UNIVERSAL ASSAY CARTRIDGE AND METHODS OF USE

Abstract

Sample cartridge, valve assembly and processing methods for providing mechanical lysis, chemical lysis or both for a given fluid sample are provided herein. Such systems can include a sample processing cartridge having a valve assembly configured for transport of the processing of fluid sample within the sample cartridge. The valve assembly can include a valve body and cap that secure a filter therebetween and facilitate inflow of mechanical or chemical lysing agents as needed for a fluid sample. Assay workflows for performing both mechanical and chemical lysis of a fluid sample within the same workflow of a single universal sample cartridge are also provided.

Claims

1. A sample cartridge for processing and/or analytical testing of a biological fluid sample, the sample cartridge comprising: a cartridge body having a plurality of chambers therein, wherein at least one chamber is configured to receive a fluid sample; a lysing chamber having an fluid inlet and a fluid outlet; and a filter disposed within the lysing chamber, wherein the filter is formed of glass fibers to facilitate affinity binding with free nucleic acids, wherein the filter has a pore size selected to accommodate correspondingly sized glass beads to facilitate mechanical lysing.

2. The sample cartridge of claim 1, wherein the cartridge includes a plurality of glass beads in a chamber of the plurality, the glass beads correspondingly sized to the pore size of the filter.

3. The sample cartridge of claim 1, wherein the pore size is between 0.2 um and 2 um.

4. The sample cartridge of claim 1, wherein the pore size is about 0.7 um.

5. The sample cartridge of claim 1, wherein the lysing chamber comprises: a valve body; and a valve cap, wherein the valve body interfaces with the valve cap to define the lysing chamber therebetween, wherein the filter is held within the lysing chamber secured between the valve body and the valve cap.

6. The sample cartridge of claim 5, further comprising: a syringe that is movable to facilitate fluid flow into and from the lysing chamber by fluctuation of pressure.

7. The sample cartridge of claim 5, further comprising: wherein the valve body comprises the outlet from the lysing chamber, and the valve cap comprises the inlet into the lysing chamber.

8. The sample cartridge of claim 7, wherein the cap is configured such that a gap between the cap and the filter is 0.003″ or greater.

9. The sample cartridge of claim 7, wherein the cap and the valve body are configured so that a fluid-flow path therebetween is without any 90 degree corners so as to inhibit any buffer carryover along the flowpath.

10. The sample cartridge of claim 7, wherein the cap and/or or the valve body include one or more support protrusions extending into the sample processing region adjacent the inlet and/or the outlet so as to facilitate consistency and uniform flow of fluid sample through the sample processing region for chemical lysing and/or infill of lysing agents across the filter to facilitate mechanical lysing of the system.

11. The sample cartridge of claim 7, wherein the one or more protrusions comprise oval-shaped posts.

12. The sample cartridge of claim 7, wherein the valve cap comprises at least a pair of protrusions or posts extending into the lysing chamber adjacent the inlet to press the filter away from the inlet to facilitate improved infill of glass beads into the lysing chamber.

13. The sample cartridge of claim 12, wherein the valve body comprises at least a pair of protrusions or posts extending into the lysing chamber adjacent the outlet to press the filter away from the outlet to facilitate improved fluid flow and reduce stress on the filter.

14. The sample cartridge of claim 7, wherein the valve body comprises at least a pair of protrusions or posts extending into the lysing chamber adjacent the outlet to press the filter away from the outlet to facilitate improved fluid flow and reduce stress on the filter.

15. A sample cartridge for processing and/or analytical testing of a biological fluid sample, the sample cartridge comprising: a cartridge body having a plurality of chambers therein, wherein at least one chamber is configured to receive a fluid sample; a syringe extending through a central region of the cartridge body to facilitate fluid flow along a flow path through the cartridge by movement of the syringe; a valve body that interfaces with the syringe; a valve cap that interfaces with the valve body to form a sample processing region; and a filter secured between the valve body and valve cap to facilitate collection of DNA from the fluid sample within the sample processing region; wherein the sample cartridge is configured for both mechanical lysing and chemical lysing of targets from the fluid sample.

16. The sample cartridge of claim 15, wherein the valve body and cap together define a lysing chamber having a fluid flow path between an inlet in the cap and an outlet in the valve body that is fluidically coupled to a fluid displacement region of the valve body, wherein the fluid displacement region is depressurizable by movement of the syringe to draw fluid into the fluid displacement region and pressurizable by movement of the syringe to expel fluid from the fluid displacement region.

17. The sample cartridge of claim 15, wherein the cap and/or the valve body include one or more support protrusions extending into the sample processing region along the inlet and/or the outlet so as to facilitate consistent and uniform flow of fluid sample through the sample processing region to facilitate chemical lysing and/or infill of lysing agents across the filter to facilitate mechanical lysing of the system.

18. The sample cartridge of claim 15, wherein the cap includes one or more support protrusions that engage the filter, thereby forming a consistent gap between the cap and the filter.

19. The sample cartridge of claim 18, wherein the cap and the one or more protrusions are configured such that the gap between the cap and the filter is 0.003″ or greater.

20. The sample cartridge of claim 18, wherein the cap is generally circular in shape and the sample processing region is generally circular in shape.

21. The sample cartridge of claim 18, wherein the one or more protrusions comprise are disposed adjacent the inlet of the cap.

22. The sample cartridge of claim 21, wherein the one or more protrusions comprise a pair of posts.

23. The sample cartridge of claim 22, wherein the posts are oval shaped with a major axis extending in a direction of flow.

24. The sample cartridge of claim 15, wherein the valve body includes one or more support protrusions to space the filter away from the outlet.

25. The sample cartridge of claim 24 wherein the valve body includes a series of ridges extending along a direction of flow between the inlet and outlet of the sample processing region.

26. The sample cartridge of claim 24, wherein the valve body is generally circular in shape and the sample processing region is generally circular in shape.

27. The sample cartridge of claim 24, wherein the one or more protrusions are disposed adjacent the outlet in the valve body.

28. The sample cartridge of claim 24, wherein the one or more protrusions comprise a pair of posts between the series of ridges and the outlet with respect to the flow path.

29. The sample cartridge of claim 15, wherein the pair of posts straddle the outlet with respect to the flow path.

30. The sample cartridge of claim 15, wherein the posts are oval shaped having a major axis extending in a direction of flow.

31. The sample cartridge of claim 15, wherein the valve body includes a plurality of ports, wherein at least one of the plurality of ports is fluidically coupled to the fluid displacement region.

32. The sample cartridge of claim 31, wherein the valve body is adjustable with respect to the housing to allow the plurality of ports to be placed selectively in fluid communication with the plurality of chamber.

33. The sample cartridge of claim 15, wherein at least one chamber of the plurality includes chemical lysing agents for lysing DNA from viral targets and at least one other chamber having lysing agents for lysing DNA from biological targets.

34. The sample cartridge of claim 33, wherein at least one other chamber of the plurality includes mechanical lysing agents for lysing targets from the fluid sample.

35. The sample cartridge of claim 33, wherein the mechanical lysing agents comprise glass beads.

36. The sample cartridge of claim 15, wherein the filter comprises glass fibers having a nominal pore size.

37. The sample cartridge of claim 36, wherein the nominal pore size of between 0.5-1 um.

38. The sample cartridge of claim 36, wherein the filter has a nominal pore size of about 0.7 um.

39. The sample cartridge of claim 35, wherein at least one other chamber of the plurality of glass beads for mechanical lysing, wherein the plurality of glass beads have a nominal diameter that corresponds to the nominal pore size of the filter.

40. A method of processing a fluid sample utilizing a sample cartridge, the sample cartridge comprising cartridge body having a plurality of chambers, a syringe configured to facilitate movement of a fluid sample along a flow path of the cartridge, and a rotatable valve assembly having a sample processing region within, wherein the method comprises: controlling flow of the fluid sample disposed in a chamber of the plurality of chamber into one or more other chambers of the plurality by coordinated movement of the syringe and rotation of the valve body thereby: exposing the fluid sample to treatment and/or chemical lysing agents disposed within another chamber of the cartridge body to release nucleic acids from the fluid sample; directing the treated and/or chemically lysed fluid sample into the sample processing region of the valve assembly and binding nucleic acids from chemically lysed target with a filter of the valve assembly; mechanically lysing the targets captured in the filter to release additional nucleic acids from the fluid sample; elute the nucleic acids to remove the nucleic acids from the filter; and performing PCR on the eluted nucleic acids.

41.-52. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 is an overview of a sample cartridge with a valve assembly having a valve, filter and valve cap, configured for performing differing sample processes, including both chemical and mechanical lysing of targets, in accordance with some embodiments of the invention.

[0030] FIG. 2 illustrates ultrasonic mechanical lysing techniques as performed by one type of conventional sample cartridge and chemical lysing techniques, as performed by conventional sample cartridges, both techniques can be performed by a universal sample cartridge in accordance with some embodiments.

[0031] FIG. 3 illustrates a comparison of valve assemblies of conventional sample cartridges A, B, C, each suited for a single type of target lysing as compared to a universal valve assembly for a universal sample cartridge capable of processing multiple differing types of assays requiring different types of lysing.

[0032] FIG. 4 illustrates a universal valve assembly, in accordance with some embodiments, that utilizes glass beads suited for mechanical lyses of certain types of targets, as compared to conventional valve assemblies in conventional sample cartridges.

[0033] FIGS. 5A-5C illustrates the components of a valve assembly, in accordance with some embodiments.

[0034] FIGS. 6A-6B compare a valve cap in accordance with some embodiments, as compared with a conventional design.

[0035] FIGS. 7A-7B compare a valve body in accordance with some embodiments, as compared with a conventional design.

[0036] FIGS. 8A-8B illustrate detail views of the filter pocket, as compared with a conventional design.

[0037] FIGS. 9A-9E illustrate various views of a valve body of a conventional sample cartridge utilizing mechanical lysing.

[0038] FIGS. 10A-10C illustrate various views of a valve cap of a conventional sample cartridge utilizing mechanical lysing.

[0039] FIGS. 11A-11B illustrate various views of a valve body of a conventional sample cartridge utilizing chemical lysing.

[0040] FIGS. 11C-11D illustrate various views of a valve cap of a conventional sample cartridge utilizing chemical lysing.

[0041] FIGS. 12A-12D illustrate various views of a valve cap of a universal sample cartridge, in accordance with some embodiments.

[0042] FIGS. 13A-13D illustrate various views of a valve body of a universal sample cartridge, in accordance with some embodiments.

[0043] FIG. 14A illustrate cross-sectional views of a valve assembly in accordance with some embodiments.

[0044] FIG. 14B illustrate cross-sectional views of a valve assembly in accordance with some embodiments.

[0045] FIG. 14C illustrates a cross-sectional view of a valve assembly of a universal sample cartridge before addition of support posts.

[0046] FIG. 14D illustrates a cross-sectional view of a valve assembly of a universal sample cartridge after addition of support posts.

[0047] FIG. 15A-15K illustrate various models of washflow analysis for a valve assembly of a universal sample cartridge, in accordance with some embodiments.

[0048] FIG. 16 illustrates stress distribution of a valve assembly of a universal sample cartridge, in accordance with some embodiments, as compared to a conventional valve design.

[0049] FIG. 17 illustrates filter displacement of a valve assembly of a universal sample cartridge, in accordance with some embodiments, as compared to a conventional valve design.

[0050] FIG. 18 illustrates washout of residual buffer of a valve assembly of a universal sample cartridge, in accordance with some embodiments, as compared to a conventional valve design.

[0051] FIG. 19 illustrates buffer carryover of a valve assembly of a universal sample cartridge, in accordance with some embodiments, as compared to a conventional valve design.

[0052] FIGS. 20A-20G show experimental results showing fill and performance data of a valve assembly of universal assay sample cartridge, in accordance with some embodiments, before and after addition of support posts.

[0053] FIG. 21 depicts various assay workflows, in accordance with some embodiments.

[0054] FIGS. 22A-22D show experimental results illustrating substantially reduced sample processing times alowed by the universal cartridge design, in accordance with some embodiments.

[0055] FIGS. 23A-23D show experimental results illustrating higher sampling volumes allowed by the universal cartridge design, in accordance with some embodiments.

DETAILED DESCRIPTION OF THE INVENTION

[0056] The present invention relates generally to a system, device and methods for fluid sample manipulation and analysis, in particular, sample cartridges that facilitate processing and analytical testing of biological samples.

I. System Overview

[0057] In one aspect, the invention pertains to a sample cartridge that utilizes a valve body platform that allows for detection of enveloped and free nucleic acid targets. In some embodiments, the valve body includes a sample processing region or lysing chamber that provides for either or both mechanical and chemical lysis. This allows a single cartridge to provide lysing for a multitude of differing types of target, thus, can be considered a “universal assay cartridge.” In some embodiments, the sample cartridge can perform processing and detection of both bacterial targets requiring mechanical lysing and viral targets suited for chemical lysing.

[0058] The sample cartridge device can be any device configured to perform one or more process steps relating to preparation and/or analysis of a biological fluid sample according to any of the methods described herein. In some embodiments, the sample cartridge device is configured to perform at least sample preparation. The sample cartridge can further be configured to perform additional processes, such as detection of a target nucleic acid in a nucleic acid amplification test (NAAT), e.g., Polymerase Chain Reaction (PCR) assay, by use of a reaction tube attached to the sample cartridge. In some embodiments, the reaction tube extends from the body of the cartridge. Preparation of a fluid sample generally involves a series of processing steps, which can include chemical, electrical, mechanical, thermal, optical or acoustical processing steps according to a specific protocol. Such steps can be used to perform various sample preparation functions, such as cell capture, cell lysis, binding of analyte, and binding of unwanted material.

[0059] A sample cartridge suitable for use with the invention, includes one or more transfer ports through which the prepared fluid sample can be transported into an attached reaction vessel for analysis. FIG. 1 illustrates an exemplary universal sample cartridge 100 suitable for sample preparation and analytics testing by PCR when received in an instrument module in accordance with some embodiments. The sample cartridge is attached with a reaction vessel 1 (“PCR tube”) adapted for analysis of a fluid sample processed within the sample cartridge 100. In some embodiments the reaction tube extends form the cartridge body. Such a sample cartridge 100 includes various components including a main housing 10 having one or more chambers for processing of the fluid sample, which typically include sample preparation before analysis. The instrument module facilitates the processing steps needed to perform sample preparation and the prepared sample is transported through one of a pair of transfer ports into fluid conduit of the reaction vessel 1 attached to the housing of the sample cartridge 100. The prepared biological fluid sample is then transported into a chamber of the reaction tube 1 where the biological fluid sample 4 undergoes nucleic acid amplification. In some embodiments, the amplification is a polymerase chain reaction. In some embodiments, concurrent with the amplification of the biological fluid sample, an excitation means and an optical detection means of the module is used to detect optical emissions that indicate the presence or absence of a target nucleic acid analyte of interest, e.g., a bacteria, a virus, a pathogen, a toxin, or other target analyte. It is appreciated that such a reaction vessel could include various differing chambers, conduits, or micro-well arrays for use in detecting the target analyte. In some embodiments, the reaction tube can comprise multiple separate reaction chambers isolated from each other for the detection of different analytes. In some embodiments, the reaction tube can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more separate isolated reaction chambers. The sample cartridge can be provided with means to perform preparation of the biological fluid sample before transport into the reaction vessel. Any chemical reagent required for viral or cell lysis, or means for binding or detecting an analyte of interest (e.g. reagent beads) can be contained within one or more chambers of the sample cartridge, and as such can be used for sample preparation.

[0060] An exemplary use of a reaction vessel for analyzing a biological fluid sample is described in commonly assigned U.S. patent application Ser. No. 6,818,185, entitled “Cartridge for Conducting a Chemical Reaction,” filed May 30, 2000, the entire contents of which are incorporated herein by reference for all purposes. Examples of the sample cartridge and associated module are shown and described in U.S. Pat. No. 6,374,684, entitled “Fluid Control and Processing System” filed Aug. 25, 2000, and U.S. Pat. No, 8,048,386, entitled “Fluid Processing and Control,” filed Feb. 25, 2002, incorporated herein by reference in their entirety for all purposes.

[0061] Various aspects of the sample cartridge 100 can be further understood by referring to U.S. Pat. No. 6,374,684, which described certain aspects of a sample cartridge in greater detail. Such sample cartridges can include a fluid control mechanism, such as a rotary fluid control valve, that is connected to the chambers of the sample cartridge. Rotation of the rotary fluid control valve permits fluidic communication between chambers and the valve so as to control flow of a biological fluid sample deposited in the cartridge into different chambers in which various reagents can be provided according to a particular protocol as needed to prepare the biological fluid sample for analysis. To operate the rotary valve, the cartridge processing module comprises a motor such as a stepper motor that is typically coupled to a drive train that engages with a feature of the valve in the sample cartridge to control movement of the valve in coordination with movement of the syringe, thereby resulting movement of the fluid sample according to the desired sample preparation protocol. The fluid metering and distribution function of the rotary valve according to a particular sample preparation protocol is demonstrated in U.S. Pat. No. 6,374,684.

II. Example Universal Assay Cartridge and Valve Assemblies

A. Overview

[0062] FIG. 1 illustrates a universal sample cartridge device 100 fluidically coupled with a reaction vessel 1. The fluid sample cartridge 100 is adapted for insertion into a bay of a module having an instrument interface configured to perform one or more processing steps on a fluid sample contained within the fluid sample cartridge through manipulation of the fluid sample cartridge. The processing steps can include any steps associated with sample preparation, transport of fluid sample, and analytical testing. An instrument interface of the module is incorporated into the module within the bay in which cartridge 100 is received. At right, an exploded view of the sample cartridge assembly is shown, which includes the cartridge body 10, gasket 20, syringe 12, valve body 30, filter 40, valve cap 50 and cartridge base 60. While the basic function of these components may be similar to those of conventional sample cartridges, the valve assembly has been modified to considerably improve performance and to perform multiple types of sample preparation (e.g. mechanical and chemical lysing) with the same sample that would otherwise not be possible with conventional cartridges.

B. Examples Assays for Universal Assay Cartridges

[0063] In one aspect, the universal sample cartridge described herein can perform sample preparation and analytical testing for assays that are currently performed by conventional sample cartridges. For example, as shown in FIG. 2, one conventional sample cartridge (Cartridge A) is configured to perform analytical detections for targets including bacteria, spores, and hardy cells, which require mechanical lysis (e.g. ultrasonics, sonication) so that the released nucleic acids can be physically captured and detected. Another conventional sample cartridge (Cartridge C) is configured to perform analytical detections for target including virus, free DNA, and fragile cells, which can be easily lysed with chemical reagents to release the nucleic acids. Typically, these cartridges utilize nucleic acid binding. Currently, the capabilities of each sample configuration are such that each can only perform sample preparation for those designated targets. Advantageously, the universal sample cartridge described herein can perform both of these sample preparation tasks, individually, sequentially or in combination, such that the sample cartridge can replace the conventional cartridges as well as perform complicated assay panels that would otherwise not be achievable with a single sample cartridge.

[0064] For example, the universal sample cartridge can be used for the simultaneous detection of the major viral, parasitic and bacterial causes of undifferentiated febrile illness (UFI) in a Tropical Fever Assay panel, all of which can be performed by a sample cartridge utilizing the improved valve assembly described herein. Lysis requirements of possible target organisms responsible for UFI include both viral targets that require chemical lysis, parasitic and bacterial targets may require mechanical lysis. An example of such a test is shown in the table below.

TABLE-US-00001 TABLE 1 Tropical Fever Test Target Organism Target Type Lysis Zika virus Viral (RNA) Chemical Dengue virus (serotypes 1-4, Viral (RNA) Chemical undifferentiated) Chikungunya virus Viral (RNA) Chemical P. vivax, P. malariae, P. knowlesi Parasitic Mechanical/Chemical* and P. ovale (undifferentiated) P. falciparum Parasitic Mechanical/Chemical* Leptospira Bacterial Mechanical/Chemical* Salmonella Typhi Bacterial Mechanical/Chemical* *Dependency on mechanical lysis for optimal detection to be evaluated during Xpert Tropical Fever Test Technical Feasibility Feasibility

[0065] Additional multi-target assay panels that can be developed for use with the universal sample cartridge may include a Gastrointestinal (GI) Panel, Breast Cancer Panel, and Bacterial Agents or any mixed-target panel. As shown, the differing targets within a single panel can include any of viral targets, fungal targets, parasitic targets, and bacterial targets, or any combination thereof.

C. Example Valve Assemblies

[0066] FIG. 3 illustrates a valve assembly of a universal sample cartridge, in accordance with aspects of the invention, as compared to valve assemblies in conventional cartridge (A, B,C). Cartridge A performs only mechanical lysing for more hardy targets, and Cartridges B and C perform only chemical lysing for viruses, free NA or more fragile targets. In all such cartridges, the valve assembly includes the syringe tube 12, valve body 30, and valve cap 50. In one aspect, the additional capabilities of the valve assembly of the universal sample cartridge rely in part on the filter, features of the valve body and cap, as well as the particular workflow sequence performed by the instrument interface of the module.

[0067] In some embodiments, the filter is configured to accommodate glass beads to further facilitate mechanical lysis of hardy targets, as shown in FIG. 4. In this embodiment, the filter 40 is formed of glass fibers and has a 0.7 um pore size. In contrast, Cartridge A utilizes a filter formed as a disk of a polymer film (i.e., PCTE), which while suitable for mechanical lysing, but not suited for chemical lysing. By utilizing a filter having a pore size of 0.7 um, the filter is suitable for receiving suitably sized glass beads for mechanical lysing. Utilizing glass fibers to form the filter facilitates affinity bonding with the free nucleic acid released by chemical lysing. Thus, this filter is suited for both mechanical and chemical lysing.

[0068] FIGS. 5A-5C illustrate the valve cap 50, valve body 30 and filter 40 of a valve assembly of a universal sample cartridge, in accordance with some embodiments. The valve cap 50 and valve body 30 include interlocking portions on their peripheries that engage with each so that interior circular regions interface to form a sample processing region or lysing chamber. The cap and valve body include interior circular regions 31, 51 that interface to form an interior sample processing region or lysing chamber. The filter 40 is sized to be secure between the valve cap and valve body

[0069] FIGS. 6A-6B illustrate valve caps in accordance with some embodiments. FIG. 6A shows a valve cap 50′ in accordance with some embodiments, and FIG. 6B shows a valve cap 50 of a similar design that further includes a pair of posts. As shown, the valve caps include an interior circular region 51 that defines the lysing chamber when interfaced with the valve body and a fluidic inlet 52 through which fluid sample and any glass bead infill enters the lysing chamber. In the design of FIG. 6B, the valve cap further includes a pair of protrusions or posts 53, which presses the filter away from the inlet so as to improve consistency of glass infill across the filter so as to improve mechanical lysing.

[0070] FIGS. 7A-7B illustrate valve bodies in accordance with some embodiments. FIG. 7A shows a valve body 30′ in accordance with some embodiments, and FIG. 7B shows a valve cap 30 of a similar design that further includes a pair of posts. As shown, the valve bodies includes the interior circular region 31 that defines the lysing chamber when interfaced with the valve cap and a fluidic outlet 32 through which fluid sample exits the lysing chamber. In the design of FIG. 7B, the valve body further includes a pair of protrusions or posts 33, which inhibits deflection of the filter toward the outlet so as to reduce pressure peaks on the filter and reduce tearing.

[0071] FIGS. 8A and 8B illustrate cross-sectional views of a valve body of a conventional valve assembly (FIG. 8A) and a valve body of an improved valve assembly (FIG. 8B) in accordance with some embodiments. As can be seen, the edges of the chamber or filter pocket have been smoothed such that sharp edges are eliminated, which reduces uneven flow and pressure distributions through the chamber and promote uniform flow through the lysing chamber.

[0072] FIGS. 9A-9E illustrate various views of a valve body of a conventional sample cartridge utilizing mechanical lysing and FIGS. 10A-10C illustrate various views of a valve cap of a conventional sample cartridge utilizing mechanical lysing, for comparison with the improved valve assemblies described herein.

[0073] FIGS. 11A-11B illustrate various views of a valve body of a conventional sample cartridge utilizing chemical lysing, and FIGS. 11C-11D illustrate various views of a valve cap of a conventional sample cartridge utilizing chemical lysing, for comparison with the improved valve assembly described herein.

[0074] FIGS. 12A-12D illustrate various views of a valve cap 50 for a universal sample cartridge, in accordance with some embodiments. The two posts 53 can be seen adjacent the inlet 52. FIGS. 13A-13D illustrate various views of a valve body 30 for a universal sample cartridge, in accordance with some embodiments. The two posts 33 can be seen adjacent the outlet 32 and a series of support ridges 34 support the filter upstream of the outlet.

[0075] FIG. 14A illustrate cross-sectional views of a valve assembly, in accordance with some embodiments, illustrating the gap between the filter and cap where any glass beads for mechanical lysing are filled, for comparison with the conventional valve assembly shown in FIG. 14B. In the design of FIG. 14A, the cap and valve body have been modified, as described previously, for improved fill in the void region between the filter and the cap. In FIG. 14B a thinner filter material has been used to create a larger void to facilitate glass fill. In the improved design in FIG. 14A, a thicker filter material has been used such that the void between the filter and cap is smaller, while including the posts on the gap maintain a suitable gap to facilitate the glass fill process.

[0076] FIG. 14C illustrate cross-sectional views of a valve assembly of a sample cartridge, before addition of the posts, demonstrating an inconsistent, minimal gap between the filter and the valve cap under some conditions. FIG. 14D shows the valve assembly with the addition of posts, which provides a more consistent, enlarged gap to facilitate glass infill.

III. Experimental Results as to Performance Characteristics

[0077] FIG. 15A-15K illustrate various models of a valve assembly for a universal sample cartridge, in accordance with some embodiments. In particular, the models demonstrate the improved washing of residual buffer through the flowpath of the valve assembly, see FIGS. 15B-15K. In some embodiments, improved washflow of buffer and reduction of buffer carryover is achieved by streamlining the fluid flow path through the inlet and outlet of the valve assembly. In this embodiment, the fluid flow path is streamlined by smoothing any transitions and eliminating any sharp (e.g. 90 degree) corners.

[0078] FIG. 16 demonstrates the marked advantages of the improved valve design support protrusions over conventional designs in regard to filter stress. As shown by the finite element analysis in FIG. 16, the peak stress in the valve body with the pair of support posts adjacent the outlet was 8% lower than in the conventional design without any support posts.

[0079] FIG. 17 demonstrates the marked advantages of the improved valve design having support protrusions over conventional designs in regard to vertical displacement of the filter. As shown by the finite element analysis in FIG. 16, the peak displacement in the filter supported by the pair of support posts adjacent the outlet was 10% lower than in the conventional design without any support posts.

[0080] FIG. 18 demonstrates that the use of support posts in the improved valve design did not significantly impact wash out of residual buffer. As can be seen in computation fluid dynamics analysis of FIG. 18, there was 0.28% buffer remaining and an outlet concentration of 0.15% in the conventional design, and in the improved valve body design, there was 0.38% and 0.28% outlet concentration. These results demonstrated comparable wash out and residual buffer between the two designs.

[0081] FIG. 19 illustrates experimental results as to buffer carryover of a valve assembly of a multi-assay sample cartridge, in accordance with some embodiments, as compared to a conventional valve designs. As shown, the results demonstrates that the addition of the posts did not significantly impact buffer carryover.

[0082] FIGS. 20A-20G show experimental results illustrating fill and performance data of a valve assembly of universal assay sample cartridge, in accordance with some embodiments, before and after introduction of support posts. FIG. 20A shows glass fill ability improves to over 90% with the valve design with the support post features that maintain the filter placement to cap gap thereby improving fill. FIG. 20B shows filter integrity and capture ability are not affected by the added support features. FIG. 20C shows similar functional performance between the two cap designs. FIG. 20D shows the glass fill ability is not negatively impacted by the addition of the posts. FIG. 20E shows that the filter integrity and capture ability are not affected by the added support features. FIG. 20F shows similar functional performance between the two valve body designs. FIG. 20G shows that maximum pressures were reduced by 5 psi with the added support features of the new design.

IV. Assay WorkFlows

[0083] In one aspect, the sample cartridge having an improved valve assembly, as described herein, is capable of a variety of workflows that perform: chemical lysing of targets, mechanical lysing of targets, or both. Accordingly, the sample cartridge can perform an existing workflow assocaited with conventional specialized cartridges, or can perform entirely new workflows that perform both.

[0084] Exemplary assay workflows that can be performed with a single universal cartridge, in accordance with some embodiments, is shown in FIG. 21. In any of these embodiments, the filter can be formed of glass filter to promote affiniting binding of the nucleic acids (NA) to the glass fibers and a pore size suited for chemical lysing as well. In any of these workflows, the nucleic acid amplification can be PCR, real-time PCR, isothermal amplification (including but not limited to nucleic acid sequence-based amplification, loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, multiple displacement amplification, whole genome amplification or recombinase polymerase amplification) or other nucleic acid amplification methods known to persons of skill in the art.

[0085] In Workflow A, the sample is optionally exposed to a sample treatment or chemically lysed, then the treated or lysed fluid sample is flowed through the filter where targets are captured. In some embodiments, the sample treatment is ued to either weaken the cell wall or to inactivate the sample or make it less viscous to facilitate being processed through the filter. The filter is then washed, leaving the targets on the filter. Next, the targets are mechanially lysed, such as by sonication, to release nucleic acid (NA). In some embodiments, mechanical lysing includes in-filling glass beads along the filter to aid in mechanical lysing of the target. Next, the NA is eluted from the filter and then nucleic acid amplification is performed is performed.

[0086] In Workflow B, the sample is chemically lysed to obtain the NA targets. In some embodiments, after chemicaly lysing, the NA is bound to the filter by the presence of precipitating and binding reagent. Next, the filter is washed with a rinse reagent while the NA remains bound to the filter. Typically, the wash reagents have some amount of salt which still promotes the binding of the NA to the filter, while allowing removal of non-target materials. Next, the filter is eluted to remove the NA targets. In some embodiments, the elution is performed with a pH neutral buffer or basic buffer fluid. The target NA is then delivered to an attached reaction vessel to perform nucleic acid amplification.

[0087] In Workflow C, the fluid sample is exposed to sample treatment and/or chemically lyse the targets. Next, the NA freed by chemical lysing is bound to the filter. This step may utilize precipitating and binding reagent. Next, the filter is washed with a rinse reagent while the NA remains bound to the filter. Typically, the wash reagents have some amount of salt which still promotes the binding of the NA to the filter, while allowing removal of non-target materials. Next, the targets captured in the filter are heat and/or mechanically lysed. This may utilize sonication, and may further utilize glass beads to facilitate mechanical lysing of select targets. Then, the lysed target NA is eluted from the filter. In some embodiments, the elution is performed with a pH neutral buffer or basic buffer fluid. The target NA is then delivered to an attached reaction vessel to perform nucleic acid amplification.. Thus, in this workflow, the workflow allows for lysing of multiple differing targets, some requiring only chemicaly lysing (e.g. viral targets), and others requiring mechanical lysing (e.g. bacteria, spores, etc.), such that all these target NAs can be released from a single sample and tested by the same sample cartridge. While the above workflow desribed mechanical lysing after chemical lysing, it is appreciated that other workflows may be utilized in which chemical lysing occurs after mechanical lysing, while in other workflows, the chemical and mechanical lysing can occur concurrently.

[0088] In some embodiments, the sample cartridge includes an identifier with information as to the appropriate workflow needed for a particular panel of assays, so that an instrument module receiving the sample cartridge operates according to the specified workflow.

[0089] FIGS. 22A-23D show experimental results illustrating marked improvements in performance of the improved cartridge design, such as that shown in FIGS. 1 and 5A-5C, as compared to a conventional cartridge for testing virus and free DNA, such as that shown in FIGS. 11A-11D. In these experiments, the universal cartridge A+ and the conventional cartridge C were used to detect NATROL SARS-CoV-2 under testing conditions that used various fluidic flow rates within the cartridge. These methods were aimed at either lowering the time to result by reducing the sample preparation time or were aimed at processing higher sample volumes in order to improve the sensitivity of the test kits. Each cartridge contained lysis buffer, wash buffer, elution buffer, polyethylene glycol and three separate beads used for the detection of SARS-CoV-2. The cartridges were run on a GeneXpert 16 Module System. Prior experiments with this assay revealed that the maximum fluid volume that could be processed with cartridge C was 300 uL at which volume pressure aborts (100 psi or greater) occurred approximately 50% of the time, thus volumes in cartridge C were reduced and limited to 125 uL to avoid pressure aborts. Flow rates for these experiments were 10 uL per second for cartridge C and 100 uL per second for CartA+.

[0090] FIGS. 22A-22D demonstrate that the universal cartridge A+ allowed for markedly faster sample processing times than conventional cartridge C. The filtration process in cartridge A+ and cartridge C separated nucleic acids from mixtures of nasal/nasopharyngeal swabs, lysis buffer and polyethylene glycol. The attribute of the cartridge A+ that allowed for this process to be faster was related to the design of the valve assembly, the filter material, and the width and depth of the filter. The combination of these attributes allowed for faster flow rates over and through the filter while maintaining pressure and performance. This material gave the filter an advantage over other filtration methods by lowering sample processing times while maintaining assay sensitivity. As can be seen in the first two data sets in FIGS. 22A-22D, when preparing a 125 uL sample, the sample processing time for universal cartridge A+ was 2 min, 25 sec, while the sample processing time for conventional cartridge C was 4 min, 8 sec. Thus, cartridge A+ processed the same volume as cartridge C in about half the time and with about half the pressure as obtained with cartridge C. As can also be seen, the universal cartridge A+ has similarly reduced sample processing times for 300 uL and 500 uL samples, while maintaining pressures that are similar to that seen cartridge C when processing 125 uL sample .

[0091] FIGS. 23A-23D demonstrate that the universal cartridge A+ design allows for processing of higher sample volumes resulting in higher sensitivity than conventional cartridge C design. The filtration process in this cartridge separated nucleic acids from mixtures of nasal/nasopharyngeal swabs, lysis buffer and polyethylene glycol. The attribute of the cartridge A+ that allowed for the processing of higher volumes was related to the valve assembly design and the filter material, including the width and depth of the filter. Flow rates are limited by pressure in both cartridges, but the configuration of cartridge A+ allows for flow rates up to at least 100 uL per second while the flow rates on cartridge C are limited to about 10 uL per second thus, more total sample was able to be processed with cartridge A+ as compared to cartridge C in less time while maintaining viable pressures below 100 psi. This results in more target available for detection as evidenced by the lower CoV Ct and higher CoV EPF in cartridge A+ as compared to cartridge C. As mentioned above, the maximum allowable pressure for these cartridges is 100 psi. Data not shown for cartridge C has previously demonstrated that for assay sample volumes of 300 uL resulted in pressure aborts approximately 50% of the time for conventional cartridge C, halting sample processing. Cartridge A+ allows for higher sample volumes (e.g. 300 uL, 700 uL and 1,000 uL) without reaching or exceeding the maximum pressure allowable (100 psi), as shown.

[0092] In the foregoing specification, the invention is described with reference to specific embodiments thereof, but those skilled in the art will recognize that the invention is not limited thereto. Various features, embodiments and aspects of the above-described invention can be used individually or jointly. Further, the invention can be utilized in any number of environments and applications beyond those described herein without departing from the broader spirit and scope of the specification. The specification and drawings are, accordingly, to be regarded as illustrative rather than restrictive. It will be recognized that the terms “comprising,” “including,” and “having,” as used herein, are specifically intended to be read as open-ended terms of art. Any references to publication, patents, or patent applications are incorporated herein by reference in their entirety for all purposes.