Multi-bead biological assay

Abstract

The present technology provides a system for the amplification of nucleic acids. The system comprises a reaction vessel, and a plurality of distinct lyophilized (or otherwise dried) reagent forms (e.g. beads, discs, or other structures) stored within the reaction vessel. Each distinct lyophilized reagent form comprises a distinct reagent mixture, and is configured to rehydrate at a specific rate and time after addition of rehydrating liquid.

Description

[0001] MULTI-BEAD BIOLOGICAL ASSAY

[0002] BACKGROUND OF THE INVENTION

[0003]

[0001] The present technology relates, in general, to the performance of multi-step biological assays (e.g., the amplification of nucleic acids) in a single reaction vessel or tube (i.e., single-pot reaction). More particularly, the present technology relates to the lyophilized or otherwise dry-storage of the necessary reaction components, for the performance of multi-step biological assays (e.g., amplification of nucleic acids), in multiple distinct lyophilized (or otherwise dried) reagent forms (e.g. beads, discs, or other structure) within a single reaction vessel.

[0004] BRIEF SUMMARY OF THE INVENTION

[0005]

[0002] In one aspect, the present technology provides a system for the amplification of nucleic acids. In this aspect, the system comprises a reaction vessel, and a plurality of distinct lyophilized (or otherwise dried) reagent forms (e.g. beads, discs, or other structure) stored within the reaction vessel. In this aspect, each distinct lyophilized (or otherwise dried) reagent forms (e.g., bead, disc, or other structure), comprises a distinct reagent mixture, and is configured to rehydrate at a specific rate and time after addition of rehydrating liquid. Further, it is understood that the plurality of distinct lyophilized (or otherwise dried) reagent forms can be either provided with the reaction vessel, or separate from the reaction vessel.

[0006]

[0003] In a further aspect, the lyophilized (or otherwise dry-stored) reagent format can be a bead, disc, or other structure. Further, the distinct lyophilized (or otherwise dried) reagent forms comprise distinct reagents (e.g., reagent blends or mixes). In this aspect, distinct reagents can include without limitation, lysis reagents, amplification reagents, control reagents, detection reagents, termination reagents, and the like.

[0007]

[0004] In yet a further aspect, the technology described herein relies on multiple distinct lyophilized (or otherwise dried) reagent forms (e.g., lyophilized reagent beads) stored within the same reaction vessel (or chamber), with each lyophilized (or otherwise dried) reagent form (e.g., bead, disc, or other structure form) configured to rehydrate at specific times during the course of a reaction. In this manner, the technology enables the sequential delivery of reagents during the course of a reaction occurring within a single reaction vessel.

[0008] BRIEF DESCRIPTION OF THE DRAWINGS

[0009]

[0005] Figure 1 is a schematic illustration of the multi reagent method disclosed herein.

[0010] DETAILED DESCRIPTION OF THE INVENTION

[0011]

[0006] The present disclosure provides for systems and methods for the amplification of a target nucleic acid. In certain aspects, the system comprises a reaction vessel, and a plurality of distinct lyophilized (or otherwise dried) reagent forms (e.g., bead, disc, or other structure form) stored in the reaction vessel. Furthermore, each lyophilized (or otherwise dried) reagent form (e.g., bead, disc or other structure form) comprises a distinct reagent mixture, and is configured to rehydrate at a specific time after addition of rehydration liquid.

[0012]

[0007] In one aspect, the plurality of beads comprises 2 or more beads, from 2 to 3 beads, from 2 to 4 beads, from 2 to 5 beads, from 2 to 6 beads, from 2 to 7 beads, from 2 to 8 beads, from 2 to 9 beads, from 2 to 10 beads, 3 beads, 4, beads, 5, beads, 6 beads, 7 beads, 8 beads, 9 beads, or 10 beads.

[0013]

[0008] In another aspect, the lyophilized (or otherwise dried) reagent form (e.g., beads, discs, or other structure form) are configured to rehydrate at different times after addition of a rehydrating liquid (e.g., water) to the reaction vessel. In a further aspect, at least two lyophilized (or otherwise dried) reagent form (e.g., beads, discs, or other structure forms) are configured to rehydrate at different times after addition of a rehydrating liquid to the reaction vessel. In this manner, the system (and method) disclosed herein relies on each distinct lyophilized (or otherwise dried) reagent form (such as a bead, disc, or other structured form) rehydrating at a specific time during the course of the reaction to enable the sequential delivery of reagents, rather than the components of all reagent forms mixing into the reaction simultaneously.

[0014]

[0009] Figure 1 provides an example of a multiple lyophilized (or otherwise dried) reagent bead system and method, in accordance with the technology disclosed herein. The reagent bead in Figure 1 can be configured in a different form (e.g., as a disc or other structure). In Figure 1 , three distinct lyophilized reagent beads are present in the same reaction vessel or tube. Each lyophilized reagent bead comprises a distinct reagent composition (e.g., lysis reagents, amplification reagents, and control reagents), and is configured to rehydrate at different times. For example, in Figure 1 the lyophilized lysis and control reagent beads are configured for fast rehydration, whereas the lyophilized amplification reagent bead is configured for slow rehydration.

[0015]

[0010] In one aspect, the lysis reagent bead can comprise reagents necessary to lyse an infectious agent or biological cell, including, for example without limitation, infectious agents such as viruses, bacterial, protozoan, or fungal cells. These agents can be broadly classified into enzymatic and chemical lysis agents. Enzymatic lysis agents, like lysozyme, break down bacterial cell walls, while chemical lysis agents, like SDS (Sodium Dodecyl Sulfate) and NaOH, disrupt the cell membrane and cell wall.

[0016]

[0011] In a still further aspect, the amplification reagent bead comprises reagents necessary for the amplification of a target nucleic acid (e.g., DNA or RNA). For example, and without limitation, amplification reagent beads can comprise: control target nucleic acids; primers (e.g., forward and reverse PGR primers); dNTPs (deoxyribonucleotide triphosphates, including adenine (dATP), cytosine (dCTP), guanine (dGTP), thymine (dTTP), and Deoxyuridine triphosphate (dUTP); DNA polymerases (including thermostable polymerases, non-thermostable polymerases (mesophilic polymerases), or combinations thereof); RNA polymerases; recombinase enzymes (e.g., RecA, UvsX, or combinations thereof); exonucleases; endonucleases; and buffer systems. In this aspect, the amplification reagent bead can be configured to mediate the amplification of a target nucleic acid using. For example, without limitation, the amplification reagent bead can be configured to mediate a Polymerase Chain Amplification (PCR), a Recombinase Polymerase Amplification (RPA), a Loop-Mediated Isothermal Amplification (LAMP), a Rolling Circle Amplification (RCA), a Helicase-Dependent Amplification (HDA), a Strand Displacement Amplification (SDA), Nicking Enzyme Amplification Reaction (NEAR), Selective Temperature Amplification Reaction (STAR and qSTAR), or a Multiple displacement amplification (MDA). In one embodiment, the amplification reaction can be nested, real-time, multiplexed, quantitative, or asymmetric. With respect to PCR, the amplification can be a nested PCR, a real-time PCR, a multiplex PCR, quantitative PCR, or an asymmetric PCR.

[0017]

[0012] In a further aspect, at least one bead can be a lyophilized detection reagent bead. In this aspect, the lyophilized detection reagent bead is configured to release, upon rehydration, reagents necessary to initiate or otherwise facilitate the detection of amplified nucleic acids. For example, without limitation, the lyophilized detection bead can comprise reagents necessary for the performance of end-point analysis, such as reagents necessary for the end-point analysis of multiplex SNP (Single Nucleotide Polymorphism) analysis. The lyophilized detection reagent bead can be configured to be rehydrated before, after, or simultaneous with the rehydration of the amplification reagent bead.

[0018]

[0013] In a still further aspect, at least one bead can be a lyophilized control bead. In this aspect, the lyophilized control bead is configured to release, upon rehydration, reagents necessary for the performance of an internal control reaction. In this aspect, the lyophilized control bead comprises a control target nucleic acid. Examples include, without limitation, lyophilized control beads comprising RNase P nucleic acid that is released upon rehydration. The lyophilized control bead can be configured to be rehydrated before, after, or simultaneously with the rehydration of the amplification reagent bead. Examples include, but are not limited to, delaying the control amplification in the presence of a low titer target which can limit competition of critical reagents helping with assay sensitivity.

[0019]

[0014] In a still further aspect, at least one bead can be a lyophilized termination reagent bead, which comprise reagents that terminate, stop a nucleic acid amplification reaction, or degrade amplicons to minimize potential contamination. Examples include, but are not limited to, termination reagent beads that comprises exonucleases, endonucleases, Uracil DNA Glycosylases, or combinations thereof. The termination reagent bead can also comprise reagents that inactive amplification reagents (e.g., humic acids, excessive amounts of salt (such as NaCI or KCI), and detergents (such as SDS)). In this aspect, the termination bead is configured to hydrate and / or otherwise dissolve slower than other beads present in the reaction chamber. The lyophilized termination reagent bead can be configured to be rehydrated after the rehydration of the amplification reagent bead, and after the amplification of target nucleic acid is complete.

[0020]

[0015] Different rehydration rates and times for each lyophilized (or otherwise dried) reagent bead present in the reaction vessel are based on the water-solubility of the different bead components (e.g., sugars (sucrose, and trehalose), gelatin, waxes, or another substance), as well as the relative concentration of these components. It is contemplated that some substances rehydrate in water given enough time (e.g., increasing concentrations of sugars), while others may only rehydrate when the solution reaches a specific temperature (such as waxes or gelatin). In this manner, the rate of rehydration can be controlled by adjusting individual component concentration and temperature over the course of the reaction. It is understood that the reagent bead in Figure 1 can be configured in any number of different forms, including, for example without limitation, a disc or other structure.

[0021]

[0016] In a further aspect, the lyophilized reagent beads described herein can be configured to be released not at a predetermined time, but under a predetermined condition (e.g., at a predetermined temperature or pH). For example, DNA amplification of a target nucleic can lead to changes in the pH of a reaction mixture (e.g., DNA amplification generates byproducts like pyrophosphates and hydrogen ions that can lead to a change in pH). Termination beads can be configured to rehydrate, for example, upon an appropriate decrease in pH.

[0022]

[0017] As illustrated in Figure 1 , the reaction vessel comprises three steps. Step A comprises providing a reaction vessel having multiple lyophilized reagent beads. In this embodiment, the reaction vessel has three lyophilized reagent beads, including a lysis reagent bead (bead with diagonal lines), a control reagent bead (bead with vertical lines), and an amplification reagent bead (bead with horizontal lines). These beads can be stored at ambient temperatures for multiple months.

[0018] In step B of Figure 1 , liquid is added to the reaction vessel (or tube) such that the lysis reagent bead (bead with diagonal lines) and the control reagent bead (bead with vertical lines) are rehydrated quickly. This is due to the low amount of sugar present in the lysis reagent bead (bead with diagonal lines) and the control reagent bead (bead with vertical lines), relative to the amplification reagent bead (bead with horizontal lines), which comprises more sugar (and does not immediately rehydrate). In this manner, the reagents stored in the lysis reagent bead (bead with diagonal lines) and the control reagent bead (bead with vertical lines) are now present in solution (in the reaction vessel or tube) and available for the biochemical reaction, while the reagents stored in the amplification reagent bead (bead with horizontal lines) are not yet participating in the reaction.

[0023]

[0019] In step C of Figure 1 , after a time interval (e.g., from about 2 to about 10 minutes), predetermined based on the type and amount of sugar comprising the amplification reagent bead (bead with horizontal lines), and the optional application of heat, the amplification reagent bead (bead with horizontal lines) eventually rehydrates. At this point, the reagents stored in the amplification reagent bead (bead with horizontal lines) will mix into the solution and participate in the reaction.

[0024]

[0020] One skilled in the art will recognize that modifications may be made in the present technology without deviating from the spirit or scope of the invention. The invention is further illustrated by the following examples, which are not to be construed as limiting the invention in spirit or scope to the specific procedures or compositions described therein.

[0025]

[0021] In one configuration, the lysis reagent bead (bead with diagonal lines) and the control reagent bead (bead with vertical lines) in Figure 1 have a low concentration of sugar causing them to rehydrate as soon as liquid is added to the system while the amplification reagent bead (bead with horizontal lines) will remain in-tact. In this manner, the reagents stored in the lysis reagent bead (bead with diagonal lines) and the control reagent bead (bead with vertical lines) are available to participate in the biochemical reaction prior to the reagents stored in the amplification reagent bead (bead with horizontal lines) bead.

[0022] The separation of reagents into distinct lyophilized reagent forms (e.g., beads, discs, or other structures) provides several advantages. In the example illustrated in Figure 2, the lysis reagent bead (bead with diagonal lines) contains lysis reagents and the control reagent bead (bead with vertical lines) bead contains the assay control materials. These components cannot be stored together because the lysis reagents will destroy the control materials during bead production. However, these reagents need to be introduced to the assay at the same time to ensure the lysis reagents can interact with the control materials at the start of the test. The amplification reagent bead (bead with horizontal lines) contains amplification reagents that cannot be present before the lysis reagents are deactivated (e.g., by a heating step or addition of specific deactivation reagents (such as salts)), otherwise, the lysis reagents will destroy the amplification reagents.

[0026]

[0023] Another advantage includes, for example and without limitation, improving the functionality and acceptable storage time of lysis reagents. For example and without limitation, the method described herein provides the ability to use more complex and effective lysis reagents in assays. Storing lysis reagents wet, significantly limits the types of reagents available for lysis to those that are compatible with liquid storage at ambient temperatures. Notably, there are multiple enzymes that lyse pathogens very effectively, but enzymes will irreversibly degrade if they are stored as a liquid at ambient temperatures. By storing lysis enzymes dry in a dried reagent format (e.g., in a lyophilized bead, disc or other structure), the potential reagents used can be expanded.

[0027]

[0024] A still further advantage provides for greater simplicity for the end user (e.g., requiring three or less user steps). Examples include without limitation, assays involving multiple, sequential steps. Such steps include, but are not limited to: (1 ) sample collection and addition to a reaction vessel or tube; (2) control material addition to the reaction vessel or tube; (3) placing the reaction vessel into a device (e.g., a thermocyler or point- of-care device); (4) sample and control lysis; (5) lysis reagent deactivation via heating; (6) amplification reagent addition; and (7) determination of assay results. By using multiple distinct lyophilized (or otherwise dried) reagent forms (e.g., beads, discs, or other structures), stored in a single reaction vessel, that rehydrate at programmed times, the required user steps are significantly reduced. Examples of a reduced step reaction include, but are not limited, to the following: (1 ) sample collection and addition to a reaction vessel or tube; and (2) placing the reaction vessel or tube into a device (e.g., thermocycler or point-of-care device).

[0028]

[0025] Another advantage includes the simplified and modular design for different assays. For example and without limitation, the lysis bead and the amplification bead can contain identical reagents used in multiple assays, while the control bead can have components that are unique to each individual assay. In this modular approach, only one component of the assay - the control bead - needs to be changed when developing a new test.

[0029]

[0026] A still further advantage of the technology disclosed herein is the potential to run multiple assay types simultaneously in the same reaction vessel or tube. For example and without limitation, the method described in Figure 1 can have two types of assays running simultaneously in a single reaction vessel or tube with controlled reagent delivery from individual lyophilized (or otherwise dried) reagent forms (e.g., beads, disc, or other structures). Examples include, but are not limited to, nucleic amplification assays that can be run simultaneously with an immunoassay (IA). The nucleic acid amplification assay reagents and temperatures are often not compatible with the IA reagents, which usually requires two separate tests run from the same sample. Using the technology disclosed herein, a biological sample to be tested could be added to a single reaction vessel or tube, and the IA test run first with fast rehydration reagents followed by the nucleic acid test running second with slow rehydration reagent reagents. The technology disclosed herein can be applied to any biological assay that requires multiple separate steps performed in a single tube.

[0030]

[0027] The embodiments and examples described here are illustrative, and do not limit the presently described technology in any way. The scope of the present technology described in this specification is the full scope defined or implied by the claims. Additionally, any references noted in the detailed description section of the instant application are hereby incorporated by reference in their entirety, unless otherwise noted.

[0031]

[0028] The present technology is now described in such full, clear and concise terms as to enable a person skilled in the art to which it pertains, to practice the same. It is to be understood that the foregoing describes preferred embodiments of the present technology and that modifications may be made therein without departing from the spirit or scope of the present technology as set forth in the appended claims. Further the examples are provided to not be exhaustive but illustrative of several embodiments that fall within the scope of the claims.

Claims

Docket No. 68212WO01What is claimed is:

1. A system for the amplification of a target nucleic acid, the system comprising: a reaction vessel; and a plurality of different types of lyophilized reagent beads stored in the reaction vessel; wherein each type of lyophilized reagent bead comprises a distinct reagent mixture; and wherein each lyophilized reagent bead is configured to rehydrate at a specific time after addition of a rehydrating liquid.

2. The system of claim 1 , wherein the target nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

3. The system of claim 2, wherein the target nucleic acid is an infectious agent.

4. The system of claim 3, wherein the infectious agent is viral, bacterial, protozoan, or fungal.

5. The system of claim 2, wherein the target nucleic is a genetic biomarker.

6. The system of claim 1 , wherein the reaction vessel is a reaction tube having a hinged lid.

7. The system of claim 1 , wherein the lyophilized reagent beads are configured to rehydrate at different times in the reaction vessel after addition of a rehydrating liquid.

8. The system of claim 1 , wherein at least two lyophilized reagent beads are configured to rehydrate at different times in the reaction vessel after addition of a rehydrating liquid.

9. The system of claim 1 , wherein any one of the plurality of lyophilized reagent bead is configured as a disc or other structure.

10. The system of claim 1 , wherein the plurality of lyophilized reagent beads comprises two or more beads.1 1 . The system of claim 1 , wherein at least one of the plurality of lyophilized reagent beads comprises a termination bead having a composition that when released terminates the amplification of the target nucleic acid.

12. The system of claim 11 , wherein the termination bead comprises an endonuclease, Uracil DNA Glycosylase, or combination thereof.

13. The system of claim 1 , wherein at least one of the plurality of lyophilized reagent beads comprises an amplification reagent.

14. The system of claim 1 , wherein at least one of the plurality of lyophilized reagent beads comprises a control reagent.

15. A system for the amplification of a target nucleic acid, the system comprising: a plurality of different types of lyophilized reagent beads;wherein each type of lyophilized reagent bead comprises a distinct reagent mixture; and wherein each lyophilized reagent bead is configured to rehydrate at a specific time after addition of a rehydrating liquid.

16. The system of claim 15, wherein the target nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

17. The system of claim 16, wherein the target nucleic acid is an infectious agent.

18. The system of claim 17, wherein the infectious agent is viral, bacterial, protozoan, or fungal.

19. The system of claim 16, wherein the target nucleic is a genetic biomarker.

20. The system of claim 15, wherein the lyophilized reagent beads are configured to rehydrate at different times in the reaction vessel after addition of a rehydrating liquid.

21. The system of claim 15, wherein at least two lyophilized reagent beads are configured to rehydrate at different times in the reaction vessel after addition of a rehydrating liquid.

22. The system of claim 15, wherein any one of the plurality of lyophilized reagent bead is configured as a disc or other structure.

23. The system of claim 15, wherein the plurality of lyophilized reagent beads comprises two or more beads.

24. The system of claim 15, wherein at least one of the plurality of lyophilized reagent beads comprises a termination bead having a composition that when released terminates the amplification of the target nucleic acid.

25. The system of claim 15, wherein the termination bead comprises an endonuclease, Uracil DNA Glycosylase, or combination thereof.

26. The system of claim 15, wherein at least one of the plurality of lyophilized reagent beads comprises an amplification reagent.

27. The system of claim 15, wherein at least one of the plurality of lyophilized reagent beads comprises a control reagent.

28. The system of claim 1 , wherein the different types of lyophilized reagent beads comprises a lysis reagent bead, an amplification reagent bead, a control reagent bead, a detection reagent bead, a termination reagent bead, or combinations thereof.

29. The system of claim 1 , wherein the different types of lyophilized reagent beads comprises a lysis reagent bead, an amplification reagent bead, a control reagent bead, a detection reagent bead, a termination reagent bead, or combinations thereof.

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