical ranges include numbers that define the ranges. In the case of enumerated value ranges, it is understood that each intermediate integer value and fraction thereof between the upper and lower limits of the enumerated ranges, and each subrange between these values ​​are also specifically disclosed. Therefore, the ranges enumerated herein are understood to be shorthand for all values ​​(including the enumerated endpoints) within the range. For example, a range of 1 to 10 is understood to include: any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0045] Where values ​​are explicitly enumerated, it is understood that values ​​of approximately the same quantity or amount as the enumerated value are also within the scope of the present disclosure. Where combinations are disclosed, each subcombination of the elements of the combination is also specifically disclosed and within the scope of the present disclosure. Conversely, where different elements or groups of elements are disclosed individually, their combinations are also disclosed.

[0046] The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any stated or listed components.

[0047] The term "about" refers to a value or composition within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 10%. In addition, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times of a value. When specific values ​​or compositions are provided in the present application and claims, unless otherwise stated, it should be assumed that the meaning of "about" is within an acceptable error range for the specific value or composition.

[0048] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer and, where appropriate, fractional values ​​thereof (such as tenths and hundredths of an integer) within the recited range. introduce:

[0049] The current standard of care for detecting and treating sepsis relies on blood culture, which has an average detection time of about 13 hours. The blood culture test provides the organism without the identification (ID) of the pathogen, and then the positive is plated on the culture dish. In the conventional blood culture process, each adult patient takes two blood culture sets, each of which consists of an aerobic bottle and an anaerobic bottle to ensure that the entire spectrum of sepsis pathogenic bacteria is captured during the culture event. Typically, each culture is obtained from a separate venipuncture (e.g., the left and right arms of the patient). This is to ensure that bacterial shedding events are captured by culture so that bacteria can be "recovered" for downstream testing (e.g., ID and AST). After cultivation, aerobic bottles and anaerobic bottles are incubated in a blood culture instrument, in which their growth is monitored in real time. Aerobic bottles and anaerobic bottles are incubated and stirred until any bacteria are allowed to undergo a hysteresis-logarithmic growth transition detected by electronics. Then, laboratory staff may be warned that the patient has a positive culture. Typically, blood cultures for most bacteria will become positive within an average of about 13 hours, while some yeasts and fungi may take longer (e.g., up to 5 days). However, many cultures are negative due to collection errors, insufficient blood volume obtained during collection, delayed transport to the laboratory, insufficient sensitivity, etc.

[0050] Due to the urgent nature of sepsis, after positive, the laboratory can immediately start inspection work to identify Gram bacteria staining (for example, Gram-positive or Gram-negative), determine important organisms, pollutants, single microbial infections or polymicrobial infections, and report intermediate information to the caregiver. In addition, the laboratory can take steps immediately to identify bacteria using rapid methods such as molecular diagnostic systems (which may require 1.5 hours to provide results). These systems can provide limited molecular information about the genetic drug resistance information of certain bacteria that exhibit these spectra. Alternatively, the laboratory can process positive blood culture (PBC) aliquots with matrix-assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometry systems in about one hour to report ID. Using ID, the caregiver can confirm or potentially adjust antibiotics that may have been prophylactically applied to the patient. However, by the time of identifying bacteria, up to 20-24 hours (at most) may have passed since the patient first cultured.

[0051] The entire process for testing positive blood culture samples and identifying pathogens can be time consuming, resulting in several days before critical antibiotic information for septic patients is reported. For example, if an infection is suspected, samples of blood, urine, sputum, etc. are collected from the patient and provided to the clinical laboratory to first determine if an infectious agent is present. This may require 18-24 hours (e.g., day 1) to allow most pathogenic bacterial species to fully grow. If the bacteria are isolated, an additional 18-24 hours (e.g., day 2) is further required to culture the isolate, and an additional 2-48 hours (e.g., day 3+) is required to identify the bacterial isolate and perform AST.

[0052] Conventional methods and systems for pathogen identification may be limited because they are growth-based, slow, expensive, require manual manipulation, and are not integrated. Current technology does not provide an integrated and comprehensive solution for the entire workflow of host response detection, pathogen identification, and antimicrobial or antibiotic sensitivity testing (AST). In some cases, some systems focus only on identifying a single aspect of the sepsis cascade, such as detection of host response or detection of pathogens. For example, a system can observe the host response of early indications of sepsis by detecting molecular leukocyte RNA markers (detecting gene expression via reverse transcription (RT)-PCR), but will not provide answers to pathogen identification or sensitivity. Immune response results can alert caregivers that the patient is entering or has entered the sepsis cascade and urgently needs treatment or intervention to prevent further possibilities of irreversible morbidity. Caregivers can respond immediately by finding the site of infection and the infectious agent using traditional methods (such as by obtaining blood cultures from the site of infection to identify the infectious agent).

[0053] In terms of pathogen detection, current technology can provide detection and identification methods directly from blood, using the PCR performed from blood samples, and then detect. However, such systems may be expensive, have limited menu options, and are difficult to serve when rapid identification results are not provided but limited molecular genetic resistance set solutions are provided. Other systems can utilize the rRNA RT-PCR of the pathogen directly from blood to carry out pathogen identification, but may also be limited to a limited menu (for example, 15 targets or less). Ultimately, current technology does not provide automated rapid identification solutions directly from blood, but relies on positive blood cultures to test, which may require 13-20 hours to report any feasible matters. For example, some systems can obtain aliquots from positive blood culture (PBC) bottles, thereby saving the time (for example, 6-24 hours) required for growing bacterial isolates from PBC bottles. However, these systems are limited to the quantity of drugs and organisms that they can report, and therefore have limited utility to health care providers.

[0054] To significantly reduce morbidity and mortality, new diagnostic methods, devices and systems are needed for rapid detection of bacteria causing infectious sepsis directly from blood samples at single cell or low copy number levels without the significant time delays required by the multiple culture steps (e.g., bioamplification) currently required by standard of care methods. Accordingly, the systems, devices and methods described herein provide a holistic and systematic approach to identifying pathogens in order to determine pathogen resistance and recommend effective and appropriate treatment for patients. Pathogen Identification System Overview:

[0055] Figure 1 A diagram of a system 101 for performing pathogen identification according to an embodiment of the present disclosure is shown. In some embodiments, the system 101 may be referred to herein as a pathogen identification system 101 or a polymerase chain reaction (PCR) system 101. The system 101 may include an analyzer 108, a sample tube 111, a sample preparation cartridge 114, a PCR cartridge 117, a processing device 116, and a plurality of databases 110 communicatively coupled via a network 112.

[0056] Analyzer 108 can be a point-of-care (POC) testing device that performs identification of pathogens in patient samples (which can be stored in sample tubes 111). In some embodiments, analyzer 108 may be referred to as an analyzer device herein. In some embodiments, the sample in sample tube 111 may include whole blood, urine, sterile body fluids, or other samples obtained from a patient. In some embodiments, analyzer 108 can receive sample tubes 111, which are placed in corresponding drawers in the housing of analyzer 108 by a user or operator of analyzer 108.

[0057] In addition to receiving sample tubes 111, analyzer 108 may also receive sample preparation cartridges 114 and PCR cartridges 117, which may similarly be placed by a user or operator of analyzer 108 into corresponding drawers of the housing of analyzer 108. In some embodiments, various components and subsystems in analyzer 108 may interface with sample tubes 111, sample preparation cartridges 114, PCR cartridges 117, processing devices 116, and / or database 110 to perform sample preparation and processing, including cell lysis, concentration of pathogens in a sample, and nucleic acid amplification using PCR and fluorescence readings.

[0058] In some embodiments, after receiving the sample tube 111, the pipetting system in the analyzer 108 can transfer the sample from the sample tube 111 to the sample preparation box 114. In some embodiments, the sample preparation box 114 can be a special consumable with a receiving container, which is configured to hold the sample and an element for performing sample processing. The sample preparation box 114 may include a processing tube 113, which has a diaphragm arranged on or in the tube for protecting the contents therein. The processing tube 113 can be configured to receive the sample from the sample tube 111 through the diaphragm by using the needle of the sample preparation box 114 that transfers the sample. Once the sample is transferred to the processing tube 113, the sample can be subjected to sample concentration, lysis, and / or other processing steps.

[0059] After sample preparation and processing (e.g., including sample concentration and lysis) using the elements and components of the sample preparation box 114, the sample is transferred from the processing tube 113 in the sample preparation box 114 to the PCR box 117 by the pipetting system in the analyzer 108. In some embodiments, the PCR box 117 can be a dedicated consumable having one or more primary reaction chambers and multiple secondary reaction chambers for performing a nucleic acid amplification step for pathogen identification. In some embodiments, one or more primary reaction chambers of the PCR box 117 can be configured to receive nucleic acids, and the multiple secondary reaction chambers of the PCR box 117 can be configured to receive aliquots of the first amplification product after amplifying nucleic acids in the one or more primary reaction chambers. In some embodiments, the analyzer 108 can perform nucleic acid amplification in the PCR box 117, and use a PCR subsystem arranged in the analyzer 108 to perform thermal cycling and detect fluorescent signals generated during amplification. In some embodiments, one or more single-step PCRs can be performed. In some embodiments, the one or more single-step PCRs can utilize one or more primary reaction chambers in the PCR box 117. In some embodiments, the one or more single-step PCRs may utilize a secondary reaction chamber in PCR cassette 117, wherein the initial sample is divided into aliquots for performing the single-step PCRs.

[0060] In some embodiments, analyzer 108 can be further docked with other boxes (such as antimicrobial sensitivity test (AST) boxes). In some embodiments, AST boxes can be special consumables with multiple reaction holes, and the multiple reaction holes are configured to hold multiple aliquots of enriched samples for AST. In some embodiments, analyzer 108 can be configured to hold one or more sample preparation boxes 114, PCR boxes 117 and / or AST boxes at the same time, to simultaneously or continuously perform sample preparation / processing, pathogen identification and / or sensitivity testing. In some embodiments, sample preparation boxes 114, AST boxes and / or PCR boxes 117 can be referred to herein as consumables or containers configured to be inserted into analyzer 108.

[0061] In some embodiments, the analyzer 108 may include a controller 109 disposed inside the housing of the analyzer 108. The controller 109 may control the movement and operation of different components within the analyzer 108, including the movement of one or more sample tubes, processing tubes, boxes, and pipetting systems in the analyzer 108. In some embodiments, the controller 109 may also control the operation of one or more centrifuges, subsystems, and modules in the analyzer 108 to perform sample preparation, pathogen identification, sensitivity testing, and / or other functions. In some embodiments, the controller 109 may include a microcontroller on an integrated circuit (IC) chip in the analyzer 108, which is programmed to turn on / off and operate one or more centrifuges, subsystems, and modules in the analyzer 108. In some embodiments, the controller 109 may be coupled to one or more stepper motors, actuators, or other motion control components in the analyzer 108 and programmed to control movement.

[0062] In some embodiments, the controller 109 can be programmed by the processing device 116. The processing device 116 can be a computing device coupled to the analyzer 108 for performing data processing and providing instructions to the controller 109 and / or other components in the analyzer 108. In some embodiments, the processing device 116 can be a personal digital assistant, a desktop workstation, a laptop or notebook computer, a netbook, a tablet computer, a smartphone, a mobile phone, a smart watch, or any combination thereof.

[0063] In some embodiments, the processing device 116 can communicate with the analyzer 108 to receive the results of the reaction occurring in the PCR cartridge 117, and perform further processing and data analysis to identify one or more pathogens in the sample. In some embodiments, the processing device 116 can receive fluorescence data from one or more signals from the amplified nucleic acids generated in the PCR cartridge 117 from the PCR subsystem in the analyzer 108, and analyze the fluorescence data to identify pathogens present in the patient sample based on the detected amplified nucleic acids.

[0064] In some embodiments, the processing device 116 can also communicate with the plurality of databases 110. In some embodiments, one or more of the plurality of databases 110 can represent any number of databases and can include various databases storing clinical parameter data, epidemiological information, or antibiotic resistance information, etc. for a variety of pathogens. In some embodiments, one or more of the plurality of databases 110 can be configured to store pathogen taxonomy data and / or outcomes from a previous pathogen identification workflow (e.g., performed by the analyzer 108). In some embodiments, one or more of the plurality of databases 110 can include electronic health record (EHR) data that includes patient health care information obtained from various health care services and health care providers (such as hospitals, clinical care facilities, laboratories, radiology providers, and pharmacies).

[0065] In some embodiments, the EHR data stored in the database 110 may include patient data and medical history data regarding the health and treatment of the patient, including each patient's demographics, medical history, medications and allergies, immune status, laboratory test results, radiology images, vital signs, personal statistics (like age and weight), and billing information. In some embodiments, the processing device 116 may use the results of pathogen identification and / or AST performed by the analyzer 108, as well as the data stored in the plurality of databases 110 (e.g., clinical parameter data, epidemiological information or antibiotic resistance information, EHR data, etc.) to determine treatment recommendations for the patient.

[0066] In some embodiments, the components in the system 101 can be communicatively coupled via a network 112. In particular, the network 112 can allow information transfer and communication between the analyzer 108, the plurality of databases 110, the processing device 116, and / or any other devices or components in the system 101. In some embodiments, the system 101 can include additional components, such as a Raman spectroscopy device and / or an electronic health record (EHR) system (not shown).

[0067] In some embodiments, the network 112 can be any one or any combination of a LAN (local area network), a WAN (wide area network), a telephone network, a wireless network, a point-to-point network, a star network, a token ring network, a hub network, or other suitable configurations. The network can comply with one or more network protocols, including Institute of Electrical and Electronics Engineers (IEEE) protocols, 3rd Generation Partnership Project (3GPP) protocols, 4th generation wireless protocols (4G) (e.g., Long Term Evolution (LTE) standards, advanced LTE, advanced LTE Pro), fifth generation wireless protocols (5G), and / or similar wired and / or wireless protocols, and can include one or more intermediate devices for routing data between the analyzer 108, the plurality of databases 110, the processing device 116, and / or any other devices or components in the system 101. Analyzer device implementation plan:

[0068] Figure 2 A diagram of an analyzer device 200 according to an embodiment of the present disclosure is shown. The analyzer device 200 represents Figure 1 108 is an exemplary embodiment of the analyzer 108 shown in . In some embodiments, the analyzer device 200 may be referred to as analyzer 200 herein. The analyzer device 200 is a desktop device having a housing 201, which contains various components and modules for sample preparation, processing and testing. In some embodiments, the housing 201 may include the body of the analyzer device 200 and / or the housing of the analyzer device 200 (which protects the modules and components therein). In some embodiments, the analyzer device 200 may include a cube, a cuboid, or a rectangular shape having various compartments for access and operation by a user of the analyzer device 200. In some embodiments, the analyzer device 200 may have a size of less than about 1 m 3 (eg, compact size of approximately 750 mm (length) x 650 mm (width) x 650 mm (height)).

[0069] In some embodiments, the analyzer device 200 can be coupled to a computing device (e.g., the processing device 116), such as a personal digital assistant, a desktop workstation, a laptop or notebook computer, a netbook, a tablet computer, a smart phone, a mobile phone, a smart watch, or any combination thereof. A user or operator of the analyzer device 200 can use the computing device to control the analyzer device 200, send / receive sample information, patient information, pathogen information, antimicrobial agent information, etc. to / from the analyzer device 200, and access / edit the results of pathogen detection from the analyzer device 200.

[0070] Figure 3A A diagram showing a front view of an analyzer device 200 according to an embodiment of the present disclosure is shown. Figure 3A The internal features disposed within the housing 201 of the analyzer device 200 are shown, including a first pipette 202, a second pipette 204, a sample drawer 210, a sample cartridge drawer 220, and a processing cartridge drawer 230.

[0071] In some embodiments, the first pipette 202 and the second pipette 204 can be pipette devices configured to manipulate liquid transfer between components within the housing 201 of the analyzer device 200. In some embodiments, the first pipette 202 and the second pipette 204 can be automated devices controlled by the controller 109 in the analyzer device 200. In some embodiments, the controller 109 can control the movement of the first pipette 202 and the second pipette 204, including vertical and / or horizontal movement of the first pipette 202 and the second pipette 204 to and / or from different components within the analyzer device 200. In some embodiments, there can be more or fewer pipettes in the analyzer device 200.

[0072] In some embodiments, the first pipette 202 and the second pipette 204 can be referred to as a high-capacity pipette and a low-capacity pipette, respectively. In some embodiments, the first pipette 202 can be configured to manipulate a volume in the range of about 50 microliters (μL) to 5 milliliters (mL), and the second pipette 204 can be configured to manipulate a volume in the range of about 1 to 200 μL. In some embodiments, the first pipette 202 can have a 5% coefficient of variation (CV) at 50 μL and can have a 1% CV at 5mL, and the second pipette 204 can have a 5% CV at 1 to 200 μL.

[0073] In some embodiments, the first pipette 202 and the second pipette 204 may be referred to herein as a pipette and / or pipette system. In some embodiments, the first pipette 202 and the second pipette 204 may each be configured to be attached to a removable and disposable needle. The first pipette 202 and the second pipette 204 may be attached to two different needles, which are configured to be used to manipulate different volume ranges as necessary for the first pipette 202 and the second pipette 204.

[0074] In addition to liquid transfer, the first pipette 202 and the second pipette 204 can be configured to move elements such as tubes, boxes, etc. within the housing 201 of the analyzer device 200. In particular, the first pipette 202 and the second pipette 204 can each include a pipette tip holder that can be attached to various components used in the analyzer device 200. In particular, the pipette tips of the first pipette 202 and the second pipette 204 can be pressed and fitted into the manipulation features of needles, tubes, boxes, spin column baskets, etc. to pick up various components and place them in different modules or areas in the analyzer device 200.

[0075] In some embodiments, the size of the first pipette 202 and the second pipette 204 can be about 325 mm (width) x 575 mm (depth) x 435 mm (height). In some embodiments, the first pipette 202 and the second pipette 204 can be arranged in the top portion of the housing 201 so that the first pipette 202 and the second pipette 204 can interact with samples, tubes, boxes, and different modules in the bottom portion of the housing 201. In particular, the first pipette 202 and the second pipette 204 can manipulate Figure 3A Liquid transfer and movement of components in sample drawer 210, sample cartridge drawer 220, and process cartridge drawer 230 are shown.

[0076] In some embodiments, the sample drawer 210, the sample box drawer 220, and the processing box drawer 230 include sliding horizontal compartments that are designed to fit into three corresponding receiving containers in the housing 201 of the analyzer device 200. In some embodiments, the sample drawer 210, the sample box drawer 220, and the processing box drawer 230 can be configured to receive special elements for sample processing and testing inserted into the analyzer device 200. In particular, the sample drawer 210 can receive a sample tube (e.g., sample tube 111) containing a sample obtained from a patient. The sample box drawer 220 can receive a sample preparation box (e.g., sample preparation box 114), into which the sample is transferred by components in the analyzer device 200.

[0077] In some embodiments, the processing box drawer 230 can receive a PCR box (e.g., a PCR box 117), which is configured to receive nucleic acid separated from a sample after sample processing, pathogen concentration and lysis and nucleic acid purification from the pathogen of the sample in the sample preparation box. In additional or alternative embodiments, the processing box drawer 230 can be used as an AST box and / or a PCR box drawer. For example, a PCR box or an AST box can be inserted into the processing box drawer 230, depending on whether the analyzer device 200 is being used to perform pathogen identification or antimicrobial susceptibility testing of a sample. In some embodiments, the analyzer device 200 can be configured to perform two functions of pathogen identification and antimicrobial susceptibility testing with a dual processing box drawer 230, which is configured to dock with a dedicated box or consumables for AST and pathogen identification.

[0078] In some embodiments, sample drawer 210, sample box drawer 220 and processing box drawer 230 can each include a reader, and the reader is configured to scan the identifier of sample tube, sample preparation box and PCR box (or AST box) respectively. In some embodiments, the reader in the drawer can scan the identifier of sample tube and / or box during the housing 201 process of each drawer being inserted into the analyzer device 200. In some embodiments, the reader in drawers 210, 220 and 230 can be configured to scan the identification code, bar code or data matrix of corresponding sample tube and / or box. In some embodiments, the reader in drawers 210, 220 and 230 can be a bar code reader, quick response (QR) code reader etc.

[0079] In some embodiments, Figure 3A Additional components are shown including one or more centrifuges disposed within housing 201 and configured for manipulating tubes and cartridges for sample processing. Figure 3B A diagram showing a top view of an analyzer device 200 according to an embodiment of the present disclosure is shown. Figure 3B A cross section of the analyzer device 200 from a top view is shown, illustrating various components, modules and / or subsystems arranged within the housing 201 of the analyzer device 200 below the first pipette 202 and the second pipette 204 .

[0080] Figure 3B The housing 201 in the embodiment includes a sample drawer 210 for accommodating a plurality of sample tubes 212, a sample box drawer 220 for accommodating a plurality of sample preparation boxes 224, and a processing box drawer 230 for accommodating an AST box 232 and a PCR box 234. The sample tubes 212, the sample preparation boxes 224, and the PCR boxes 234 represent Figure 1An exemplary embodiment of a sample tube 111, a sample preparation cartridge 114, and a PCR cartridge 117 is shown.

[0081] In some embodiments, the processing box drawer 230 can be configured to hold both the AST box 232 and the PCR box 234 for pathogen identification and antimicrobial sensitivity testing. In some embodiments, there can be a predefined number of sample tubes 212, sample preparation boxes 224, AST boxes 232, and / or PCR boxes 234, which are simultaneously held in their respective drawers in the housing 201. In some embodiments, the sample preparation box 224, AST box 232, and / or PCR box 234 can be disposable after a single use or can be reused for testing additional samples.

[0082] Figure 3B Also shown are a first centrifuge 240 , a second centrifuge 242 , a PCR subsystem 244 , a homogenization subsystem 246 , a mechanical device 248 , and an AST subsystem 250 disposed within the housing 201 .

[0083] In some embodiments, the first centrifuge 240 can be a high-speed centrifuge configured to centrifuge a processing tube (e.g., processing tube 113) and / or a spin column basket placed in the first centrifuge 240 by the first pipette 202. In some embodiments, the second centrifuge 242 can be a low-speed centrifuge configured to centrifuge a PCR cartridge 234 placed in the second centrifuge 242 by the second pipette 204. In some embodiments, the first centrifuge 240 and the second centrifuge 242 can centrifuge processing tubes, spin column baskets, and / or PCR cartridges 234 in a swing-bucket configuration. In some embodiments, the first centrifuge 240 and the second centrifuge 242 can include a cylindrical shape having a diameter of about 250 mm and a height of about 175 mm.

[0084] In some embodiments, the PCR subsystem 244 can include a thermal cycler equipped with an optical detection module or optical system to measure the fluorescent signal generated due to the binding of one or more fluorophores to the target sequence in the nucleic acid during each amplification cycle in the PCR cartridge 234. In some embodiments, the thermal cycler can be configured to control the temperature while performing PCR (PCR), and the optical system can be configured to perform optical interrogation of the primary and / or secondary reaction chambers in the PCR cartridge 234 by fluorescence.

[0085] In some embodiments, the thermal cycler of the PCR subsystem 244 can control the temperature in the range of about 35°C to 100°C. In some embodiments, the optical system of the PCR subsystem 244 can perform fluorescence readings from the bottom of the PCR box 234 in the housing 201 to detect signals from amplified nucleic acid products generated by PCR. In some embodiments, the PCR box 234 can be placed in the PCR subsystem 244 and removed from the PCR subsystem 244 by the second pipette 204. In some embodiments, the PCR subsystem 244 can hold up to two PCR boxes 234 at a time, wherein the PCR boxes 234 are independently subjected to thermal cycles in the PCR subsystem 244. In some embodiments, the size of the PCR subsystem 244 can be about 70 mm (width) x 125 mm (depth) x 250 mm (height).

[0086] In some embodiments, the homogenization subsystem 246 can be configured to hold multiple processing tubes in a trough for applying a rocking motion to the processing tubes to allow oscillation or mixing of the sample. In some embodiments, the first pipette 202 can be configured to load the tube vertically into the trough in the homogenization subsystem 246. In some embodiments, the homogenization subsystem 246 can change the orientation of the tube from a vertical position to a horizontal position and apply a rocking motion of ±15° around the horizontal position at a frequency of 1 Hz. In some embodiments, the homogenization subsystem 246 can hold up to about four 15 mL processing tubes at a time, each of which represents a different sample.

[0087] In some embodiments, the homogenization subsystem 246 can be equipped with a movable magnet. The movable magnet can engage the process tube when in a vertical position in the homogenization subsystem 246. In some embodiments, the homogenization subsystem 246 can apply 37°C temperature control to the process tube held in the homogenization subsystem 246. In some embodiments, the dimensions of the homogenization subsystem 246 can be approximately 150 mm (width) x 175 mm (depth) x 115 mm (height).

[0088] In some embodiments, mechanical device 248 can be configured to stir the processing tube to carry out the cell lysis of the pathogen in the sample. In some embodiments, the first pipette 202 can be configured to be used for vertically loading the tube into the groove in the mechanical device 248. In some embodiments, the mechanical device 248 can include an agitator device or a cell disruption device, and the agitator device or the cell disruption device are configured to apply rapid vibration motion to the processing tube. In some embodiments, the mechanical device 248 can apply rapid vibration motion by applying reciprocating motion along a predetermined axis by the agitator when the processing tube is in a vertical position.

[0089] In some embodiments, the mechanical device 248 can hold up to two processing tubes at a time and apply ±2° of angular motion to the processing tubes. In some embodiments, the mechanical device 248 can oscillate the processing tubes at about 5,000-30,000 cycles / minute. In some embodiments, the size of the mechanical device 248 can be about 75mm (width) x 175mm (depth) x100mm (height). In additional or alternative embodiments, the mechanical device 248 can include a sonicator configured to sonicate the processing tubes to agitate the sample.

[0090] In some embodiments, the AST subsystem 250 may include a heater and an imaging subsystem, the heater being configured to incubate a sample in the AST box 232 in the housing 201, and the imaging subsystem being used to image the reaction chamber of the AST box 232. In some embodiments, the heater of the AST subsystem 250 may be used to control the incubation temperature of the sample in the AST box 232. In some embodiments, pathogens may be concentrated and enriched in the sample and transferred to the reaction wells in the AST box 232 for imaging by the AST subsystem 250. In some embodiments, the imaging subsystem of the AST subsystem 250 may include a microscope configured to acquire an image by scanning the bottom of each reaction chamber of the AST box 232 using a motorized XYZ translation stage. In some embodiments, the imaging subsystem of the AST subsystem 250 may include a fluorescence sensor and two optical channels for detecting different fluorescence signals (e.g., green and red).

[0091] In some embodiments, the AST subsystem 250 can identify the antimicrobial phenotypic resistance of the microorganism (e.g., pathogen) based on the acquired image and / or signal. In some embodiments, the AST box 232 can be placed in the AST subsystem 250 and removed from the AST subsystem 250 by the second pipette 204.

[0092] In some embodiments, the AST subsystem 250 can hold up to five AST cassettes 232 at a time. In some embodiments, the AST subsystem 250 can apply temperature control to the AST cassettes 232 held in the AST subsystem 250, such as by using a heat block. In some embodiments, the AST subsystem 250 can apply temperature control of about 37° C. In some embodiments, the dimensions of the AST subsystem 250 can be about 200 mm (width) x 185 mm (depth) x 285 mm (height).

[0093] Figure 4 A diagram of an analyzer device 200 having three open compartments according to an embodiment of the present disclosure is shown. In some embodiments, Figure 4 1 shows a sample drawer 210, a sample box drawer 220, and a processing box drawer 230 extending from a housing 201 in an open position for loading and / or unloading samples and / or boxes. The sample drawer 210, the sample box drawer 220, and the processing box drawer 230 can be pushed into the housing 201 in a closed position, wherein the compartments fit into three corresponding receiving containers in the housing 201. In some embodiments, the opening and closing of the sample drawer 210, the sample box drawer 220, and the processing box drawer 230 can be controlled by a controller (e.g., controller 109) of the analyzer device 200 and / or by the processing device 116. In some embodiments, a user of the processing device 116 can control the opening and closing of the sample drawer 210, the sample box drawer 220, and the processing box drawer 230 by using software installed on the processing device 116. In some embodiments, the sample drawer 210 , the sample box drawer 220 , and the processing box drawer 230 can be manually pulled out and pushed into the housing 202 by a user to load and / or unload sample tubes, boxes, and / or other elements into the analyzer device 200 .

[0094] In some embodiments, sample drawer 210 can hold multiple samples. In some embodiments, sample drawer 210 can hold up to 10 samples at a time, including samples stored in sample tubes 212. In some embodiments, Figure 4 The sample tube 212 shown can represent one or more blood sample tubes, urine sample tubes and / or blood culture bottles. In some embodiments, the sample box drawer 220 can hold multiple sample preparation boxes at a time, such as up to 10 sample preparation boxes. In some embodiments, the processing box drawer 230 can hold multiple processing boxes at a time, such as up to 12 PCR boxes, 6 AST boxes or a combination thereof.

[0095] In some embodiments, the dimensions of the sample drawer 210 can be about 40 mm (width) x 155 mm (height) x 600 mm (depth). In some embodiments, the dimensions of the sample box drawer 220 can be about 85 mm (width) x 155 mm (height) x 615 mm (depth). In some embodiments, the dimensions of the process box drawer 230 can be about 140 mm (width) x 155 mm (height) x 270 mm (depth). Implementation of sample processing, concentration, lysis and nucleic acid purification in the analyzer device:

[0096] In some embodiments, pathogen identification includes using a pipetting system to transfer the sample to a consumable (e.g., a processing tube 113 and / or a spin column) for further processing (e.g., nucleic acid extraction, purification, and amplification), pathogen concentration, pathogen lysis (by mechanical disruption), purification of nucleic acid from the pathogen, and amplification and identification of the pathogen by PCR. In some embodiments, the first pipette 202 of the analyzer device 200 can transfer the sample from the sample tube 111 to the processing tube 113, and perform steps to process, concentrate, separate and lyse the pathogen in the sample and purify nucleic acid from the lysed pathogen, and then perform PCR.

[0097] In some embodiments, sample processing can include the step of hemocyte lysis. In the embodiment where sample is a blood sample, the first pipette 202 can be configured to perform hemocyte lysis of the blood sample. The first pipette 202 can add one or more lysis reagents to the treatment tube 113. The mixer (for example, such as in the homogenization subsystem 246) arranged in the housing 201 of the analyzer device 200 can be used to mix one or more lysis reagents with the blood sample in the treatment tube 113 to make the hemocyte lysis in the blood sample. In some embodiments, one or more lysis reagents can include one or more buffers based on saponin. In some embodiments, one or more lysis reagents can include one or more detergents, surfactants or proteases.

[0098] After the blood cells in the blood sample are lysed, the first pipette 202 can transfer the processing tube 113 to the centrifuge 240 (or Fig.15A 1502). In some embodiments, centrifuge 240 can apply centrifugal force to processing tube 113 to concentrate pathogens in the sample. In some embodiments, processing tube 113 can be centrifuged from a high volume (e.g., up to 10 mL) to a low volume (e.g., 2 mL or less), wherein pathogens are concentrated in processing tube 113.

[0099] After centrifugation, the first pipette 202 or the second pipette 204 can be used to remove fluid from the processing tube 113, thereby leaving the concentrated pathogens in the processing tube 113. In some embodiments, the first pipette 202 or the second pipette 204 can add one or more lysis buffers to the concentrated pathogens in the processing tube 113. In some embodiments, the first pipette 202 or the second pipette 204 can retrieve one or more lysis buffers from one or more reservoirs in the sample preparation box and distribute the one or more lysis buffers to the processing tube 113 through a needle coupled to the first pipette 202 or the second pipette 204. In some embodiments, a plurality of lysis beads can be added to the processing tube 113 together with the one or more lysis buffers to perform lysis of the concentrated pathogens in the processing tube 113. In some embodiments, a plurality of lysis beads can be added to the processing tube 113 of the sample preparation box during manufacturing and / or assembly.

[0100] In some embodiments, the processing tube 113 can be agitated or sonicated in the analyzer device 200 to mechanically lyse the pathogen using lysis beads and one or more lysis buffers. In some embodiments, the rapid movement of the lysis beads (caused by agitation or sonication in the analyzer device 200) can mechanically disrupt the cell walls of the pathogens in the processing tube 113, resulting in the release of nucleic acids from the pathogens.

[0101] In some embodiments, after the pathogen is lysed, the processing tube 113 can be subjected to an additional centrifugation step (via centrifugation 240) to drive the liquid in the processing tube 113 to the bottom of the tube. In some embodiments, the first pipette 202 or the second pipette 204 can remove a portion of the liquid from the processing tube 113, wherein the portion of the liquid includes nucleic acid from the pathogen. In some embodiments, cells, lysis beads, and debris produced by lysis remain in the processing tube 113.

[0102] After lysing and removing the nucleic acid from the processing tube 113, the nucleic acid can be purified by using a spin column or magnetic beads. In some embodiments, the first pipette 202 or the second pipette 204 can transfer the portion of liquid from the processing tube 113 to the spin column in the analyzer device 200 after cell lysis. In some embodiments, the spin column can extract and purify nucleic acids from the portion of liquid. In some embodiments, the eluate (e.g., purified nucleic acid) produced by the spin column can be collected in a basket of the spin column.

[0103] In additional or alternative embodiments, the first pipette 202 or the second pipette 204 can transfer the portion of liquid from the processing tube 113 to one or more reservoirs in the sample preparation box after cell lysis. In some embodiments, the one or more reservoirs can be configured to store magnetic beads for nucleic acid extraction and purification. In some embodiments, the magnetic beads can be configured to be attached to nucleic acids. In some embodiments, the analyzer device 200 can apply magnetic force to retain the nucleic acids attached to the magnetic beads. In some embodiments, the first pipette 202 or the second pipette 204 can then remove irrelevant liquid from the nucleic acids attached to the magnetic beads, while the nucleic acids attached to the magnetic beads are retained in one or more reservoirs. The removal of irrelevant liquid can cause one or more reservoirs to accommodate purified nucleic acids.

[0104] After purification, the nucleic acid obtained from the sample may then be ready for transfer to the PCR cartridge 234 via a pipetting system in the analyzer 200 for nucleic acid amplification and pathogen identification via PCR. Sample preparation kit implementation plan:

[0105] Figure 5 A diagram of a sample preparation kit 500 according to an embodiment of the present disclosure is shown. The sample preparation kit 500 represents Figure 3B 224. The sample preparation box 500 can be a consumable inserted into the sample drawer 210 of the analyzer device 200 for preparing and processing samples in the sample tube. In some embodiments, the sample preparation box 500 can be made of a polypropylene (PP) material by injection molding. The sample preparation box 500 can include a housing 502, a plurality of reservoirs 504, a cover 506, and an identifier 508.

[0106] In some embodiments, the housing 502 may have an elongated rectangular shape with rounded edges. The housing 502 may be configured to hold a Fig. 6AIn some embodiments, a plurality of reservoirs 504 can be separate reservoirs or tubes molded together. A plurality of reservoirs 504 can be configured to store materials for carrying out sample concentration, cracking and / or nucleic acid amplification. In some embodiments, the material stored in the reservoir 504 can include one or more buffers (for example, buffers based on NaCl, phosphate buffered saline (PBS, etc.), detergents, surfactants, proteases, growth medium, etc. In some embodiments, the reservoir 504 can store one or more lysis reagents, such as one or more buffers based on saponin, detergents, surfactants or proteases, for carrying out the cell lysis of a blood sample. In some embodiments, the reservoir 504 can store one or more washing materials, and the one or more washing materials can include a combination of one or more buffers, detergents, surfactants and proteases.

[0107] In some embodiments, the cover 506 of the sample preparation box 500 is a protective cover that extends across and covers the housing 502 and the plurality of reservoirs 504. In some embodiments, the identifier 508 is an identifier of the sample preparation box 500 that can be scanned by the analyzer device 200 for pathogen identification. In some embodiments, the identifier 508 can be at least one of an identification code, a barcode, or a data matrix (such as a QR code). In some embodiments, the size of the sample preparation box 500 can be as follows: Figure 5 Approximately 80mm (width) x 55mm (depth) x 155mm (height) as shown.

[0108] Fig. 6A A diagram showing an exploded view of a sample preparation cartridge 500 having a processing tube 510 and other components to be inserted therein according to an embodiment of the present disclosure is shown. The sample preparation cartridge 500 may include the processing tube 510, a first removable needle 512, two second removable needles 514, a spin column 522, a spin column basket 524, and reagent tubes 526, which are stored in corresponding receiving containers in the housing 502 of the sample preparation cartridge 500. The processing tube 510 represents Figure 1 An exemplary embodiment of a process tube 113 is shown.

[0109] In some embodiments, the processing tube 510 may include, for example, a 15 ml tube with a conical bottom. In some embodiments, the processing tube 510 may be removed from the sample preparation box 500 for processing in other modules in the analyzer device 200. In some embodiments, the processing tube 510 may be a tube into which the sample is transferred after the sample tube and the sample preparation box 500 are loaded into the sample drawer 210 and the sample box drawer 220 of the analyzer device 200, respectively.

[0110] In some embodiments, a sample can be transferred from a sample tube in a sample drawer 210 to a processing tube 510 of a sample preparation cartridge 500 in a sample cartridge drawer 220 via a first removable needle 512. In some embodiments, a first pipette 202 can be attached with the first removable needle 512, which is configured for transferring a sample to and / or from a processing tube 510 by inserting the first removable needle 512 through a septum of the processing tube 510. In some embodiments, two second removable needles 514 can be used to manipulate small volumes, and the second pipette 204 can be configured for attachment with the two second removable needles 514.

[0111] Fig. 6A Also shown are openings 520 of the plurality of reservoirs 504 of the sample preparation cartridge 500. In some embodiments, there may be 12 reservoirs 504 in the sample preparation cartridge 500, wherein each reservoir 504 holds a volume of approximately 2.5 mL. In some embodiments, the sample preparation cartridge 500 may include a pierceable membrane 516 covering the receiving container of the housing 502 and / or a foil seal 518 covering the openings 520 of the plurality of reservoirs 504. In some embodiments, the pierceable membrane 516 may be a polyester film, and the foil seal 518 may include an aluminum foil, wherein both the pierceable membrane 516 and the foil seal 518 may be pierced by the first removable needle 512 and / or the second removable needle 514.

[0112] Fig. 6A Further shown are a spin column 522, a spin column basket 524, and a reagent tube 526 stored in the sample preparation box 500. In some embodiments, the spin column 522 and the spin column basket 524 are used to perform nucleic acid extraction and purification from nucleic acids obtained from the sample. In some embodiments, the reagent tube 526 stores one or more reagents for performing nucleic acid amplification of purified nucleic acids from the sample. In some embodiments, the reagent tube 526 can be sealed with aluminum foil. In some embodiments, the spin column 522 can be configured to be inserted and stored in the spin column basket 524. In some embodiments, the spin column basket 524 and the reagent tube 526 are both configured to fit in a corresponding receiving container in the sample preparation box 500.

[0113] Figure 6B A diagram showing an exploded view of a sample preparation cartridge 500' having a processing tube 510 and other components to be inserted therein according to an embodiment of the present disclosure. Sample preparation cartridge 500' is similar to sample preparation cartridge 500, but has a more linear form factor and a peelable film 506' instead of lid 506. Processing tubes, reagent tubes, needles and spin columns implementation:

[0114] Fig. 7A and Figure 7BA diagram of a process tube 700 is shown according to an embodiment of the present disclosure. The process tube 700 represents Fig. 6A An exemplary embodiment of a processing tube 510 is shown. In particular, Fig. 7A The assembled process tube 700 is shown, and Figure 7B An exploded view of the components in process tube 700 is shown. Process tube 700 includes cap 702, septum 704, and tube 706.

[0115] In some embodiments, the septum 704 can be affixed to the interior of the tube 706, with the cap 702 fitting over the septum 704 of the processing tube 700. In some embodiments, the septum 704 can be configured for insertion and removal of a needle (e.g., needles 512 and 514) to transfer liquid to and from the processing tube 700 without removing the cap 702 from the tube 706. In some embodiments, the septum 704 can provide an airtight seal within the processing tube 700 and prevent contamination of the contents of the processing tube 700.

[0116] In some embodiments, the processing tube 700 may include a manipulation feature at the top of the processing tube 700 that is compatible with being manipulated by a pipette (e.g., the first pipette 202 and / or the second pipette 204). In some embodiments, the manipulation feature of the processing tube 700 may be a cylindrical cavity in the cap 702 that is compatible with being inserted into the tip of the pipette. In some embodiments, the tip of the pipette may be referred to as a core tube herein. In some embodiments, the pipette tips of the first pipette 202 and the second pipette 204 may be pressed and fitted into the cylindrical cavity in the cap 702 to pick up and move the processing tube around the analyzer device 200.

[0117] In some embodiments, cap 702 can be made of high density polyethylene (HDPE) material, and tube 706 can be made of polypropylene (PP) material. In some embodiments, diaphragm 704 can include at least one of rubber, polytetrafluoroethylene (PTFE), thermoplastic elastomer (TPE), silicone, butyl rubber or its combination. In some embodiments, diaphragm 704 can include a double layer of polytetrafluoroethylene (PTFE) and another material, and the another material is selected from silicone, rubber and butyl rubber. In some embodiments, the size of treatment tube 700 after assembly can include, for example, a height of about 110mm and a diameter of about 20mm. In some embodiments, diaphragm 704 can have a thickness ranging from about 1 to 2mm.

[0118] In some embodiments, the processing tube 700 may include a plurality of lysis beads disposed within the tube 706 and configured to perform lysis. In some embodiments, the plurality of lysis beads may have a predetermined size and material configured to perform lysis of at least one of the following pathogens: yeast, fungi, gram-positive bacteria, or gram-negative bacteria, among others. In some embodiments, the processing tube 700 may be transferred to the mechanical device 248 of the analyzer apparatus 200, where a rapid vibration motion may be applied to the plurality of lysis beads to perform lysis of the pathogen in the sample in the processing tube 700. In some embodiments, the movement of the lysis beads may mechanically disrupt the cell walls of the pathogen in the sample, resulting in the release of nucleic acids from the pathogen.

[0119] In some embodiments, after cell lysis, the first pipette 202 or the second pipette 204 can transfer a portion of the liquid from the processing tube 700 to one or more reservoirs 504 in the sample preparation box 500, in which a plurality of magnetic beads can be stored. In some embodiments, the plurality of magnetic beads in the one or more reservoirs 504 can be configured to attach to nucleic acids in the portion of liquid obtained from the processing tube 700. In some embodiments, the plurality of magnetic beads can be used to extract and purify nucleic acids from the portion of liquid obtained from the processing tube 700.

[0120] Figure 7C and Fig.7D A diagram of an exemplary reagent tube 710 is shown according to an embodiment of the present disclosure. The processing tube 710 represents Fig. 6A710. In some embodiments, the reagent tube 710 may be referred to as a master mixture tube. The reagent tube 710 may hold a first set of reagents for performing nucleic acid amplification. In some embodiments, the first set of reagents may include one or more of the following: DNA polymerase, deoxyribonucleotide triphosphate (dNTP), magnesium chloride (MgCl2), etc. In some embodiments, the reagents stored in the reagent tube 710 may be dried or freeze-dried. In some embodiments, the reagent tube 710 may include a cap 712 and a tube 714. The cap 712 may include a diaphragm, such as a top across the cap. In some embodiments, the diaphragm on the cap 712 of the reagent tube 710 may be similar to the diaphragm 704 of the processing tube 700. In some embodiments, the cap 712 may be configured to be attached to the tube 714 to provide an airtight seal within the reagent tube 710 and prevent contamination of the contents of the reagent tube 710. In some embodiments, the cap 712 may include a depression 716 to facilitate evacuation of air during the freeze drying process by a freeze dryer. In some embodiments, when inserted into the lyophilizer, the cap 712 can be slightly pressed into the tube 714 so that there is air through the recess 716. The cap 712 can then be sealed within the lyophilizer by the pressure of the cover, ensuring that the air within the tube 714 is free of oxygen.

[0121] In some embodiments, tube 714 can be made of polypropylene (PP) material, and the diaphragm on cap 712 can include at least one of the following: rubber, polytetrafluoroethylene (PTFE), thermoplastic elastomer (TPE), silicone, butyl rubber or its combination. In some embodiments, diaphragm can include a double layer of polytetrafluoroethylene (PTFE) and another material, and the another material is selected from silicone, rubber and butyl rubber. In some embodiments, after assembly (for example, after cap 712 is attached to tube 714), the size of reagent tube 710 can include, for example, a height of about 40 mm and a diameter of about 10 mm.

[0122] In some embodiments, the processing tube 700 and / or the reagent tube 710 can be configured to receive a needle, such as Fig. 8A , Figure 8B and Figure 8C as shown in . Fig. 8A , Figure 8B and Figure 8C A diagram of a needle 800 configured for insertion into a processing tube 700 and / or a reagent tube 710 is shown according to some embodiments of the present disclosure.

[0123] in particular, Fig. 8AA needle 800 including a plastic body 810, a cannula 820, and a plurality of grooves 825 is shown. In some embodiments, the plastic body 810 can be attached to the cannula 820 by bonding. In some embodiments, one or more pipettes (e.g., the first pipette 202) in the analyzer device can be attached to the proximal end of the plastic body 810 of the needle 800. In some embodiments, the plastic body 810 of the needle 800 includes an aerosol filter configured to prevent contamination of the pipettes in the analyzer device. In some embodiments, the needle 800 can be a venting needle configured to ventilate the processing tube 700 when the needle is inserted. In some embodiments, the ventilation of the needle 800 can help release the pressure in the sealed processing tube 700. In some embodiments, the plastic body 810 of the needle 800 includes a predetermined number of grooves 825, which are configured to provide an air connection between the inside and the outside of the processing tube 706 when the needle 800 is inserted through the septum 704 and inserted into the tube 700. For example, there may be four slots 825 in the injection portion of the retaining sleeve 820 of the needle 800. In some embodiments, the slots 825 may be produced by injection molding. In some embodiments, the needle 800 may be inserted into the reagent tube 710, similar to inserting the needle 800 into the processing tube 700. In some embodiments, the sleeve 820 of the needle 800 may be configured to pierce the septum to be inserted into the tube 706 of the reagent tube 710.

[0124] Figure 8B The needle 800 is shown during insertion into the processing tube 700, and Figure 8C The needle 800 is shown after being fully inserted into the treatment tube 700. In some embodiments, the cannula 820 of the needle 800 can be inserted through the septum 704, into the cap 702, and into the tube 706 of the treatment tube 700. In some embodiments, when the cannula 820 is fully inserted into the tube 706, the distal end of the plastic body 810 can fit into the cap 702 of the treatment tube 700.

[0125] Fig.9A and Fig. 9B A diagram showing a high volume needle 900 and a low volume needle 910 according to an embodiment of the present disclosure is shown, respectively. In some embodiments, the high volume needle 900 and the low volume needle 910 can be coupled to the first pipette 202 and the second pipette 204 in the analyzer device 200, respectively.

[0126] like Fig.9AAs shown, the high-capacity needle 900 can include a plastic body 902 and a cannula 904. In some embodiments, the plastic body 902 can be a reservoir configured to hold a volume of about 50 μL to 5 mL during liquid transfer in an analyzer device. In some embodiments, the plastic body 902 can include a filter 903 disposed therein to prevent contamination of a pipette coupled to the high-capacity needle 900.

[0127] In some embodiments, the plastic body 902 can be made of a polypropylene (PP) material. In some embodiments, the plastic body 902 of the needle 900 can have a diameter of about 16 mm and a length of about 50 mm. In some embodiments, the cannula 904 can be made of stainless steel. In some embodiments, the cannula 904 can have a length of about 100 mm. In some embodiments, the plastic body 902 and the cannula 904 can have a length of about 150 mm when assembled together. In some embodiments, the high capacity needle 900 can be a 17 gauge needle with an inner diameter (ID) of about 1.05 mm, an outer diameter (OD) of about 1.60 mm, and a cannula diameter (CD) of about 2.50 mm.

[0128] In some embodiments, the cannula 904 can further include a secondary cannula disposed around the inner core of the needle 900. In some embodiments, the cannula 904 includes one or more vents 906. In some embodiments, the cannula 904 of the needle 900 can be in fluid communication with the vents 906. In some embodiments, the cannula 904 can be a slotted cannula having slots around the needle.

[0129] like Fig. 9B As shown, the low-capacity needle 910 may include a plastic body 912 and a needle valve stem 914. In some embodiments, the plastic body 912 may be a reservoir configured to maintain a volume of about 1 to 200 μL during liquid transfer in an analyzer device. In some embodiments, the plastic body 912 may include a filter 913 arranged therein to prevent contamination of a pipette coupled to the low-capacity needle 910. In some embodiments, the plastic body 912 may be made of a polypropylene (PP) material. In some embodiments, the plastic body 912 of the needle 910 may have a diameter of about 7.25 mm and a length of about 45 mm. In some embodiments, the needle valve stem 914 may have a length of about 10 mm. In some embodiments, the needle valve stem 914 may be made of stainless steel. In some embodiments, the low-capacity needle 910 may be a 29-gauge needle with an inner diameter (ID) of about 0.20 mm and an outer diameter (OD) of about 0.30 mm.

[0130] Fig.10 A diagram showing an example of a high capacity needle 900 according to an embodiment of the present disclosure is shown. In particular, Fig.10 Various examples of cannulas for high capacity needles 900 having vents, slots, etc. are shown. In some embodiments, Fig.10 The first needle in the needle can be vented by having two connected orifices or vents in the cannula of the needle. In some embodiments, Fig.10 The second and fourth needles shown in FIG. 7 can be vented when the tip of each needle is inserted into the septum (eg, septum 704) of the processing tube.

[0131] Fig.11A and Fig. 11B A diagram showing a spin column 1100 and a spin column basket 1102 according to an embodiment of the present disclosure is shown. The spin column 1100 and the spin column basket 1102 represent Fig. 6A 100 and 100. In particular, the spin column 1100 can be configured to perform nucleic acid extraction and purification from nucleic acids obtained from a sample. In some embodiments, after cell lysis is performed in the processing tube 700, the first pipette 202 or the second pipette 204 can transfer a portion of the liquid from the processing tube 700 to the spin column 1100. In some embodiments, the spin column 1100 can be configured to extract and purify nucleic acids from the portion of liquid. In some embodiments, the spin column 1100 can be configured to move to an elution position in the spin column basket 1102 to obtain nucleic acids from the sample. In some embodiments, the spin column basket 1102 can be configured to collect purified nucleic acids passing through the spin column 1100. In some embodiments, the spin column basket 1102 can include one or more tubular receiving containers, which are configured to receive purified nucleic acids, store the spin column 1100, and / or receive a needle coupled to the first pipette 202 or the second pipette 204.

[0132] In some embodiments, the spin column 1100 can have a height H of about 34 mm. In some embodiments, the upper portion of the spin column 1100 can have a diameter D of about 17 mm. In some embodiments, the spin column basket 1102 can have a length L of about 50 mm and a height H of about 60 mm. In some embodiments, the diameter of the tubular receiving container in the spin column basket 1102 can be about 19 mm. In some embodiments, the spin column 1100 and the spin column basket 1102 can be made of a polypropylene (PP) material.

[0133] Fig. 12AA diagram of a low-volume needle 910 docked with a sample preparation box 500 according to an embodiment of the present disclosure is shown. In particular, FIG. 12 shows an example of a low-volume needle 910 disposed in one of a plurality of reservoirs 504 of the sample preparation box 500, such as for transferring materials for sample concentration and / or lysis from the reservoir 504 to a sample in a processing tube. In some embodiments, the sample preparation box 500 can be configured to receive one needle, such as the low-volume needle 910. In other embodiments, the sample preparation box 500 can be configured to receive two needles, such as both a high-volume needle 900 and a low-volume needle 910.

[0134] Fig. 12B A low volume needle 910 is shown docked with a spin column basket 1102 according to an embodiment of the present disclosure. In some embodiments, the low volume needle 910 can be inserted into one of the tubular receiving receptacles of the spin column basket 1102. In some embodiments, the spin column 1100 can also be disposed in one of the tubular receiving receptacles of the spin column basket 1102. PCR cassette implementation plan:

[0135] Fig.13A and Fig. 13B A diagram of a PCR cassette 1300 according to an embodiment of the present disclosure is shown. PCR cassette 1300 represents an exemplary embodiment of PCR cassette 234. In particular, Fig.13A The assembled PCR cassette 1300 is shown, and Fig. 13B An exploded view of the components in PCR cassette 1300 is shown. PCR cassette 1300 includes lid 1302, septum 1308, and base 1312. In some embodiments, lid 1302 is disposed on septum 1308, and septum is disposed on base 1312.

[0136] In some embodiments, base 1312 includes two primary reaction chambers 1313 and multiple secondary reaction chambers 1314. In some embodiments, there can be at least one primary reaction chamber 1313 in base 1312. In some embodiments, the number of secondary reaction chambers 1313 in base 1312 can be in the range of about 10 to 50, for example, 20 secondary reaction chambers. In some embodiments, each primary reaction chamber 1313 can be maintained in a volume in the range of about 50 to 500 μL, for example, 100 μL. In some embodiments, each secondary reaction chamber 1314 can be maintained in a volume in the range of about 2 to 50 μL, for example, 30 μL. In some embodiments, each primary reaction chamber 1313 and multiple secondary reaction chambers 1314 can be sealed with a septum 1308, wherein septum 1308 is configured to receive a needle (e.g., needle 900 or 910).

[0137] In some embodiments, the PCR box 1300 may include an additional receiving container configured to receive a reagent tube 710 storing a first set of reagents for performing nucleic acid amplification. In other embodiments, the first set of reagents for performing nucleic acid amplification may be stored in a reservoir in the PCR box 1300 (such as in a reaction chamber in the PCR box 1300). For example, a primary reaction chamber 1313 of the PCR box 1300 may store a first set of reagents (e.g., a master mix) for performing nucleic acid amplification. In some embodiments, the needle 900 or 910 may dispense one or more reagents from the first set of reagents (e.g., stored in a primary reaction chamber 1313 in the PCR box 1300 or stored in the reagent tube 710) into a primary reaction chamber 1313 for performing nucleic acid amplification.

[0138] In some embodiments, at least one primary reaction chamber 1313 can be configured to receive nucleic acid from a sample (e.g., after concentration, lysis, and purification steps), which can be transferred from processing tube 700 after resuspending the master mix through needle 900 or 910 coupled to first pipette 202 or second pipette 204. In some embodiments, primary reaction chamber 1313 can be configured to perform a first amplification of the nucleic acid, thereby generating a first amplification product.

[0139] In some embodiments, one set of primers is present in primary reaction chamber 1313. In some embodiments, one set of dried primers is present in primary reaction chamber 1313. In some embodiments, one set of primers can be transferred into primary reaction chamber 1313 along with the nucleic acid used for the first amplification. In some embodiments, the one set of primers can be compatible with amplification of the target sequence to be detected. In some embodiments, a different set of primers can be transferred into primary reaction chamber 1313 along with the nucleic acid used for the first amplification. In some embodiments, a combination of different sets of primers can be compatible with amplification of the target sequence to be detected.

[0140] In some embodiments, secondary reaction chamber 1314 can be configured to receive a corresponding aliquot of the first amplification product from primary reaction chamber 1313 via needle 900 or 910. In some embodiments, multiple secondary reaction chambers 1314 can be configured to perform a second amplification of the product nucleic acid. In some embodiments, an aliquot of the first amplification product from primary reaction chamber 1313 can be diluted with the contents of one reservoir 504 of sample preparation cartridge 500.

[0141] In some embodiments, each secondary reaction chamber 1314 may include a set of reagents for reacting with a corresponding aliquot of the first amplification product. In some embodiments, the reagents disposed in each secondary reaction chamber 1314 may be in liquid form, or in a dried or freeze-dried form at the bottom of each secondary reaction chamber 1314. In some embodiments, the reagents disposed in each secondary reaction chamber 1314 may be added to each secondary reaction chamber 1314 in the base 1312 by the first pipette 202 or the second pipette 204, and then a plurality of aliquots of the first amplification product are dispensed into the secondary reaction chamber 1314. In some embodiments, the set of reagents in each secondary reaction chamber 1314 may correspond to the internal sequence of the amplicon in the first amplification product and may be specific to one or more target sequences to be detected. In some embodiments, the set of reagents in each secondary reaction chamber 1314 may include a fluorescent probe specific to one or more target sequences to be detected.

[0142] In some embodiments, after pathogen concentration, pathogen lysis to release nucleic acid, and nucleic acid purification in the processing tube 700, the first pipette 202 or the second pipette 204 in the analyzer device 200 can dispense nucleic acid from the sample into the primary reaction chamber 1313 in the PCR cartridge 1300. After the first amplification of the nucleic acid in the primary reaction chamber 1313, the first pipette 202 or the second pipette 204 in the analyzer device 200 can then dispense a plurality of aliquots of the first amplification product into the secondary reaction chamber 1314, wherein each aliquot corresponds to a respective secondary reaction chamber 1314. In some embodiments, the first amplification product can be referred to as a pre-amplification product.

[0143] In some embodiments, each secondary reaction chamber 1314 can be configured to receive an aliquot of the first amplification product by non-contact dispensing of the aliquot by a needle (e.g., needle 900 or 910 coupled to the first pipette 202 or the second pipette 204). In some embodiments, the needle can penetrate the septum 1308 of each secondary reaction chamber 1314 to dispense each aliquot without the needle contacting the bottom surface of each secondary reaction chamber 1314. In some embodiments, the volume of each aliquot dispensed to each secondary reaction chamber 1314 by the needle 900 or 910 can be in the range of about 0.5 μL to about 5 μL. In some embodiments, the first pipette 202 or the second pipette 204 can add a set of reagents prepared for nucleic acid amplification in a reagent tube (e.g., reagent tube 710) to the first amplification product, and then dispense multiple aliquots of the first amplification product into the secondary reaction chambers 1314 in the PCR box 1300.

[0144] In some embodiments, at least one primary reaction chamber 1313 and multiple secondary reaction chambers 1314 may include mineral oil to prevent at least one of evaporation, aerosol formation, and cross contamination. In some embodiments, each secondary reaction chamber 1314 may be configured to receive mineral oil before receiving a corresponding aliquot of the first amplification product from needle 900 or 910. In some embodiments, the mineral oil in each secondary reaction chamber 1314 may be stored separately from the set of reagents in each secondary reaction chamber 1314. In some embodiments, at least one primary reaction chamber 1313 and multiple secondary reaction chambers 1314 may receive another set of reagents for nucleic acid amplification (e.g., in addition to the reagents stored in the secondary reaction chamber 1314). In some embodiments, the other set of reagents may be dried or freeze-dried and stored in a reagent tube (such as reagent tube 710).

[0145] In some embodiments, each primary reaction chamber 1313 and secondary reaction chamber 1314 includes a conical shape and a bottom wall. In some embodiments, each primary reaction chamber 1313 can have a width greater than the individual width of each secondary reaction chamber 1314. In some embodiments, the diameter of the bottom wall of each of at least one primary reaction chamber 1313 and multiple secondary reaction chambers 1314 can be less than about 2.5 mm. In some embodiments, non-contact dispensing of multiple aliquots to each secondary reaction chamber 1314 can include using a needle 900 or 910 in the sample preparation box 500 for jet dispensing to avoid cross-contamination of the reaction chambers of the aliquots, the needle penetrating the corresponding septum of the secondary reaction chamber 1314 and the needle 900 or 910 not contacting the bottom wall of each secondary reaction chamber 1314.

[0146] In some embodiments, the bottom wall of each primary reaction chamber 1313 and secondary reaction chamber 1314 can be optically transparent and configured for optical interrogation. In some embodiments, the bottom wall of each primary reaction chamber 1313 and secondary reaction chamber 1314 can be configured for optical interrogation, such as by PCR subsystem 244 in analyzer device 200. In some embodiments, the bottom wall of each primary reaction chamber 1313 and secondary reaction chamber 1314 can be configured for fluorescence detection, such as by PCR subsystem 244 in analyzer device 200. In some embodiments, multiple primary reaction chambers 1313 and secondary reaction chambers 1314 can be configured to fit into corresponding chambers in a temperature control block in PCR subsystem 244, and the temperature control block can be configured to heat the sides of multiple primary reaction chambers 1313 and secondary reaction chambers 1314.

[0147] The septum 1308 seals each primary reaction chamber 1313 and each secondary reaction chamber 1314 of the base 1312. In some embodiments, the septum 1308 can be referred to as a sealing cap. In some embodiments, the septum 1308 can include a single body (unibody) that extends across the primary reaction chamber 1313 and multiple secondary reaction chambers 1314 of the base 1312. In some embodiments, the septum 1308 can include a plurality of parts assembled together, wherein each part covers one or more corresponding primary reaction chambers 1313 and secondary reaction chambers 1314 of the base 1312. The septum 1308 can be configured to receive a needle, such as a needle 900 or 910 coupled to the first pipette 202 or the second pipette 204. In some embodiments, the needle can create an orifice in the septum 1308 during insertion, and the orifice in the septum 1308 can be closed due to the material of the septum 1308 when the needle is removed.

[0148] In some embodiments, the diaphragm 1308 may include at least one of rubber, polytetrafluoroethylene (PTFE), thermoplastic elastomer (TPE), silicone, butyl rubber, or a combination thereof. In some embodiments, the diaphragm 1308 may include a double layer of polytetrafluoroethylene (PTFE) and another material selected from silicone, rubber, and butyl rubber. In some embodiments, the diaphragm 1308 may have a thickness in the range of about 1 to 2 mm. In some embodiments, the diaphragm 1308 may be designed so that optical interrogation (e.g., fluorescence) can be performed from above by the PCR subsystem 244.

[0149] In some embodiments, cover 1302 may include a plurality of openings 1306, wherein each opening 1306 is aligned with a corresponding primary reaction chamber 1313 or secondary reaction chamber 1314 in a plurality of primary reaction chambers 1313 and secondary reaction chambers 1314 in base 1312. In some embodiments, cover 1302 may also include identifier 1310. Identifier 1310 may be an identifier of PCR cartridge 1300, which is scanned by analyzer device 200 for pathogen identification. In some embodiments, identifier 1310 may be at least one of an identification code, a barcode, or a data matrix.

[0150] In some embodiments, the cover 1302 can be fitted onto the septum 1308 and the base 1312 to form the assembled PCR cartridge 1300. In some embodiments, the septum 1308 can be overmolded into the cover 1302 to form a combined component, and the combined component can be assembled onto the base 1312 by at least one of a snap-fit ​​joint or a mechanical fastener. In some embodiments, the cover 1302, the septum 1308, and the base 1312 can be interlocked or clamped together by one or more mechanical fasteners.

[0151] In some embodiments, the base 1312, the diaphragm 1308, and the cover 1302 each include an opening 1304 in the center of the PCR box 1300. The size or shape of the opening 1304 can be designed to engage with an object manipulator within the system. For example, in some embodiments, the opening 1304 can be a circular hole that is compatible with being inserted by a pipette tip holder (e.g., a first pipette 202 and a second pipette 204) to move the PCR box 1300 in the analyzer device 200. In some embodiments, the openings 1304 in the base 1312, the diaphragm 1308, and the cover 1302 can be aligned with each other when the PCR box 1300 is assembled. In some embodiments, the base 1312 and the cover 1302 can be made of polypropylene (PP) or polycarbonate (PC) material. In some embodiments, the size of the assembled PCR box 1300 can be about 135 mm (length) x 15 mm (width) x 10 mm (height).

[0152] In some embodiments, assembled PCR box 1300 can be configured to receive a heated cover that is disposed on cover 1302 and on primary reaction chamber 1313 and secondary reaction chamber 1314 during PCR performed within PCR subsystem 244. In some embodiments, heated cover can be configured to heat the upper portion of at least one primary reaction chamber 1313 and multiple secondary reaction chambers 1314 to prevent condensation during thermal cycling.

[0153] Fig.14 A diagram of a low volume needle 910 inserted into a PCR cassette 1300 according to an embodiment of the present disclosure is shown. In particular, Fig.14 The needle stem 914 of the needle 910 is shown piercing the septum 1308 and entering the secondary reaction chamber 1314 of the PCR cartridge 1300. In some embodiments, the needle stem 914 can create an orifice in the septum 1308 during insertion. When the needle stem 914 is removed, the orifice in the septum 1308 can close due to the material of the septum 1308. Embodiment of the PCR method in a PCR cassette:

[0154] In diagnostic applications where there is a high sensitivity requirement (due to the low copy number available in the original sample) and a large number of target microorganisms, two-stage PCR can provide an attractive combination to meet both extremes. However, due to the need to retrieve the amplification product after the first amplification step (pre-amplification) and deliver the amplification product to a second container for further amplification, the two-stage PCR method may not be often used. In some embodiments, the use of the amplification product may involve the risk of contaminating the test equipment as well as the laboratory facilities, which may result in extensive cleaning or even closure of the laboratory facilities.

[0155] In some embodiments, PCR cassette 1300 includes primary reaction chamber 1313 and secondary reaction chamber 1314 sealed by septum 1308, which can allow transfer of pre-amplification liquid volumes to subsequent PCR reactions while minimizing contamination risk without the need to work with open tubes. In some embodiments, the configuration of PCR cassette 1300 can be compatible with real-time PCR, where fluorescence readings are performed by PCR subsystem 244 from below reaction chambers 1313, 1314. In some embodiments, septum cover 1308 can be designed so that fluorescence optical interrogation can be performed from above.

[0156] In some embodiments, reagents for the secondary reaction in secondary reaction chamber 1314 can be dried at the bottom of secondary reaction chamber 1314. In some embodiments, jet dispensing can be used to deliver the pre-amplification product to secondary reaction chamber 1314 (e.g., with a volume in the range of 5 uL or less). In some embodiments, the jet dispensing method can prevent contact dispensing, which may involve cross-contamination issues, and a spin step that may slow down the overall process.

[0157] In some embodiments, the PCR assay strategy can include (1) a first-stage PCR primer set to increase the copy number of the pathogen of interest, (2) a dilution step from the first stage to the second stage, and (3) a second-stage PCR performed by multiple reaction chambers (such as in a Taqman-based assay) to provide further specificity and semi-quantitative information.

[0158] In some embodiments, a method for performing contamination-free two-stage PCR may include: (1) delivering a template to at least one primary reaction chamber 1313 in a PCR box 1300 through a membrane 1308, wherein the template includes a set of reagents for nucleic acid amplification; (2) performing a first amplification step in at least one primary reaction chamber 1313; (3) removing a first amplification product from at least one primary reaction chamber 1313 through the membrane 1308; and (4) performing non-contact distribution of corresponding aliquots of the first amplification product to multiple secondary reaction chambers 1314 through the membrane 1308, wherein each secondary reaction chamber 1314 includes reagents for performing a specific reaction from the multiple secondary reaction chambers 1314. Implementation of modules and subsystems in the analyzer:

[0159] Fig.15A and Fig. 15B A diagram of an exemplary centrifuge for use in an analyzer device 200 according to an embodiment of the present disclosure is shown. In particular, Fig.15A A first centrifuge 1502 is shown that can be used to centrifuge samples in tubes 1504 or spin column baskets 1510, while Fig. 15B A second centrifuge 1512 is shown that can be used to centrifuge samples in a PCR box 1514. The first centrifuge 1502 and the second centrifuge 1512 are respectively Figure 3B 1504 and PCR box 1514 represent an exemplary embodiment of the first centrifuge 240 and the second centrifuge 242 shown in FIG. Figure 7A-7B and Figure 13A-13B 15. In some embodiments, the spin column basket 1510 may include a spin column stored in a receiving container therein. The spin column basket 1510 represents Fig.11A , Fig. 11B and Fig. 12B An exemplary embodiment of a spin column basket 1102 is shown in FIG.

[0160] In some embodiments, the first centrifuge 1502 can be a high-speed centrifuge configured to apply a relative centrifugal force (RCF) or g-force of about 12,000 G to the processing tubes 1504 and / or the spin column basket 1510. In some embodiments, the second centrifuge 1512 can be a low-speed centrifuge configured to apply a relative centrifugal force (RCF) or g-force of about 3,000 G to the PCR box 1514.

[0161] In some embodiments, the first centrifuge 1502 can hold the processing tubes 1504 and / or the spin column baskets 1510 in a first orientation and apply a 45° swing bucket centrifugation to the processing tubes 1504 and / or the spin column baskets 1510. In some embodiments, the second centrifuge 1512 can hold the PCR cassettes 1514 in another orientation and apply a 90° swing bucket centrifugation such that the PCR cassettes 1514 move to a vertical position in the second centrifuge 1512.

[0162] In some embodiments, the first centrifuge 1502 and the second centrifuge 1512 can centrifuge multiple processing tubes 1504, spin column baskets 1510, and PCR boxes 1514 at a time. For example, the first centrifuge 1502 can be configured to hold two processing tubes 1504 and two spin column baskets 1510 at a time for centrifugation together. In another example, the second centrifuge 1512 can be configured to hold two PCR boxes 1514 at a time for centrifugation together. In some embodiments, the processing tubes 1504 can be moved to the first centrifuge 1502 during the concentration and lysis steps to separate nucleic acids from samples with pathogens.

[0163] Fig.16A , Fig. 16B , Fig. 16C and Fig.16DA diagram of an exemplary homogenization subsystem 1600 for use in an analyzer device 200 according to an embodiment of the present disclosure is shown. The homogenization subsystem 1600 represents Figure 3B 246 . In some embodiments, the homogenization subsystem 1600 can apply a rocking motion to the processing tube 700 to allow the sample to oscillate and mix with other materials. In some embodiments, the processing tube 700 can be placed in the homogenization subsystem 1600 by a pipetting system (e.g., a first pipette 202 or a second pipette 204) for mixing the pathogen in the processing tube 700 with any reagent, such as for performing blood cell lysis. In some embodiments, the processing tube 700 can be placed in the homogenization subsystem 1600 for mixing one or more lysis reagents or buffers with the blood sample in the processing tube 700 to lyse the blood cells in the blood sample. In some embodiments, the mixing function of the homogenization subsystem 1600 can be used for sample processing to perform both pathogen identification and / or antimicrobial sensitivity testing in the analyzer device 200.

[0164] In some embodiments, the homogenization subsystem 1600 can be a mixer so that it rotates one or more processing tubes 700 in a horizontal position by rocking the processing tubes 700 back and forth at an angle of ±30°. In some embodiments, four processing tubes 700 can be loaded into the homogenization subsystem 1600 at a time. In some embodiments, the homogenization subsystem 1600 can include a magnet 1601 that moves back and forth between an upward position and a downward position. In some embodiments, one or more processing tubes 700 can include magnetic beads configured to attach to nucleic acids in the one or more processing tubes 700. In some embodiments, the magnet 1601 can be used to retain nucleic acids attached to the magnetic beads in the one or more processing tubes 700.

[0165] In some embodiments, the homogenization subsystem 1600 can include an additional or alternative independent magnet station that can be used to retain the nucleic acid attached to the magnetic beads in the processing tube 700 and / or the PCR box 1300. In some embodiments, the magnet station in the homogenization subsystem 1600 can be used to move the nucleic acid attached to the magnetic beads to the bottom or side of the processing tube 700 so that the remaining liquid in the processing tube can be removed and the nucleic acid is retained.

[0166] Fig.17A , Fig. 17B and Fig. 17C A diagram of an exemplary mechanical device 1700 for use in an analyzer apparatus 200 according to an embodiment of the present disclosure is shown. The mechanical device 1700 represents Figure 3B. In some embodiments, the mechanical device 1700 can hold two processing tubes 700 in a vertical position and provide rapid vibratory movement to the processing tubes 700, such as for lysis of microorganisms in a sample. In some embodiments, the mechanical device 1700 can be an agitator. In additional or alternative embodiments, the mechanical device 1700 can include a sonicator configured to sonicate the processing tubes 700 to agitate the sample.

[0167] In some embodiments, there may be a plurality of lysis beads disposed within each processing tube 700 for lysis. In some embodiments, the plurality of lysis beads may have a predetermined size and material configured to lyse at least one of the following pathogens: yeast, fungi, gram-positive bacteria, or gram-negative bacteria, etc. In some embodiments, the plurality of lysis beads may be a mixture of a plurality of predetermined sizes and materials configured to lyse a variety of pathogen types, including yeast, fungi, gram-positive bacteria, gram-negative bacteria, etc. In some embodiments, a rapid vibrating motion applied to the processing tube 700 may excite the plurality of lysis beads in the processing tube 700, causing the lysis beads to lyse the pathogen in the sample in the processing tube 700. In some embodiments, the rapid movement of the lysis beads (applied by the mechanical device 1700) may mechanically disrupt the cell walls of the pathogens in the sample, resulting in the release of nucleic acids from the pathogens.

[0168] Figures 18A-18C 1 is a diagram showing different views of a PCR cartridge 1300 docked with a PCR subsystem 1800 in an analyzer according to an embodiment of the present disclosure. Figure 3B 1800 is an exemplary embodiment of the PCR subsystem 244 shown in . In some embodiments, the PCR subsystem 1800 can be a module configured to perform PCR of nucleic acids in the PCR box 1300, optical detection of fluorescent signals from PCR amplification, and pathogen identification based on fluorescent signals. In some embodiments, the PCR subsystem 1800 can include one or more heat blocks, a heat sink 1802, a fan 1804, a heated cover 1806, a sensor unit 1810, and a linear translation stage 1814. In some embodiments, the PCR subsystem 1800 can include one or more heat blocks, thermoelectric cooler (TEC) elements, conductive elements, Peltier cells, and / or the like.

[0169] In some embodiments, the PCR box 1300 can be pressed down to ensure contact with the heat block, which can be part of a thermal cycler configured to control temperature when performing nucleic acid amplification. In some embodiments, the heat block can control the temperature within a range of about 35°C to 100°C with a set resolution of about 0.1°C. In some embodiments, the heat block can have a temperature accuracy of about ±0.25°C relative to the setting (e.g., steady state). In some embodiments, the temperature ramp rate of the heat block can be greater than about 10 degrees (in °C) / second. In some embodiments, the temperature spectrum of the heat block can be configured or customized by the user of the analyzer device 200 for performing thermal cycling.

[0170] In some embodiments, the PCR subsystem 1800 includes a thermoelectric cooler (TEC) element, a heat sink 1802, and one or more fans 1804 disposed below each thermal block and below the PCR cartridge 1300. In some embodiments, the TEC element, the heat sink 1802, and the fans 1804 can be used to heat and / or cool the PCR cartridge 1300 to maintain a specific temperature during the amplification process of PCR. In some embodiments, the fans 1804 can be distributed along the length of the heat sink 1802, such as Fig.18A and Fig.18C 1304. In some embodiments, the heating cover 1806 may be disposed on the cover 1302 of the PCR box 1300 and on the primary reaction chamber 1313 and the secondary reaction chamber 1314. In some embodiments, the heating cover 1806 may be configured to heat the upper portion of at least one primary reaction chamber 1313 and a plurality of secondary reaction chambers 1314 to prevent condensation during thermal cycling in the PCR subsystem 1800. In some embodiments, the heating cover 1806 may control the temperature within a range of about 35°C to 100°C with a setting resolution of about 0.1°C. In some embodiments, the heating cover 1806 may have a temperature accuracy of about ±0.25°C relative to a setting (e.g., steady state).

[0171] In some embodiments, the sensor unit 1810 (in Fig.18A and Fig.18B 1300) can include one or more fluorescence sensors configured to take fluorescence readings from the bottom of the secondary reaction chamber 1314 in the PCR cartridge 1300 through the thermal block. In some embodiments, the sensor unit 1810 can be configured to detect fluorescence from a plurality of fluorescent dyes (such as FAM TM , ROX TM , In some embodiments, the sensor unit 1810 may include one or more solid-state based optical elements (e.g., LED / PIN diodes). In some embodiments, one or more key parameters of the sensor unit 1810 (e.g., LED power, read time, etc.) may be configured by software (e.g., installed on the processing device 116).

[0172] In some embodiments, linear translation stage 1814 can be a translation stage that allows positioning / scanning sensor unit 1810 below primary reaction chamber 1313 and secondary reaction chamber 1314 of PCR box 1300. In some embodiments, the bottom wall of each primary reaction chamber 1313 and secondary reaction chamber 1314 can be optically transparent so that sensor unit 1810 can optically interrogate reaction chambers 1313, 1314 and detect fluorescent signals. In some embodiments, a set of optical fibers can be used to deliver excitation to primary reaction chamber 1313 and secondary reaction chamber 1314 of PCR box 1300 and collect emission therefrom.

[0173] In some embodiments, light can be prevented from entering the PCR subsystem 1800 during thermal cycling. In some embodiments, the PCR cassette 1300 can be pushed down during operation (e.g., by positioning between thermal blocks and under the heated cover 1806) to ensure optimal thermal contact.

[0174] Figure 19A-19B FIG. 18 is a diagram illustrating an exemplary fluorescence sensor subsystem 1900 in an analyzer according to an embodiment of the present disclosure. The fluorescence sensor subsystem 1900 represents an exemplary embodiment of the PCR subsystem 1800 shown in FIG. Fig.19A As shown, fluorescence sensor subsystem 1900 includes heat block 1902, Peltier cells 1912, heat sink 1916, fluorescence sensor 1910, and linear translation stage 1914. In some embodiments, PCR cartridge 1300 can be pressed down on heat block 1902 and adjacent to Peltier cells 1912. In some embodiments, fluorescence sensor 1910 can be configured to take fluorescence readings from the bottom of secondary reaction chamber 1314 in PCR cartridge 1300. In some embodiments, at least heat block 1902 and Peltier cells 1912 are surrounded by housing 1920, such as Fig.19B Housing 1920 may include one or more identifiers 1922 containing information about the PCR cartridge (eg, sample identifier, date, etc.). Exemplary operation method:

[0175] Fig. 20A flow chart of a method 2000 for performing sample processing (including concentration and lysis of a sample) prior to pathogen identification according to an embodiment of the present disclosure is shown. In some embodiments, the method 2000 may describe steps for performing sample processing using various components in a pathogen identification system, including an analyzer 108, 200; a sample preparation cartridge 114, 224, 500; and a processing tube 113, 510, 700, as described above with reference to Figure 1-Figure 1 9. It should be understood that the operations shown in method 2000 are not exhaustive, and other operations may be performed before, after, or between any of the operations shown. In various embodiments of the present disclosure, the operations of method 2000 may be performed in different orders and / or variations.

[0176] Fig. 20 Method 2000 of begins at step 2002, in which a sample preparation box and a sample tube are received in an analyzer device. In some embodiments, the analyzer device 200 may receive a sample preparation box 224 and a sample tube, which are placed in a sample box drawer 220 and a sample drawer 210 of the analyzer device 200 by a user or operator of the analyzer device 200, respectively. In some embodiments, the sample box drawer 220 and the sample drawer 210 may be referred to as the first compartment and the second compartment of the analyzer device 200, respectively. In some embodiments, the sample tube may be a sample obtained from a patient, wherein the sample comprises a pathogen. In some embodiments, the sample in the sample tube may include whole blood, urine, sterile body fluids or other samples obtained from a patient.

[0177] In step 2004, the first needle from the sample preparation cartridge is installed in the pipette system in the analyzer device. In some embodiments, the needle 512 from the sample preparation cartridge 500 can be installed in the first pipette 202 by moving the pipette tip of the first pipette 202 over the sample preparation cartridge 500, pressing and fitting into the plastic body portion of the needle (e.g., plastic body 902).

[0178] At step 2006, the first needle is inserted into the sample tube using the pipette system. In some embodiments, after the needle 512 is installed, the first pipette 202 can be moved over the sample tube in the sample drawer 210, and then the needle 512 is pressed down and inserted into the sample tube.

[0179] In step 2008, the sample is transferred from the sample tube to the processing tube in the sample preparation box by the first needle. In some embodiments, the needle 512 can draw the sample into the plastic body reservoir of the first pipette 202, and the first pipette 202 can be moved to the sample preparation box 500 to transfer the sample to the processing tube 510 in the sample preparation box 500.

[0180] To transfer the sample from the plastic body reservoir of the first or second pipette 202 to the processing tube 510, the needle 512 (coupled to the first pipette 202) can pierce the septum of the processing tube (e.g., the septum 704 of the processing tube 700). The first pipette 202 can then dispense the sample into the processing tube through the needle 512.

[0181] In step 2010, one or more lysis reagents may be added to the processing tube to lyse the blood cells in the sample. In some embodiments, the first pipette 202 may add one or more lysis reagents, such as a saponin-based lysis buffer, to the processing tube 700 through the needle 512. The one or more lysis reagents may be mixed with the sample in the processing tube 700 (e.g., by the homogenization subsystem 246) to lyse the blood cells in the sample.

[0182] After the blood cells are lysed, the processing tube is moved to a centrifuge in the analyzer device in step 2012. In some embodiments, the first pipette 202 or the second pipette 204 can be configured to move the processing tube 700 to the first centrifuge 1502 by manipulating the top end (e.g., the cylindrical cavity in the cap 702) of the processing tube 700 compatible with the pipette system. In some embodiments, the pipette tip of the first pipette 202 or the second pipette 204 can be pressed and fitted into the cylindrical cavity in the cap 702 to pick up and move the processing tube to the first centrifuge 1502 in the analyzer device 200.

[0183] In step 2014, the treatment tube is centrifuged in a centrifuge to concentrate one or more pathogens in the sample. In some embodiments, the treatment tube 700 is centrifuged in the first centrifuge 1502 to concentrate the pathogens in the treatment tube 700 from a high volume (e.g., 10 mL) to a low volume (e.g., 0.5 mL or less). In some embodiments, the volume of the sample in the sample tube is in the range of about 0.5 mL to about 10 mL, and the volume of the concentrated pathogens in the treatment tube 700 after centrifugation is in the range of about 0.1 mL to about 2 mL. In some embodiments, after the centrifugation, the volume of the concentrated pathogens in the treatment tube is in the range of about 0.1 mL to about 1 mL.

[0184] In step 2016, a pipette system is used to remove fluid from the treatment tube, thereby leaving concentrated pathogens in the treatment tube. In some embodiments, a needle 512 coupled to the first pipette 202 can collect and remove fluid from the treatment tube 700, thereby leaving concentrated pathogens at the bottom of the treatment tube 700. In step 2018, a lysis buffer can be added to the treatment tube using a pipette system. In some embodiments, a needle 512 coupled to the first pipette 202 can retrieve one or more lysis buffers from one or more reservoirs 504 in the sample preparation box 500, and the one or more lysis buffers are dispensed into the treatment tube 700 by the needle 512.

[0185] In some embodiments, reagents can be added to the processing tube 700 using a needle 512 coupled to the first pipette 202 to perform a DNA depletion step, and then a lysis buffer is added to the processing tube 700. In some embodiments, the reagents for performing the DNA depletion step can include at least one of DNase I (deoxyribonuclease I), DNase II (deoxyribonuclease II), or Benzonase.

[0186] In step 2020, the processing tube can be moved to the device in the analyzer device using the pipette system. In some embodiments, the first pipette 202 or the second pipette 204 can move the processing tube 700 to the mechanical device 1700 in the analyzer device 200. In some embodiments, the first pipette 202 or the second pipette 204 can be configured to move the processing tube 700 to the mechanical device 1700 by manipulating the top end of the processing tube 700 compatible with the pipette system (e.g., the cylindrical cavity in the cap 702). In some embodiments, the pipette core tube of the first pipette 202 or the second pipette 204 can be pressed and fitted into the cylindrical cavity in the cap 702 to pick up and move the processing tube to the mechanical device 1700 in the analyzer device 200.

[0187] In step 2022, the treatment tube can be agitated with an apparatus to perform cell lysis of concentrated pathogens. In some embodiments, the mechanical device 1700 can apply a rapid vibration motion to the treatment tube 700 to perform cell lysis of concentrated pathogens in the treatment tube 700. In some embodiments, the mechanical device 1700 can be an agitator. In some embodiments, the treatment tube 700 can be maintained in the agitator in a vertical position. In some embodiments, applying a rapid vibration motion can include applying a reciprocating motion along a predetermined axis by the agitator. In additional or alternative embodiments, agitating the treatment tube 700 can include using an ultrasonicator in the analyzer device 200 to ultrasonicate the treatment tube 700.

[0188] Fig.21 A flow chart of a method 2100 for performing PCR for pathogen identification according to an embodiment of the present disclosure is shown. In some embodiments, method 2100 may describe performing PCR for pathogen identification in a PCR cassette (such as PCR cassette 234, 1300), as described above with reference to Figure 3B-Figure 1 9. It should be understood that the operations shown in method 2100 are not exhaustive, and other operations may also be performed before, after, or between any of the operations shown. In various embodiments of the present disclosure, the operations of method 2100 may be performed in different orders and / or variations.

[0189] Fig.21 The method 2100 of the present invention begins at step 2102, wherein nucleic acid is transferred to at least one primary reaction chamber in a PCR cartridge inserted into the analyzer device by a pipette system in the analyzer device. In some embodiments, the PCR cartridge 234 may be inserted into the analyzer device 200 when it is placed in the processing cartridge drawer 230 by a user or operator of the analyzer device 200. In some embodiments, a low-volume needle 910 coupled to the second pipette 204 may transfer nucleic acid from the spin column basket 1102 or from one or more reservoirs 504 of the sample preparation cartridge 500 after extraction and purification. In some embodiments, the low-volume needle 910 coupled to the second pipette 204 may transfer nucleic acid to at least one primary reaction chamber 1313 in the PCR cartridge 1300. In some embodiments, the first pipette 202 or the second pipette 204 may add a set of reagents prepared for nucleic acid amplification in a reagent tube (e.g., reagent tube 710) to the nucleic acid, and then transfer the nucleic acid to at least one primary reaction chamber 1313 in the PCR cartridge 1300. In some embodiments, the set of reagents for nucleic acid amplification may be stored in one primary reaction chamber 1313 in the PCR box 1300, and may be dispensed into the second primary reaction chamber 1313 to be suspended together with the nucleic acid in the sample.

[0190] In step 2104, a first amplification of the nucleic acid is performed in at least one primary reaction chamber to produce a first amplification product. In some embodiments, the first amplification of the nucleic acid is performed in at least one primary reaction chamber 1313 of the PCR box 1300 using PCR technology. In some embodiments, a set of primers can be transferred to the primary reaction chamber 1313 together with the nucleic acid used for the first amplification. In some embodiments, the set of primers can be compatible with the amplification of the target sequence to be detected. In some embodiments, different sets of primers can be transferred to the primary reaction chamber 1313 together with the nucleic acid used for the first amplification. In some embodiments, the combination of different sets of primers can be compatible with the amplification of the target sequence to be detected.

[0191] In step 2106, a needle of the analyzer device is inserted through the septum of at least one primary reaction chamber to remove the first amplification product. In some embodiments, a low volume needle 910 coupled to the second pipette 204 can be inserted through the septum 1308 disposed on the primary reaction chamber 1313 to collect the first amplification product from the primary reaction chamber 1313 of the PCR box 1300.

[0192] In step 2108, a plurality of aliquots of the first amplification product are dispensed to a plurality of secondary reaction chambers in the PCR cartridge through the respective septa of the plurality of secondary reaction chambers. In some embodiments, a plurality of aliquots of the first amplification product can be dispensed by inserting a low volume needle 910 coupled to the second pipette 204 through one or more septa 1308 into a plurality of secondary reaction chambers 1314 in the PCR cartridge 1300. In some embodiments, each aliquot can correspond to a respective secondary reaction chamber 1314 in the PCR cartridge 1300. In some embodiments, an aliquot of the first amplification product from the primary reaction chamber 1313 can be diluted with the contents of one reservoir 504 of the sample preparation cartridge 500.

[0193] In some embodiments, each secondary reaction chamber 1314 may include a set of reagents for reacting with a corresponding aliquot of the first amplification product. In some embodiments, the set of reagents in each secondary reaction chamber 1314 may correspond to an internal sequence of an amplicon in the first amplification product and may be specific to one or more target sequences to be detected. In some embodiments, the set of reagents in each secondary reaction chamber 1314 may include a fluorescent probe specific to one or more target sequences to be detected.

[0194] In some embodiments, the first pipette 202 or the second pipette 204 can add a set of reagents prepared for nucleic acid amplification in a reagent tube (e.g., reagent tube 710) to the first amplification product, and then dispense a plurality of aliquots of the first amplification product into the secondary reaction chambers 1314 in the PCR box 1300. In some embodiments, dispensing the plurality of aliquots into the plurality of secondary reaction chambers 1314 can include performing non-contact dispensing of the aliquots using a jet dispenser through a needle 910 that penetrates a corresponding septum 1308 of the secondary reaction chamber 1314 and the needle 910 does not contact the bottom surface of each secondary reaction chamber 1314.

[0195] In step 2110, a second amplification of an aliquot of the first amplification product is performed in a plurality of secondary reaction chambers. In some embodiments, a second amplification of an aliquot of the first amplification product is performed in a plurality of secondary reaction chambers 1314 of PCR cassette 1300 using PCR techniques.

[0196] In some embodiments, the fluorescence at the bottom of each secondary reaction chamber 1314 can be detected by the PCR subsystem 1800 to monitor the second amplification and PCR and detect the amplified nucleic acid. In some embodiments, the processing device 116 can receive fluorescence data from the monitoring second amplification and PCR workflow from the PCR subsystem 1800. In some embodiments, the processing device 116 can then use the fluorescence data to detect the amplified nucleic acid in the secondary reaction chamber 1314, and identify the pathogens present in the patient sample based on the detected amplified nucleic acid. Experimental Example:

[0197] As discussed below, several experiments were conducted to test various embodiments of the pathogen ID systems and methods described above. Example 1: Optimization of sample preparation and DNA extraction protocol

[0198] The experiments shown in this example were performed to improve DNA purification by increasing the buffer extraction volume and represent certain embodiments disclosed in this patent application.

[0199] 10 mL of human whole blood was spiked with Klebsiella pneumoniae (K. pneumoniae) at a concentration of 1 CFU / mL and the following sample treatments were performed:

[0200] Blood spiked with Klebsiella pneumoniae was transferred to a 15 mL conical tube (treatment tube) containing 0.7 mL saponin 2.8% w / v (Sigma-Aldrich, S4521), 0.5 mL cushioning agent FC-40 (Sigma-Aldrich, F9755) and a mixture of zirconium oxide-silica beads containing: 0.25 mL 0.1 mm + 0.25 mL 0.5 mm + 10 beads of 1 mm diameter and 3-4 beads of 2.7 mm diameter. Blood and reagent were mixed for 30 seconds (five inversions) at a speed of 10 rpm on a PTR-35 platform under a rotating orbital motion for blood cell homogenization and lysis. In order to separate the supernatant from the remainder, the sample was centrifuged at 12000 x g for 10 minutes in a fixed angle rotor FA-45-6-30 in a 5810 Eppendorf centrifuge. Keeping the tube upright, carefully remove and discard the supernatant, leaving a total volume of 1 mL in the tube containing the bead mixture, cushion, and concentrated blood sample.

[0201] To test pathogen DNA purification improvements, tests were performed with increasing volumes of lysis buffer and / or binding buffer.

[0202] The lysis buffer is homogenized, and the buffer of different volumes (0.45mL and 0.9mL) is added to the mixture of pearl and sample. The test condition thing (test condition) is mixed for 5 seconds by vortex. Next, the sample tube is fixed on the customized stirring platform (reciprocating motion, 2 degrees, 16.000rpm), it is opened for 30 seconds so that sample cracking. Carry out 4 pulses of 30 seconds, wait 5 seconds between them, continue the cracking of 2min altogether. In this process, pipe is maintained in vertical position. Once mechanical cracking ends, the sample tube is centrifuged for 5min with 3200x g in swing bucket rotor A-4-62, to separate pearl and cell debris from lysate. Pipe is maintained in vertical position, by pipetting, lysate is transferred and fully mixed with the binding buffer of different volumes (1mL and 2mL). Each test condition uses two spin columns, and 0.5mL lysate is loaded on each post and with 10000x g (FA24X 2 rotors) centrifuged for 1 minute. Repeat this step until all lysates pass through the column. The spin column is washed twice with 0.5mL wash buffer, centrifuged at 10000x g for 1 minute, and dried by centrifugation at 13000x g for 2 minutes. The spin column is transferred to a 1.5mL tube, and 60 μL elution buffer is loaded thereon. The sample is incubated at room temperature for 5 minutes to ensure proper contact of DNA with the elution buffer and optimal DNA recovery, and then centrifuged at 10000xg for 1 minute. The volumes of the two eluates belonging to the same sample obtained in the two columns are mixed in the last tube. It should be noted that this process can be considered equivalent to using a single spin column, the binding surface of the single spin column being equal to the sum of the binding surfaces of the separate spin columns used in this experiment.

[0203] The results are Fig.23 These results indicate that increasing the volume of both lysis buffer and binding buffer improves the amount of DNA extracted.

[0204] Based on the observed results, in another example, the lysis buffer and binding buffer volumes were increased by x4 compared to the nominal volumes (1.8 mL and 4 mL, respectively), and the sample preparation protocol shown above was performed. Fig.24 The results presented in show that the amount of total DNA obtained is increased compared to the protocol using the nominal lysis buffer volume. In addition, pathogen detection is 1 Ct earlier than samples treated with the recommended volume of buffer.

[0205] Since each single column in this example consisted of three silica membranes and two columns improved the extraction yield, the number of layers in the spin column was changed from 3 to 6 in order to increase the total DNA obtained. In this assay, all conditions tested produced good DNA quality and no differences were observed related to pathogen detection. However, slightly lower DNA amounts were obtained from those samples processed using the 6-layer column. In another experiment, two 6-layer columns were used and the yields were higher compared to those using only one 6-layer column, as shown in Figure 2. Fig.25 As a conclusion, the more columns and silica layers used, i.e. the more DNA binding surface, the higher the amount of DNA recovered. Example 2: End-to-end protocol using a single tip in sample preparation

[0206] This example demonstrates how to perform the operations disclosed in the method of this patent using a single device for transferring fluids along all operations, which can be performed with a needle in the disclosed method embodiment. In this test example, this is simulated with a single pipette tip.

[0207] 10 mL of human whole blood spiked with 1.3 CFU / mL of Klebsiella pneumoniae was added to a flask containing 0.7 mL of 2.8% saponin (w / v), 0.5 mL of Fluorinert TM The 100 μl flow cytometer was used to collect the blood samples. ...

[0208] All steps of adding buffer and removing supernatant were performed using the same pipette filter tip (Eppendorf reference 30078624) to simulate the conditions met by embodiments of the device disclosed herein in the laboratory, where all manipulation steps were performed using a single needle. Before adding wash buffer, the pipette tip was rinsed with 2.5 mL or 5 mL elution buffer for comparison. Addition of 60 μL of elution buffer to each spin column was also performed with the same used and rinsed pipette tip.

[0209] For comparison of results, real-time PCR was performed. As a summary, Fig.26The results shown show no significant differences between PCR assays (Ct values ​​obtained) between samples using either 2.5 mL or 5 mL elution buffer volumes to rinse the pipette filter tips. The assay Ct values ​​obtained using only one tip for sample preparation were comparable to Ct values ​​observed in other experiments using multiple filter tips. Example 3: Sample preparation and DNA extraction for high sensitivity detection by PCR

[0210] In the following examples, a whole blood pool was spiked with an inoculum of a microorganism (bacteria or yeast) at a known concentration. Depending on the growth characteristics of the microorganism being tested, the inoculum was prepared from an exponential culture or from a microorganism suspension from a fresh strike on an agar plate.

[0211] 10mL of spiked whole blood was transferred to a 15mL conical tube containing 0.7mL of saponin 2.8% w / v (Sigma-Aldrich, S4521), 0.5mL of demulcent FC-40 (Sigma-Aldrich, F9755) and a mixture of zirconium oxide-silica beads containing: 0.25mL of 0.1mm + 0.25mL of 0.5mm + 10 beads of 1mm diameter and 3-4 beads of 2.7mm diameter. The blood and reagents were mixed for 30s (five turns) at a speed of 10rpm under a rotating orbital motion on a PTR-35 platform for blood cell homogenization and lysis. Pathogens were collected by centrifugation at 12000x g for 10 minutes in a fixed angle rotor. The tube was kept vertical, and the supernatant was carefully removed and discarded, leaving a total volume of 1mL containing the bead mixture, demulcent and concentrated blood sample in the tube.

[0212] The lysis buffer is homogenized, and 1.8mL is added to the mixture of pearl and sample, and briefly mixed for 5 seconds by vortex. Next, the sample tube is fixed on the customized stirring platform (reciprocating motion, 2 degrees, 16.000rpm), it is opened with speed 1 and continues for 30 seconds to carry out pearl beating (bead beating) so that sample cracking. Carry out 4 pulses of 30 seconds, wait 5 seconds between them, continue the cracking of 2 minutes in total. In this process, the pipe is maintained in a vertical position. Once mechanical lysis ends, the sample tube is centrifuged for 5 minutes with 3200x g in a swing bucket rotor, to separate pearl and cell debris from lysate. The pipe is maintained in a vertical position, lysate is transferred and fully mixed with 4mL binding buffer by pipetting. Each sample uses two spin columns, and 0.5mL lysate is loaded on each post and centrifuged for 1 minute with 10000x g (FA 24X 2 rotor). Repeat this step until all lysates pass through the post. The spin column is washed twice with 0.5mL washing buffer, centrifuged for 1 minute with 10000x g, and dried by centrifuging for 2 minutes with 13000x g. The spin column is transferred in a 1.5mL tube, and 60 μL elution buffers are loaded thereon. The sample is incubated at room temperature for 5 minutes to ensure that the appropriate contact of DNA with elution buffer and the best DNA recovery, and then centrifuged for 1 minute with 10000x g. The volume of the two eluates belonging to the same test conditions obtained in the two posts is mixed in the last tube.

[0213] To confirm specific pathogen recovery after sample preparation, detection was performed in a single-plex PCR reaction containing a specific primer pair and probe for the spiked pathogen. Each reaction included the following: 50 μL 2X TaqPath Bactopure Microbial Detection Master Mix (ThermoFisher Scientific), 0.9 μM Fw primer, 0.9 μM Rv primer, 0.25 μM probe, and 50 μL of the eluate obtained in the previous step (see explanation above). The cycling conditions were as follows: 2 minutes at 95°C, followed by 45 cycles of 10 seconds at 95°C and 30 seconds at 60°C for annealing and extension. A pre-reading step and a post-reading step at 60°C were included.

[0214] The microorganisms shown in the table below were tested according to this PCR procedure, including Gram-positive bacteria, Gram-negative bacteria and yeast, several of which are considered difficult to lyse. The results are shown in Table 1 below (Table 1). It can be observed that pathogens can be detected at concentrations of the order of a few CFU / mL, which is within the sensitivity range required for detecting pathogens in bloodstream infections. Table 1 Example 4: Detection of Staphylococcus aureus in samples containing high white blood cell counts (WBC).

[0215] Human DNA is a strong interfering substance that can act as a potential PCR inhibitor. In healthy adults, normal white blood cell counts range from 3 to 11 x 10E6 white blood cells / mL of blood. In sepsis patients, white blood cell counts can be overwhelmingly increased.

[0216] To test PCR performance in the presence of elevated WBC counts, 10 mL of blood containing high white blood cell counts (20-30 x 10E6 white blood cells / mL) was spiked with 1.3 CFU / mL of S. aureus (13 CFU / tube). Samples were processed as described in the previous examples and pathogen detection was performed by PCR in a singleplex format. Figure 27A-27B The results obtained are shown. 4 out of 6 replicates of the S. aureus spiked sample were detected. Example 5: Two-stage multiplex PCR for detecting low concentrations in the presence of high concentrations of pathogens.

[0217] In Example 5, 100 genome copies of Pseudomonas aeruginosa (low concentration) are combined with high concentrations of Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae. The detection of 100 genome copies / reaction of Pseudomonas aeruginosa is assessed in a two-stage PCR format against the increasing DNA background of 10E4, 10E5, 10E6 genome copies / reaction for the remaining pathogens mentioned. 0.2mL PCR strips are used as primary reaction chambers. 25 μL of PCR volume is used in each amplification stage. Primary PCR consists of 20 cycles of PCR mixtures containing primer pairs for detecting all mentioned pathogens.

[0218] 25 μL PCR reaction mixture was prepared by mixing 12.5 μL 2x TaqPath Bactopure Microbial Detection Master Mix (ThermoFisherScientific), 0.36 μM of each primer, 5 μL sample and water. The following cycling conditions were applied in this first PCR: 2 minutes at 95°C, then 20 cycles of 10 seconds and 30 seconds at 95°C for annealing and extension.

[0219] At the end of the primary PCR, the amplified products of the primary PCR reaction were extracted with a pipette and aliquoted into the secondary reaction chambers.

[0220] Secondary PCR was performed in 0.2 mL PCR strips. Using Prime Time gene expression master mix (IDT), 0.36 μM of each primer and 0.25 μM targeting probe of the oprL gene from Pseudomonas aeruginosa, secondary PCR was performed in a final volume of 25 μL. The primary PCR reaction of 5 μL volume was used as a template for secondary PCR. Cycling conditions were: 3 minutes at 95 ° C, then 45 cycles of 5 seconds at 95 ° C and 30 seconds at 60 ° C to anneal and extend.

[0221] Fig.28 Detection of the Pseudomonas aeruginosa target (Ct value) from the second stage PCR is shown. No amplification of Pseudomonas aeruginosa was observed in the control that included DNA from the high concentration sample but did not have Pseudomonas aeruginosa DNA, indicating that the observed amplification signal was pathogen specific. Fig.28 In, HSS4P100 references Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus (each 10E4 copies / reaction) and Pseudomonas aeruginosa (10E2 copies / reaction); HSS5P100 references Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus (each 10E5 copies / reaction) and Pseudomonas aeruginosa (10E2 copies / reaction); and HSS6P100 references Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus (each 10E6 copies / reaction) and Pseudomonas aeruginosa (10E2 copies / reaction); HSS4, HSS5 and HSS6 are controls and reference Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus (respectively 10E4, 10E5 and 10E6 copies / reaction). Pseudomonas aeruginosa is not expected to amplify under these test conditions. Example 6: Effect of PCR product dilution between the first-stage PCR and the second-stage PCR on performance.

[0222] In Example 6, the effect of diluting the first stage PCR product before the second stage PCR was evaluated using the same pathogen mixture combination shown above (low concentration of Pseudomonas aeruginosa combined with high concentrations of Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae). The detection of 10 or 100 genome copies of Pseudomonas aeruginosa was evaluated in two-stage PCR for the remaining pathogens mentioned in the context of increasing DNA of 10E4, 10E5, 10E6 genome copies. A PCR volume of 25 μL was used in each amplification stage. The two-stage PCR consists of 20 cycles of the first PCR, which contains primers for detecting all pathogens mentioned above, and uses 2x TaqPath Bactopure microbial detection master mix (ThermoFisher Scientific). The following cycling conditions are applied in this first PCR: 2 minutes at 95°C, then 20 cycles of 10 seconds and 30 seconds at 95°C to anneal and extend.

[0223] The following dilutions (1 / 5, 1 / 20 and 1 / 200) of the PCR products obtained in the first-stage PCR were prepared and loaded in the second-stage PCR.

[0224] The second stage PCR was performed as a single PCR containing primers and probes for detecting the oprL gene of Pseudomonas aeruginosa and 2X Prime Time gene expression master mix (IDT). The first stage PCR reaction was used as a template for the second stage PCR using a 5 μL volume. The cycle conditions were: 3 minutes at 95°C, then 45 cycles of 5 seconds at 95°C and 30 seconds at 60°C for annealing and extension.

[0225] The results are Fig.29 In Fig.29 In the experiment, HSS4P10 was used for reference to Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus (each 10E4 copies / reaction) and Pseudomonas aeruginosa (10E2 copies / reaction); HSS5P100 was used for reference to Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus (each 10E5 copies / reaction) and Pseudomonas aeruginosa (10E2 copies / reaction). In summary, for the 1 / 20 condition, a slight Ct difference was observed between the undiluted condition and the diluted condition. For the 1 / 200 dilution condition, a difference of 4 Ct was observed between the undiluted condition and the diluted condition. Exemplary Computer System:

[0226] Fig. 222200. For example, one or more computer systems 2200 may be used to implement any of the embodiments discussed herein and combinations and sub-combinations thereof. In some embodiments, one or more computer systems 2200 may be used for fluorescence data and / or image acquisition, data analysis, and data processing, such as for a fluorescence sensor subsystem or processing device 116 in a PCR subsystem 1800, as described herein. In some embodiments, one or more computer systems 2200 may also be used in a controller 109 to program and operate the movement of various components in an analyzer device 200. The computer system 2200 may include one or more processors (also referred to as central processing units or CPUs), such as a processor 2204. The processor 2204 may be connected to a communication infrastructure or bus 2206.

[0227] The computer system 2200 may also include one or more user input / output interfaces 2202 , such as a monitor, keyboard, pointing device, etc., which may communicate with the communication infrastructure 2206 via one or more user input / output devices 2203 .

[0228] One or more processors 2204 may be a graphics processing unit (GPU). In an embodiment, a GPU may be a processor that is a dedicated electronic circuit designed to process mathematically intensive applications. A GPU may have a parallel structure that efficiently processes large blocks of data in parallel, such as mathematically intensive data commonly found in computer graphics applications, images, videos, etc.

[0229] The computer system 2200 may also include a main or primary memory 2208, such as a random access memory (RAM). The main memory 2208 may include one or more levels of cache. The main memory 2208 may store control logic (i.e., computer software) and / or data therein. In some embodiments, the main memory 2208 may include optical logic configured to perform sepsis detection, sepsis likelihood prediction, pathogen identification, and sensitivity testing, and generate recommendations for treatment of the patient accordingly.

[0230] The computer system 2200 may also include one or more secondary storage devices or memories 2210. The secondary storage 2210 may include, for example, a hard drive 2212 and / or a removable storage drive 2214.

[0231] Removable storage drive 2214 can interact with removable storage unit 2218. Removable storage unit 2218 can include a computer usable or readable storage device having computer software (control logic) and / or data stored thereon. Removable storage unit 2218 can be a program cartridge and cartridge interface (such as found in a video game device), a removable storage chip (such as an EPROM or PROM) and associated sockets, a memory stick and USB port, a memory card and associated memory card slot, and / or any other removable storage unit and associated interface. Removable storage drive 2214 can read from and / or write to removable storage unit 2218.

[0232] Secondary storage 2210 may include other means, devices, components, tools, or other methods for allowing access to computer programs and / or other instructions and / or data by computer system 2200. Such means, devices, components, tools, or other methods may include, for example, a removable storage unit 2222 and an interface 2220. Examples of removable storage unit 2222 and interface 2220 may be a program cartridge and cartridge interface (such as found in video game devices), a removable storage chip (such as an EPROM or PROM) and associated socket, a memory stick and USB port, a memory card and associated memory card slot, and / or any other removable storage unit and associated interface.

[0233] The computer system 2200 may further include a communication or network interface 2224. The communication interface 2224 may enable the computer system 2200 to communicate and interact with any combination of external devices, external networks, external entities, etc. (individually and collectively referred to by reference numeral 2228). For example, the communication interface 2224 may allow the computer system 2200 to communicate with an external or remote device 2228 via a communication path 2226, which may be wired and / or wireless (or a combination thereof) and may include any combination of a LAN, a WAN, the Internet, etc. Control logic and / or data may be transmitted to and from the computer system 2200 via the communication path 2226.

[0234] Computer system 2200 may also be any of the following: a personal digital assistant (PDA), a desktop workstation, a laptop or notebook computer, a netbook, a tablet computer, a smart phone, a smart watch or other wearable device, an appliance, part of the Internet of Things, and / or an embedded system (to name a few non-limiting examples) or any combination thereof.

[0235] Computer system 2200 can be a client or server, accessing or hosting any application and / or data through any delivery paradigm, including but not limited to remote or distributed cloud computing solutions; local or on-premises software ("on-premises" cloud-based solutions); "as a service" models (e.g., content as a service (CaaS), digital content as a service (DCaaS), software as a service (SaaS), hosted software as a service (MSaaS), platform as a service (PaaS), desktop as a service (DaaS), framework as a service (FaaS), backend as a service (BaaS), mobile backend as a service (MBaaS), infrastructure as a service (IaaS), etc.); and / or a hybrid model including any combination of the foregoing examples or other services or delivery paradigms.

[0236] Any applicable data structures, file formats, and schemas in computer system 2200 may be derived from standards, including but not limited to JavaScript Object Notation (JSON), Extensible Markup Language (XML), another markup language (YAML), Extensible Hypertext Markup Language (XHTML), Wireless Markup Language (WML), message packages, XML User Interface Language (XUL), or any other functionally similar representation (alone or in combination). Alternatively, proprietary data structures, formats, or schemas may be used alone or in combination with known or open standards.

[0237] In some embodiments, a tangible non-transitory device or article of manufacture comprising a tangible non-transitory computer usable or readable medium having control logic (software) stored thereon may also be referred to herein as a computer program product or program storage device. This includes, but is not limited to, computer system 2200, main memory 2208, secondary memory 2210, and removable storage units 2218 and 2222, and tangible articles of manufacture embodying any combination of the foregoing. When executed by one or more data processing devices (such as computer system 2200), such control logic may cause such data processing devices to operate as described herein.

[0238] Based on the teachings contained in this disclosure, it will be apparent to one or more persons skilled in the relevant art how to use the Fig. 22 It will be clear that data processing devices, computer systems, and / or computer architectures other than those shown can be used to make and use the embodiments of the present disclosure. In particular, the embodiments can be operated using software, hardware, and / or operating system implementations other than those described herein.

[0239] It should be understood that the detailed description section (rather than the summary and abstract sections) is intended to be used to interpret the claims. The summary and abstract sections may set forth one or more but not all exemplary embodiments of the present disclosure as contemplated by one or more inventors, and therefore are not intended to limit the present disclosure and the appended claims in any way.

[0240] The embodiments of the present disclosure have been described above with the help of functional building blocks, which show the implementation of specified functions and their relationships. For ease of description, the boundaries of these functional building blocks are arbitrarily defined herein. As long as the specified functions and their relationships are properly performed, alternative boundaries can be defined.

[0241] The above description of specific embodiments will fully reveal the general nature of the present disclosure, and others can easily modify and / or adjust such specific embodiments for various applications without undue experimentation by applying knowledge within the technical scope of the art without departing from the general concept of the present disclosure. Therefore, based on the teachings and guidance given herein, such adjustments and modifications are intended to be included in the meaning and range of equivalents of the disclosed embodiments. It should be understood that the words or terms in this article are for the purpose of description rather than limitation, and therefore the terms or words of this specification will be interpreted by those skilled in the art based on the teachings and guidance.

[0242] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

1. A sample preparation kit, comprising: case; a removable processing tube disposed within the housing and comprising a septum and configured to hold a sample; a first removable needle disposed within the housing and configured for transferring the sample into and / or from the removable processing tube by inserting the first removable needle through the septum; as well as One or more reservoirs are coupled to the housing and configured to store materials for performing sample concentration, lysis, and nucleic acid amplification.

2. The sample preparation kit according to claim 1, further comprising: A protective cover is configured to cover the housing and the one or more reservoirs.

3. The sample preparation kit according to claim 1, further comprising: One or more spin columns configured to perform nucleic acid extraction and purification from the sample.

4. The sample preparation kit according to claim 3, further comprising: A basket or receiving container having the one or more spin columns disposed therein is configured to be moved to an elution position to obtain the nucleic acid from the sample. 5 . The sample preparation kit of claim 1 , wherein at least one of the one or more reservoirs is configured to store magnetic beads for extracting and purifying nucleic acids from the sample.

6. The sample preparation kit according to claim 1, further comprising: A reagent tube configured to hold a first set of reagents for nucleic acid amplification.

7. The sample preparation kit of claim 6, wherein the reagent tubes and / or the one or more reservoirs are sealed with aluminum foil.

8. The sample preparation kit of claim 1, wherein the septum in the removable processing tube is disposed at a top end of the removable processing tube, and wherein a plurality of lysing beads are disposed within the removable processing tube for performing the lysing.

9. The sample preparation kit of claim 8, wherein the plurality of lysis beads comprises a plurality of predetermined sizes and materials configured for lysis of at least one of the following pathogens: yeast, fungi, gram-positive bacteria, or gram-negative bacteria.

10. The sample preparation kit of claim 1, wherein the first removable needle is a vent needle comprising a cannula and a plastic body attached to the cannula, wherein the vent needle is configured to vent the removable processing tube.

11. The sample preparation kit of claim 10, wherein the plastic body of the vent needle comprises a filter configured to prevent contamination.

12. A sample preparation box according to claim 10, wherein the plastic body of the vent needle includes a predetermined number of grooves, and the predetermined number of grooves are configured to provide an air connection between the interior and exterior of the removable processing tube when the vent needle is inserted into the removable processing tube through the septum.

13. The sample preparation kit of claim 10, wherein the first removable needle comprises a cannula disposed around an inner core.

14. The sample preparation kit of claim 10, wherein the cannula of the vent needle comprises a slotted cannula.

15. The sample preparation kit of claim 10, wherein the cannula of the vent needle is in fluid communication with the vent hole.

16. The sample preparation kit of claim 1, further comprising: A second removable needle configured to transfer nucleic acid to a second cartridge for use in amplifying the nucleic acid.

17. The sample preparation kit of claim 16, wherein the first removable needle is configured for coupling with a higher volume pipette, and wherein the removable second needle is configured for coupling with a lower volume pipette.

18. The sample preparation kit of claim 17, wherein the higher volume pipette is configured for manipulating volumes in the range of about 50 microliters to 5 milliliters.

19. The sample preparation kit of claim 17, wherein the lower volume pipette is configured for manipulating volumes in the range of about 1 to 200 microliters.

20. The sample preparation kit of claim 1, wherein the removable processing tube includes a manipulation feature at a top end of the removable processing tube, the manipulation feature being compatible with being manipulated by a pipette coupled to the first removable needle.

21. The sample preparation kit of claim 20, wherein the manipulation feature comprises a cap having a cylindrical cavity compatible with insertion by a core tube of a pipette coupled to the first removable needle, wherein the cap is positioned above the septum at the top end of the removable processing tube.

22. A system for analyzing a sample, the system comprising: a housing configured to receive a sample tube containing a sample containing one or more pathogens; a pipette system, the pipette system being arranged inside the housing; one or more centrifuges, the one or more centrifuges being disposed within the housing; a mechanical agitator disposed inside the housing; as well as A controller, wherein the controller is configured to: transferring the sample from the sample tube to a processing tube using the pipette system; centrifuging the processing tubes using the one or more centrifuges to concentrate the one or more pathogens in the sample; removing fluid from the processing tube using the pipette system, thereby leaving concentrated pathogens in the processing tube; adding lysis buffer to the processing tube using the pipette system; using the pipette system to move the processing tube to the mechanical agitator; and using the mechanical agitator to agitate the processing tube to perform cell lysis of the concentrated pathogen.

23. The system of claim 22, wherein the controller is further configured to: transferring a portion of the liquid from the processing tube to a spin column after the cell lysis by the pipette system; and Nucleic acids are extracted and purified from the portion of the liquid using the spin column.

24. The system of claim 22, further comprising: One or more reservoirs configured to store magnetic beads for nucleic acid extraction and purification, wherein the controller is further configured to: transferring, by the pipette system, a portion of the liquid from the processing tube to the one or more reservoirs after lysis of the cells; as well as Nucleic acids are extracted and purified from the portion of the liquid using the magnetic beads in the one or more reservoirs.

25. The system of claim 22, wherein the pipette system is configured to move a sample tube within the housing by manipulating a top end of the sample tube that is compatible with the pipette system.

26. The system of claim 22, wherein the pipette system comprises: A first pipette is configured to manipulate volumes in the range of about 50 microliters to about 5 milliliters.

27. The system of claim 26, wherein the pipette system further comprises: A second pipette configured to manipulate volumes in the range of about 1 microliter to about 200 microliters.

28. The system of claim 26, wherein the sample tube and the processing tube each include a septum that allows insertion of a first needle coupled to the first pipette.

29. The system of claim 28, wherein transferring the sample from the sample tube to the processing tube comprises inserting the first needle into a septum of the processing tube and dispensing the sample through the first needle.

30. The system of claim 29, wherein the first needle comprises a vent needle attached to the plastic body and configured to vent the processing tube.

31. The system of claim 30, wherein the plastic body comprises a filter configured to prevent contamination of the sample.

32. The system of claim 30, wherein the plastic body includes a predetermined number of slots configured to provide an air connection between an interior and an exterior of the processing tube when the ventilation needle is inserted into the processing tube through the septum.

33. The system of claim 22, wherein the system further comprises: A first subsystem is disposed within the housing and is configured to perform a polymerase chain reaction (PCR) on the sample using nucleic acid extracted from the sample.

34. The system of claim 33, wherein the first subsystem comprises a thermal cycler configured to control one or more temperatures during PCR of the sample in a plurality of reaction chambers in a PCR box.

35. The system of claim 33, wherein the system further comprises: A second subsystem is disposed within the housing and is configured to perform an antimicrobial susceptibility test (AST) on the sample.

36. The system of claim 35, wherein the second subsystem comprises a microscope configured to acquire one or more images of the concentrated pathogens after enriching and transferring the concentrated pathogens to a plurality of reaction wells in an AST cartridge.

37. A method comprising: receiving, by an analyzer device, a sample preparation cartridge and a sample tube containing a sample comprising one or more pathogens; installing a first needle from the sample preparation cartridge in a pipette system in the analyzer device; inserting the first needle into the sample tube using the pipette system; transferring the sample from the sample tube to a processing tube in the sample preparation kit through the first needle; adding one or more lysis reagents to the processing tube using the pipette system; mixing the one or more lysing reagents with the sample in the processing tube to lyse blood cells in the sample; moving the processing tube to a centrifuge in the analyzer device; centrifuging the processing tube in the centrifuge to concentrate one or more pathogens in the sample; removing fluid from the processing tube using the pipette system, thereby leaving concentrated pathogens in the processing tube; adding lysis buffer to the processing tube using the pipette system; means for moving said processing tube into said analyzer device using said pipetting system; as well as The device is used to agitate the process tube to effect lysis of the concentrated pathogen.

38. The method of claim 37, further comprising: After lysis of the concentrated pathogen, transferring a portion of the liquid from the processing tube to a spin column in the analyzer device by the pipette system; as well as Nucleic acids are extracted and purified from the portion of the liquid using the spin column.

39. The method of claim 37, further comprising: After lysis of the concentrated pathogen, transferring a portion of the liquid from the processing tube to one or more reservoirs in the sample preparation cartridge by the pipette system; and Nucleic acids are extracted and purified from the portion of the liquid using the magnetic beads stored in the one or more reservoirs.

40. The method of claim 37, wherein transferring the sample from the sample tube to the processing tube through the first needle comprises inserting the first needle into a septum of the processing tube and dispensing the sample through the first needle.

41. The method of claim 37, wherein agitating the process tube comprises applying a rapid vibrating motion to the process tube using an apparatus in the analyzer device, wherein the apparatus comprises an agitator.

42. The method of claim 41, wherein the processing tube is in a vertical position, and wherein applying the rapid vibrating motion comprises applying a reciprocating motion along a predetermined axis by the agitator.

43. The method of claim 37, wherein agitating the process tube comprises sonicating the process tube using an apparatus in the analyzer device, wherein the apparatus comprises a sonicator.

44. The method of claim 37, wherein moving the processing tube to the first centrifuge comprises using the pipette system to move the processing tube by manipulating a top end of the processing tube that is compatible with the pipette system.

45. The method of claim 37, wherein moving the processing tube to the device comprises using the pipette system to move the processing tube by manipulating a tip of the processing tube that is compatible with the pipette system.

46. ​​The method of claim 37, wherein the volume of the sample in the sample tube is in the range of about 0.5 mL to about 10 mL, and wherein the volume of the concentrated pathogens in the processing tube after the centrifugation is in the range of about 0.1 mL to about 2 mL.

47. The method of claim 37, wherein after said centrifugation, the volume of said concentrated pathogens in said processing tube is in the range of about 0.1 mL to about 1 mL.

48. The method of claim 37, wherein receiving the sample preparation cartridge and the sample tube comprises: receiving the sample preparation cartridge in a first compartment of the analyzer device; as well as The sample tube is received in a second compartment of the analyzer device.

49. The method of claim 37, further comprising: Prior to adding the lysis buffer to the processing tubes, reagents were added to the processing tubes using the pipetting system for the DNA depletion step.

50. The method of claim 37, wherein the one or more lysis reagents comprise one or more saponin-based buffers.

51. The method of claim 37, further comprising: extracting and purifying nucleic acid from a portion of the liquid in the processing tube after lysis of the concentrated pathogen; transferring the nucleic acid to at least one primary reaction chamber in a polymerase chain reaction (PCR) cartridge inserted into the analyzer device; Performing a first amplification of the nucleic acid in the at least one primary reaction chamber to generate a first amplification product; inserting a needle of the analyzer device through the septum of the at least one primary reaction chamber to remove the first amplification product; distributing a plurality of aliquots of the first amplification product to the plurality of secondary reaction chambers in the PCR cartridge through respective membranes of the plurality of secondary reaction chambers, wherein each aliquot corresponds to a respective secondary reaction chamber, and wherein each secondary reaction chamber comprises a set of reagents for reacting with the respective aliquot of the first amplification product; and A second amplification of an aliquot of the first amplification product is performed in the plurality of secondary reaction chambers.

52. A method comprising: transferring the nucleic acid by a pipette system in the analyzer device to at least one primary reaction chamber in a polymerase chain reaction (PCR) cartridge inserted into the analyzer device; Performing a first amplification of the nucleic acid in the at least one primary reaction chamber to generate a first amplification product; inserting a needle of the analyzer device through the septum of the at least one primary reaction chamber to remove the first amplification product; distributing a plurality of aliquots of the first amplification product to the plurality of secondary reaction chambers in the PCR cartridge through respective membranes of the plurality of secondary reaction chambers, wherein each aliquot corresponds to a respective secondary reaction chamber, and wherein each secondary reaction chamber comprises a set of reagents for reacting with the respective aliquot of the first amplification product; and A second amplification of an aliquot of the first amplification product is performed in the plurality of secondary reaction chambers.

53. The method of claim 52, further comprising: Before transferring the nucleic acid to at least one primary reaction chamber in the PCR box, the nucleic acid is mixed with a prepared set of reagents for nucleic acid amplification in a reagent tube by the pipette system.

54. The method of claim 52, further comprising: Before dispensing a plurality of aliquots of the first amplification product into the secondary reaction chambers in the PCR cartridge, the first amplification product is mixed with a prepared set of reagents for nucleic acid amplification in a reagent tube by the pipette system.

55. The method of claim 52, wherein the set of reagents in each secondary reaction chamber is dried or freeze-dried at the bottom of each secondary reaction chamber.

56. The method of claim 52, further comprising: The second amplification is monitored by detecting fluorescence at the bottom of each secondary reaction chamber.

57. A method according to claim 52, wherein dispensing the plurality of aliquots to the plurality of secondary reaction chambers comprises performing non-contact dispensing of the aliquots through the needle using jet dispensing, the needle penetrating the corresponding diaphragms of the secondary reaction chambers and the needle not contacting the bottom surface of each secondary reaction chamber.

58. The method of claim 52, wherein performing the first amplification and the second amplification comprises using PCR techniques.

59. The method of claim 58, further comprising: A set of primers and the nucleic acid are transferred to at least one primary reaction chamber in the PCR cassette, wherein the set of primers is compatible with amplification of a target sequence to be detected.

60. The method of claim 58, further comprising: Different sets of primers are transferred with the nucleic acid to at least one primary reaction chamber in the PCR cassette, wherein the combination of the different sets of primers is compatible with amplification of a target sequence to be detected.

61. The method of claim 58, wherein the set of reagents in each secondary reaction chamber corresponds to an internal sequence of an amplicon in the first amplification product and is specific to one or more target sequences to be detected.

62. The method of claim 58, wherein the set of reagents in each secondary reaction chamber comprises fluorescent probes specific for one or more target sequences to be detected.

63. A polymerase chain reaction (PCR) kit, comprising: at least one primary reaction chamber configured to perform a first amplification of the nucleic acid to produce a first amplification product; as well as A plurality of secondary reaction chambers, wherein the plurality of secondary reaction chambers are configured for performing a second amplification of the product nucleic acid, wherein the at least one primary reaction chamber and the plurality of secondary reaction chambers are each sealed by a diaphragm, wherein the diaphragm is configured for receiving a needle, each of the plurality of secondary reaction chambers is configured for receiving a corresponding aliquot of the first amplification product from the needle, and each secondary reaction chamber includes a set of reagents for reacting with a corresponding aliquot of the first amplification product.

64. The PCR box of claim 63, wherein the volume of the at least one primary reaction chamber is in the range of about 50 to about 500 microliters.

65. A PCR box according to claim 63, wherein the at least one primary reaction chamber and the multiple secondary reaction chambers are configured to receive a heating cover arranged on the at least one primary reaction chamber and the multiple secondary reaction chambers, wherein the heating cover heats the upper portion of the at least one primary reaction chamber and the multiple secondary reaction chambers to prevent condensation.

66. A PCR box according to claim 63, wherein the diameter of the bottom wall of the at least one primary reaction chamber and each of the plurality of secondary reaction chambers is less than about 2.5 mm.

67. The PCR cassette of claim 63, further comprising: A reagent tube or reservoir configured to hold an additional set of reagents for nucleic acid amplification, wherein the additional set of reagents is dried or freeze-dried.

68. A PCR box according to claim 63, wherein the at least one primary reaction chamber and each of the multiple secondary reaction chambers include a transparent bottom surface, and the transparent bottom surface is configured to allow optical interrogation of the at least one primary reaction chamber and the at least one secondary reaction chamber by fluorescence.

69. A PCR box according to claim 63, wherein the at least one primary reaction chamber and each of the plurality of secondary reaction chambers include mineral oil to prevent at least one of evaporation, aerosol formation, and cross contamination.

70. The PCR box of claim 69, wherein each secondary reaction chamber is configured to receive the mineral oil prior to receiving a corresponding aliquot of the first amplification product.

71. A PCR box according to claim 69, wherein the mineral oil in each secondary reaction chamber is stored separately from the set of reagents in each secondary reaction chamber.

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