Systems, methods and apparatus for pathogen identification - Patents.com

Abstract

Described herein are systems, methods, and devices for pathogen identification. The system includes a housing configured to receive a sample containing a pathogen, a pipetter system disposed within the housing, a centrifuge disposed within the housing, a mechanical agitator disposed within the housing, and a controller. The controller is configured to transfer the sample to a processing tube using the pipetter system, centrifuge the processing tube using the centrifuge to concentrate the pathogens in the sample, remove liquid from the processing tube using the pipetter system to leave the concentrated pathogens in the processing tube, add a lysis buffer to the processing tube using the pipetter system, move the processing tube to a mechanical agitator using the pipetter system, and agitate the processing tube using the mechanical agitator to lyse the concentrated pathogens. The system is further configured to perform PCR using nucleic acids extracted from the sample.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 299,611, filed January 14, 2022, the entire contents of which are incorporated herein by reference. This application is related to co-pending PCT Patent Application No. ___, filed January 13, 2023 (Attorney Docket No. 4518.002PC01), entitled "SYSTEMS, METHODS, AND APPARATUS FOR ANTIMICROBIAL SUSCEPTIBILITY TESTING," by Bru Gibert et al., and PCT Application No. PCT / US2022 / 039290, filed August 3, 2022, the disclosures of which are incorporated herein by reference in their entireties.

[0002] SUMMARY OF THE DISCLOSURE Embodiments of the present disclosure relate to systems, methods, and devices for pathogen identification and / or resistance gene identification from whole blood or other samples to aid in determining patient treatment. [Background technology]

[0003] Sepsis is defined as life-threatening organ dysfunction caused by an uncontrolled host response to infection. Patients with or developing sepsis may begin with a localized infection, such as pneumonia, to which the patient's immune system overreacts, causing inflammation throughout the body. If left untreated, inflammation can eventually lead to organ failure and patient death. Sepsis causes 11 million deaths each year, many of which are preventable with early diagnosis, appropriate clinical management, and treatment.

[0004] The diagnosis of sepsis is performed by laboratory testing of cultured blood samples to identify infection. Current testing can take days to produce test results due to the time required to collect and process blood cultures for the bacteria causing sepsis to determine their susceptibility to antimicrobial agents that will ultimately be effective in treating them. However, for patients in hospitals, detection of sepsis is a race against time, as sepsis can lead to septic shock and death within hours if not properly identified and treated in time. Furthermore, blood culture testing is time-consuming and does not always produce reliable results in detecting bacteria and fungi in patients suspected of sepsis, especially for patients who have already been treated with antibiotics. Patients have a low probability of obtaining a positive blood culture result, and patients may still have sepsis even without identification of a positive result.

[0005] Without remedial measures, patients may continue to suffer and often worsen while physicians treat with empirical antibiotics until they receive more actionable information from laboratories about the causative agents (e.g., bacterial pathogens) causing the infection and about antimicrobial agents to treat highly resistant pathogens in patients with sepsis. Summary of the Invention

[0006] The embodiments of the present disclosure provide a cost-effective solution for improved diagnostic methods, systems, and devices for isolating and identifying pathogens to provide appropriate treatment to patients to improve their prognosis.

[0007] Described herein are systems, methods, and devices for identifying pathogens directly from blood samples or other samples such as urine, sterile body fluids, etc. In the embodiments presented herein, pathogen identification systems, analyzers, polymerase chain reaction (PCR) cartridges, sample preparation cartridges, and processing tubes are provided that perform pathogen identification and resistance gene determination from whole blood samples without a culture step, using a multiplex PCR approach, significantly reducing analysis time. Pathogen identification includes transferring the sample to consumables for further processing (e.g., nucleic acid extraction, purification, amplification) using a pipetting system, concentrating the pathogen, lysing the pathogen (e.g., by mechanical disruption), purifying nucleic acids from the pathogen, and amplifying and identifying the pathogen by nested PCR. In some embodiments, the systems, methods, and devices for pathogen identification described herein can be used to treat patients with sepsis and / or other underlying diseases.

[0008] In an embodiment, an example sample preparation cartridge is described. The sample preparation cartridge includes a housing, a removable processing tube disposed within the housing, a first removable needle disposed within the housing, and one or more reservoirs coupled to the housing. The removable processing tube includes a septum and is configured to hold a sample. The first removable needle is configured to transfer the sample to and / or from the removable processing tube upon insertion of the first removable needle through the septum. The one or more reservoirs are configured to store materials used to concentrate, lyse, and amplify the sample.

[0009] In another embodiment, an example of a system for analyzing a sample is described. The system includes a housing configured to receive a sample tube containing a sample including one or more pathogens, a pipetter 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 transfers the sample from the sample tube to a processing tube using the pipetter system, centrifuges the processing tube using the one or more centrifuges to concentrate one or more pathogens in the sample, removes fluid from the processing tube using the pipetter system to leave the concentrated pathogens in the processing tube, adds a lysis buffer to the processing tube using the pipetter system, transfers the processing tube to the mechanical agitator using the pipetter system, and agitates the processing tube using the mechanical agitator to perform cell lysis of the concentrated pathogens.

[0010] In another embodiment, an example method is described, which includes receiving, by an analytical device, a sample preparation cartridge and a sample tube, the sample tube containing a sample including one or more pathogens, and the method also includes attaching a first needle from the sample preparation cartridge to a pipettor system in the analytical device, inserting the first needle into the sample tube using the pipettor system, transferring the sample from the sample tube through the first needle to a processing tube in the sample preparation cartridge, adding one or more lysis reagents to the processing tube using the pipettor system, and mixing the one or more lysis reagents in the processing tube with the sample to lyse blood cells in the sample. The method further includes transferring the processing tube to a centrifuge in the analytical device, centrifuging the processing tube in the centrifuge to concentrate one or more pathogens in the sample, removing liquid from the processing tube using the pipettor system to leave the concentrated pathogens in the processing tube, adding a lysis buffer to the processing tube using the pipettor system, transferring the processing tube to an apparatus in the analytical device using the pipettor system, and agitating the processing tube using the apparatus to perform cell lysis of the concentrated pathogens.

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

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

[0013] Further features and advantages, as well as the 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 limiting. Such embodiments are presented herein for illustrative purposes only. Further embodiments will be apparent to one of ordinary skill in the relevant art based on the teachings set forth herein. [Brief description of the drawings]

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

[0015] [Figure 1] FIG. 1 shows a diagram of a system 101 for performing pathogen discrimination, according to an embodiment of the present disclosure. [Diagram 2] FIG. 2 illustrates an analytical device according to an embodiment of the present disclosure. [Figure 3A] FIG. 3A shows a front view of an analytical device according to an embodiment of the present disclosure. [Figure 3B] FIG. 3B illustrates a top view of an analytical device according to an embodiment of the present disclosure. [Figure 4] FIG. 4 shows a diagram of an analytical device with three open compartments according to an embodiment of the present disclosure. [Diagram 5] FIG. 5 shows a diagram of a sample preparation cartridge according to an embodiment of the present disclosure. [Figure 6A] FIG. 6A shows a processing tube and other components in a sample preparation cartridge according to an embodiment of the present disclosure. [Figure 6B] FIG. 6B shows processing tubes and other components in a linear sample preparation cartridge according to an embodiment of the present disclosure. [Figure 7A] FIG. 7A shows a diagram of a processing tube according to an embodiment of the present disclosure. [Figure 7B] FIG. 7B shows a diagram of a processing tube according to an embodiment of the present disclosure. [Figure 7C] FIG. 7C shows a diagram of an example of a reagent tube according to an embodiment of the present disclosure. [Figure 7D] FIG. 7D shows a diagram of an example of a reagent tube according to an embodiment of the present disclosure. [Figure 8A] FIG. 8A shows a diagram of a needle 800 configured for insertion into a process and / or reagent tube according to some embodiments of the present disclosure. [Figure 8B] FIG. 8B shows a diagram of a needle 800 configured for insertion into a process tube and / or a reagent tube according to some embodiments of the present disclosure. [Figure 8C] FIG. 8C shows a diagram of a needle 800 configured for insertion into a process tube and / or a reagent tube according to some embodiments of the present disclosure. [Figure 9A] FIG. 9A shows a diagram of a high volume needle according to an embodiment of the present disclosure. [Figure 9B] FIG. 9B shows a diagram of a low volume needle according to an embodiment of the present disclosure. [Figure 10] FIG. 10 shows a diagram of an example high volume needle according to an embodiment of the present disclosure. [Figure 11A] FIG. 11A shows a diagram of a spin column and spin column basket according to an embodiment of the present disclosure. [Figure 11B] FIG. 11B shows a diagram of a spin column and spin column basket according to an embodiment of the present disclosure. [Figure 12A] FIG. 12A shows a diagram illustrating how a low volume needle interfaces with a sample preparation cartridge, according to an embodiment of the present disclosure. [Figure 12B] FIG. 12B shows a low volume needle interfacing with a spin column basket according to an embodiment of the present disclosure. [Figure 13A] FIG. 13A shows a diagram of a PCR cartridge according to an embodiment of the present disclosure. [Figure 13B] FIG. 13B shows a diagram of a PCR cartridge according to an embodiment of the present disclosure. [Figure 14]FIG. 14 illustrates a low volume needle being inserted into a PCR cartridge according to an embodiment of the present disclosure. [Figure 15A] FIG. 15A shows a diagram of an example centrifuge for use in an analytical device according to an embodiment of the present disclosure. [Figure 15B] FIG. 15B shows a diagram of an example centrifuge for use in an analytical device according to an embodiment of the present disclosure. [Figure 16A] FIG. 16A shows a diagram of an example homogenization subsystem for use in an analytical device according to an embodiment of the present disclosure. [Figure 16B] FIG. 16B illustrates a diagram of an example homogenization subsystem for use in an analyzer according to an embodiment of the present disclosure. [Figure 16C] FIG. 16C illustrates a diagram of an example homogenization subsystem for use in an analyzer according to an embodiment of the present disclosure. [Figure 16D] FIG. 16D shows a diagram of an example of a homogenization subsystem for use in an analyzer according to an embodiment of the present disclosure. [Figure 17A] FIG. 17A shows a diagram of an example of a mechanical device used in an analysis device according to an embodiment of the present disclosure. [Figure 17B] FIG. 17B shows a diagram of an example of a mechanical device used in an analysis device according to an embodiment of the present disclosure. [Figure 17C] FIG. 17C shows a diagram of an example of a mechanical device used in an analysis device according to an embodiment of the present disclosure. [Figure 18A] FIG. 18A shows different views of a PCR cartridge that interfaces with a PCR subsystem in an analyzer, according to an embodiment of the present disclosure. [Figure 18B] FIG. 18B shows a different view of a PCR cartridge that interfaces with a PCR subsystem within an analyzer, according to an embodiment of the present disclosure. [Figure 18C] FIG. 18C shows a different view of a PCR cartridge that interfaces with a PCR subsystem within an analyzer, according to an embodiment of the present disclosure. [Figure 19A]FIG. 19A illustrates an example of a fluorescent sensor subsystem in an analytical device according to an embodiment of the present disclosure. [Figure 19B] FIG. 19B illustrates an example of a fluorescent sensor subsystem within an analytical device according to an embodiment of the present disclosure. [Figure 20] FIG. 20 is a flow chart diagram of a method of performing sample processing including concentration and lysis of a sample prior to pathogen identification according to an embodiment of the present disclosure. [Figure 21] FIG. 21 is a flow chart diagram of a method for performing PCR for pathogen discrimination according to an embodiment of the present disclosure. [Figure 22] FIG. 22 is a block diagram of exemplary components of a computer system 2200. [Diagram 23] FIG. 23 shows experimental results of tests according to embodiments of the present disclosure. [Figure 24] FIG. 24 shows experimental results of tests according to embodiments of the present disclosure. [Diagram 25] FIG. 25 shows experimental results of tests according to embodiments of the present disclosure. [Figure 26] FIG. 26 shows experimental results of tests according to embodiments of the present disclosure. [Figure 27] FIG. 27 shows experimental results of tests according to embodiments of the present disclosure. [Figure 28] FIG. 28 shows experimental results of tests based on embodiments of the present disclosure. [Figure 29] FIG. 29 shows experimental results of tests based on embodiments of the present disclosure.

[0016] Embodiments of the present disclosure will be described with reference to the accompanying drawings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] While specific configurations and arrangements are described, it should be understood that this is done for illustrative purposes only. A person skilled in the art will recognize that other configurations and arrangements can be used without departing from the spirit and scope of the present disclosure. It will be apparent to one skilled in the art that the present disclosure can be employed in a variety of other applications.

[0018] Units, prefixes, and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges are intended to be inclusive of the numbers defining the range. When a range of numerical values ​​is stated, it is understood that each integer and fraction between the numerical values ​​stated as the upper and lower limits of the range is also specifically disclosed, along with each subrange between the numerical values. Thus, ranges described herein are understood to be shorthand for all values ​​within the range, including the stated endpoints. For example, a range from 1 to 10 is understood to include every numerical value, combination of numerical values, or subranges in the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0019] Where a numerical value is explicitly recited, it is understood that numerical values ​​that are approximately the same amount or quantity as that numerical value are also within the scope of the disclosure. Where combinations are disclosed, subcombinations of each element of the combination are also specifically disclosed and are within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed.

[0020] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of the described or listed components.

[0021] The term "about" refers to a value or composition that one of ordinary skill in the art would determine to be within an acceptable error range for a particular value or composition, depending on the method of measuring or determining the value or composition, i.e., the limitations of the measurement system. For example, "about" can mean within 1× standard deviation or more than 1× standard deviation, as is customary in the art. Alternatively, "about" can mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5-fold. When a particular value or composition is described in the present application and claims, unless otherwise indicated, the meaning of "about" should be assumed to be within an acceptable error range for that particular value or composition.

[0022] As used herein, concentration ranges, percentage ranges, ratio ranges, and integer ranges are understood to include any integer value within the stated range, and fractions thereof, where appropriate (such as tenths or hundredths of an integer), unless otherwise specified.

[0023] Introduction: The current standard of care in the detection and treatment of sepsis relies on blood cultures, with a median time to detection of approximately 13 hours. Blood culture tests detect microorganisms without pathogen identification (ID), and positive samples are subsequently plated in petri dishes. In the traditional blood culture process, two sets of blood cultures are taken per adult patient, with each set consisting of an aerobic and an anaerobic bottle to ensure that the full spectrum of sepsis-causing bacteria is captured during culture. Typically, each culture is taken from a separate venipuncture (e.g., the patient's left arm and right arm). This is to ensure that bacterial shedding events are captured by culture and that bacteria are "recovered" for subsequent testing (e.g., ID and AST). After incubation, the aerobic and anaerobic bottles are incubated in the blood culture machine and growth is monitored in real time. The aerobic and anaerobic bottles are incubated and agitated until the bacteria complete the lag-log growth transition, at which point they are electronically detected. Laboratory personnel may then be notified that the patient's culture is positive. Cultures typically become positive in an average of about 13 hours for most bacteria, but may take longer (up to 5 days) for yeasts and fungi. However, many cultures remain negative due to sampling errors, insufficient blood volume, delays in transport to the laboratory, or lack of sensitivity.

[0024] Due to the urgency of sepsis, after a positive test, the laboratory immediately performs a gram stain (e.g., gram-positive or gram-negative bacteria) to identify the key microorganism, contaminant, monomicrobial, or polymicrobial infection, and reports intermediate information to the healthcare provider. In addition, the laboratory can take immediate measures to identify the bacteria using rapid methods such as molecular diagnostic systems, the results of which can be obtained in 1.5 hours. These systems may provide limited molecular information on the genetic drug resistance of specific bacteria that show a specific profile. Alternatively, the laboratory can process the positive blood culture (PBC) aliquot with a matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry system and report the ID in about 1 hour. The healthcare provider can use this ID to confirm and possibly adjust antibiotics that may have been administered prophylactically to the patient. However, by the time the bacteria is identified, up to 20–24 hours (at best) may have passed since the patient's culture was first taken.

[0025] The entire process from detecting a positive blood culture sample to identifying the pathogen can take time, and it can take several days for important antibiotic information to be reported for a septic patient. For example, if an infection is suspected, blood, urine, sputum, or other samples are collected from the patient and submitted to a clinical laboratory to first determine whether an infectious agent is present. Most pathogenic bacterial species may require 18–24 hours (e.g., day 1) to grow sufficiently. If bacteria are isolated, an additional 18–24 hours (e.g., day 2) is required to culture the isolate, and an additional 2–48 hours (e.g., day 3 or later) is required to identify the bacterial isolate and perform AST.

[0026] Traditional pathogen identification methods and systems may be limited because they are growth-based, time-consuming, expensive, require manual labor, and are not integrated. Current technology cannot provide a comprehensive solution that integrates the entire workflow of host response detection, pathogen identification, and antimicrobial or antibiotic susceptibility testing (AST). In some cases, some systems focus only on a single aspect of the sepsis cascade, such as host response detection or pathogen detection. For example, a system can examine the host response as an early sign of sepsis by detecting molecular leukocyte RNA markers (via detection of gene expression by reverse transcription (RT)-PCR), but cannot provide answers regarding pathogen identification or susceptibility. Immune response results can alert healthcare professionals that a patient has entered or is entering the sepsis cascade and indicate the urgent need for treatment or intervention to prevent potential irreversible sequelae. Healthcare professionals identify the site of infection and the infectious agent in a traditional manner, such as taking a blood culture from the infection site to identify the infectious agent, and respond immediately.

[0027] In terms of pathogen detection, current technology offers detection and identification methods using PCR directly from blood samples. However, such systems are expensive, have limited menu options, are difficult to service, may not provide a solution for rapid identification results, and may only offer limited molecular genetic resistance panels. Other systems may utilize rRNA RT-PCR methods for pathogen identification that detect pathogens directly from blood, but may be constrained by a limited menu (e.g., 15 or fewer targets). Ultimately, current technology does not offer a solution for rapid automated identification directly from blood, instead relying on positive blood cultures that can take 13–20 hours to report any useful results. For example, some systems can obtain aliquots from positive blood culture bottles (PBCs), reducing the time (e.g., 6–24 hours) required to grow bacteria isolated from PBC bottles. However, these systems are limited in the types of drugs and bacteria they can report, limiting their usefulness to healthcare professionals.

[0028] To significantly reduce morbidity and mortality, new diagnostic methods, devices, and systems are needed to rapidly detect infectious sepsis-causing bacteria at single cell or low copy number levels from blood samples without the significant time delays caused by multiple culture steps (e.g., biological amplification) currently required by standard treatment methods. Thus, the systems, devices, and methods described herein provide a comprehensive and systematic approach to identify pathogens, determine pathogen resistance, and recommend effective and appropriate treatments for patients.

[0029] Pathogen Identification System Overview: 1 shows a diagram of a system 101 for performing pathogen identification, according to an embodiment of the present disclosure. 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 number of databases 110 communicatively coupled via a network 112.

[0030] The analyzer 108 may be a point-of-care (POC) testing device that performs identification of pathogens in a patient sample, which may be stored in a sample tube 111. In some embodiments, the analyzer 108 may be referred to herein as an analyzer. In some embodiments, the sample in the sample tube 111 may consist of whole blood, urine, sterile bodily fluid, or other sample obtained from a patient. In some embodiments, the analyzer 108 may receive the sample tube 111 placed by a user or operator of the analyzer 108 into a corresponding drawer in the housing of the analyzer 108.

[0031] In addition to receiving sample tubes 111, the analyzer 108 can also receive sample preparation cartridges 114 and PCR cartridges 117, which can likewise be placed by a user or operator of the analyzer 108 into corresponding drawers in the housing of the analyzer 108. In some embodiments, various components and subsystems within the analyzer 108 can interface with the sample tubes 111, the sample preparation cartridge 114, the PCR cartridge 117, the processing device 116, and / or the database 110 to perform sample preparation and processing including cell lysis, enrichment of pathogens in the sample, and nucleic acid amplification using PCR or fluorescence values.

[0032] In some embodiments, after receiving the sample tube 111, a pipetting system in the analytical device 108 may transfer the sample from the sample tube 111 to the sample preparation cartridge 114. In some embodiments, the sample preparation cartridge 114 may be a dedicated consumable with a receptacle configured to hold the sample and elements for performing sample processing. The sample preparation cartridge 114 may include a processing tube 113 having a septum disposed on or within the tube to protect its contents. The processing tube 113 may be configured to receive the sample from the sample tube 111 through the septum by transferring the sample using a needle of the sample preparation cartridge 114. Once the sample is transferred to the processing tube 113, the sample may undergo sample concentration, lysis, and / or other processing steps.

[0033] After sample preparation and processing (e.g., including sample concentration and lysis) using elements and components of the sample preparation cartridge 114, the sample is transferred from the processing tube 113 in the sample preparation cartridge 114 to the PCR cartridge 117 by a pipetting system in the analyzer 108. In some embodiments, the PCR cartridge 117 may be a specialized consumable with one or more primary reaction chambers and multiple secondary reaction chambers for performing a nucleic acid amplification step for pathogen identification. In some embodiments, the one or more primary reaction chambers of the PCR cartridge 117 may be configured to receive nucleic acid and the multiple secondary reaction chambers of the PCR cartridge 117 may be configured to receive an aliquot of a first amplification product after amplification of the nucleic acid in the one or more primary reaction chambers. In some embodiments, the analyzer 108 may perform nucleic acid amplification in the PCR cartridge 117 and thermal cycling and detection of a fluorescent signal generated during amplification using a PCR subsystem disposed in the analyzer 108. In some embodiments, one or more single-step PCRs may be performed. In some embodiments, the one or more single-step PCRs may utilize one or more primary reaction chambers in the PCR cartridge 117. In some embodiments, the one or more single-step PCRs may utilize a secondary reaction chamber in the PCR cartridge 117 in which the initial sample is divided into aliquots for performing the single-step PCR.

[0034] In some embodiments, the analytical device 108 may further interface with additional cartridges, such as an antimicrobial susceptibility testing (AST) cartridge. In some embodiments, the AST cartridge may be a specialized consumable with multiple reaction wells configured to hold multiple aliquots of concentrated sample to perform AST. In some embodiments, the analytical device 108 may be configured to simultaneously hold one or more sample preparation cartridges 114, PCR cartridges 117, and / or AST cartridges to perform sample preparation / processing, pathogen identification, and / or susceptibility testing simultaneously or sequentially. In some embodiments, the sample preparation cartridge 114, AST cartridge, and / or PCR cartridge 117 may be referred to herein as consumables or containers configured to be inserted into the analytical device 108.

[0035] In some embodiments, the analyzer 108 can include a controller 109 disposed within a housing of the analyzer 108. The controller 109 can control the movement and operation of different components within the analyzer 108, including the movement of one or more sample tubes, processing tubes, cartridges, and pipetting systems within the analyzer 108. In some embodiments, the controller 109 can also control the operation of one or more centrifuges, subsystems, and modules within the analyzer 108 to perform sample preparation, pathogen identification, susceptibility testing, and / or other functions. In some embodiments, the controller 109 can include a microcontroller on an integrated circuit (IC) chip within the analyzer 108 that is programmed to turn on / off and operate one or more centrifuges, subsystems, and modules within the analyzer 108. In some embodiments, the controller 109 can be coupled to and programmed to control the operation of one or more stepper motors, actuators, or other motion control components within the analyzer 108.

[0036] In some embodiments, the controller 109 may be programmed by the processing unit 116. The processing unit 116 may be a computing device coupled to the analyzer 108 to perform data processing and provide instructions to the controller 109 and / or other components within the analyzer 108. In some embodiments, the processing unit 116 may be a personal digital assistant, a desktop workstation, a laptop or notebook computer, a netbook, a tablet, a smartphone, a mobile phone, a smartwatch, or any combination thereof.

[0037] In some embodiments, the processing device 116 can communicate with the analytical device 108 to receive results of reactions occurring within 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 fluorescent data of one or more signals generated within the PCR cartridge 117 from the amplified nucleic acid from a PCR subsystem within the analytical device 108 and analyze the fluorescent data to identify pathogens present in a patient sample based on the detected amplified nucleic acid.

[0038] In some embodiments, the processing device 116 may also be in communication with the plurality of databases 110. In some embodiments, one or more of the plurality of databases 110 may represent any number of databases and may include various databases, such as those storing clinical parameter data, epidemiological information, or antibiotic resistance information for a plurality of pathogens. In some embodiments, one or more of the plurality of databases 110 may be configured to store pathogen taxonomy data and / or results of past pathogen identification workflows (e.g., performed by the analysis device 108). In some embodiments, one or more of the plurality of databases 110 may include electronic health record (EHR) data including patient healthcare information obtained from various healthcare services and healthcare providers, such as hospitals, clinical care facilities, laboratories, radiology departments, pharmacies, etc.

[0039] In some embodiments, the EHR data stored in database 110 includes patient and medical history data regarding the patient's health and treatment, including demographics, medical history, medications and allergies, immunization status, laboratory test results, radiology images, vital signs, personal statistics such as age and weight, and billing information for each patient. In some embodiments, processing device 116 can use the pathogen identification and / or AST results performed by analyzer 108 along with data stored in multiple databases 110 (e.g., clinical parameter data, epidemiological or antibiotic resistance information, EHR data, etc.) to determine treatment recommendations for the patient.

[0040] In some embodiments, the components in the system 101 may be communicatively coupled via a network 112. In particular, the network 112 may enable the transmission and communication of information between the analytical device 108, the multiple databases 110, the processing device 116, and / or any other devices or components in the system 101. In some embodiments, the system 101 may include additional components, such as a Raman spectroscopy device and / or an electronic health record (EHR) system (not shown).

[0041] In some embodiments, the network 112 may be any or a 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 any other suitable configuration. The network may conform to one or more network protocols (including IEEE (Institute of Electrical and Electronics Engineers) protocols, 3GPP (3rd Generation Partnership Project) protocols, Fourth Generation Wireless Protocols (4G) (e.g., LTE (Long Term Evolution) standards, LTE Advanced, LTE Advanced Pro), Fifth Generation Wireless Protocols (5G), and / or similar wired and / or wireless protocols) and may include one or more intermediate devices for routing data between the analysis device 108, the multiple databases 110, the processing device 116, and / or any other devices or components in the system 101.

[0042] Embodiments of the analysis device: FIG. 2 shows a diagram of an analytical device 200 according to an embodiment of the present disclosure. The analytical device 200 represents an exemplary embodiment of the analytical device 108 shown in FIG. 1. In some embodiments, the analytical device 200 may be referred to herein as an analyzer 200. The analytical device 200 is a benchtop device with a housing 201 that houses various components and modules for sample preparation, processing, and testing. In some embodiments, the housing 201 may include the body of the analytical device 200 and / or an exterior case of the analytical device 200 that protects the internal modules and components. In some embodiments, the analytical device 200 may be configured in a cubic, cuboid, or rectangular shape with various compartments for user access and manipulation of the analytical device 20 ... 3 It may have a compact size with dimensions less than 10 mm.

[0043] In some embodiments, the analytical device 200 may be coupled to a computing device (e.g., processing device 116), such as a personal digital assistant, desktop workstation, laptop or notebook computer, netbook, tablet, smartphone, mobile phone, smartwatch, or any combination thereof. A user or operator of the analytical device 200 can use the computing device to control the analytical device 200, to send and receive sample information, patient information, pathogen information, antimicrobial information, etc. to the analytical device 200, and to access and edit pathogen identification results from the analytical device 200.

[0044] 3A shows a front view of an analytical device 200 according to an embodiment of the present disclosure. FIG 3A shows the internal features located within the housing 201 of the analytical device 200, including a first pipettor 202, a second pipettor 204, a sample drawer 210, a sample cartridge drawer 220, and a processing cartridge drawer 230.

[0045] In some embodiments, the first pipettor 202 and the second pipettor 204 may be pipettor devices configured to handle liquid transfer between components within the housing 201 of the analytical device 200. In some embodiments, the first and second pipettors 202, 204 may be automated devices controlled by a controller 109 in the analytical device 200. In some embodiments, the controller 109 may control the movement of the first and second pipettors 202, 204, including vertical and / or horizontal movement of the first and second pipettors 202, 204 relative to different components within the analytical device 200. In some embodiments, there may be more or fewer pipettors in the analytical device 200.

[0046] In some embodiments, the first pipettor 202 and the second pipettor 204 may be referred to as a high-volume pipettor and a low-volume pipettor, respectively. In some embodiments, the first pipettor 202 may be configured to handle volumes ranging from about 50 microliters (μL) to 5 milliliters (mL), while the second pipettor 204 may be configured to handle volumes ranging from about 1 to 200 μL. In some embodiments, the first pipettor 202 may have a coefficient of variation (CV) of 5% at 50 μL and a CV of 1% at 5 mL, and the second pipettor 204 may have a CV of 5% at 1 to 200 μL.

[0047] In some embodiments, the first and second pipettors 202, 204 may be referred to herein as a pipette and / or a pipettor system. In some embodiments, the first and second pipettors 202, 204 may each be configured to be attached to a removable, disposable needle. The first and second pipettors 202, 204 may be attached to two different needles configured to handle different volume ranges as required by the first and second pipettors 202, 204.

[0048] In addition to transferring liquids, the first and second pipettors 202, 204 may be configured to move elements, such as tubes, cartridges, etc., within the housing 201 of the analytical device 200. In particular, the first and second pipettors 202, 204 may each be configured with a pipette tip holder that can be attached to various components used in the analytical device 200. In particular, the pipette tips of the first and second pipettors 202, 204 can be pressed into and engaged with handling features, such as needles, tubes, cartridges, spin column baskets, etc., to pick up and place various components in different modules or areas within the analytical device 200.

[0049] In some embodiments, the first and second pipettors 202, 204 may have dimensions of approximately 325 mm (width) x 575 mm (depth) x 435 mm (height). In some embodiments, the first and second pipettors 202, 204 may be located in an upper section of the housing 201 such that the first and second pipettors 202, 204 interact with samples, tubes, cartridges, and different modules in a lower section of the housing 201. In particular, the first and second pipettors 202, 204 may handle liquid transfer and component movement in the sample drawer 210, sample cartridge drawer 220, and processing cartridge drawer 230 shown in FIG. 3A.

[0050] In some embodiments, the sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 are configured as sliding horizontal compartments designed to fit within three corresponding receptacles in the housing 201 of the analytical device 200. In some embodiments, the sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 may be configured to receive specialized elements that are inserted into the analytical device 200 for sample processing and testing. In particular, the sample drawer 210 can receive a sample container (e.g., sample tube 111) that contains a sample obtained from a patient. The sample cartridge drawer 220 can receive a sample preparation cartridge (e.g., sample preparation cartridge 114) through which the sample is transferred by components within the analytical device 200.

[0051] In some embodiments, the processing cartridge drawer 230 may receive a PCR cartridge (e.g., PCR cartridge 117) configured to receive nucleic acids isolated from a sample after sample processing, pathogen concentration and lysis, and purification of nucleic acids from pathogens of the sample in the sample preparation cartridge. In additional or alternative embodiments, the processing cartridge drawer 230 may be used as an AST cartridge and / or a PCR cartridge drawer. For example, a PCR cartridge or an AST cartridge may be inserted into the processing cartridge drawer 230 depending on whether the analytical device 200 is being used to perform pathogen identification or antimicrobial susceptibility testing of a sample. In some embodiments, the analytical device 200 may be configured to perform both pathogen identification and antimicrobial susceptibility testing functions with the dual processing cartridge drawer 230 configured to interface with dedicated cartridges or consumables for AST and pathogen identification.

[0052] In some embodiments, the sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 may include readers configured to scan identifiers of the sample tubes, the sample preparation cartridges, and the PCR cartridges (or AST cartridges), respectively. In some embodiments, a reader in the drawer may scan the identifiers of the sample tubes and / or cartridges as each drawer is inserted into the housing 201 of the analytical device 200. In some embodiments, the readers in the drawers 210, 220, and 230 may be configured to scan identification codes, bar codes, or data matrices of the corresponding sample tubes and / or cartridges. In some embodiments, the readers in the drawers 210, 220, and 230 may be bar code readers, quick response (QR) code readers, or the like.

[0053] In some embodiments, Figure 3A illustrates additional components including one or more centrifuges disposed within the housing 201 and configured to process tubes and cartridges for sample processing. Figure 3B illustrates a top view of an analyzer 200 according to an embodiment of the present disclosure. Figure 3B illustrates a cross-sectional view from the top view of the analyzer 200, illustrating various components, modules, and / or subsystems disposed below the first and second pipettors 202, 204 within the housing 201 of the analyzer 200.

[0054] The housing 201 of Figure 3B includes a sample drawer 210 containing a plurality of sample tubes 212, a sample cartridge drawer 220 containing a plurality of sample preparation cartridges 224, and a processing cartridge drawer 230 containing both an AST cartridge 232 and a PCR cartridge 234. The sample tubes 212, sample preparation cartridges 224, and PCR cartridges 234 represent exemplary embodiments of the sample tubes 111, sample preparation cartridges 114, and PCR cartridges 117, respectively, shown in Figure 1.

[0055] In some embodiments, the processing cartridge drawer 230 may be configured to house both AST cartridges 232 and PCR cartridges 234 for performing pathogen identification and antimicrobial susceptibility testing. In some embodiments, each drawer in the housing 201 may house a predetermined number of sample tubes 212, sample preparation cartridges 224, AST cartridges 232, and / or PCR cartridges 234 at one time. In some embodiments, the sample preparation cartridges 224, AST cartridges 232, and / or PCR cartridges 234 may be disposable after a single use or may be reused for testing additional samples.

[0056] FIG. 3B also shows 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 .

[0057] In some embodiments, the first centrifuge 240 may be a high-speed centrifuge configured to centrifuge the processing tubes (e.g., processing tubes 113) and / or spin column baskets placed in the first centrifuge 240 by the first pipettor 202. In some embodiments, the second centrifuge 242 may be a low-speed centrifuge configured to centrifuge the PCR cartridges 234 placed in the second centrifuge 242 by the second pipettor 204. In some embodiments, the first and second centrifuges 240, 242 may centrifuge the processing tubes, spin column baskets, and / or PCR cartridges 234 in a swinging bucket configuration. In some embodiments, the first and second centrifuges 240, 242 may configure a cylindrical shape with a diameter of about 250 mm and a height of about 175 mm.

[0058] In some embodiments, the PCR subsystem 244 may comprise a thermal cycler with an optical detection module or optics for measuring fluorescent signals generated during each amplification cycle in the PCR cartridge 234 upon binding of one or more fluorescent entities to target sequences in the nucleic acid. In some embodiments, the thermal cycler may be configured to control the temperature during PCR (polymerase chain reaction), and the optical system may be configured to optically interrogate primary and / or secondary reaction chambers in the PCR cartridge 234 by fluorescence.

[0059] In some embodiments, the thermal cycler of the PCR subsystem 244 may control the temperature in the range of about 35° C. to 100° C. In some embodiments, the optical system of the PCR subsystem 244 may perform fluorescent measurements from the bottom of the PCR cartridge 234 in the housing 201 to detect signals from the amplified nucleic acid products resulting from the PCR. In some embodiments, the PCR cartridge 234 is placed in and removed from the PCR subsystem 244 by the second pipettor 204. In some embodiments, the PCR subsystem 244 may hold up to two PCR cartridges 234 at a time, in which the PCR cartridges 234 undergo thermal cycling independently within the PCR subsystem 244. In some embodiments, the dimensions of the PCR subsystem 244 are approximately 70 mm (width) by 125 mm (depth) by 250 mm (height).

[0060] In some embodiments, the homogenization subsystem 246 may be configured to hold multiple processing tubes in slots to apply a swinging motion to the processing tubes to allow vibration or mixing of the samples. In some embodiments, the first pipettor 202 may be configured to vertically mount the tubes in the slots of the homogenization subsystem 246. In some embodiments, the homogenization subsystem 246 reorients the tubes from a vertical position to a horizontal position and applies a swinging motion of ±15° about 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, with each processing tube representing a different sample.

[0061] In some embodiments, the homogenization subsystem 246 may include a moving magnet. The moving magnet may engage the process tube when in a vertical position within the homogenization subsystem 246. In some embodiments, the homogenization subsystem 246 may apply a 37° C. temperature control of the process tube held within the homogenization subsystem 246. In some embodiments, the homogenization subsystem 246 may have dimensions of approximately 150 mm (width) by 175 mm (depth) by 115 mm (height).

[0062] In some embodiments, the mechanical device 248 may be configured to agitate the processing tube to effect cell lysis of pathogens in the sample. In some embodiments, the first pipettor 202 may be configured to vertically load the tube into a slot of the mechanical device 248. In some embodiments, the mechanical device 248 may include an agitator device or cell disrupting device configured to apply a high-speed vibrational motion to the processing tube. In some embodiments, the mechanical device 248 may apply the high-speed vibrational motion by applying a reciprocating motion along a predetermined axis by the agitator device while the processing tube is in a vertical position.

[0063] In some embodiments, the mechanical device 248 can hold up to two process tubes at a time and apply ±2° angular motion to the process tubes. In some embodiments, the mechanical device 248 can vibrate the process tubes at about 5,000-30,000 cycles per minute. In some embodiments, the dimensions of the mechanical device 248 can be about 75 mm (width) x 175 mm (depth) x 100 mm (height). In additional or alternative embodiments, the mechanical device 248 can include a sonicator configured to sonicate the process tubes to agitate the sample.

[0064] In some embodiments, the AST subsystem 250 may include a heater configured for incubation of the sample in the AST cartridge 232 in the housing 201 and an imaging subsystem for imaging the reaction chambers of the AST cartridge 232. In some embodiments, the heater of the AST subsystem 250 may be used for temperature control for incubation of the sample in the AST cartridge 232. In some embodiments, the pathogens may be concentrated and enriched in the sample and transferred to a reaction well in the AST cartridge 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 scan the bottom of each reaction chamber of the AST cartridge 232 using a motorized XYZ translation stage to acquire images. In some embodiments, the imaging subsystem of the AST subsystem 250 may include a fluorescent sensor with two optical channels for detecting different fluorescent signals (e.g., green and red).

[0065] In some embodiments, the AST subsystem 250 can identify antimicrobial phenotypic resistance of a microorganism (e.g., a pathogen) based on the acquired images and / or signals. In some embodiments, the AST cartridge 232 can be placed into and removed from the AST subsystem 250 by the second pipettor 204.

[0066] In some embodiments, the AST subsystem 250 can hold up to five AST cartridges 232 at a time. In some embodiments, the AST subsystem 250 can apply temperature control, such as by using a thermal block, to the AST cartridges 232 held within the AST subsystem 250. In some embodiments, the AST subsystem 250 may apply a temperature control of approximately 37° C. In some embodiments, the dimensions of the AST subsystem 250 may be approximately 200 mm (width) by 185 mm (depth) by 285 mm (height).

[0067] FIG. 4 shows a diagram of an analytical device 200 with three open compartments, according to an embodiment of the present disclosure. In some embodiments, the analytical device 200 of FIG. 4 shows a sample drawer 210, a sample cartridge drawer 220, and a processing cartridge drawer 230 extending from the housing 201 in an open position for loading and / or removing samples and / or cartridges. The sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 may be pushed into the housing 201 in a closed position, in which case the compartments fit into three corresponding receptacles in the housing 201. In some embodiments, the opening and closing of the sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 may be controlled by a controller (e.g., controller 109) and / or the processing device 116 of the analytical device 200. In some embodiments, a user of the processing device 116 can control the opening and closing of the sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 using software installed on the processing device 116. In some embodiments, the sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 can be manually pulled out and pushed into the housing 202 by a user to load and / or unload sample tubes, cartridges, and / or other elements into the analysis device 200.

[0068] In some embodiments, the sample drawer 210 can hold multiple samples. In some embodiments, the sample drawer 210 can hold up to 10 samples at a time, including samples stored in sample tubes 212. In some embodiments, the sample tubes 212 shown in FIG. 4 can represent one or more blood sample tubes, urine sample tubes, and / or blood culture bottles. In some embodiments, the sample cartridge drawer 220 can accommodate multiple sample preparation cartridges, for example, up to 10 sample preparation cartridges at a time. In some embodiments, the processing cartridge drawer 230 can accommodate multiple processing cartridges, for example, up to 12 PCR cartridges, 6 AST cartridges, or a combination thereof at a time.

[0069] In some embodiments, the dimensions of the sample drawer 210 may be approximately 40 mm (width) x 155 mm (height) x 600 mm (depth). In some embodiments, the dimensions of the sample cartridge drawer 220 may be approximately 85 mm (width) x 155 mm (height) x 615 mm (depth). In some embodiments, the dimensions of the processing cartridge drawer 230 may be approximately 140 mm (width) x 155 mm (height) x 270 mm (depth).

[0070] Embodiments of sample processing, concentration, lysis, and purification of nucleic acids in an analytical device: In some embodiments, pathogen identification includes transferring the sample to a consumable (e.g., processing tube 113 and / or spin column) for further processing (e.g., nucleic acid extraction, purification, amplification) using a pipetting system, concentrating the pathogens, lysing the pathogens (by mechanical disruption), purifying nucleic acids from the pathogens, and amplifying and identifying the pathogens by PCR. In some embodiments, a first pipettor 202 of the analyzer 200 may transfer the sample from the sample tube 111 to the processing tube 113 and perform steps to process, concentrate, separate, and lyse the pathogens in the sample, and purify nucleic acids from the lysed pathogens, before performing PCR.

[0071] In some embodiments, the sample processing may include a step of blood cell lysis. In embodiments where the sample is a blood sample, the first pipettor 202 may be configured to perform blood cell lysis of the blood sample. The first pipettor 202 may add one or more lysis reagents to the processing tube 113. The one or more lysis reagents may be mixed with the blood sample in the processing tube 113 using a mixer (e.g., homogenization subsystem 246, etc.) disposed in the housing 201 of the analyzer 200 to lyse blood cells in the blood sample. In some embodiments, the one or more lysis reagents may include one or more saponin-based buffers. In some embodiments, the one or more lysis reagents may include one or more detergents, or proteases.

[0072] After lysis of blood cells in the blood sample, the first pipettor 202 may transfer the processing tube 113 to the centrifuge 240 (or 1502 in FIG. 15A). In some embodiments, the centrifuge 240 may apply centrifugal force to the processing tube 113 to concentrate pathogens in the sample. In some embodiments, the processing tube 113 may be centrifuged from a high volume (e.g., up to 10 mL) to a low volume (e.g., 2 mL or less) with the pathogens concentrated in the processing tube 113.

[0073] After centrifugation, the first or second pipettor 202, 204 may be used to remove liquid from the processing tube 113, leaving the concentrated pathogens in the processing tube 113. In some embodiments, the first or second pipettor 202, 204 may add one or more lysis buffers to the concentrated pathogens in the processing tube 113. In some embodiments, the first or second pipettor 202, 204 may take one or more lysis buffers from one or more reservoirs in the sample preparation cartridge and dispense the one or more lysis buffers into the processing tube 113 via a needle coupled to the first or second pipettor 202, 204. In some embodiments, a plurality of lysis beads may be added to the processing tube 113 along with the one or more lysis buffers to effect lysis of the concentrated pathogens in the processing tube 113. In some embodiments, a plurality of lysis beads may be added to the processing tube 113 of the sample preparation cartridge during manufacturing and / or assembly.

[0074] In some embodiments, the processing tube 113 may be agitated or sonicated in the analyzer 200 to mechanically lyse the pathogens using lysis beads and one or more lysis buffers. In some embodiments, the cell walls of the pathogens in the processing tube 113 may be mechanically disrupted by the rapid movement of the lysis beads (by agitation or sonication in the analyzer 200) within the processing tube 113, resulting in the release of nucleic acids from the pathogens.

[0075] In some embodiments, after lysis of the pathogens, the processing tube 113 may undergo an additional centrifugation step (by a centrifuge 240) to drive the liquid in the processing tube 113 to the bottom of the tube. In some embodiments, the first or second pipettor 202, 204 removes a portion of the liquid from the processing tube 113, which portion of the liquid includes nucleic acid from the pathogens. In some embodiments, cells, lysis beads, and debris resulting from lysis are left behind in the processing tube 113.

[0076] After lysis and removal of the nucleic acids from the processing tube 113, the nucleic acids may be purified using either a spin column or magnetic beads. In some embodiments, the first or second pipettor 202, 204 may transfer a portion of the liquid from the processing tube 113 after cell lysis to a spin column in the analysis device 200. In some embodiments, the spin column can perform extraction and purification of the nucleic acids from the portion of the liquid. In some embodiments, the eluate (e.g., purified nucleic acids) resulting from the spin column can be collected in the basket of the spin column.

[0077] In additional or alternative embodiments, the first or second pipettor 202, 204 may transfer a portion of the liquid from the processing tube 113 after cell lysis to one or more reservoirs in the sample preparation cartridge. In some embodiments, the one or more reservoirs may be configured to store magnetic beads for nucleic acid extraction and purification. In some embodiments, the magnetic beads may be configured to attach to the nucleic acids. In some embodiments, the analysis device 200 may apply a magnetic force to retain the nucleic acids attached to the magnetic beads. In some embodiments, the first or second pipettor 202, 204 may remove excess liquid from the nucleic acids attached to the magnetic beads while the nucleic acids attached to the magnetic beads are retained in the one or more reservoirs. Removal of the excess liquid may result in the one or more reservoirs containing purified nucleic acids.

[0078] After purification, the nucleic acid from the sample is then ready to be transferred by a pipetting system within the analyzer 200 to a PCR cartridge 234 for nucleic acid amplification and pathogen identification by PCR.

[0079] Sample Preparation Cartridge Embodiments: FIG. 5 shows a diagram of a sample preparation cartridge 500 according to an embodiment of the present disclosure. The sample preparation cartridge 500 represents an exemplary embodiment of the sample preparation cartridge 224 shown in FIG. 3B. The sample preparation cartridge 500 may be a consumable item that is inserted into the sample drawer 210 of the analytical device 200 to prepare and process samples in sample tubes. In some embodiments, the sample preparation cartridge 500 may be manufactured by injection molding from a polypropylene (PP) material. The sample preparation cartridge 500 may include a housing 502, a plurality of reservoirs 504, a lid 506, and an identifier 508.

[0080] In some embodiments, the housing 502 may have an elongated rectangular shape with rounded edges. The housing 502 may be configured to hold additional elements used in sample preparation, as shown in FIG. 6A. In some embodiments, the multiple reservoirs 504 may be separate reservoirs or tubes molded together. The multiple reservoirs 504 may be configured to store materials used to perform sample concentration, lysis, and / or nucleic acid amplification. In some embodiments, the materials stored in the reservoirs 504 may include one or more buffers (e.g., a salt-based buffer, a phosphate-buffered saline (such as PBS), a surfactant, a protease, a growth medium, etc.). In some embodiments, the reservoirs 504 may store one or more lysis reagents, such as one or more saponin-based buffers, surfactants, or proteases for performing cell lysis of the blood sample. In some embodiments, the reservoirs 504 may store one or more wash materials, which may include a combination of one or more buffers, surfactants, and proteases.

[0081] In some embodiments, the lid 506 of the sample preparation cartridge 500 is a protective lid that extends over the housing 502 and the multiple reservoirs 504. In some embodiments, the identifier 508 is an identifier of the sample preparation cartridge 500 that may be scanned by the analysis device 200 to perform pathogen identification. In some embodiments, the identifier 508 may be at least one of an identification code, a barcode, or a data matrix such as a QR code. In some embodiments, the dimensions of the sample preparation cartridge 500 may be approximately 80 mm (width) x 55 mm (depth) x 155 mm (height), as shown in FIG. 5.

[0082] 6A shows an exploded perspective view of a sample preparation cartridge 500 with a processing tube 510 and other components inserted therein, according to an embodiment of the present disclosure. The sample preparation cartridge 500 may include a 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 housed in corresponding receptacles within the housing 502 of the sample preparation cartridge 500. The processing tube 510 represents an exemplary embodiment of the processing tube 113 shown in FIG.

[0083] In some embodiments, the processing tube 510 may comprise, for example, a 15 ml tube with a conical bottom. In some embodiments, the processing tube 510 may be removable from the sample preparation cartridge 500 for processing in other modules within the analytical device 200. In some embodiments, the processing tube 510 may be a tube to which the sample is transferred after the sample tube and sample preparation cartridge 500 are loaded into the sample drawer 210 and sample cartridge drawer 220 of the analytical device 200, respectively.

[0084] In some embodiments, a sample may be transferred from a sample tube in the sample drawer 210 to a processing tube 510 of the sample preparation cartridge 500 in the sample cartridge drawer 220 by a first removable needle 512. In some embodiments, the first pipettor 202 is adapted to be attached to the first removable needle 512 and configured to transfer the sample to and / or from the 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 are used for processing small volumes and the second pipettor 204 may be configured to be attached to the two second removable needles 514.

[0085] 6A also shows the openings 520 of the plurality of reservoirs 504 of the sample preparation cartridge 500. In some embodiments, the sample preparation cartridge 500 has 12 reservoirs 504, with each reservoir 504 holding a volume of approximately 2.5 mL. In some embodiments, the sample preparation cartridge 500 may include a pierceable film 516 covering a receptacle 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 film 516 may be a polyester film and the foil seal 518 may include aluminum foil, and both the pierceable film 516 and the foil seal 518 are pierceable by the first and / or second removable needles 512, 514.

[0086] FIG. 6A further illustrates a spin column 522, a spin column basket 524, and a reagent tube 526 housed in the sample preparation cartridge 500. In some embodiments, the spin column 522 and the spin column basket 524 are used to perform extraction of nucleic acids obtained from a sample and purification of the nucleic acids. In some embodiments, the reagent tube 526 stores one or more reagents used to perform nucleic acid amplification of the purified nucleic acids obtained from the sample. In some embodiments, the reagent tube 526 may be sealed with aluminum foil. In some embodiments, the spin column 522 may be configured to be inserted and housed within the spin column basket 524. In some embodiments, the spin column basket 524 and the reagent tube 526 are both configured to fit within corresponding receptacles in the sample preparation cartridge 500.

[0087] 6B shows an exploded perspective view of a sample preparation cartridge 500' with a processing tube 510 and other components inserted therein, according to an embodiment of the present disclosure. Sample preparation cartridge 500' is similar to sample preparation cartridge 500, but with a more linear shape and a peelable film 506' in place of lid 506.

[0088] Embodiments of processing tubes, sample tubes, needles, and spin columns: 7A and 7B show views of a process tube 700 according to an embodiment of the present disclosure. Process tube 700 represents an exemplary embodiment of process tube 510 shown in FIG. 6A. In particular, FIG. 7A shows process tube 700 after assembly, while FIG. 7B shows an exploded view of the components within process tube 700. Process tube 700 includes cap 702, septum 704, and tube 706.

[0089] In some embodiments, septum 704 may be secured inside tube 706 with cap 702 attached over septum 704 of process tube 700. In some embodiments, septum 704 may be configured to allow insertion and removal of needles (e.g., needles 512 and 514) for transfer of liquids into process tube 700 without having to remove cap 702 from tube 706. In some embodiments, septum 704 provides an airtight seal within process tube 700 to prevent contamination of the contents of process tube 700.

[0090] In some embodiments, the process tube 700 may include a handling structure at the top of the process tube 700 that is compatible for handling by a pipettor (e.g., the first and / or second pipettors 202, 204). In some embodiments, the handling structure of the process tube 700 may be a cylindrical cavity in the cap 702 that is compatible for insertion by the tip of a pipettor. In some embodiments, the tip of the pipettor may be referred to herein as a mandrel. In some embodiments, the pipette tips of the first and second pipettors 202, 204 may press against and fit into the cylindrical cavity of the cap 702, allowing the process tube to be picked up and moved within the analyzer 200.

[0091] In some embodiments, the cap 702 may be made from a high density polyethylene (HDPE) material and the tube 706 may be made from a polypropylene (PP) material. In some embodiments, the septum 704 may include at least one of rubber, polytetrafluoroethylene (PTFE), thermoplastic elastomer (TPE), silicone, butyl rubber, or a combination thereof. In some embodiments, the septum 704 may include a double layer of polytetrafluoroethylene (PTFE) and another material selected from the group consisting of silicone, rubber, and butyl rubber. In some embodiments, the dimensions of the assembled process tube 700 may comprise, for example, a height of about 110 mm and a diameter of about 20 mm. In some embodiments, the septum 704 may have a thickness in the range of about 1 mm to 2 mm.

[0092] 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 be of a predetermined size and material configured to perform lysis of at least one of yeast, fungi, gram-positive bacteria, gram-negative bacteria, or the like. In some embodiments, the processing tube 700 may be transferred to the mechanical device 248 of the analytical device 200, where a high-speed vibrational motion may be applied to the plurality of lysis beads to perform lysis of pathogens in the sample within the processing tube 700. In some embodiments, the motion of the lysis beads may mechanically disrupt cell walls of pathogens in the sample, resulting in the release of nucleic acids from the pathogens.

[0093] In some embodiments, after cell lysis, the first or second pipettor 202 or 204 may transfer a portion of the liquid from the processing tube 700 to one or more reservoirs 504 in the sample preparation cartridge 500 where a plurality of magnetic beads may be stored. In some embodiments, the plurality of magnetic beads in the one or more reservoirs 504 may be configured to bind to nucleic acids in the portion of the liquid obtained from the processing tube 700. In some embodiments, the plurality of magnetic beads may be used to extract and purify nucleic acids from the portion of the liquid obtained from the processing tube 700.

[0094] 7C and 7D show diagrams of an example of a reagent tube 710 according to an embodiment of the present disclosure. Reagent tube 710 represents an embodiment of an example of reagent tube 526 shown in FIG. 6A. In some embodiments, reagent tube 710 may be referred to as a master mix tube. Reagent tube 710 may contain a first set of reagents used to perform nucleic acid amplification. In some embodiments, the first set of reagents may include DNA polymerase, deoxyribonucleotide triphosphates (dNTPs), magnesium chloride (MgCl 2), etc. In some embodiments, the reagent contained in the reagent tube 710 may be desiccated or lyophilized. In some embodiments, the reagent tube 710 may include a cap 712 and a tube 714. The cap 712 may include a septum, such as over the top of the cap. In some embodiments, the septum on the cap 712 of the reagent tube 710 may be similar to the septum 704 of the process tube 700. In some embodiments, the cap 712 may be configured to attach 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 recess 716 to facilitate the evacuation of air during the freeze-drying process by a lyophilizer. In some embodiments, the cap 712 may be slightly pressed into the tube 714 when inserted into the freeze-drying machine, allowing air to pass through the recess 716. The cap 712 may then be closed in the freeze dryer by lid pressure, rendering the air within the tube 714 oxygen-free.

[0095] In some embodiments, the tube 714 is made from a polypropylene (PP) material and the septum on the cap 712 may be comprised of at least one of rubber, polytetrafluoroethylene (PTFE), thermoplastic elastomer (TPE), silicone, butyl rubber, or combinations thereof. In some embodiments, the septum may include a double layer of polytetrafluoroethylene (PTFE) and another material selected from the group consisting of silicone, rubber, and butyl rubber. In some embodiments, the dimensions of the reagent tube 710 after assembly (e.g., after the cap 712 is attached to the tube 714) may comprise, for example, a height of about 40 mm and a diameter of about 10 mm.

[0096] In some embodiments, the process tube 700 and / or the reagent tube 710 may be configured to receive a needle, as shown in Figures 8A, 8B, and 8C, which show diagrams of a needle 800 configured for insertion into the process tube 700 and / or the reagent tube 710 according to some embodiments of the present disclosure.

[0097] In particular, FIG. 8A shows a needle 800 comprising a plastic body 810, a cannula 820, and a number of slots 825. In some embodiments, the plastic body 810 may be attached to the cannula 820 by bonding. In some embodiments, a pipettor (e.g., the first pipettor 202) in the analytical device may 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 pipettor in the analytical device. In some embodiments, the needle 800 may be a venting needle configured to ventilate the process tube 700 upon insertion of the needle. In some embodiments, venting the needle 800 facilitates relieving pressure in the sealed process tube 700. In some embodiments, the plastic body 810 of the needle 800 includes a number of slots 825 configured to provide an air communication path between the interior and exterior of the process tube 700 when the needle 800 is inserted through the septum 704 and into the tube 706. For example, four slots 825 may be provided in the injection portion of the needle 800 that holds the cannula 820. In some embodiments, the slots 825 may be produced by injection molding. In some embodiments, inserting the needle 800 into the reagent tube 710 may be performed similarly to inserting the needle 800 into the process tube 700. In some embodiments, the cannula 820 of the needle 800 may be configured to pierce the septum for insertion into the tube 706 of the reagent tube 710.

[0098] 8B shows needle 800 during insertion into process tube 700, and FIG. 8C shows needle 800 after full insertion into process tube 700. In some embodiments, cannula 820 of needle 800 may be inserted through septum 704, into cap 702, and into tube 706 of process tube 700. In some embodiments, the distal end of plastic body 810 may fit into cap 702 of process tube 700 when cannula 820 is fully inserted into tube 706.

[0099] 9A and 9B show diagrams of a high-volume needle 900 and a low-volume needle 910, respectively, according to embodiments of the present disclosure. In some embodiments, the high-volume needle 900 and the low-volume needle 910 may be coupled to a first pipettor 202 and a second pipettor 204, respectively, within an analytical device 200.

[0100] 9A, the high volume needle 900 may include a plastic body 902 and a cannula 904. In some embodiments, the plastic body 902 may be a reservoir configured to hold a volume of about 50 μL to 5 mL during transfer of liquid in an analytical device. In some embodiments, the plastic body 902 may include a filter 903 disposed therein to prevent contamination of a pipettor coupled to the high volume needle 900.

[0101] In some embodiments, the plastic body 902 may be made of a polypropylene (PP) material. In some embodiments, the plastic body 902 of the needle 900 may have a diameter of about 16 mm and a length of about 50 mm. In some embodiments, the cannula 904 may be made of stainless steel. In some embodiments, the cannula 904 may have a length of about 100 mm. In some embodiments, the overall length of the plastic body 902 and the cannula 904 when assembled may be about 150 mm. In some embodiments, the high volume needle 900 may 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.

[0102] In some embodiments, the cannula 904 may further include a secondary cannula disposed about the inner core of the needle 900. In some embodiments, the cannula 904 includes one or more vent holes 906. In some embodiments, the cannula 904 of the needle 900 may be in fluid communication with the vent holes 906. In some embodiments, the cannula 904 may be a slotted cannula that includes a slot around the needle.

[0103] As shown in FIG. 9B, the low-volume needle 910 may include a plastic body 912 and a needle shaft 914. In some embodiments, the plastic body 912 may be a reservoir configured to hold a volume of about 1-200 μL during transfer of liquid in an analytical device. In some embodiments, the plastic body 912 may include a filter 913 disposed therein to prevent contamination of a pipettor coupled to the low-volume needle 910. In some embodiments, the plastic body 912 may be manufactured from 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 shaft 914 may have a length of about 10 mm. In some embodiments, the needle shaft 914 may be made of stainless steel. In some embodiments, the low-volume 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.

[0104] FIG. 10 shows a diagram of an example high volume needle 900 according to an embodiment of the present disclosure. In particular, FIG. 10 shows various examples of the cannula of the high volume needle 900 with vent holes, slots, etc. In some embodiments, the first needle shown in FIG. 10 can be vented by having two communicating orifices or vent holes in the needle cannula. In some embodiments, the second and fourth needles shown in FIG. 10 can be vented when the tip of each needle is inserted into a septum (e.g., septum 704) of a processing tube.

[0105] 11A and 11B show diagrams of a spin column 1100 and a spin column basket 1102 according to an embodiment of the present disclosure. The spin column 1100 and the spin column basket 1102 represent exemplary embodiments of the spin column 522 and the spin column basket 524 shown in FIG. 6A, respectively. In particular, the spin column 1100 may be configured to extract and purify nucleic acids obtained from a sample. In some embodiments, after cell lysis in the processing tube 700, the first pipettor 202 or the second pipettor 204 may transfer a portion of the liquid from the processing tube 700 to the spin column 1100. In some embodiments, the spin column 1100 may be configured to extract and purify nucleic acids from the portion of the liquid. In some embodiments, the spin column 1100 may 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 may be configured to collect the purified nucleic acids that have passed through the spin column 1100. In some embodiments, the spin column basket 1102 may include one or more tubular receptacles configured to receive purified nucleic acids, store the spin column 1100, and / or receive needles coupled to the first or second pipettors 202, 204.

[0106] In some embodiments, the spin column 1100 may have a height H of about 34 mm. In some embodiments, the top of the spin column 1100 may have a diameter D of about 17 mm. In some embodiments, the spin column basket 1102 may have a length L of about 50 mm and a height H of about 60 mm. In some embodiments, the diameter of the tubular receptacle in the spin column basket 1102 is about 19 mm. In some embodiments, the spin column 1100 and the spin column basket 1102 may be made of polypropylene (PP) material.

[0107] 12A illustrates how a low-volume needle 910 interfaces with a sample preparation cartridge 500, according to an embodiment of the present disclosure. In particular, FIG. 12 illustrates an example of a low-volume needle 910 disposed in one of a plurality of reservoirs 504 of the sample preparation cartridge 500, for use, for example, to transfer materials used for sample enrichment and / or lysis from the reservoir 504 to a sample in a processing tube. In some embodiments, the sample preparation cartridge 500 may be configured to receive one needle, such as the low-volume needle 910. In other embodiments, the sample preparation cartridge 500 may be configured to receive two needles, such as both a high-volume needle 900 and a low-volume needle 910.

[0108] 12B shows a low-volume needle 910 interfacing with a spin column basket 1102, according to an embodiment of the present disclosure. In some embodiments, the low-volume needle 910 may be inserted into one of the tubular receptacles of the spin column basket 1102. In some embodiments, the spin column 1100 may also be disposed within one of the tubular receptacles of the spin column basket 1102.

[0109] PCR cartridge embodiment: 13A and 13B show diagrams of a PCR cartridge 1300 according to an embodiment of the present disclosure. The PCR cartridge 1300 represents an exemplary embodiment of the PCR cartridge 234. In particular, FIG. 13A shows the PCR cartridge 1300 after assembly, while FIG. 13B shows an exploded view of the components within the PCR cartridge 1300. The PCR cartridge 1300 includes a cover 1302, a septum 1308, and a base 1312. In some embodiments, the cover 1302 is disposed over the septum 1308, which is disposed over the base 1312.

[0110] In some embodiments, the base 1312 includes two primary reaction chambers 1313 and a plurality of secondary reaction chambers 1314. In some embodiments, there may be at least one primary reaction chamber 1313 in the base 1312. In some embodiments, the number of secondary reaction chambers 1313 present in the base 1312 may range from about 10 to 50, such as, for example, 20 secondary reaction chambers. In some embodiments, each primary reaction chamber 1313 may accommodate a volume in the range of about 50-500 μL, such as a volume of 100 μL. In some embodiments, each secondary reaction chamber 1314 may accommodate a volume in the range of about 2-50 μL, such as a volume of 30 μL. In some embodiments, each primary reaction chamber 1313 and the plurality of secondary reaction chambers 1314 may be sealed with a septum 1308, where the septum 1308 is configured to receive a needle (e.g., needle 900 or 910).

[0111] In some embodiments, the PCR cartridge 1300 may include an additional receptacle configured to receive a reagent tube 710 that stores a first set of reagents used to perform the nucleic acid amplification. In other embodiments, the first set of reagents used to perform the nucleic acid amplification may be stored in a reservoir in the PCR cartridge 1300, such as in one of the reaction chambers in the PCR cartridge 1300. For example, one of the primary reaction chambers 1313 of the PCR cartridge 1300 may store the first set of reagents (e.g., a master mix) used to perform the nucleic acid amplification. In some embodiments, the needle 900 or 910 may dispense one or more reagents for performing the nucleic acid amplification from the first set of reagents (e.g., stored in one of the primary reaction chambers 1313 in the PCR cartridge 1300 or stored in the reagent tube 710) to one of the primary reaction chambers 1313.

[0112] In some embodiments, at least one primary reaction chamber 1313 may be configured to receive nucleic acids from a sample (e.g., after enrichment, lysis, and purification steps) and may be transferred from the processing tube 700 by a needle 900 or 910 coupled to the first pipettor 202 or the second pipettor 204 after resuspending the master mix. In some embodiments, the primary reaction chamber 1313 may be configured to perform a first amplification of the nucleic acids to generate a first amplification product.

[0113] In some embodiments, a set of primers is present in the primary reaction chamber 1313. In some embodiments, a dried set of primers is present in the primary reaction chamber 1313. In some embodiments, a set of primers may be transferred to the primary reaction chamber 1313 together with the nucleic acid for the primary amplification. In some embodiments, the primer set may be compatible with the amplification of the target sequence to be detected. In some embodiments, different primer sets may be transferred to the primary reaction chamber 1313 together with the nucleic acid for the first amplification. In some embodiments, different primer set combinations may be compatible with the amplification of the target sequence to be detected.

[0114] In some embodiments, the secondary reaction chamber 1314 may be configured to receive a respective aliquot of the first amplification product from the primary reaction chamber 1313 by needle 900 or 910. In some embodiments, the multiple secondary reaction chambers 1314 may be configured to perform a second amplification of the product nucleic acid. In some embodiments, the aliquot of the first amplification product from the primary reaction chamber 1313 may be diluted with the contents of one of the reservoirs 504 of the sample preparation cartridge 500.

[0115] In some embodiments, each secondary reaction chamber 1314 may include a set of reagents for reacting with a respective aliquot of the first amplification product. In some embodiments, the reagents disposed in each secondary reaction chamber 1314 may be in liquid form or may be in a dried or lyophilized 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 before the first or second pipettor 202, 204 dispenses the multiple aliquots of the first amplification product into the secondary reaction chamber 1314. 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.

[0116] In some embodiments, a first or second pipettor 202, 204 in the analyzer 200 may dispense nucleic acid from the sample into a primary reaction chamber 1313 in the PCR cartridge 1300 after concentrating the pathogens, lysing the pathogens to release the nucleic acids, and purifying the nucleic acids in the process tube 700. After initial amplification of the nucleic acids in the primary reaction chamber 1313, a first or second pipettor 202, 204 in the analyzer 200 may then dispense multiple aliquots of the first amplification product into secondary reaction chambers 1314, each aliquot corresponding to a respective secondary reaction chamber 1314. In some embodiments, the first amplification products may be referred to as pre-amplification products.

[0117] In some embodiments, each secondary reaction chamber 1314 may be configured to receive an aliquot of the first amplification product by a needle (e.g., a needle 900 or 910 coupled to the first or second pipettor 202, 204) by non-contact dispensing. In some embodiments, the needle may pierce the septum 1308 of each secondary reaction chamber 1314 to dispense each aliquot without contacting the bottom surface of each secondary reaction chamber 1314. In some embodiments, each aliquot volume dispensed by the needle 900 or 910 into each secondary reaction chamber 1314 may be in the range of about 0.5 μL to about 5 μL. In some embodiments, the first or second pipettor 202 or 204 may add to the first amplification product a spare set of reagents used for nucleic acid amplification in a reagent tube (e.g., reagent tube 710) before dispensing multiple aliquots of the first amplification product into the secondary reaction chambers 1314 in the PCR cartridge 1300.

[0118] In some embodiments, the at least one primary reaction chamber 1313 and the plurality of 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 prior to receiving a respective aliquot of the first amplification product from the 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, the at least one primary reaction chamber 1313 and the plurality of secondary reaction chambers 1314 may receive an additional set of reagents (e.g., in addition to the reagents stored in the secondary reaction chamber 1314) used for nucleic acid amplification. In some embodiments, the additional set of reagents may be dried or lyophilized and stored in a reagent tube, e.g., the reagent tube 710.

[0119] In some embodiments, each of the primary and secondary reaction chambers 1313, 1314 comprises a conical shape and a bottom wall. In some embodiments, each of the primary reaction chambers 1313 may have a width that is greater than the individual widths of each of the secondary reaction chambers 1314. In some embodiments, the diameter of the bottom wall of at least one of the primary reaction chambers 1313 and each of the multiple secondary reaction chambers 1314 may be less than about 2.5 mm. In some embodiments, non-contact dispensing of multiple aliquots into each of the secondary reaction chambers 1314 may include the use of jet dispensing, where a needle 900 or 910 in the sample preparation cartridge 500 pierces a septum of each of the secondary reaction chambers 1314, so that the needle 900 or 910 does not contact the bottom wall of each of the secondary reaction chambers 1314, avoiding cross-contamination.

[0120] In some embodiments, the bottom wall of each of the primary and secondary reaction chambers 1313, 1314 may be optically transparent and configured for optical interrogation. In some embodiments, the bottom wall of each of the primary and secondary reaction chambers 1313, 1314 may be configured for optical interrogation, such as by a PCR subsystem 244 in the analyzer 200. In some embodiments, the bottom wall of each of the primary and secondary reaction chambers 1313, 1314 may be configured for fluorescent detection, such as by a PCR subsystem 244 in the analyzer 200. In some embodiments, the multiple primary and secondary reaction chambers 1313, 1314 may be configured to fit into corresponding chambers in a temperature control block in the PCR subsystem 244, and the temperature control block may be configured to heat the sides of the multiple primary and secondary reaction chambers 1313, 1314.

[0121] 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 may be referred to as a sealing cap mat. In some embodiments, the septum 1308 may be comprised of a unitary structure that extends across the primary reaction chamber 1313 and the multiple secondary reaction chambers 1314 of the base 1312. In some embodiments, the septum 1308 may be comprised of an assembled multiple parts, each part covering one or more respective primary and secondary reaction chambers 1313, 1314 of the base 1312. The septum 1308 may be configured to receive a needle, such as the needle 900 or 910 coupled to the first or second pipettor 202, 204. In some embodiments, the needle forms a hole in the septum 1308 during insertion, and the hole in the septum 1308 may become blocked when the needle is removed as a result of the material of the septum 1308.

[0122] In some embodiments, the septum 1308 may be constructed of at least one of rubber, polytetrafluoroethylene (PTFE), thermoplastic elastomer (TPE), silicone, butyl rubber, or combinations thereof. In some embodiments, the septum 1308 may be constructed of a bilayer of polytetrafluoroethylene (PTFE) and another material selected from the group consisting of silicone, rubber, and butyl rubber. In some embodiments, the septum 1308 may have a thickness in the range of about 1-2 mm. In some embodiments, the septum 1308 may be designed to be optically interrogated (e.g., fluorescent) from above by the PCR subsystem 244.

[0123] In some embodiments, the cover 1302 includes a plurality of openings 1306, each opening 1306 aligning with a respective primary or secondary reaction chamber 1313, 1314 of the plurality of primary and secondary reaction chambers 1313, 1314 in the base 1312. In some embodiments, the cover 1302 may also include an identifier 1310. The identifier 1310 may be an identifier of the PCR cartridge 1300 that is scanned by the analyzer 200 to perform pathogen identification. In some embodiments, the identifier 1310 may be at least one of an identification code, a bar code, or a data matrix.

[0124] In some embodiments, the cover 1302 may fit over the septum 1308 and base 1312 to form the assembled PCR cartridge 1300. In some embodiments, the septum 1308 may be overmolded onto the cover 1302 to form a unitary piece, which may be assembled onto the base 1312 by at least one of a snap-fit ​​connection or mechanical fasteners. In some embodiments, the cover 1302, septum 1308, and base 1312 may be coupled or secured together by one or more mechanical fasteners.

[0125] In some embodiments, the base 1312, septum 1308, and cover 1302 each include an opening 1304 in the center of the PCR cartridge 1300. The opening 1304 may be sized or shaped to engage with an object treatment tool in the system. For example, in some embodiments, the opening 1304 may be a circular hole that is compatible with insertion by a pipette tip holder (e.g., first and second pipettors 202, 204) for moving the PCR cartridge 1300 within the analyzer 200. In some embodiments, the openings 1304 in the base 1312, septum 1308, and cover 1302 are aligned with each other upon assembly of the PCR cartridge 1300. In some embodiments, the base 1312 and cover 1302 may be manufactured from polypropylene (PP) or polycarbonate (PC) materials. In some embodiments, the dimensions of the assembled PCR cartridge 1300 may be approximately 135 mm (length) x 15 mm (width) x 10 mm (height).

[0126] In some embodiments, the assembled PCR cartridge 1300 may be configured to receive a heated lid to be placed over the cover 1302 and the primary and secondary reaction chambers 1313, 1314 during PCR performed in the PCR subsystem 244. In some embodiments, the heated lid may be configured to heat the tops of at least one primary reaction chamber 1313 and multiple secondary reaction chambers 1314 to prevent condensation / condensation during thermal cycling.

[0127] 14 is a diagram illustrating how a low-volume needle 910 is inserted into a PCR cartridge 1300, according to an embodiment of the present disclosure. In particular, FIG. 14 illustrates how a needle shaft 914 of the needle 910 penetrates a septum 1308 and is inserted into a secondary reaction chamber 1314 of the PCR cartridge 1300. In some embodiments, the needle shaft 914 may form an opening in the septum 1308 during insertion. The opening in the septum 1308 may close when the needle shaft 914 is removed as a result of the material of the septum 1308.

[0128] Embodiments of the PCR method in a PCR cartridge: In diagnostic applications, where high sensitivity requirements (due to low available copy numbers in the original sample) and high numbers of microorganisms of interest are required, two-step PCR may provide an attractive combination of both extremes. However, the need to collect the amplified product after the first amplification step (pre-amplification) and transfer the amplified product to a secondary container for further amplification may make the two-step PCR approach less popular. In some embodiments, the use of the amplified product may entail a risk of contamination of testing equipment and laboratory facilities, which may result in extensive cleaning and / or closure of the laboratory.

[0129] In some embodiments, the PCR cartridge 1300 includes primary and secondary reaction chambers 1313 and 1314 sealed by a septum 1308, which allows the transfer of pre-amplification liquid volumes to a subsequent PCR reaction without the need to work with open tubes and with minimal contamination risk. In some embodiments, the configuration of the PCR cartridge 1300 is compatible with real-time PCR, and fluorescence measurements are made by the PCR subsystem 244 from below the reaction chambers 1313, 1314. In some embodiments, the septum lid 1308 is designed in such a way that fluorescence optical measurements can be performed from above.

[0130] In some embodiments, reagents for the secondary reaction in the secondary reaction chamber 1314 can be dried at the bottom of the secondary reaction chamber 1314. In some embodiments, jet dispensing may be used to deliver the pre-amplification products to the secondary reaction chamber 1314 (e.g., in volumes in the range of 5 μL or less). In some embodiments, jet dispensing may avoid contact dispensing, which may involve cross-contamination issues, which may also include a spin step that may slow down the overall process.

[0131] In some embodiments, the assay strategy for PCR may 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 with multiple reaction chambers, such as in a Taqman-based assay, to provide additional specificity and semi-quantitative information.

[0132] In some embodiments, a method for performing contamination-free two-step PCR may include: (1) delivering a template containing a set of reagents used for nucleic acid amplification to at least one primary reaction chamber 1313 in a PCR cartridge 1300 through a septum 1308; (2) performing a first amplification step in the at least one primary reaction chamber 1313; (3) removing a first amplification product from the at least one primary reaction chamber 1313 through the septum 1308; and (4) non-contact dispensing respective aliquots of the first amplification product through the septum 1308 to a plurality of secondary reaction chambers 1314, where each secondary reaction chamber 1314 contains reagents for performing a specific reaction from the plurality of secondary reaction chambers 1314.

[0133] Embodiments of modules and subsystems in an analytical device: 15A and 15B show diagrams of example centrifuges used in an analytical device 200 according to embodiments of the present disclosure. In particular, FIG. 15A shows a first centrifuge 1502 that may be used to centrifuge samples in a processing tube 1504 or a spin column basket 1510, while FIG. 15B shows a second centrifuge 1512 that may be used to centrifuge samples in a PCR cartridge 1514. The first and second centrifuges 1502, 1512 represent exemplary embodiments of the first and second centrifuges 240, 242, respectively, shown in FIG. 3B. The processing tube 1504 and the PCR cartridge 1514 represent exemplary embodiments of the processing tube 700 and the PCR cartridge 1300, respectively, shown in FIGS. 7A-7B and 13A-13B. In some embodiments, the spin column basket 1510 may include a spin column housed within a receptacle therein. The spin column basket 1510 represents an exemplary embodiment of the spin column basket 1102 shown in Figures 11A, 11B, and 12B.

[0134] In some embodiments, the first centrifuge 1502 may be a high-speed centrifuge configured to apply a relative centrifugal acceleration (RCF) or g-force of about 12,000 G to the processing tubes 1504 and / or spin column basket 1510. In some embodiments, the second centrifuge 1512 may be a low-speed centrifuge configured to apply a relative centrifugal acceleration (RCF) or g-force of about 3,000 G to the PCR cartridge 1514.

[0135] In some embodiments, the first centrifuge 1502 may hold the processing tubes 1504 and / or spin column basket 1510 in a first orientation and subject the processing tubes 1504 and / or spin column basket 1510 to 45 degree swing rotor centrifugation. In some embodiments, the second centrifuge 1512 may hold the PCR cartridges 1514 in a different orientation and subject them to 90 degree swing rotor centrifugation such that the PCR cartridges 1514 move to a vertical position within the second centrifuge 1512.

[0136] In some embodiments, the first centrifuge 1502 and the second centrifuge 1512 can each centrifuge multiple processing tubes 1504, spin column baskets 1510, and PCR cartridges 1514 at one time. For example, the first centrifuge 1502 can be configured to hold two processing tubes 1504 and two spin column baskets 1510 at one time for centrifugation together. In another example, the second centrifuge 1512 can be configured to hold two PCR cartridges 1514 at one 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 for isolating nucleic acids from a sample containing a pathogen.

[0137] 16A, 16B, 16C, and 16D show diagrams of an example of a homogenization subsystem 1600 used in the analyzer 200 according to an embodiment of the present disclosure. The homogenization subsystem 1600 represents an embodiment of an example of the homogenization subsystem 246 shown in FIG. 3B. In some embodiments, the homogenization subsystem 1600 may apply a swinging motion to the processing tube 700 to allow vibration and mixing of the sample with other materials. In some embodiments, the processing tube 700 may be placed in the homogenization subsystem 1600 by a pipetting system (e.g., the first or second pipettor 202, 204) to mix pathogens in the processing tube 700 with any reagents, for example, to perform blood cell lysis. In some embodiments, the processing tube 700 may be placed in the homogenization subsystem 1600 to mix one or more lysis reagents or buffers with the blood sample in the processing tube 700 to lyse blood cells in the blood sample. In some embodiments, the mixing functionality of the homogenization subsystem 1600 can be used for sample processing in the analyzer 200 for both pathogen identification and / or antimicrobial susceptibility testing.

[0138] In some embodiments, the homogenization subsystem 1600 may be a mixer that 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 may be loaded into the homogenization subsystem 1600 at one time. In some embodiments, the homogenization subsystem 1600 may include a magnet 1601 that reciprocates between an up position and a down position. In some embodiments, one or more processing tubes 700 may include magnetic beads configured to attach to nucleic acids in the one or more processing tubes 700. In some embodiments, the magnet 1601 may be used to retain the nucleic acids attached to the magnetic beads in the one or more processing tubes 700.

[0139] In some embodiments, the homogenization subsystem 1600 may include an additional or alternative separate magnet station that is used to retain nucleic acids attached to magnetic beads within the processing tube 700 and / or PCR cartridge 1300. In some embodiments, the magnet station in the homogenization subsystem 1600 may be used to move nucleic acids attached to magnetic beads to the bottom or sides of the processing tube 700, allowing the remaining liquid in the processing tube to be removed and the nucleic acids to be retained.

[0140] 17A, 17B, and 17C show diagrams of an example of a mechanical device 1700 used in the analytical device 200 according to an embodiment of the present disclosure. The mechanical device 1700 represents an embodiment of the example of the mechanical device 248 shown in FIG. 3B. In some embodiments, the mechanical device 1700 can hold two processing tubes 700 in a vertical position and provide a high-speed vibrational motion to the processing tubes 700, for example, to perform lysis of microorganisms in the sample. In some embodiments, the mechanical device 1700 can be an agitator. In additional or alternative embodiments, the mechanical device 1700 can include an ultrasonic generator configured to sonicate the processing tubes 700 to agitate the sample.

[0141] In some embodiments, there may be a plurality of lysis beads disposed within each processing tube 700 to effect lysis. In some embodiments, the plurality of lysis beads may be of a predetermined size and material configured to effect lysis of at least one pathogen, such as yeast, fungus, gram-positive bacteria, gram-negative bacteria, or the like. In some embodiments, the plurality of lysis beads may be a mix of a plurality of predetermined sizes and materials configured to effect lysis of a variety of pathogen types, including yeast, fungus, gram-positive bacteria, gram-negative bacteria, or the like. In some embodiments, a high speed vibration motion applied to the processing tube 700 may excite the plurality of lysis beads within the processing tube 700, which may cause the lysis beads to effect lysis of pathogens in the sample within the processing tube 700. In some embodiments, the rapid movement of the lysis beads (applied by the mechanical device 1700) may mechanically disrupt cell walls of pathogens in the sample, resulting in the release of nucleic acids from the pathogens.

[0142] 18A-C are diagrams illustrating different views of a PCR cartridge 1300 interfacing with a PCR subsystem 1800 in an analyzer according to an embodiment of the present disclosure. The PCR subsystem 1800 represents an exemplary embodiment of the PCR subsystem 244 shown in FIG. 3B. In some embodiments, the PCR subsystem 1800 may be a module configured to perform PCR of nucleic acids in the PCR cartridge 1300, optical detection of fluorescent signals from the PCR amplification, and pathogen identification based on the fluorescent signals. In some embodiments, the PCR subsystem 1800 may include one or more thermal blocks, heat sinks 1802, fans 1804, heated lids 1806, sensor units 1810, and linear motion stages 1814. In some embodiments, the PCR subsystem 1800 may include one or more thermal blocks, thermoelectric cooling (TEC) elements, conductive elements, Peltier elements, and / or the like.

[0143] In some embodiments, the PCR cartridge 1300 may be depressed to ensure contact with a thermal block that is part of a thermal cycler configured to control temperature during nucleic acid amplification. In some embodiments, the thermal block may control temperatures in the range of about 35° C. to about 100° C. with a setting resolution of about 0.1° C. In some embodiments, the thermal block may have a temperature accuracy of about ±0.25° C. about a setting (e.g., steady state). In some embodiments, the thermal block may have temperature ramp rates greater than about 10° C. (in ° C.) per second. In some embodiments, the thermal block temperature profile may be configured or customized by a user of the analytical device 200 to perform thermal cycling.

[0144] In some embodiments, the PCR subsystem 1800 includes a thermoelectric cooling (TEC) element, a heat sink 1802, and one or more fans 1804 disposed below each of the thermal block and the PCR cartridge 1300. In some embodiments, the TEC element, the heat sink 1802, and the fans 1804 may be used to heat and / or cool the PCR cartridge 1300 for maintenance of a particular temperature during the PCR amplification process. In some embodiments, the fans 1804 may be distributed along the length of the heat sink 1802, as shown in FIGS. 18A and 18C. In other embodiments, the fans 1804 may be located at one or more ends of the heat sink 1802 arranged as end caps. In some embodiments, a heated lid 1806 may be disposed over the cover 1302 and the primary and secondary reaction chambers 1313, 1314 of the PCR cartridge 1300. In some embodiments, the heated lid 1806 may be configured to heat the tops of the at least one primary reaction chamber 1313 and the plurality of secondary reaction chambers 1314 to prevent condensation during thermal cycling in the PCR subsystem 1800. In some embodiments, the heated lid 1806 may control a temperature in the range of about 35° C. to about 100° C. with a setting resolution of about 0.1° C. In some embodiments, the heated lid 1806 may have a temperature accuracy of about ±0.25° C. about a setting (e.g., steady state).

[0145] In some embodiments, the sensor unit 1810 (shown in FIGS. 18A and 18B) may include one or more fluorescent sensors configured to perform fluorescent measurements from the bottom of the secondary reaction chamber 1314 in the PCR cartridge 1300 via a thermal block. In some embodiments, the sensor unit 1810 may be configured to detect fluorescent signals from multiple fluorescent dyes, such as FAM™, VIC™, ROX™, SYBR™, Cy5 (cyanine dye), Cy5.5, or analogs thereof. In some embodiments, the sensor unit 1810 may include one or more optical elements based on solid state (e.g., LED / PIN diode). In some embodiments, one or more key parameters of the sensor unit 1810 (e.g., LED power, read time, etc.) may be configurable by software (e.g., installed in the processing unit 116).

[0146] In some embodiments, the linear translation stage 1814 may be a translation stage that can position / scan the sensor unit 1810 under the primary and secondary reaction chambers 1313, 1314 of the PCR cartridge 1300. In some embodiments, the bottom wall of each primary and secondary reaction chamber 1313, 1314 is optically transparent, allowing the sensor unit 1810 to optically interrogate the reaction chambers 1313, 1314 and detect fluorescent signals. In some embodiments, a set of optical fibers may be used to provide excitation to and collect emission from the primary and secondary reaction chambers 1313, 1314 of the PCR cartridge 1300.

[0147] In some embodiments, during thermal cycling, light can be avoided from entering the PCR subsystem 1800. In some embodiments, the PCR cartridge 1300 can be pushed downwards (e.g., placed between a thermal block and under a heated lid 1806) during operation to ensure optimal thermal contact.

[0148] 19A-B are diagrams illustrating an example of a fluorescent sensor subsystem 1900 in an analyzer according to an embodiment of the present disclosure. The fluorescent sensor subsystem 1900 represents an exemplary embodiment of the PCR subsystem 1800 shown in FIG. 18. As shown in FIG. 19A, the fluorescent sensor subsystem 1900 includes a thermal block 1902, a Peltier cell 1912, a heat sink 1916, a fluorescent sensor 1910, and a linear translation stage 1914. In some embodiments, the PCR cartridge 1300 may be pressed against the thermal block 1902 and positioned adjacent to the Peltier cell 1912. In some embodiments, the fluorescent sensor 1910 may be configured to make a fluorescent measurement from the bottom of the secondary reaction chamber 1314 in the PCR cartridge 1300. In some embodiments, at least the thermal block 1902 and the Peltier cell 1912 are surrounded by a housing 1920, as shown in FIG. 19B. The housing 1920 may include one or more identifiers 1922 that contain information about the PCR cartridge (eg, a sample identifier, a date, etc.).

[0149] Example of operation: 20 is a flow chart diagram of a method 2000 of performing sample processing including concentration and lysis of the sample prior to pathogen identification, according to embodiments of the present disclosure. In some embodiments, the method 2000 may describe steps for performing sample processing using various components in a pathogen identification system, including the analyzer 108, 200, the sample preparation cartridge 114, 224, 500, and the processing tube 113, 510, 700, as previously described with reference to FIGS. 1-19. It should be understood that the operations shown in the method 2000 are not exhaustive and that other operations may be performed before, after, or between the operations shown as well. In various embodiments of the present disclosure, the operations of the method 2000 may be performed in different orders and / or may be different.

[0150] The method 2000 of FIG. 20 begins at step 2002, where a sample preparation cartridge and a sample tube are received into the analytical device. In some embodiments, the analytical device 200 may receive the sample preparation cartridge 224 and the sample tube that are placed into the sample cartridge drawer 220 and the sample drawer 210, respectively, of the analytical device 200 by a user or operator of the analytical device 200. In some embodiments, the sample cartridge drawer 220 and the sample drawer 210 may be referred to as the first compartment and the second compartment, respectively, of the analytical device 200. In some embodiments, the sample tube is a sample taken from a patient, the sample including a pathogen. In some embodiments, the sample in the sample tube may include whole blood, urine, sterile bodily fluids, or other samples taken from a patient.

[0151] In step 2004, a first needle from the sample preparation cartridge is placed into the pipettor system of the analytical device. In some embodiments, the needle 512 from the sample preparation cartridge 500 is placed into the first pipettor 202 by moving the pipette tip of the first pipettor 202 up the sample preparation cartridge 500 and pressing against and engaging a plastic body portion of the needle (e.g., plastic body 902).

[0152] In step 2006, a first needle is inserted into a sample tube using a pipettor system. In some embodiments, after attachment of the needle 512, the first pipettor 202 may move above the sample tube in the sample drawer 210 before pushing the needle 512 down to insert it into the sample tube.

[0153] In step 2008, the sample from the sample tube is transferred through the first needle to a processing tube in the sample preparation cartridge. In some embodiments, the needle 512 may draw the sample up into a plastic body reservoir of the first pipettor 202, which moves to the sample preparation cartridge 500 and transfers the sample to a processing tube 510 in the sample preparation cartridge 500.

[0154] To transfer a sample from a plastic body reservoir of the first or second pipettor 202 to a processing tube 510, a needle 512 (coupled to the first pipettor 202) may pierce a septum of the processing tube (e.g., septum 704 of processing tube 700). The first pipettor 202 may then dispense the sample through the needle 512 into the processing tube.

[0155] In step 2010, one or more lysis reagents may be added to the processing tube to lyse blood cells in the sample. In some embodiments, the first pipettor 202 may add one or more lysis reagents, such as a saponin-based lysis buffer, to the processing tube 700 via 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 blood cells in the sample.

[0156] After blood cell lysis, in step 2012, the processing tube is moved to a centrifuge in the analyzer. In some embodiments, the first or second pipettor 202, 204 may be configured to move the processing tube 700 to the first centrifuge 1502 by handling the upper end of the processing tube 700 (e.g., a cylindrical cavity in the cap 702) that fits into the pipettor system. In some embodiments, the pipette tip of the first or second pipettor 202, 204 may press-fit into the cylindrical cavity in the cap 702 to pick up and move the processing tube to the first centrifuge 1502 in the analyzer 200.

[0157] In step 2014, the processing tube is centrifuged in a centrifuge to concentrate one or more pathogens in the sample. In some embodiments, the processing tube 700 is centrifuged in a first centrifuge 1502 to concentrate the pathogens in the processing tube 700 from a large volume (e.g., 10 mL) to a small volume (e.g., 0.5 mL or less). In some embodiments, the volume of the sample in the sample tube ranges from about 0.5 mL to about 10 mL, and the volume of the concentrated pathogens in the processing tube 700 after centrifugation ranges from about 0.1 mL to about 2 mL. In some embodiments, the volume of the concentrated pathogens in the processing tube after centrifugation is in the range of about 0.1 mL to about 1 mL.

[0158] In step 2016, fluid is removed from the processing tube using a pipettor system, leaving the concentrated pathogens in the processing tube. In some embodiments, a needle 512 coupled to the first pipettor 202 can collect and remove fluid from the processing tube 700, leaving the concentrated pathogens at the bottom of the processing tube 700. In step 2018, a lysis buffer can be added to the processing tube using a pipettor system. In some embodiments, a needle 512 coupled to the first pipettor 202 can withdraw one or more lysis buffers from one or more reservoirs 504 in the sample preparation cartridge 500 and dispense the one or more lysis buffers into the processing tube 700 via the needle 512.

[0159] In some embodiments, a needle 512 coupled to the first pipettor 202 can be used to add reagents to the processing tube 700 to perform a DNA depletion step prior to adding the lysis buffer to the processing tube 700. In some embodiments, the reagents used to perform the DNA depletion step can include at least one of DNaseI (deoxyribonuclease I), DNaseII (deoxyribonuclease II), or benzonase.

[0160] In step 2020, a pipettor system can be used to move the process tube 700 to a machine 1700 in the analytical device. In some embodiments, the first or second pipettor 202, 204 can move the process tube 700 to the machine 1700 in the analytical device 200. In some embodiments, the first or second pipettor 202, 204 can be configured to move the process tube 700 to the machine 1700 by handling the top end of the process tube 700 (e.g., a cylindrical cavity in the cap 702) that fits into the pipettor system. In some embodiments, a pipette mandrel of the first or second pipettor 202, 204 can press fit into the cylindrical cavity of the cap 702 to pick up and move the process tube to the machine 1700 in the analytical device 200.

[0161] In step 2022, the processing tube can be agitated using an apparatus to effect cell lysis of the concentrated pathogens. In some embodiments, the mechanical apparatus 1700 can apply a high-speed vibrational motion to the processing tube 700 to effect cell lysis of the concentrated pathogens in the processing tube 700. In some embodiments, the mechanical apparatus 1700 can be an agitator. In some embodiments, the processing tube 700 can be held in a vertical position in the agitator. In some embodiments, applying the high-speed vibrational motion can include applying a reciprocating motion along a predetermined axis by the agitator. In additional or alternative embodiments, agitating the processing tube 700 can include sonicating the processing tube 700 using an ultrasonic generator in the analysis device 200.

[0162] FIG. 21 shows a flow chart diagram of a method 2100 for performing PCR for pathogen identification, according to an embodiment of the present disclosure. In some embodiments, the method 2100 may describe performing PCR for pathogen identification in a PCR cartridge, such as PCR cartridge 234, 1300, as described above with reference to FIGS. 3B-19. It should be understood that the operations shown in the method 2100 are not exhaustive and that other operations may be performed before, after, or between the operations shown as well. In various embodiments of the present disclosure, the operations of the method 2100 may be performed in different orders and / or may be varied.

[0163] The method 2100 of FIG. 21 begins at step 2102, where nucleic acids are transferred by a pipettor system in the analyzer to at least one primary reaction chamber in a PCR cartridge inserted into the analyzer. In some embodiments, the PCR cartridge 234 may be inserted into the analyzer 200 when placed in the processing cartridge drawer 230 by a user or operator of the analyzer 200. In some embodiments, a low-volume needle 910 coupled to a second pipettor 204 may transfer nucleic acids after extraction and purification from the spin column basket 1102 or from one or more reservoirs 504 of the sample preparation cartridge 500. In some embodiments, a low-volume needle 910 coupled to a second pipettor 204 may transfer nucleic acids to at least one primary reaction chamber 1313 in the PCR cartridge 1300. In some embodiments, the first or second pipettor 202 or 204 can add a preliminary set of reagents used for nucleic acid amplification in a reagent tube (e.g., reagent tube 710) to the nucleic acids prior to transferring the nucleic acids to at least one primary reaction chamber 1313 in the PCR cartridge 1300. In some embodiments, the set of reagents used for nucleic acid amplification can be stored in one of the primary reaction chambers 1313 in the PCR cartridge 1300 and dispensed into the second primary reaction chamber 1313 for suspension with the nucleic acids in the sample.

[0164] In step 2104, a first amplification of the nucleic acid is performed in at least one primary reaction chamber to obtain 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 cartridge 1300 using a PCR technique. In some embodiments, a set of primers may be transferred to the primary reaction chamber 1313 together with the nucleic acid for the first amplification. In some embodiments, the set of primers may be adapted for the amplification of a target sequence to be detected. In some embodiments, different sets of primers may be transferred to the primary reaction chamber 1313 together with the nucleic acid for the first amplification. In some embodiments, a combination of different sets of primers may be adapted for the amplification of a target sequence to be detected.

[0165] In step 2106, a needle of an analytical device is inserted through a septum of at least one primary reaction chamber to remove the first amplification product. In some embodiments, a low-volume needle 910 coupled to a second pipettor 204 can be inserted through a septum 1308 located above the primary reaction chamber 1313 to collect the first amplification product from the primary reaction chamber 1313 of the PCR cartridge 1300.

[0166] In step 2108, multiple aliquots of the first amplification product are dispensed into multiple secondary reaction chambers in the PCR cartridge through the septum of each of the multiple secondary reaction chambers in the PCR cartridge. In some embodiments, the multiple aliquots of the first amplification product may be dispensed by inserting a low volume needle 910 coupled to a second pipettor 204 through the septum 1308 into multiple secondary reaction chambers 1314 in the PCR cartridge 1300. In some embodiments, each aliquot may 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 may be diluted with the contents of one of the reservoirs 504 of the sample preparation cartridge 500.

[0167] In some embodiments, each secondary reaction chamber 1314 can include a set of reagents for reacting with a respective aliquot of the first amplification product. In some embodiments, the set of reagents in each secondary reaction chamber 1314 can correspond to an internal sequence of an amplicon in the first amplification product and can be specific to one or more target sequences to be detected. In some embodiments, the set of reagents in each secondary reaction chamber 1314 can include fluorescent probes specific to one or more target sequences to be detected.

[0168] In some embodiments, the first or second pipettor 202 or 204 can add a preliminary set of reagents used for nucleic acid amplification in a reagent tube (e.g., reagent tube 710) to the first amplification product prior to dispensing the multiple aliquots of the first amplification product into the secondary reaction chambers 1314 in the PCR cartridge 1300. In some embodiments, dispensing the multiple aliquots into the multiple secondary reaction chambers 1314 can include using jet dispensing to perform non-contact dispensing of the aliquots without the needle 910 penetrating the septum 1308 of each of the secondary reaction chambers 1314 and the needle 910 contacting the bottom surface of each secondary reaction chamber 1314.

[0169] In step 2110, a second amplification of the aliquots of the first amplification products is carried out in a plurality of secondary reaction chambers. In some embodiments, the second amplification of the aliquots of the first amplification products is carried out in a plurality of secondary reaction chambers 1314 of the PCR cartridge 1300 using PCR techniques.

[0170] 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 the fluorescence data from monitoring the 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 chambers 1314 and identify pathogens present in the patient sample based on the detected amplified nucleic acid.

[0171] Experimental Example: As described below, several experiments were performed to test various embodiments of the pathogen identification systems and methods described above.

[0172] Example 1: Optimization of sample preparation and DNA extraction protocols The experiments presented in this example were performed to improve DNA purification by increasing the volume of buffer extraction and are representative of specific embodiments disclosed in this patent application.

[0173] 10 mL of human whole blood was spiked with Klebsiella pneumoniae at a concentration of 1 CFU / mL and the samples were treated as follows:

[0174] The Klebsiella pneumoniae spiked blood was transferred to a 15 mL conical tube (processing tube) containing 0.7 mL of saponin 2.8% w / v (Sigma-Aldrich, S4521), 0.5 mL of cushioning agent FC-40 (Sigma-Aldrich, F9755), and a zirconia-silica bead mix containing 0.25 mL of 0.1 mm diameter beads, 10 1 mm beads, and 3-4 2.7 mm diameter beads. The blood and reagents were mixed for 30 seconds (5 turnovers) in a rotating orbital motion on a PTR-35 platform at 10 rpm to homogenize and lyse the blood cells. The supernatant was separated from the debris by centrifugation at 12,000 x g for 10 minutes in a fixed angle rotor FA-45-6-30 in a 5810 Eppendorf centrifuge. Holding the tube vertically, the supernatant was carefully removed and discarded, leaving a total volume of 1 mL in the tube containing the bead mix, cushion, and concentrated blood sample.

[0175] To test for improved purification of pathogen DNA, increasing volumes of lysis buffer and binding buffer were tested.

[0176] The lysis buffer was homogenized and different volumes of buffer (0.45 mL and 0.9 mL) were added to the bead-sample mixture. The test conditions were briefly mixed with a vortex mixer for 5 seconds. The sample tubes were then fixed on a custom-made stirring platform (reciprocating motion, 2 degree angle, 16,000 rpm) and switched on for 30 seconds to lyse the samples. Four 30-second pulses with 5-second waits in between were used for a total of 2 minutes of lysis. The tubes were kept vertical during this time. Once mechanical lysis was over, the sample tubes were centrifuged at 3200 x g for 5 minutes in a swing-out rotor A-4-62 to separate beads and cell debris from the lysate. While the tubes were upright, the lysate was transferred and mixed thoroughly with different volumes (1 mL and 2 mL) of binding buffer by pipetting. Two spin columns were used per test condition, each loaded with 0.5 mL of lysate and centrifuged at 10000 x g for 1 min (FA 24 x 2 rotor). This step was repeated until all lysate had passed through the column. The spin columns were washed twice with 0.5 mL of wash buffer, centrifuged at 10000 x g for 1 min, and dried by centrifugation at 13000 x g for 2 min. The spin columns were transferred to 1.5 mL tubes and 60 μL of elution buffer was added. The samples were incubated at room temperature for 5 min to ensure proper contact of the DNA with the elution buffer and optimal DNA recovery, followed by centrifugation at 10000 x g for 1 min. The volumes of elution buffer obtained in the two columns belonging to the same sample were mixed into a single final tube. It should be noted that this process can be considered equivalent to using a single spin column with a binding surface equal to the sum of the binding surfaces of the individual spin columns used in this experiment.

[0177] The results are shown in Figure 23. These results show that increasing the volumes of both the lysis and binding buffers improves the amount of DNA extracted.

[0178] Based on the observed results, in another example, the volumes of lysis buffer and binding buffer were increased 4-fold compared to the standard volumes (1.8 mL and 4 mL, respectively) and the above sample preparation protocol was performed. The results shown in Figure 24 show an increase in the amount of total DNA obtained compared to the protocol using the standard lysis buffer volume. Moreover, detection of pathogens occurred 1 Ct earlier than samples treated with the recommended amount of buffer.

[0179] In this example, each column was composed of three silica membranes, and because two columns provided better extraction yields, the number of layers in the spin column was changed from three to six layers to increase the total DNA obtained. The assay yielded good DNA quality in all conditions tested, and no relevant differences in pathogen detection were observed. However, a slight drop in DNA amount was obtained from samples processed with the six-layer column. In another experiment, two six-layer columns were used, which gave a higher yield compared to the use of one six-layer column, as shown in Figure 25. In conclusion, the more columns and silica layers used, i.e., the more DNA binding surface, the more DNA will be recovered.

[0180] Example 2: Use of a single chip in sample preparation to perform an end-to-end protocol This example illustrates how the operations disclosed in the method of this patent can be performed using a single device for the transfer of fluids along with all operations. In the disclosed method example, the device is a needle. In this test example, this was simulated with a single pipette tip.

[0181] Ten mL of human whole blood spiked with K. pneumoniae at a concentration of 1.3 CFU / mL was added to a sample tube containing 0.7 mL of 2.8% saponin (w / v), 0.5 mL of Fluorinert™ FC-40, and a mixture of zirconia-silica beads consisting of 0.25 mL of 0.1 mm beads, 0.25 mL of 0.5 mm beads, ten 1 mm beads, and two to three 2.7 mm beads. The blood and reagents were mixed for 30 seconds (five turnovers) under rotary orbital motion at a speed of 10 rpm on a PTR-35 platform to homogenize and lyse blood cells. Pathogens were collected by centrifugation at 12000 x g for 10 minutes in a fixed-angle rotor. Holding the tube vertically, the supernatant was carefully removed and discarded, leaving a total volume of 1 mL in the tube containing the bead mix, cushion, and concentrated blood sample.

[0182] All steps of buffer addition and supernatant removal were performed with the same pipette filter tip (Eppendorf Ref.30078624) to simulate in the laboratory the conditions met by the embodiments of the device disclosed herein, i.e. handling with a single needle. Before adding the wash buffer, the pipette tip was rinsed with 2.5 mL or 5 mL of elution buffer for comparison. The addition of 60 μL of elution buffer to each spin column was also performed with the same used and rinsed pipette tip.

[0183] Real-time PCR was performed for comparison of the results. In summary, as shown in Figure 26, no significant differences were observed in PCR detection (Ct values ​​obtained) between samples in which the pipette filter tips were rinsed using 2.5 mL or 5 mL elution buffer volumes. The detection Ct values ​​obtained using only one tip for sample preparation are comparable to those observed in other experiments using multiple filter tips.

[0184] Example 3: Sample preparation and DNA extraction for sensitive detection by PCR In the following examples, a pool of whole blood was spiked with an inoculum of known concentration of microorganism (bacteria or yeast). Depending on the growth characteristics of the tested microorganism, the inoculum was prepared from a microbial suspension from an exponential culture or from a fresh spike on an agar plate.

[0185] 10 mL of spiked whole blood was transferred to a 15 mL conical tube that already contained 0.7 mL of saponin 2.8% w / v (Sigma-Aldrich, S4521), 0.5 mL of cushioning agent FC-40 (Sigma-Aldrich, F9755), 0.25 mL of 0.1 mm beads, 0.25 mL of 0.5 mm beads, 10 1 mm beads, and 3–4 2.7 mm beads. The blood and reagents were mixed for 30 s (5 turnovers) under rotary orbital motion on a PTR-35 platform at a speed of 10 rpm to homogenize and lyse blood cells. Pathogens were collected by centrifugation at 12000 x g for 10 min in a fixed-angle rotor. Holding the tube vertically, the supernatant was carefully removed and discarded, leaving a total volume of 1 mL in the tube containing the bead mix, cushion, and concentrated blood sample.

[0186] Lysis buffer was homogenized and 1.8 mL was added to the bead-sample mixture and mixed on a vortex mixer for 5 seconds. The sample tube was then fixed on a custom-made stirring platform (reciprocating motion, 2 degree angle, 16.000 rpm) and turned on at speed 1 for 30 seconds to perform bead beating to lyse the sample. Four 30-second pulses with 5-second waits in between were performed for a total of 2 minutes of lysis. The tube was kept vertical during this time. Once mechanical lysis was over, the sample tube was centrifuged at 3200 x g for 5 minutes in a swing-out rotor to separate the beads and cell debris from the lysate. With the tube still upright, the lysate was transferred and mixed thoroughly by pipetting with 4 mL of binding buffer. Two spin columns per sample were used, each filled with 0.5 mL of lysate and centrifuged at 10000 x g for 1 minute (FA 24 X 2 rotor). This step was repeated until all the lysate had passed through the columns. The spin column was washed twice with 0.5 mL of wash buffer, centrifuged at 10000 x g for 1 min, and dried by centrifugation at 13000 x g for 2 min. The spin column was transferred to a 1.5 mL tube and 60 μL of elution buffer was added. The sample was incubated at room temperature for 5 min to ensure proper contact of the DNA with the elution buffer and optimal DNA recovery, followed by centrifugation at 10000 x g for 1 min. The volumes of elution buffer obtained in the two columns belonging to the same sample were combined into a single final tube.

[0187] To confirm recovery of specific pathogens after sample preparation, detection was performed in singleplex PCR reactions containing primer pairs and probes specific for the spiked pathogens. Each reaction contained: 50 μL of 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 eluate from the previous step (see explanation above). Cycling conditions were as follows: 95°C for 2 min, followed by 45 cycles of annealing and extension at 95°C for 10 s and 60°C for 30 s, including a pre-read and post-read step at 60°C.

[0188] Following this PCR method, the microorganisms shown in the table below were tested, including gram-positive bacteria, gram-negative bacteria, and yeasts, some of which are considered difficult to lyse. The results are shown in Table 1 below (Table 1). It was observed that pathogens were detected at concentrations of a few CFU / mL, which is within the sensitivity range required for detection of pathogens in bloodstream infections.

[0189] [Table 1]

[0190] Example 4: Detection of Staphylococcus aureus in a sample containing a high white blood cell count (WBC). Human DNA is a strong interfering agent and can inhibit PCR. A normal leukocyte count is 3-11×10E6 leukocytes / mL in blood in healthy adults. In septic patients, the leukocyte count increases dramatically.

[0191] To test the performance of PCR in the presence of elevated white blood cell counts, 10 mL of blood with high white blood cell counts (20-30 x 10E6 white blood cells / mL) was spiked with S. aureus at a concentration of 1.3 CFU / mL (13 CFU / tube). Samples were treated as in the previous examples and detection of pathogens by PCR was performed in singleplex. The results obtained are shown in Figures 27A-B. 4 / 6 replicates of the sample spiked with S. aureus were detected.

[0192] Example 5: Two-step multiplex PCR to detect low levels of pathogens in the presence of high levels In example 5, 100 genome copies of P. aeruginosa (low concentration) were combined with high concentrations of Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae. Detection of P. aeruginosa 100 genome copies / rxn in increasing DNA backgrounds of 10E4, 10E5, and 10E6 genome copies / rxn was evaluated in a two-step PCR format. As primary reaction chambers, 0.2 mL PCR strips were used. A PCR volume of 25 μL was used in each amplification step. Primary PCR was performed with 20 cycles of a PCR mix containing primer pairs for the detection of all aforementioned pathogens.

[0193] A 25-μL PCR reaction mixture was prepared by mixing 12.5 μL of 2x TaqPath Bactopure Microbial detection Master Mix (ThermoFisher Scientific), 0.36 μM of each primer, 5 μL of sample, and water. The following cycling conditions were applied in this first PCR: 95°C for 2 min, followed by 20 cycles of 95°C for 10 s and annealing and extension for 30 s.

[0194] At the end of the primary PCR, the amplified product of the primary PCR reaction was extracted with a pipette and dispensed into the secondary reaction chamber.

[0195] Secondary PCR was performed in 0.2 mL PCR strips. Secondary PCR was performed in a final volume of 25 μL using Prime Time Gene Expression Master Mix (IDT), 0.36 μM of each primer, and 0.25 μM of the probe targeting the oprL gene from P. aeruginosa. Five μL of the primary PCR reaction was used as template for secondary PCR. Cycling conditions were as follows: 95°C for 3 min, followed by 45 cycles of annealing and extension at 95°C for 5 s and 60°C for 30 s.

[0196] FIG. 28 shows detection of P. aeruginosa target by second-stage PCR (Ct value). No amplification of P. aeruginosa is observed in the control containing DNA from the high concentration sample, indicating that the amplification signal observed is pathogen specific in the absence of P. aeruginosa DNA. In FIG. 28, HSS4P100 is the reference name for the condition of 10E4 copies / rxn for H. influenzae, S. pneumoniae, and S. aureus, respectively, and 10E2 copies / rxn for P. aeruginosa, HSS5P100 is the condition of 10E5 copies / rxn for H. influenzae, S. pneumoniae, and S. aureus, respectively, and 10E2 copies / rxn for P. aeruginosa, and HSS6P100 is the condition of 10E6 copies / rxn for H. influenzae, S. pneumoniae, and S. aureus, respectively, and 10E2 copies / rxn for P. aeruginosa. HSS4, HSS5, and HSS6 are controls, referencing Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus at 10E4, 10E5, and 10E6 copies / rxn, respectively. Amplification of Pseudomonas aeruginosa was not expected under these test conditions.

[0197] Example 6: Effect of dilution of PCR products between first-stage and second-stage PCR on performance In Example 6, the effect of diluting the first-stage PCR product prior to the second-stage PCR was evaluated using the same pathogen mix combinations shown above (low concentrations of P. aeruginosa combined with high concentrations of S. aureus, S. pneumoniae, and H. influenzae). Detection of 10 or 100 genome copies of P. aeruginosa in an increasing DNA background of 10E4, 10E5, and 10E6 genome copies of the remaining pathogens described was evaluated in a two-stage PCR. A PCR volume of 25 μL was used for each amplification step. The two-stage PCR consisted of a first PCR containing primer pairs for the detection of all pathogens listed above, run for 20 cycles using 2x TaqPath Bactopure Microbial detection Master Mix (ThermoFisher Scientific). In this first PCR, the following cycling conditions were applied: 95°C for 2 min, followed by 20 cycles of 95°C for 10 s, annealing and extension for 30 s.

[0198] The following dilutions (1 / 5, 1 / 20, 1 / 200) of the PCR product obtained in the first stage PCR were prepared and loaded into the second stage PCR.

[0199] The second stage PCR was performed as a singleplex PCR containing primers and probe for the P. aeruginosa oprL gene detection and 2X Prime Time Gene Expression Master Mix (IDT). Five microliters of the first stage PCR reaction mixture was used as a template for the second stage PCR. The cycling conditions were as follows: 95°C for 3 min, followed by 45 cycles of annealing and extension at 95°C for 5 s and 60°C for 30 s.

[0200] The results are shown in Figure 29. In Figure 29, HSS4P10 is the reference name for the condition of 10E4 copies / rxn for H. influenzae, Streptococcus pneumoniae, and Staphylococcus aureus, respectively, and 10E2 copies / rxn for P. aeruginosa, and HSS5P100 is the condition of 10E5 copies / rxn for H. influenzae, Streptococcus pneumoniae, and Staphylococcus aureus, respectively, and 10E2 copies / rxn for P. aeruginosa. In summary, at the 1 / 20 dilution condition, a slight Ct difference was observed between the non-diluted and diluted conditions. At the 1 / 200 dilution condition, a 4 Ct difference was observed between the non-diluted and diluted conditions.

[0201] Examples of computer systems: FIG. 22 is a block diagram of exemplary components of a computer system 2200. One or more computer systems 2200 may be used to implement, for example, any of the embodiments described herein, and combinations and subcombinations thereof. In some embodiments, one or more computer systems 2200 may be used for fluorescent data or image acquisition, data analysis, and data processing, such as the fluorescent sensor in the PCR subsystem 1800, or the processing device 116, as described herein. In some embodiments, one or more computer systems 2200 may be used in the controller 109 to program and operate the operation of various components in the analysis device 200. The computer system 2200 may include one or more processors (also referred to as central processing units, or CPUs), such as processor 2204. The processor 2204 may be connected to a communication infrastructure or bus 2206.

[0202] The computer system 2200 also includes a user input / output interface 2202 , such as a monitor, keyboard, pointing device, etc., and can communicate with a communications infrastructure 2206 via user input / output devices 2203 .

[0203] One or more of the processors 2204 may be a graphics processing unit (GPU). In one embodiment, a GPU may be a processor that is a specialized electronic circuit designed to handle mathematically intensive applications. GPUs may have a parallel structure that is efficient for parallel processing of large blocks of data, such as mathematically intensive data common in computer graphics applications, images, videos, etc.

[0204] 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 susceptibility testing, and may generate patient treatment recommendations accordingly.

[0205] The computer system 2020 may also include one or more secondary storage devices or memories 2210. The secondary memory 2210 may include, for example, a hard disk drive 2212 and / or a removable storage drive 2214.

[0206] The removable storage drive 2214 may interface with a removable storage unit 2218. The removable storage unit 2218 may include available or readable computer readable storage devices having computer software (control logic) and / or data stored therein. The removable storage device 2218 may be a program cartridge and cartridge interface (such as found in a video game device), a removable memory 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 other removable storage device and associated interface. The removable storage drive 2214 may read from and / or write to the removable storage device 2218.

[0207] Secondary storage 2210 may include other means, devices, components, equipment or other approaches that allow computer programs and / or other instructions and / or data to be accessed by computer system 2200. Such means, devices, components, equipment or other approaches could include, for example, removable storage units 2222 and interfaces 2220. Examples of removable storage units 2222 and interfaces 2220 include program cartridges and cartridge interfaces (such as found in video game devices), removable memory chips (such as EPROMs or PROMs) and associated sockets, memory sticks and USB ports, memory cards and associated memory card slots, and / or other removable storage devices and associated interfaces.

[0208] The computer system 2200 may further include a communication interface 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 number 2228). For example, the communication interface 2224 may enable the computer system 2200 to communicate with external or remote devices 2228 via a communication path 2226, which may be wired and / or wireless (or a combination thereof) and include any combination of a LAN, a WAN, the Internet, etc. Control logic and / or data may be transmitted to or received from the computer system 2200 via the communication path 2226.

[0209] The computer system 2200 may be, but is not limited to, a personal digital assistant (PDA), a desktop workstation, a laptop or notebook computer, a netbook, a tablet, a smartphone, a smart watch, other wearable, a consumer electronics device, part of the Internet of Things (IOT), and / or an embedded system, or any combination thereof.

[0210] Computer system 2200 may be a client or a server and may be a remote or distributed cloud computing solution, a local or on-premise software (an "on-premise" cloud-based solution), an "as a service" model (e.g., Content as a Service (CaaS), Digital Content as a Service (DCaaS), Software as a Service (SaaS), Managed 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 service or delivery paradigms.

[0211] Applicable data structures, file formats, and schemas in the computer system 2200 may use JavaScript Object Notation (JSON), Extensible Markup Language (XML), Yet Another Markup Language (YAML), Extensible Hypertext Markup Language (XHTML), Wireless Markup Language (WML), MessagePack, XML User Interface Language (XUL), or other functionally similar representations, either alone or in combination, or may use proprietary data structures, formats, or schemas exclusively or in combination with known or open standards.

[0212] In some embodiments, tangible, non-transitory devices or articles of manufacture including usable or readable, tangible, non-transitory computer media having control logic (software) stored thereon may also be referred to herein as computer program products or program storage devices. This includes, but is not limited to, computer system 2200, main memory 2208, secondary memory 2210, and removable storage units 2218 and 2222, as well as tangible articles of manufacture embodying any combination of the foregoing. Such control logic, when executed by one or more data processing devices (such as computer system 2200), may cause such data processing devices to operate as described herein.

[0213] Based on the teachings contained herein, it will be apparent to one skilled in the relevant art how to make and use embodiments of the present disclosure using data processing devices, computer systems, and / or computer architectures other than those shown in Figure 22. In particular, embodiments may operate with software, hardware, and / or operating system implementations other than those described herein.

[0214] It is to be understood that the Detailed Description section, and not the Summary and Abstract sections, is used to interpret the claims. The Summary and Abstract sections present one or more exemplary embodiments of the present disclosure as intended by the inventors, but are not exhaustive, and thus are not intended to limit the present disclosure and the appended claims in any manner.

[0215] The embodiments of the present disclosure are described above with the aid of functional building blocks illustrating the implementation of certain functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of description. Other boundaries may be defined as long as the certain functions and relationships thereof are appropriately performed.

[0216] The foregoing description of the specific embodiments sufficiently reveals the general nature of the disclosure, so that those skilled in the art can easily modify and / or adapt the specific embodiments to various applications without undue experimentation and without departing from the general concept of the disclosure. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the terms and expressions in this specification are for the purpose of explanation and not for the purpose of limitation. The terms and expressions in this specification should be interpreted by those skilled in the art in light of the teaching and guidance.

[0217] 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. 1. A sample preparation cartridge comprising: Housing and a removable process tube disposed within the housing, the process tube including a septum and configured to hold the sample; a first removable needle disposed within the housing and configured to transfer the sample to and / or from the removable processing tube upon insertion of the first removable needle through the septum; one or more reservoirs coupled to the housing and configured to store materials used to concentrate, lyse, and amplify the nucleic acid of the sample; Including, Sample preparation cartridge.

2. further comprising a protective lid configured to cover the housing and the one or more reservoirs; The sample preparation cartridge of claim 1 .

3. further comprising one or more spin columns configured to extract and purify nucleic acids from the sample; The sample preparation cartridge of claim 1 .

4. further comprising a basket or receptacle having one or more spin columns disposed therein; the one or more spin columns are configured to move to an elution position to obtain nucleic acids from the sample; The sample preparation cartridge of claim 3 .

5. At least one of the one or more reservoirs is configured to store magnetic beads for extraction and purification of nucleic acids from a sample; The sample preparation cartridge of claim 1 .

6. further comprising a reagent tube configured to store a first set of reagents used to perform nucleic acid amplification; The sample preparation cartridge of claim 1 .

7. The reagent tubes and / or one or more reservoirs are sealed with aluminum foil; The sample preparation cartridge of claim 6.

8. a septum within the removable process tube is disposed at the upper end of the removable process tube; A plurality of lysis beads are placed in a removable processing tube to perform lysis; The sample preparation cartridge of claim 1 .

9. the plurality of lysis beads comprise a plurality of predetermined sizes and materials configured to effect lysis of at least one of yeast, fungi, gram-positive bacteria, or gram-negative bacteria; The sample preparation cartridge of claim 8.

10. the first removable needle is a ventilation needle including a cannula and a plastic body attached to the cannula; The ventilation needle is configured to ventilate the removable treatment tube. The sample preparation cartridge of claim 1 .

11. the plastic body of the ventilation needle includes a filter configured to prevent contamination; The sample preparation cartridge of claim 10.

12. the plastic body of the ventilation needle includes a predetermined number of slots configured to provide an air communication path between the interior and exterior of the removable processing tube when the ventilation needle is inserted through the septum into the removable processing tube; The sample preparation cartridge of claim 10.

13. The removable needle includes a cannula disposed about an inner core. The sample preparation cartridge of claim 10.

14. the cannula of the ventilation needle comprises a slotted cannula; The sample preparation cartridge of claim 10.

15. a cannula of the ventilation needle in fluid communication with the ventilation hole; The sample preparation cartridge of claim 10.

16. further comprising a second removable needle configured to transfer the nucleic acid to a second cartridge for nucleic acid amplification; The sample preparation cartridge of claim 1 .

17. The first removable needle is configured to couple with a high-volume pipettor and the second removable needle is configured to couple with a low-volume pipettor; 17. The sample preparation cartridge of claim 16.

18. High volume pipettors are configured to handle volumes ranging from about 50 microliters to 5 milliliters.

18. The sample preparation cartridge of claim 17.

19. The low volume pipettor is configured to handle volumes ranging from about 1 to 200 microliters.

18. The sample preparation cartridge of claim 17.

20. the removable processing tube includes a handling structure at an upper end of the removable processing tube adapted for handling by a pipettor coupled with the first removable needle; The sample preparation cartridge of claim 1 .

21. the handling structure includes a cap having a cylindrical cavity adapted for insertion of a mandrel of a pipettor coupled to the first removable needle; The cap is placed over the septum at the upper end of the removable processing tube; 21. The sample preparation cartridge of claim 20.

22. 1. A system for analyzing a sample, comprising: a housing configured to receive a sample tube containing a sample including one or more pathogens; a pipettor system disposed within the housing; one or more centrifuges disposed within the housing; a mechanical agitator disposed within the housing; a controller; The controller Transfer the sample from the sample tube to the processing tube using a pipettor system. centrifuging the processing tube using one or more centrifuges to concentrate one or more pathogens in the sample; Use a pipettor system to remove fluid from the processing tube, leaving concentrated pathogens in the processing tube; Add lysis buffer to the processing tube using a pipettor system. Transfer the processing tubes to a mechanical stirrer using a pipettor system, agitating the processing tube using a mechanical agitator to effect cell lysis of the concentrated pathogen; system.

23. The controller After cell lysis, a portion of the liquid from the processing tube is transferred to the spin column using a pipettor system. further configured to extract and purify nucleic acids from the portion of the liquid using a spin column; 23. The system of claim 22.

24. further comprising one or more reservoirs configured to store magnetic beads for nucleic acid extraction and purification; The controller transferring a portion of the liquid from the processing tube after cell lysis into one or more reservoirs using a pipettor system; further configured to extract and purify nucleic acids from the portion of the liquid using magnetic beads in the one or more reservoirs; 23. The system of claim 22.

25. the pipettor system is configured to move the sample tube into the housing by handling an upper end of the sample tube that fits into the pipettor system; 23. The system of claim 22.

26. The pipetter system a first pipettor configured to handle volumes ranging from about 50 microliters to 5 milliliters; 23. The system of claim 22.

27. The pipetter system a second pipettor configured to handle volumes in the range of about 1 to 200 microliters; 27. The system of claim 26.

28. the sample tube and the processing tube each include a septum that allows insertion by a first needle connected to a first pipettor; 27. The system of claim 26.

29. The step of transferring the sample from the sample tube to the processing tube includes inserting a first needle into a septum of the processing tube and dispensing the sample through the first needle; 29. The system of claim 28.

30. a first needle attached to the plastic body and including a ventilation needle configured to ventilate the processing tube; 30. The system of claim 29.

31. the plastic body includes a filter configured to prevent contamination of the sample; 31. The system of claim 30.

32. the plastic body includes a predetermined number of slots configured to provide an air communication path between the interior and exterior of the processing tube when the ventilation needle is inserted into the processing tube through the septum; 31. The system of claim 30.

33. a first subsystem disposed within the housing and configured to perform a polymerase chain reaction (PCR) of the sample using nucleic acids extracted from the sample; 23. The system of claim 22.

34. the first subsystem including a thermal cycler configured to control one or more temperatures during PCR of samples in a plurality of reaction chambers in the PCR cartridge; 34. The system of claim 33.

35. a second subsystem disposed within the housing and configured to perform antimicrobial susceptibility testing (AST) of the sample; 34. The system of claim 33.

36. the second subsystem includes a microscope configured to acquire one or more images of the concentrated pathogen after concentration and transfer of the concentrated pathogen to the plurality of reaction wells in the AST cartridge; 36. The system of claim 35.

37. receiving, by the analytical device, a sample preparation cartridge and a sample tube, the sample tube containing a sample including one or more pathogens; attaching a first needle from the sample preparation cartridge to a pipettor system in the analytical device; inserting a first needle into a sample tube using a pipettor system; transferring the sample from the sample tube through a first needle to a processing tube within the sample preparation cartridge; adding one or more lysis reagents to the processing tube using a pipettor system; mixing one or more lysis reagents in a processing tube with the sample to lyse blood cells in the sample; transferring the processing tube to a centrifuge within the analyzer; centrifuging the processing tube in a centrifuge to concentrate one or more pathogens in the sample; removing the liquid from the processing tube using a pipettor system, leaving the concentrated pathogens in the processing tube; adding lysis buffer to the processing tube using a pipettor system; transferring the processing tube to a device within the analytical device using a pipettor system; agitating the processing tube using the device to effect lysis of the concentrated pathogens. method.

38. After lysis of concentrated pathogens, transferring a portion of the liquid from the processing tube to a spin column of the analytical device using a pipettor system; extracting and purifying nucleic acids from a portion of the liquid using a spin column; further comprising:

38. The method of claim 37.

39. After lysis of concentrated pathogens, transferring a portion of the liquid from the processing tube into one or more reservoirs of the sample preparation cartridge using a pipettor system; extracting and purifying nucleic acids from a portion of the liquid using magnetic beads stored in one or more reservoirs; further comprising:

38. The method of claim 37.

40. The step of transferring the sample from the sample tube through the first needle to the processing tube includes inserting the first needle into a septum of the processing tube and dispensing the sample through the first needle; 38. The method of claim 37.

41. The step of agitating the processing tube includes applying a high speed vibrational motion to the processing tube using a device within the analytical device, the device including an agitator.

38. The method of claim 37.

42. The processing tube is in a vertical position applying high-speed vibrational motion includes applying a reciprocating motion along a predetermined axis by the agitator; 42. The method of claim 41.

43. the step of agitating the process tube includes sonicating the process tube using a device within the analytical device, the device including an ultrasonic generator; 38. The method of claim 37.

44. the step of transferring the processing tube to the first centrifuge includes using a pipettor system to transfer the processing tube by manipulating an upper end of the processing tube that fits into the pipettor system; 38. The method of claim 37.

45. The step of transferring the processing tube to the device includes using a pipettor system to transfer the processing tube by manipulating an upper end of the processing tube that fits into the pipettor system; 38. The method of claim 37.

46. the volume of the sample in the sample tube ranges from about 0.5 mL to about 10 mL, and the volume of the concentrated pathogen in the processing tube after centrifugation ranges from about 0.1 mL to about 2 mL; 38. The method of claim 37.

47. the volume of concentrated pathogens in the processing tube after centrifugation ranges from about 0.1 mL to about 1 mL; 38. The method of claim 37.

48. The step of receiving the sample preparation cartridge and the sample tube comprises: receiving a sample preparation cartridge in a first compartment of an analytical device; receiving the sample tube in a second compartment of the analytical device; 38. The method of claim 37.

49. further comprising the step of adding reagents to the processing tube using a pipettor system to perform a DNA depletion step before adding the lysis buffer to the processing tube; 38. The method of claim 37.

50. the one or more lysis reagents include one or more saponin-based buffers; 38. The method of claim 37.

51. extracting and purifying nucleic acids from a portion of the liquid from the processing tube after lysis of the concentrated pathogens; transferring nucleic acids to at least one primary reaction chamber within a polymerase chain reaction (PCR) cartridge inserted into an analytical device; conducting a first amplification of nucleic acids in at least one primary reaction chamber to obtain a first amplification product; inserting a needle of the analytical device through a septum of at least one primary reaction chamber to remove a first amplification product; Dispensing a plurality of aliquots of the first amplification product into a plurality of secondary reaction chambers in the PCR cartridge through respective septa of the plurality of secondary reaction chambers, each aliquot corresponding to a respective secondary reaction chamber, each secondary reaction chamber including a set of reagents for reacting with a respective aliquot of the first amplification product; and subjecting each aliquot of the first amplification product to a second amplification in a plurality of secondary reaction chambers.

38. The method of claim 37.

52. transferring nucleic acids to at least one primary reaction chamber within a polymerase chain reaction (PCR) cartridge inserted into the analytical device using a pipettor system of the analytical device; conducting a first amplification of nucleic acids in at least one primary reaction chamber to obtain a first amplification product; inserting a needle of the analytical device through a septum of at least one primary reaction chamber to remove a first amplification product; Dispensing a plurality of aliquots of the first amplification product into a plurality of secondary reaction chambers in the PCR cartridge through respective septa of the plurality of secondary reaction chambers, each aliquot corresponding to a respective secondary reaction chamber, each secondary reaction chamber including a set of reagents for reacting with a respective aliquot of the first amplification product; and subjecting each aliquot of the first amplification product to a second amplification in a plurality of secondary reaction chambers. method.

53. The method further includes a step of mixing the nucleic acid with a spare set of reagents used for nucleic acid amplification in a reagent tube by a pipettor system before transferring the nucleic acid to at least one primary reaction chamber of the PCR cartridge.

53. The method of claim 52.

54. mixing, by a pipettor system, the first amplification products with a spare set of reagents used for nucleic acid amplification in a reagent tube before dispensing the plurality of aliquots of the first amplification products into secondary reaction chambers in the PCR cartridge; 53. The method of claim 52.

55. The reagent set in each secondary reaction chamber is dried or lyophilized at the bottom of each secondary reaction chamber; 53. The method of claim 52.

56. detecting fluorescence at the bottom of each secondary reaction chamber to monitor the second amplification; 53. The method of claim 52.

57. the step of dispensing the plurality of aliquots into the plurality of secondary reaction chambers includes using jet dispensing to perform non-contact dispensing of the aliquots with a needle piercing a septum of each of the secondary reaction chambers without the needle contacting a bottom surface of each secondary reaction chamber; 53. The method of claim 52.

58. The first and second amplifications include using PCR techniques; 53. The method of claim 52.

59. further comprising the step of transferring the set of primers together with the nucleic acid to at least one primary reaction chamber of the PCR cartridge; The primer set is compatible with the amplification of the target sequence to be detected.

59. The method of claim 58.

60. further comprising the step of transferring a different set of primers together with the nucleic acid to at least one primary reaction chamber of the PCR cartridge; The combination of different sets of primers is compatible with the amplification of the target sequence to be detected.

59. The method of claim 58.

61. a set of reagents in each secondary reaction chamber corresponding to an internal sequence of the amplification product in the first amplification product and specific to one or more target sequences to be detected; 59. The method of claim 58.

62. The reagent set in each secondary reaction chamber includes a fluorescent probe specific to one or more target sequences to be detected.

59. The method of claim 58.

63. 1. A polymerase chain reaction (PCR) cartridge comprising: at least one primary reaction chamber configured to perform a first amplification of nucleic acids to obtain a first amplification product; a plurality of secondary reaction chambers configured to perform a second amplification of the product nucleic acid; the at least one primary reaction chamber and the plurality of secondary reaction chambers are each sealed with a septum; the septum is configured to receive a needle; each secondary reaction chamber of the plurality of secondary reaction chambers configured to receive a respective aliquot of the first amplification product from the needle; each secondary reaction chamber containing a set of reagents for reacting with a respective aliquot of the first amplification product; PCR cartridge.

64. The volume of the at least one primary reaction chamber ranges from about 50 to about 500 μL; 64. The PCR cartridge of claim 63.

65. The at least one primary reaction chamber and the plurality of secondary reaction chambers include: configured to receive a heated lid disposed over the at least one primary reaction chamber and the plurality of secondary reaction chambers; a heated lid for heating the top of the at least one primary reaction chamber and the plurality of secondary reaction chambers to prevent condensation / condensation; 64. The PCR cartridge of claim 63.

66. the diameter of the bottom wall of each of the at least one primary reaction chamber and the plurality of secondary reaction chambers is less than about 2.5 mm; 64. The PCR cartridge of claim 63.

67. further comprising a reagent tube or reservoir configured to hold an additional set of reagents used in nucleic acid amplification; The additional reagent set is dried or lyophilized.

64. The PCR cartridge of claim 63.

68. the at least one primary reaction chamber and the plurality of secondary reaction chambers include a transparent bottom configured to allow optical interrogation of the at least one primary and secondary reaction chamber by fluorescence; 64. The PCR cartridge of claim 63.

69. the at least one primary reaction chamber and the plurality of secondary reaction chambers comprise mineral oil to prevent at least one of evaporation, aerosol formation, and cross-contamination; 64. The PCR cartridge of claim 63.

70. each secondary reaction chamber is configured to receive mineral oil prior to receiving a respective aliquot of the first amplification product; 70. The PCR cartridge of claim 69.

71. The mineral oil in each secondary reaction chamber is stored separately from the reagent set in each secondary reaction chamber; 70. The PCR cartridge of claim 69.

72. The proximal end of the plastic body is configured to couple with a pipettor. The sample preparation cartridge of claim 10.

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