Reaction tube and optical detection system for biological reactions

Abstract

Among other things, the present application provides simplified optical detection systems for nucleic acid detection having optical performance sufficient to permit spectrophotometric (e.g., fluorescent) detection of reaction products within a reaction tube. The present application further provides a reaction tube that is amenable to production using injection molding, while maintaining optical performance sufficient to permit spectrophotometric (e.g., fluorescent) detection of reaction products within the tube. In some embodiments, a reaction tube comprises: a bottom wall comprising a closed, molded lens; four side walls joined to the bottom portion and forming a reservoir for accommodating a reaction mixture; and an open upper portion for receiving a cap, wherein one of the four side walls comprises or consists of a flat, optically transmissive side wall.

Description

REACTION TUBE AND OPTICAL DETECTION SYSTEM FOR BIOLOGICAL REACTIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application number 63 / 693,854 filed on September 12, 2024, the disclosure of which is incorporated by reference herein in its entirety.FIELD OF THE INVENTION

[0002] The present application pertains to the field of optical detection systems. More particularly, the present application relates to optical detection systems for performance of diagnostic assays, including nucleic acid amplification-based assays.BACKGROUND

[0003] Since the discovery of DNA, many nucleic acid amplification technologies have been developed to detect the presence, absence, or amount of specific DNA or RNA sequences. The Polymerase Chain Reaction (PCR) is a technique used to amplify and, thereby, detect or quantify a specific nucleic acid sequence of interest. This technique has formed the basis of numerous assays for detecting particular nucleic acid sequences. Multiplex PCR employs PCR to amplify and detect multiple target sequences simultaneously. While other nucleic acid amplification technologies have been developed, PCR remains the most commonly used in molecular diagnostic assays.

[0004] Following the invention of PCR, optical detection technologies were developed to enable fluorescent detection of PCR amplicons, utilizing a method known as real-time PCR. Incorporation of such fluorescent detection into these assays further required the development of optical detection systems to enable visualization and quantification of the PCR assay results. Consequently, real-time thermal cyclers combine a thermal cycling apparatus with a system for optical detection of fluorescence generated by a successful PCR.

[0005] Optical detection systems include an excitation light path including a light source, for example light-emitting diode (LED), a filter, and a lens. The utilization of a lens in front of thelight source is common in detection assemblies. For example, U.S. Patent 6,818,185 teaches that its "detection assembly 218 includes one or more lenses for focusing and collimating the emitted light." This patent describes a reaction tube in which the side walls are optically transmissive to permit excitation of the reaction mixture in the chamber through one side wall and detection of light emitted from the chamber through the other side wall (see Figure 22 of U.S. 6,818,185). The two side walls are flat, optically transmissive, and positioned at approximately a 90-degree angle to each other.

[0006] In another example, described in U.S. Patent 8,835,118, a thermal cycling system was developed comprising a movable optical detection system in the lid of the reaction tubes. One disadvantage of this technology is the requirement for complicated optics because the excitation light and emission light are both transmitted through the tops of the reaction tubes, which include transparent sample caps. Another disadvantage is the requirement for a bulky lid containing the optical detection system.

[0007] Disadvantages of U.S. Patent 8,835,118 are addressed in U.S. Patents 8,528,777 and 10,391,498. Specifically, U.S. Patent 8,528,777 describes a tube for DNA reactions that has a flat, optically-clear bottom region, which "enables better optical detection by minimizing light reflections and refractions...and improves the signal-to-noise ratio." One skilled in the art can appreciate that if the tube did not have a flat, optically-clear bottom, and instead had the curved bottom of a standard PCR tube, there would be greater light reflections and refractions. This would also be the case if the excitation light was focused on the side curvature of the reaction tube as described by Mulberry et al. (PLoS One. (2017). 12(6): e0179133, see Figure 3), even if there were a lens in front of the light source.

[0008] U.S. Patent 10,391,498 describes an apparatus incorporating the tube of U.S. Patent 8,528,777 into an optical detection system, whereby "a bottom surface of a housing includes a cutout, so that when a base is received by a housing, a receptacle portion of a nucleic acid amplification reaction vessel is optically accessible." In this manner, excitation light may be shone from the side of the tube and emission light may detected through the flat, optically-clear bottom of the tube. Compared to U.S. Patent 8,835,118, this significantly simplifies the optics and eliminates the requirement for a bulky lid.

[0009] However, a disadvantage of U.S. Patent 8,528,777 is that it is difficult to manufacture a tube with a flat, optically-clear bottom using conventional injection molding techniques. One skilled in the art would appreciate that the distal end of a mold is the most difficult region in which to maintain the tight tolerances required for a flat, optically clear bottom that does not have artefacts that could interfere with optical detection.

[0010] Thus, there remains a need for alternative optical systems including reaction tubes for nucleic acid amplification reaction that overcome at least some of the drawbacks of previous systems.

[0011] The above information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present disclosure.SUMMARY OF THE INVENTION

[0012] An object of the present application is to provide a simplified optical detection system for nucleic acid detection, while maintaining optical performance sufficient to permit spectrophotometric (e.g., fluorescent) detection of reaction products within a reaction tube. A further or alternative object of the present application is to provide a reaction tube that is amenable to production using injection molding, while maintaining optical performance sufficient to permit spectrophotometric (e.g., fluorescent) detection of reaction products within the tube.

[0013] In accordance with one aspect of the present application, there is provided a reaction tube comprising: a bottom wall comprising a closed, molded lens; four side walls joined to the bottom portion and forming a reservoir for accommodating a reaction mixture; and an open upper portion for receiving a cap, wherein one of the four side walls comprises or consists of a flat, optically transmissive side wall.

[0014] In some embodiments, the optically transmissive side wall is angularly offset from the closed, molded lens by an angle of from approximately 90° to approximately 120°, preferably about 90°.

[0015] In some embodiments, the four side walls consist of the flat, optically transmissive side wall, an opposing side wall having the same or approximately the same dimensions as the flat, optically transmissive side wall and two connecting side walls.

[0016] In some embodiments, the four side walls define a square or rectangular pyramidal shape truncated by the bottom wall to form the reservoir for accommodating the reaction mixture.

[0017] In some embodiments, the four side walls terminate at a top edge that defines an opening of the open upper portion and the opening is for receiving the cap.

[0018] In some embodiments, the four side walls terminate at a top edge that defines an opening, the open upper portion comprises a receiving reservoir connected to the top edge and in fluid communication with the reservoir for accommodating the reaction mixture.

[0019] In some embodiments, the reaction tube is formed of a thermally stable plastic.

[0020] In accordance with another aspect of the present application, there is provided an optical detection system comprising: a reaction tube as defined herein; a light source for providing excitation light to excite the reaction mixture; and a photodetector for detecting light emitted from the reaction mixture.

[0021] In some embodiments, the excitation light passes through the flat, optically transmissive side wall of the reaction tube and the light emitted is collimated through the lens of the reaction tube to the photodetector.

[0022] In some embodiments, the excitation light is collimated by the lens of the reaction tube and into the reaction mixture and the light emitted passes through the flat, optically transmissive side wall of the reaction tube to the photodetector.

[0023] In accordance with another aspect of the present application, there is provided a method for monitoring a reaction comprising: (a) mixing reactants in a reaction tube, as described herein, to form a reaction mixture; (b) inserting the reaction tube into a receptacle in an optical detection apparatus, wherein said optical detection apparatus comprises a light source and a photodetector and wherein the reaction tube is positioned inthe receptacle to allow light from the light source to pass through the optically transmissive side wall or through the lens; (c) illuminating the reaction mixture with light from the light source; and (d) measuring, using the photodetector, light emitted from the reaction mixture through the optically transmissive side wall or the molded lens, wherein the measured light emitted is used to detect or quantify one or more products of the reaction and / or one or more reactants of the reaction.

[0024] In some embodiments, the reaction is a nucleic acid amplification reaction and the measured light emitted is used to detect or quantify one or more target amplicons.Optionally, the nucleic acid amplification reaction is a non-isothermal amplification reaction.

[0025] In some embodiments, the optical detection apparatus further comprises a heatexchanging module for thermally controlling the reaction mixture when the reaction tube is in the receptacle.

[0026] In some embodiments, the reaction tube is positioned in the receptacle to allow light from the light source to pass through the optically transmissive side wall and the method comprises measuring, using the photodetector, light emitted from the reaction mixture through the molded lens.BRIEF DESCRIPTION OF THE FIGURES

[0027] For a better understanding of the application as described herein, as well as other aspects and further features thereof, reference is made to the following description which is to be used in conjunction with the accompanying drawings, where:

[0028] Figure 1 is a side view of a reaction tube according to one embodiment of the present application;

[0029] Figure 2 is a side view of a reaction tube including a reagent reservoir according to another embodiment of the present application; and

[0030] Figure 3 is a cross-section of the reaction tube depicted in Figure 2.DETAILED DESCRIPTION

[0031] Definitions

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which technologies disclosed herein belong.

[0033] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0034] The term "comprising," as used herein, will be understood to mean that the list following is non-exhaustive and may or may not include any other additional suitable items, for example one or more further feature(s), component(s) and / or ingredient(s) as appropriate.

[0035] The term "consisting of," as used herein, refers to compositions, methods, devices, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the present disclosure. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about."

[0036] Reference throughout this specification to "one embodiment," "an embodiment," "another embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "an additional embodiment," or "a further embodiment" or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0037] The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A," and "B".

[0038] The terms "about" and "approximately" are used herein as equivalents. Any numerals used in this application with or without about / approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0039] The term "amplification reaction" is used herein to refer to a reaction in which multiple copies of an original nucleic acid sequence are generated, typically by repeating an enzymatic duplication process for a number of cycles. When additional copies can be made from each of the duplicate copies made in an earlier cycle, the amplification process is said to be exponential with respect to the number of cycles. In certain embodiments, an amplification reaction is an isothermal amplification reaction. In certain embodiments, an amplification reaction is a non-isothermal amplification reaction.

[0040] Some amplification reactions, for example PCR and LCR, involve cycles of alternately high and low set temperatures, a process known as "thermal cycling." Amplification reactions which use thermal cycling are referred to as non-isothermal amplification reactions. PCR is an amplification reaction in which a polymerase enzyme, usually thermostable, generates multiple copies of the original sequence by extension of a primer using the original nucleic add as a template. PCR is described in more detail in U.S. Patent Nos 4,683,202 and 4,683,195 and elsewhere. LCR or "Ligase Chain Reaction" is a nucleic add amplification reaction in which a ligase enzyme, usually thermostable, generates multiple copies of the original sequence by ligating two or more oligonucleotide probes while they are hybridized to the target. LCR, and its variation, Gap LCR, are described in more detail in EP-A-320-308, EP-A-439-182 and WO 93 / 100447 and elsewhere.

[0041] Some amplification reactions, for example Loop-Mediated Isothermal Amplification (LAMP), Whole Genome Amplification (WGA), Strand Displacement Amplification (SDA), Helicase-Dependent Amplification (HDA), Recombinase Polymerase Amplification (RPA), Nucleic Acid Sequences Based Amplification (NASBA), and Transcription Mediated Amplification (TMA), do not involve cycling temperatures. In certain embodiments, an isothermal amplification reaction maintains a substantially constant or fixed temperature during amplification.

[0042] The term "amplicon," or "amplicon molecule," as used herein, refers to a target piece of nucleic acid (DNA or RNA), that is the source and / or product of nucleic acid amplification. In certain embodiments, an amplicon molecule is a nucleic acid molecule generated by transcription from a template nucleic acid molecule, or a nucleic acid molecule having a sequence complementary thereto, or a double-stranded nucleic acid including any such nucleic acid molecule. Transcription can be initiated from a primer.

[0043] The term "lens", as used herein, refers to a transparent substance with curved sides for concentrating or dispersing light rays. In some embodiments, a lens is transparent (e.g., transmissive) to light, for example, light having a visible, near infrared, infrared, and / or ultraviolet (UV) spectrum.

[0044] As used herein, in its broadest sense, the term "nucleic acid" refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments e.g., as set forth herein, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments e.g., as set forth herein, the term nucleic acid refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside), and in some embodiments e.g., as set forth herein refers to a polynucleotide chain comprising a plurality of individual nucleic acid residues. A nucleic acid can be or include DNA, RNA, or a combination thereof. A nucleic acid can include natural nucleic acid residues, nucleic acid analogs, and / or synthetic residues. In some embodiments e.g., as set forth herein, a nucleic acid includes natural nucleotides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments e.g., as set forth herein, a nucleic acid is or includes of one or more nucleotideanalogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof).

[0045] In some embodiments e.g., as set forth herein, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments e.g., as set forth herein, a nucleic acid includes one or more introns. In some embodiments e.g., as set forth herein, a nucleic acid includes one or more genes. In some embodiments e.g., as set forth herein, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.

[0046] In some embodiments e.g., as set forth herein, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments e.g., as set forth herein, a nucleic acid can include one or more peptide nucleic acids, which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone. Alternatively or additionally, in some embodiments e.g., as set forth herein, a nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments e.g., as set forth herein, a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'- deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids.

[0047] In some embodiments, e.g., as set forth herein, a nucleic acid is or includes at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues. In some embodiments, e.g., as set forth herein, a nucleic acid is partly or wholly single stranded, or partly or wholly double stranded.

[0048] As used herein the terms "sample" and "biological sample" means any sample, including, but not limited to cells, organisms, lysed cells, cellular extracts, nuclear extracts, components of cells or organisms, extracellular fluid, media in which cells are cultured, blood, plasma, serum, gastrointestinal secretions, tissues, homogenates of tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid, tears, vaginal fluids and / or secretions, and prostatic fluid. In addition, a sample can be a viral or bacterial sample, a sample obtained from an environmental source or a body of water (e.g., a lake, a reservoir, a well, ground water, wastewater), a forensic sample, a food sample (e.g., a food source believed to be contaminated), soil extracts, pesticide residues, or airborne spores.

[0049] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.

[0050] The term "thermal cycler" or "thermocycler" is used herein to refer to a device used to heat, cool and / or hold a nucleic acid amplification reaction mixture between or at a set temperature for a set time duration over a set number of cycles. Thermocyclers used for real-time and / or quantitative PCR or LCR additionally comprise detection systems, typically optical detection systems, for monitoring reaction product generation. Real-time PCR (RT- PCR) detection systems are typically optical detection systems for measuring fluorescence signal generated during each amplification cycle as the fluorophore binds to the target sequence in amplicons.

[0051] Reaction Tube

[0052] Among other things, the present application provides a reaction tube, or vessel or cartridge, useful for conveniently monitoring reactions performed therein using optical detection. The reaction tube can be used for performing nucleic acid amplification assays, such as PCR assays, and monitoring the assay, such as monitoring in real time. The present application encompasses a recognition that standard reaction tubes are not well suited for real-time monitoring (including detection and quantification) and those that are specifically designed for real-time monitoring are not amenable to simple manufacturing.

[0053] The present application provides a reaction tube comprising a bottom portion comprising a closed, molded lens and side walls connected to the bottom portion, wherein the side walls and the bottom portion form a reservoir for accommodating a reaction mixture, and wherein at least one of the side walls comprises or consists of a flat, optically transmissive material. As a consequence of this configuration, the present reaction tube provides a simplified system for nucleic acid detection and / or quantification, while maintaining optical performance sufficient to permit spectrophotometric (e.g., fluorescent) detection of reaction products within the tube. The present reaction tube is amenable to production using injection molding, while also providing optical performance sufficient to permit optical (e.g., fluorescent) detection of reaction products within the tube.

[0054] In accordance with some embodiments, the flat, optically transmissive (e.g., optically transparent) surface on one side of the reaction tube is angularly offset at an angle of from about 90° to about 120° to the molded lens, or preferably about 90°.

[0055] In some embodiments, the reaction tube comprises a reservoir for accommodating a reaction mixture, for example a nucleic acid amplification reaction mixture, that is sized to accommodate a reaction mixture having a volume in a range of: from about less than 1 nanoliter to about 1 milliliter; from about 1 nL to about 10 nL; from about 1 nL to about 1 pL; from about 1 pL to about 1 mL; from about 1 pL to about 10 pL; from about 1 nL to about 1.5 nL; from about 2 nL to about 2.5 nL; from about 3 nL to about 3.5 nL; from about 4 nL to about 4.5 nL; from about 5 nL to about 5.5 nL; from about 6 nL to about 6.5 nL; from about 7 nL to about 7.5 nL; from about 8 nL to about 8.5 nL; from about 9 nL to about 9.5 nL; from about 10 nL to about 20 nL; from about 30 nL to about 40 nL; from about 50 nL to about 60 nL; from about 70 nL to about 80 nL; from about 90 nL to about 100 nL; from about 200 nL to about 300 nL; from about 400 nL to about 500 nL; from about 600 nL to about 700 nL; from about 800 nL to about 900 nL; from about 1 pL to about 10 pL; from about 20 pL to about 30 pL; from about 40 pL to about 50 pL; from about 60 pL to about 70 pL; from about 80 pL to about 90 pL; from about 100 pL to about 200 pL; from about 300 pL to about 400 pL; from about 500 pL to about 600 pL; from about 700 pL to about 800 pL; or from about 900 pL to about 1 mL or more. In some embodiments, the reservoir for the reaction mixture is sized to contain a volume in the range of: from less than about 1 nanoliter to about 1 nL; from about 1.5 nL to about 2 nL; from about 2.5 nL to about 3 nL;from about 3.5 nL to about 4 nL; from about 4.5 nL to about 5 nL; from about 5.5 nL to about 6 nL; from about 6.5 nL to about 7 nL; from about 7.5 nL to about 8 nL; from about 8.5 nL to about 9 nL; from about 9.5 nL to about 10 nL; from about 20 nL to about 30 nL; from about 40 nL to about 50 nL; from about 60 nL to about 70 nL; from about 80 nL to about 90 nL; from about 100 nL to about 200 nL; from about 300 nL to about 400 nL; from about 500 nL to about 600 nL; from about 700 nL to about 800 nL; from about 900 nL to about 1 pL; from about 10 pL to about 20 pL; from about 30 pL to about 40 pL; from about 50 pL to about 60 pL; from about 70 pL to about 80 pL; from about 90 pL it to about 100 pL; from about 200 pL to about 300 pL; from about 400 pL to about 500 pL; from about 600 pL to about 700 pL; from about 800 pL to about 900 pL; or from about 1 mL to more than about 1 mL.

[0056] In some embodiments, walls of the reaction tube comprise an inner surface.

[0057] In some embodiments, walls of the reaction tube terminate at a top edge that defines an opening to the reaction reservoir of the reaction tube. The top edge optionally comprises a lip. In some embodiments, the optional lip extends outward, for example to provide an operator with something to grasp.

[0058] In some embodiments, the reaction tube additionally comprises a cap or a lid, which is fixedly or removably attached to the reaction tube and which is for closing or sealing the reaction reservoir of the reaction tube. In some embodiments, the cap or lid can be engaged within the opening defined by the top edge thereby sealing the reaction reservoir.

[0059] In some embodiments, the reaction tube or cartridge further comprises an upper element including a receiving reservoir for receiving a sample and / or one or more reagents. The upper element can be generally in the form of an open-ended funnel or cylinder that is fixedly or removably attached at an opening to a reaction reservoir of the reaction tube to provide fluid communication between the receiving reservoir and the reaction reservoir. In some embodiments there is a temporary and removable barrier between the receiving reservoir and the reaction reservoir. Such a barrier can be disrupted to allow passage of a sample and / or one or more reagent from the receiving reservoir into the reaction reservoir

[0060] In some embodiments, the reaction tube is in whole or in part made from plastic, glass, natural polymers, synthetic polymers, metal, or combinations thereof. In some embodiments, the reaction tube is made of any material suitable for conditions for nucleic acid amplification reactions, such as nucleic acid amplification reaction mixtures, chemical reagents and / or thermal cycling, such as, a thermally stable plastic. In some embodiments, the reaction tube is made, at least in part, of polypropylene.

[0061] In some embodiments, the reaction tube is for use in a thermal cycler having a thermal well with openings for light transmission. In some embodiments, a thermal well configured to conform to a side wall of a reaction reservoir allows for a more efficient transfer of heat to the reaction reservoir.

[0062] Figures 1 to 3 depict illustrative, non-limiting examples of the reaction tube of the present application.

[0063] Figure 1 is a schematic structural diagram of a reaction tube 10 according to one embodiment of the present application. As shown in Figure 1, reaction tube 10 includes a reaction reservoir 20 formed by connecting side walls 22, 24, 26, and 28 and bottom wall 30, and having an upper open end 40. In some embodiments, reaction reservoir 20 has a rectangular pyramidal or square pyramidal shape truncated by bottom wall 30. All or a portion of bottom wall 30 forms lens 32. At least one of side walls 22, 24, 26, and 28 is formed of or comprises an optically transmissive material.

[0064] Reaction tube 10 can be closed by a cap or lid (not shown) that mates with upper open end 40. The cap or lid can be attached to reaction tube 10 or can be a separate element. In some embodiments, the cap or lid seals a nucleic acid amplification reaction mixture in reaction reservoir 20.

[0065] In one embodiment, light from a light source (not shown) that passes through the at least one optically transmissive side wall can be used to excite a reaction mixture within reaction reservoir 20 and the resulting emission light can then be collimated by lens 32 and detected by a photodetector (not shown), for example, in performing real-time PCR.

[0066] In another embodiment, light from a light source (not shown) is collimated by lens 32 and can be used to excite a reaction mixture within reaction reservoir 20. The resulting emission light then exits through the at least one optically transmissive side wall and is detected by a photodetector (not shown), for example, in performing real-time PCR.

[0067] Figure 2 is a schematic structural diagram of a reaction tube 100 according to another embodiment of the present application. Figure 3 is a cross-section of reaction tube 100. As shown in Figures 2 and 3, reaction tube 100 includes a reaction reservoir 120 formed by four connecting side walls 122 (not shown), 124, 126, and 128 and bottom wall 130. Reaction tube 100 further includes receiving reservoir 150 for receiving a sample and / or reagent(s). Receiving reservoir 150 is removably or fixedly attached to upper open end 140 of reaction reservoir 120.

[0068] In the embodiments in which receiving reservoir 150 is removably attached to upper open end 140 of reaction reservoir 120, receiving reservoir 150 can be removed following addition of a sample and / or reagent(s) to allow a reaction mixture to be sealed within reaction reservoir 120 with a cap or lid (not shown).

[0069] In the embodiments in which receiving reservoir 150 is fixedly attached to upper open end 140 of reaction reservoir 120, a cap or a lid can be used to seal top, open end 155 of receiving reservoir 150. Alternatively, a plug-type element (not shown) can be matingly received in receiving reservoir 150 and thereby allow a reaction mixture to be sealed within reaction reservoir 120.

[0070] In some embodiments, reaction reservoir 120 has a rectangular pyramidal or square pyramidal shape truncated by bottom wall 130. All or a portion of bottom wall 130 forms lens 132. At least one of side walls 122, 124, 126, and 128 is formed of or comprises an optically transmissive material.

[0071] In one embodiment, light from a light source (not shown) that passes through the at least one optically transmissive side wall can be used to excite a reaction mixture within reaction reservoir 120 and the resulting emission light can then be collimated by lens 132 and detected by a photodetector (not shown), for example, in performing real-time PCR.

[0072] In another embodiment, light from a light source (not shown) is collimated by lens 132 and can be used to excite a reaction mixture within reaction reservoir 120. The resulting emission light then exits through the at least one optically transmissive side wall and is detected by a photodetector (not shown), for example, in performing real-time PCR.

[0073] Optical Detection System

[0074] The present application further provides a system for optical detection of a reaction. The system comprises a reaction tube comprising a reaction reservoir for a reaction mixture, the reservoir having a bottom portion comprising a closed, molded lens and side walls, wherein at least one of the side walls comprises or consists of a flat, optically transmissive material. The system further comprises an excitation light source and a photodetector.

[0075] In one embodiment, excitation light from the light source enters the reaction reservoir through the flat, optically transmissive material of one of the side walls of the reaction tube and light generated by excitation of the reaction mixture is collimated by the molded lens that is part of the reaction tube and the photodetector detects the emitted light collimated by the molded lens.

[0076] In another embodiment, excitation light from the light source is collimated by the lens as it enters the reaction reservoir of the reaction tube. Light generated by excitation of the reaction mixture passes through the flat, optically transmissive material of one of the side walls of the reaction tube and is detected by the photodetector.

[0077] The present reaction tube and system for optical detection have one or more of the following, non-limiting, advantages.

[0078] A reaction tube as described herein, with a molded lens, can be relatively simple to manufacture by injection molding, especially in comparison to a reaction tube having a flat, optically-clear bottom.

[0079] A reaction tube as described herein, having a molded lens at the bottom and one side comprising or consisting of a flat, optically transmissive (e.g., optically transparent) material can be easier to manufacture than a tube requiring two flat, optically transmissive sides.

[0080] Incorporating a lens in the reaction tube minimizes light reflections and refractions and improves the signal-to-noise ratio in comparison to a standard conical reaction tube. This is true if the excitation light source has a lens in front of it, because light passing through the lens will be reflected and refracted by the bottom curvature (or side curvature) of the standard conical reaction tube.

[0081] There is no requirement in the present system for a lens in front of the excitation light source as part of the detection system because the lens is incorporated into the reaction tube. This saves cost and reduces complexity for this component.

[0082] The overall optical detection system is relatively simple to manufacture and, therefore, can be easier and more inexpensive to manufacture than previous optical detection systems.

[0083] Method for Monitoring a Reaction

[0084] Among other things, the present application further provides a method for monitoring a reaction (e.g., an isothermal reaction or a non-isothermal reaction) using optical detection, for example, optical detection of products or reactants.

[0085] In some embodiments, the method for monitoring a reaction comprises mixing reagents in a reaction tube as described herein, to form a reaction mixture and optically interrogating the reaction mixture using an optical detection system to detect reactants and / or products of the reaction.

[0086] In some embodiments, the method is for monitoring a nucleic acid amplification reaction by detecting amplicons or amplicon molecules generated by the amplification reaction. In some embodiments, a method is for nucleic acid amplification of a target sequence (e.g., a target nucleic acid sequence) or combination of target sequences.

[0087] In some embodiments the reaction to be monitored is a non-isothermal reaction (e.g., a non-isothermal nucleic acid amplification reaction), and the method comprises providing an apparatus for receiving the present reaction tube and thermally controlling and optically interrogating a reaction mixture, e.g., a sample mixed with one or more chemicals or reagents, within the reaction reservoir of the reaction tube.

[0088] In some embodiments, a non-isothermal reaction is for detection of the presence, absence, and / or amount of a target nucleic acid sequence in a sample comprising one or more nucleic acid sequences. In some embodiments, an amplification reaction can detect the presence, absence, and / or amount of two, three, four, five, six, or more target nucleic acid sequences.

[0089] Non-isothermal amplification reactions for detection of the presence, absence, and / or amount of a target nucleic acid are advantageous over isothermal amplification reactions as cycling temperatures in non-isothermal amplification reactions provide for predictable estimators of amplification progress. For non-isothermal reactions, each cycle of heating and cooling provides for a predictable degree or amount of nucleic acid amplification.

[0090] The sample may also be mixed with diluents or buffers in the reaction tube. The sample may be an aqueous solution containing particles, cells, microorganisms, ions, or small and large molecules, such as proteins and nucleic acids, etc. In a particular use, the sample may be a bodily fluid (e.g., blood, urine, saliva, sputum, vaginal fluids and / or secretions, seminal fluid, cerebrospinal fluid, mucus, or other bodily fluids), faeces, or an extraction or component thereof. Alternatively, the sample may be a solid or tissue made soluble in a liquid or the sample may be an environmental sample (e.g., pool water, water from a body of water such as a lake, an ocean, or a river, well water, ground or wastewater, soil extracts, pesticide residues, or airborne spores placed in a liquid).

[0091] In certain embodiments, a sample is from an organism. An organism can be, for example, a human, a non-human animal, a micro-organism, or a plant.

[0092] In some embodiments, the apparatus includes a heat-exchanging module or thermal well into which a reaction tube is inserted for thermal processing and optical detection. A heat-exchanging module can include one or more heating or cooling elements for heating or cooling surfaces of a receptacle that are in contact the reaction tube, and optics for optically interrogating a reaction mixture contained in the reaction tube. Optics of the apparatus can include a light source for providing excitation light to the reaction mixture in the reaction tube (through one of the lens or the flat, optically transmissive side wall) and aphotodetector for detection of light emitted (through the other of the lens or the flat, optically transmissive side wall) from the reaction mixture following excitation. Light emission is used to detect and / or quantify target reaction products (e.g., amplicons) or reactants. An increase in products or a decrease in reactants can be used as an indicator of reactions progression. The apparatus may also include a controller, such as a personal computer or network computer, that provides a user interface to the apparatus and controls the operation of the apparatus.

[0093] The apparatus optionally includes a base unit with processing electronics for receiving a plurality of such heat-exchanging modules and for independently controlling each module.

[0094] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill of those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication, patent, or patent applications was specifically and individually indicated to be incorporated by reference.

[0095] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

WE CLAIM:

1. A reaction tube comprising: a. a bottom wall comprising a closed, molded lens; b. four side walls joined to the bottom portion and forming a reservoir for accommodating a reaction mixture; and c. an open upper portion for receiving a cap, wherein one of the four side walls comprises or consists of a flat, optically transmissive side wall.

2. The reaction tube according to claim 1, wherein the optically transmissive side wall is angularly offset from the closed, molded lens by an angle of from approximately 90° to approximately 120°, preferably about 90°.

3. The reaction tube according to claim 1 or 2, wherein the four side walls consist of the flat, optically transmissive side wall, an opposing side wall having the same or approximately the same dimensions as the flat, optically transmissive side wall and two connecting side walls.

4. The reaction tube according to claim 3, wherein the four side walls define a square or rectangular pyramidal shape truncated by the bottom wall to form the reservoir for accommodating the reaction mixture.

5. The reaction tube according to any one of claims 1 to 4, wherein the four side walls terminate at a top edge that defines an opening of the open upper portion and the opening is for receiving the cap.

6. The reaction tube according to any one of claims 1 to 4, wherein the four side walls terminate at a top edge that defines an opening, the open upper portion comprises a receiving reservoir connected to the top edge and in fluid communication with the reservoir for accommodating the reaction mixture.

7. The reaction tube according to any one of claims 1 to 6, wherein the reaction tube is formed of a thermally stable plastic.

8. An optical detection system comprising: a. a reaction tube as defined in any one of claims 1 to 7; b. a light source for providing excitation light to excite the reaction mixture; and c. a photodetector for detecting light emitted from the reaction mixture.

9. The optical detection system according to claim 8, wherein the excitation light passes through the flat, optically transmissive side wall and the light emitted is collimated through the lens to the photodetector.

10. The optical detection system according to claim 8, wherein the excitation light is collimated by the lens and into the reaction mixture and the light emitted passes through the flat, optically transmissive side wall to the photodetector.

11. A method for monitoring a reaction comprising: a. mixing reactants in a reaction tube according to any one of claims 1 to 7 to form a reaction mixture; b. inserting the reaction tube into a receptacle in an optical detection apparatus, wherein said optical detection apparatus comprises a light source and a photodetector and wherein the reaction tube is positioned in the receptacle to allow light from the light source to pass through the optically transmissive side wall or through the lens; c. illuminating the reaction mixture with light from the light source; and d. measuring, using the photodetector, light emitted from the reaction mixture through the optically transmissive side wall or the molded lens, wherein the measured light emitted is used to detect or quantify one or more products of the reaction and / or one or more reactants of the reaction.

12. The method according to claim 11, wherein the reaction is a nucleic acid amplification reaction and the measured light emitted is used to detect or quantify one or more target amplicons.

13. The method according to claim 12, wherein the nucleic acid amplification reaction is a non-isothermal amplification reaction.

14. The method according to claim 13, wherein the optical detection apparatus further comprises a heat-exchanging module for thermally controlling the reaction mixture when the reaction tube is in the receptacle.

15. The method according to any one of claims 11 to 14, wherein the reaction tube is positioned in the receptacle to allow light from the light source to pass through the optically transmissive side wall and the method comprises measuring, using the photodetector, light emitted from the reaction mixture through the molded lens.

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