Control of the nucleic acid amplification process

Abstract

Methods, systems, and computer-readable media for monitoring fluorescent signals from nucleic acid amplification reactions for the detection of abnormal fluorescence. This specification discloses materials, methods, and systems for detecting abnormal fluorescence detection. Methods disclosed herein can detect abnormal fluorescence detection that may occur during nucleic acid amplification, for example, due to equipment failure. Exemplary methods include determining fluorescence-based values ​​from a fluorophore-containing nucleic acid amplification reaction mixture, and determining whether those values ​​meet a predetermined threshold and / or range, or whether they indicate abnormality.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of European Patent Application No. 23315136.4 filed on April 28, 2023, which is incorporated herein by reference for all purposes.

[0002] Field[[ID= (12)]] The present disclosure relates to the field of nucleic acid amplification. More particularly, the present disclosure relates to methods, materials, devices, and systems for monitoring fluorescence signals from nucleic acid amplification reactions for the detection of abnormal fluorescence.

[0003] Introduction Nucleic acid amplification reactions are widely used in research and clinical laboratories for the detection of genetic disorders and infectious diseases. Nucleic acid amplification reactions utilize enzymes such as polymerase or ligase, either separately or in combination, to generate multiple copies of a target nucleic acid sequence in a primer extension reaction that incorporates nucleotides or by ligation of adjacent probes complementary to the target nucleic acid sequence. In such reactions, each template generates more copies, and the copies themselves can serve as templates. The nucleic acid copies are called "amplicons". The production of amplicons can be tracked in real - time using fluorescence. For example, probe oligonucleotides containing a quencher and a fluorophore can be configured in an amplicon - dependent manner to undergo cleavage, such as in the TaqMan® assay, or a conformational change, such as in a molecular torch. The cleavage or conformational change reduces or eliminates quenching of the fluorophore, thereby increasing fluorescence, which functions as a read - out of amplicon production.

[0004] Fluorescence-based detection methods require the excitation of fluorophores and the detection of emitted fluorescence. Failure of either the light source responsible for excitation or the fluorescence detector responsible for detecting emitted light can occur during nucleic acid amplification. Such anomalies can lead to unreliable or inconsistent results, such as false negatives, and wasted reagents. Existing amplification assays may not adequately control such anomalies and failure modes. Therefore, improved nucleic acid amplification process control is needed.

[0005] This specification discloses materials, methods, and systems for detecting anomalies in fluorescence detection. The methods disclosed herein can detect anomaly fluorescence detection that may occur during nucleic acid amplification, for example, due to equipment failure. This disclosure is partly based on the finding that the fluorescence intensity measured over time from a nucleic acid amplification reaction does not fall below a threshold level when fluorescence detection is functioning correctly. This allows for the setting of a predetermined threshold, and the fluorescence intensity measured from the nucleic acid amplification reaction to be compared to this threshold. Fluorescence intensity measured from a reaction below the threshold results in the detection of an anomaly in fluorescence detection. [Overview of the Initiative]

[0006] overview The following embodiments are among those provided in this disclosure.

[0007] Embodiment 1 is a method for monitoring a fluorescence signal from a nucleic acid amplification reaction for abnormal fluorescence detection, a) Measure the fluorescence signal from the nucleic acid amplification reaction at multiple time points during the period in which the nucleic acid amplification reaction is occurring. b) Determining multiple fluorescence intensities for each or some of multiple time points from the measured fluorescence signal, c) A method comprising comparing each of a plurality of fluorescence intensities with a predetermined threshold, wherein abnormal fluorescence detection is detected when the fluorescence intensity falls below the predetermined threshold.

[0008] Embodiment 2 is a nucleic acid amplification system comprising a docking station configured to accept a container comprising at least one nucleic acid amplification reaction chamber, a fluorescence detector configured to measure fluorescence from a nucleic acid amplification reaction in the nucleic acid reaction chamber, and a processor operably coupled to the fluorescence detector and memory, the memory including instructions, when executed by the processor, causing the nucleic acid amplification system to perform a method of monitoring the fluorescence signal from the nucleic acid amplification reaction for abnormal fluorescence detection, the method being: a) Measure the fluorescence signal from the nucleic acid amplification reaction at multiple time points during the period in which the nucleic acid amplification reaction is occurring. b) Determining multiple fluorescence intensities for each or some of multiple time points from the measured fluorescence signal, c) A nucleic acid amplification system that includes comparing each of a plurality of fluorescence intensities with a predetermined threshold, wherein abnormal fluorescence detection is detected when the fluorescence intensity falls below the predetermined threshold.

[0009] Embodiment 3 includes a computer-readable medium, which, when executed by the processor of a nucleic acid amplification system, includes instructions causing the nucleic acid amplification system to perform a method for monitoring a fluorescence signal from a nucleic acid amplification reaction for abnormal fluorescence detection, wherein the method is a) Measure the fluorescence signal from the nucleic acid amplification reaction at multiple time points during the period in which the nucleic acid amplification reaction is occurring. b) Determining multiple fluorescence intensities for each or some of multiple time points from the measured fluorescence signal, c) A computer-readable medium that includes comparing each of a plurality of fluorescence intensities with a predetermined threshold, wherein abnormal fluorescence detection is detected when the fluorescence intensity falls below the predetermined threshold.

[0010] Embodiment 4 is a method, system, or computer-readable medium of any one of the prior embodiments, wherein the nucleic acid amplification reaction is a thermal cycle nucleic acid amplification reaction.

[0011] Embodiment 5 is the method, system, or computer-readable medium of the preceding embodiment, wherein the period begins after the start of thermal cycling or after the first thermal cycle.

[0012] Embodiment 6 is a method, system, or computer-readable medium of any of the preceding embodiments in which multiple fluorescence intensities are determined by smoothing fluorescence signals measured at multiple time points.

[0013] Embodiment 7 is a method, system, or computer-readable medium of any one of the preceding embodiments in which multiple fluorescence intensities are determined as a moving average or moving median of fluorescence signals measured at multiple time points.

[0014] Embodiment 8 is a method, system, or computer-readable medium of any of the preceding embodiments in which the fluorescence signal is measured by a fluorescence detector, and optionally the fluorescence detector includes a photomultiplier tube or a photodiode.

[0015] Embodiment 9 is a method, system, or computer-readable medium of the preceding embodiment in which a defect in the fluorescence detector is detected when the fluorescence intensity falls below a predetermined threshold.

[0016] Embodiment 10 is a method, system, or computer-readable medium of any of the prior embodiments, in which a nucleic acid amplification reaction is housed in a microfluidic cartridge.

[0017] Embodiment 11 is a microfluidic cartridge, a) Multiple functional areas including a sample preparation area, a nucleic acid amplification area, and a waste disposal area, b) Central distribution hub, and c) comprising a pump, multiple valves, and a microchannel fluid network connecting the functional areas to a hub, A fluid network of pumps, multiple valves, and microchannels can drive the movement of fluid from a first functional area through a central distribution hub to a second functional area among multiple functional areas, and the nucleic acid amplification reaction is the method, system, or computer-readable medium of the immediately preceding embodiment that is within the nucleic acid amplification area.

[0018] Embodiment 12 is the method, system, or computer-readable medium of any one of the preceding embodiments, where a fluorescence signal is measured from multiple nucleic acid amplification reactions in step a).

[0019] Embodiment 13 is the method, system, or computer-readable medium of the immediately preceding embodiment, where step b) includes determining multiple fluorescence intensities for each or some of multiple time points from the fluorescence signals measured for each of the multiple nucleic acid amplification reactions.

[0020] Embodiment 14 is the method, system, or computer-readable medium of the immediately preceding embodiment, where step c) includes comparing each of the multiple fluorescence intensities with a predetermined threshold for each of the multiple fluorescence intensities corresponding to the multiple nucleic acid amplification reactions.

[0021] Embodiment 15 is the method, system, or computer-readable medium of any one of Embodiments 12 to 14, where multiple fluorophore-containing nucleic acid amplification reactions are included in a multi-well plate or multiple tubes.

[0022] Embodiment 16 is the method, system, or computer-readable medium of any one of the preceding embodiments, where the nucleic acid amplification reaction includes a fluorophore.

[0023] Embodiment 17 is the method, system, or computer-readable medium of the immediately preceding embodiment, where the fluorophore associates with an oligonucleotide probe, and optionally, the oligonucleotide probe further includes a quencher.

[0024] Embodiment 18 is any one of the methods, systems or computer-readable media of Embodiments 1 to 15, in which the nucleic acid amplification reaction does not contain a fluorophore associated with an oligonucleotide, and a fluorescence signal including intrinsic fluorescence from one or more reagents or templates in the nucleic acid amplification reaction is measured, and optionally, the one or more reagents include dNTPs and / or one or more primers.

[0025] Embodiment 19 is any one of the methods of Embodiment 1 or 4 to 18, in which the fluorescence intensity is below a predetermined threshold and abnormal fluorescence detection is detected.

[0026] Embodiment 20 is the method of Embodiment 19, further including aborting the nucleic acid amplification reaction after detecting the abnormal fluorescence detection.

[0027] Embodiment 21 is any one of the methods of Embodiment 1 or 4 to 18, in which each of the plurality of fluorescence intensities exceeds a predetermined threshold and abnormal fluorescence detection is not detected.

[0028] Embodiment 22 is any one of the systems of Embodiment 2 or 4 to to 17, in which the system is configured to abort the nucleic acid amplification reaction when abnormal fluorescence detection is detected.

[0029] Embodiment 23 is any one of the systems or computer-readable media of Embodiments 2 to 17 or 22, or the method further includes aborting the nucleic acid amplification reaction when an abnormality is detected.

[0030] Additional objectives and advantages will be partially described in the following description, partially understood from the description, or learned through practice. The objectives and advantages will be realized and achieved by the elements and combinations particularly pointed out in the appended claims.

[0031] It should be understood that both the foregoing general description and the following detailed description are merely exemplary and explanatory and do not limit the claims. [Brief explanation of the drawing]

[0032] Brief explanation of the drawing [Figure 1] Figure 1 is an exemplary graph of fluorescence over time from a nucleic acid amplification reaction. Under normal qPCR conditions, non-null fluorescence intensity (relative fluorescence units, measured in RFUs) is provided. The LED, the light source responsible for fluorescence excitation, was switched off for a specified time to simulate an anomaly such as a failure of the light source or detector. The measured fluorescence intensity was substantially lower than the fluorescence intensity measured before the LED was switched off. Relative fluorescence units (signal; RFU; y-axis) are plotted against time (seconds on the x-axis).

[0033] [Figure 2] Figure 2 shows an exemplary distribution of minimum fluorescence intensity (determined as a 5-point average of individual measurements) for two qPCR channels from multiple reactions under normal and various abnormal conditions. Each circle represents a fluorescence measurement (measured in RFU) observed in a single nucleic acid amplification run. The "NEG LED off" condition shows the result when the LED light source was inoperable during the run and represents abnormal fluorescence detection. To distinguish abnormal fluorescence detection from normal conditions and other anomalies unrelated to fluorescence detection, a threshold can be set for each channel, for example, at approximately 100 RFU (shown as a vertical line), as shown in the figure.

[0034] [Figure 3] Figure 3 shows an exemplary nucleic acid amplification system 300 with a microfluidic cartridge 601 loaded into a docking station 303. System 300 includes a heat sink 305, a fluorometer 306, a press 301, and a press / heater 304 used to interact with and measure the sample loaded in cartridge 601.

[0035] [Figure 4]Figure 4 is a side view of the nucleic acid amplification system 300, including the docking station 303 of Figure 3. The side view shows an optical fiber 403 extending from the cartridge 601 to the light source 307 (e.g., one or more LEDs) of the fluorometer 306, and a hole 404 formed in an aluminum block 402 for guiding the fluorescence from the cartridge 601 to a photodetector 401 used to perform fluorescence measurements, the photodetector 401 being a component of the fluorometer 306.

[0036] [Figure 5] Figure 5 shows a cross-sectional view of the nucleic acid amplification system 300, including the docking station 303 and microfluidic cartridge 601 shown in Figures 3 and 4. The cross-sectional view shows internal components such as a thermal cycler 508 including a thermal block 505, a Peltier module 506, and a heat sink 507, an array detector 511, and a rotary valve system 501 including a piston 502, a ball 503, and a cam 504 useful for moving and analyzing the liquid in different functional areas of the cartridge 601. The system 300 also includes a clamp 509 for securing a detection tip (not shown) and an illuminator 510.

[0037] [Figure 6-1]Figures 6A–6D show various diagrams of an exemplary microfluidic cartridge 601. Figure 6A shows a top perspective view of the microfluidic cartridge 601 containing multiple chambers for holding samples, reagents, or other liquids. Figure 6B shows a bottom perspective view of an exemplary microfluidic cartridge 601, showing the fluid network of microchannels 606 connecting the various chambers to other areas within the cartridge 601. For example, chambers 602, 603, 604, and 605 may be the sample chamber 602, the weighing chamber 603, the waste chamber 604, and the PCR chamber 605. Figure 6C shows a bottom perspective view of a fully constructed microfluidic cartridge 601, including a bottom cover 613 and a microarray slide 614, as shown in Figure 6D, which include a sample preparation area 611, a nucleic acid amplification area 610, and a nucleic acid analysis area 609. The nucleic acid amplification area 610 may also constitute a nucleic acid detection area capable of detecting signals from real-time PCR reactions. These functional areas are connected by a series of microchannels 606, which are shown in detail in Figure 6B. The sample preparation area 611 contains liquids in adjacent chambers. The chambers within the sample preparation area 611 are arranged around a central distribution hub 608. The system can be programmed to combine the liquids contained in these chambers in separate volumes in a specific order. When docked to the system, the nucleic acid amplification area 610 is in close proximity to the thermal cycler 508 in Figure 5. Similarly, when docked to the system, the nucleic acid analysis area 609 is in close proximity to the array detector 511, as shown in Figure 5. Figure 6D shows an exploded view of a fully constructed exemplary microfluidic cartridge 601, which includes a cartridge body 612, a vent cap 623 for covering the sample after input, a sample filter 624, and components for the cartridge 601 to interact with the system's mechanical components to move and combine the liquids in the sample preparation area 611, including a stopper 618, a nucleic acid binding matrix column 619, a protective cover 621, a blocker 622, and a blocker ring 625.The exploded view also shows how the microarray slide 614 is attached to the cartridge body 612 via microarray adhesive tape 615 in the nucleic acid amplification analysis area 609. The cartridge 601 also includes polypropylene (PP) coverings 613, 616 on the bottom and top of each cartridge 601, and optionally includes a pre-printed sticker 617. [Figure 6-2] Same as above. [Figure 6-3] Same as above.

[0038] [Figure 7] Figure 7 shows an exploded view of an exemplary microfluidic cartridge 601 and plunger 701 of a nucleic acid amplification system, in which the plunger 701 interacts with blocker 622 to move and combine the liquid within the microfluidic cartridge 601. [Modes for carrying out the invention]

[0039] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art relating to the methods and compositions described herein. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety. If any definition in this section contradicts or is inconsistent with any definition in any patent, application, published application and other publication incorporated herein by reference, the definition in this section shall prevail over the definition incorporated herein by reference.

[0040] As used herein, “fluorescence detector” or “fluorescence sensor” refers to an optical detector capable of obtaining fluorescence measurements. Such measurements may be obtained before, during, and after nucleic acid amplification. The fluorescence detector may be a photodiode or a photomultiplier tube. The fluorescence detector receives light (fluorescence) emitted from a sample.

[0041] "Nucleic acids" and "polynucleotides" refer to multimeric compounds containing two or more covalently bonded nucleosides or nucleoside analogs or base analogs having nitrogen heterocyclic bases, where nucleosides are bonded together by phosphodiester bonds or other bonds to form polynucleotides. Nucleic acids include RNA, DNA, and combinations and analogs thereof such as "peptide nucleic acids" or PNAs (see, e.g., WO95 / 32305) and "locked nucleic acids" (LNAs), where one or more nucleotide monomers have a bicyclic furanose unit locked to RNA mimicking sugar conformation (see, e.g., Vester et al., Biochemistry 43:13233-41, 2004). Nitrogen bases include conventional bases (A, G, C, T, U), purine or pyrimidine base derivatives (e.g., N 4 These may include analogs such as methyl-9-deoxyguanosine, deaza- or aza-purine, deaza- or aza-pyrimidine (see U.S. Patent No. 5,378,825, U.S. Patent No. 6,949,367, and International Publication No. 93 / 13121) (e.g., inosine, 5-methylisocytosine, isoguanine, e.g., The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992; Abraham et al., 2007, BioTechniques 43: 617-24), and / or "decayable" residues (e.g., see U.S. Patent No. 5,585,481).

[0042] As used herein, the “target” material is a material to be detected or quantified. The target material may be a nucleic acid. Other embodiments of the target material include cells, viruses, and other biomolecules. As used herein, the “target nucleic acid” is a nucleic acid containing a target sequence to be detected or quantified, for example, by amplification. The target nucleic acid or the target nucleic acid of interest may be DNA or RNA or a combination or analog thereof as described herein, and may be single-stranded or double-stranded. The target nucleic acid may contain other sequences besides the target sequence that may not be detected or quantified.

[0043] As used herein, the term “region” refers to a portion of a nucleic acid, which may be less than the entire nucleic acid. For example, the term “region” may be used to refer to a smaller target hybridize portion of an entire oligonucleotide. A particular oligonucleotide, such as a primer, may consist entirely of one region (e.g., a target hybridize region) or may contain multiple regions (e.g., a promoter sequence region and a target hybridize region).

[0044] "Sample" refers to any composition that may or is suspected to contain the target material. A sample may be a composite mixture of its components. Examples of samples include "biological samples" containing any tissue or material derived from living or dead mammals or organisms, including, for example, feces, blood, plasma, serum, blood cells, saliva, mucus, and cerebrospinal fluid. Samples may also include samples of cell culture components in vitro, including, for example, conditioned media resulting from the growth of cells and tissues in culture media. Samples may also include foods containing any material intended for or suitable for consumption, including solids, suspensions, emulsions, gels, and liquids (i.e., gelatin, milk, soups, beverages, ice cream, fruit smoothies, emulsified cheese dips, fruit purees, nut butters, processed and / or textured proteins, as well as bread, fruits, vegetables, and meat). Samples may also include water and aqueous solutions. Samples may be treated chemically, physically, or mechanically to disrupt tissue or cellular structures and release intracellular nucleic acids into solution. The sample can be processed to release nucleic acids into a solution containing enzymes, buffers, salts, detergents, etc.

[0045] The interchangeable terms “oligomer,” “oligo,” and “oligonucleotide” refer to nucleic acids generally having fewer than 1,000 nucleotides (nt), including polymers with a lower limit of about 5 nt residues and an upper limit of about 500–900 nt residues. In some embodiments, oligonucleotides are in a size range with a lower limit of about 12–15 nt and an upper limit of about 50–600 nt, while in other embodiments, they are in a range with a lower limit of about 15–20 nt and an upper limit of about 22–100 nt. Oligonucleotides can perform one or more of a variety of different functions, such as primers and / or promoters, detection probes, and capture oligomers.

[0046] A "primer," "amplifying oligonucleotide," or "oligonucleotide primer" refers to an oligonucleotide, generally containing a "target" binding moiety, that is designed to selectively hybridize with the corresponding primer binding site of a target nucleic acid flanking sequence or amplification product under appropriate stringency conditions, and to act as a starting point for the synthesis of a nucleotide sequence complementary to the corresponding polynucleotide template from its 3' end.

[0047] "Amplifying" refers to any known procedure for obtaining multiple copies of a target nucleic acid sequence or its complement or fragment. These multiple copies may be referred to as amplicons or amplification products. Known amplification methods include both thermal cycling and isothermal amplification methods. Polymerase chain reaction (PCR), replicase-mediated amplification, ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-associated amplification (e.g., transcription-mediated amplification (TMA) or NASBA) are non-exclusive examples of nucleic acid amplification methods. See, for example, U.S. Patents 4,868,105, 5,124,246, 5,130,238, 5,399,491, 5,437,990, 5,554,516, and 7,374,885, as well as PCT Publications 88 / 01302, 88 / 10315, and 95 / 03430 (TMA), U.S. Patent 4,786,600 (RCA), U.S. Patents 5,427,930, and 5,516,663 (LCR), as well as U.S. Patents 5,422,252, 5,547,861, and U.S. Patent 5,648,211 (SDA). See, for example, Compton, Nature 350:91-92, 1991; Malek et al., Methods Mol. Biol. 28:253-260, 1994 (NASBA). Briefly, PCR amplification uses DNA polymerase, primer pairs, and thermal cycling to synthesize multiple copies of two complementary strands from dsDNA or cDNA (see, for example, U.S. Patents 4,683,195, 4,683,202, and 4,800,159).

[0048] As used herein, the term “real-time amplification” refers to the amplification of a target nucleic acid monitored by real-time detection. Real-time PCR amplification includes, for example, what is commonly called TaqMan® PCR (see, e.g., Holland et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, 1991; and Livak et al., US Pat. No. 6,030,787). TaqMan® PCR is a type of real-time PCR that uses a nucleic acid probe complementary to the internal segment of the target DNA. The probe is labeled with two fluorescent moieties. The emission spectrum of one overlaps with the excitation spectrum of the other, resulting in the “quenching” of the first fluorophore by the second fluorophore.

[0049] As used herein, “thermal cycling” is a process of periodic heating and cooling of a nucleic acid amplification mixture to facilitate the amplification of nucleic acids, for example, through continuous denaturation by a thermally stable polymerase, primer annealing, and primer extension. In many examples, thermal cycling involves holding the reaction mixture at two or more different temperatures, each for a predetermined duration, and performing several cycles of those two or more temperatures to induce nucleic acid amplification. A nucleic acid amplification reaction mixture subjected to thermal cycling is referred to as a “thermal cycling reaction mixture.”

[0050] As used herein, “nucleic acid amplification system” refers to a device or apparatus that can be used to carry out nucleic acid amplification. In many examples, the nucleic acid amplification system includes a fluorescence detector, and fluorescence measurements can be obtained before, during, and after nucleic acid amplification. In many examples, the nucleic acid amplification system includes a temperature controller, which can provide or transfer heat to the nucleic acid amplification reaction mixture via one or more heating elements, which may include, for example, a heat block. The nucleic acid amplification system is typically programmable and can maintain a temperature over different time lengths. The nucleic acid amplification system may be configured to house one or more of a variety of reaction vessels, such as tubes, multiwell strips, multiwell plates, microfluidic chips, and microfluidic cartridges, which may contain one or more nucleic acid amplification mixtures.

[0051] As used herein, the terms “amplicon” or “amplification product” refer to nucleic acid molecules generated during an amplification procedure that are complementary to or homologous to the sequence contained within the target sequence. These terms may be used to refer to single-stranded amplification products, double-stranded amplification products, or one of the strands of a double-stranded amplification product.

[0052] "Complementary" means that the nucleotide sequences of similar regions of two single-stranded nucleic acids, or two different regions of the same single-stranded nucleic acid, have a nucleotide base composition that allows the single-stranded regions to hybridize together within a stable double-stranded hydrogen-bonded region under stringent hybridization or amplification conditions. Sequences that hybridize with each other may be fully or partially complementary to the target sequence intended by standard nucleic acid base pairing (e.g., G:C, A:T, or A:U pairing). "Well-complementary" means a sequence that can hybridize to another sequence by hydrogen bonding between a series of complementary bases, which may contain one or more residues that are complementary at each position in the sequence by standard base pairing, or debasic residues that are not complementary. Well-complementary sequence sequences are typically at least 80% or at least 90% complementary to the sequence in which the oligomer is intended to specifically hybridize. A sequence that is "sufficiently complementary" allows for stable hybridization between a nucleic acid oligomer and its target sequence under appropriate hybridization conditions, even if the sequences are not perfectly complementary. A nucleotide sequence is "perfectly" complementary if a sequence of nucleotides in one single-stranded region can form a series of "canonical" or "Watson-Crick" hydrogen bond base pairs with a similar sequence of nucleotides in the other single-stranded region, such that A pairs with U or T and C pairs with G (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ndSee sections §§1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, in particular §§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57 of the ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Appropriate hybridization conditions are well known in the art and can be predicted based on sequence composition or determined by using routine testing methods (see, for example, Sambrook et al., supra.).

[0053] As used herein, “label” or “detection label” refers to a moiety or compound that can detect or generate a detectable signal and is directly or indirectly linked to a molecule such as a probe. Direct labeling may occur via a bond or interaction that links the label to the molecule, including covalent or non-covalent interactions, e.g., hydrogen bonds, hydrophobic and ionic interactions, or the formation of chelates or coordination complexes. Indirect labeling may occur through the use of a crosslinking moiety or “linker,” e.g., a binding pair member, an antibody, or an additional oligomer, which is directly or indirectly labeled and can amplify a detectable signal. Examples of labels include any detectable moiety such as radionuclides, ligands (e.g., biotin, avidin), enzymes or enzyme substrates, reactive groups, or chromophores (e.g., dyes, particles, or beads that impart a detectable color), luminescent compounds (e.g., bioluminescent labels, phosphorescent labels, or chemiluminescent labels), or fluorophores. Common labels used with TaqMan® probes include fluorophores and quenchers. As used herein, “fluorophore” refers to any label whose presence can be detected by its fluorescence properties. As used herein, “quencher” refers to a portion that absorbs at least a portion of the intensity of fluorescence emission. Quenchers include fluorescent quenchers and dark quenchers (sometimes called non-fluorescent quenchers). Dark quenchers are substances that absorb excitation energy from fluorophores and dissipate that energy as heat, while fluorescent quenchers re-emit much of this energy as light. A fluorescent quencher is a portion that can absorb the fluorescence signal emitted from a fluorescence source at a first wavelength, typically a fluorophore, such as, for example, a nucleic acid dye associated with the double-stranded segment of a nucleic acid, and after absorbing sufficient fluorescence energy, the fluorescent quencher can emit fluorescence at a second wavelength characteristic of the quencher, a process called “fluorescence resonance energy transfer” or FRET.Examples of fluorophores include FAM, SYBR® Green, ATTO fluorescent labels, VIC, JOE, NED, Cy3, ROX, Texas Red, and Cy5 dyes (all available from numerous commercial sources). The synthesis and methods for attaching labels to nucleic acids and detecting the labels are known in the art (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2). nd (ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10; see U.S. Patents Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333). More than one label and more than one type of label may be present on a particular probe, or detection may be performed using a mixture of probes in which each probe is labeled with a compound that produces a different detectable signal (see, for example, U.S. Patents Nos. 6,180,340 and 6,350,579).

[0054] The terms "detection probe," "detection oligonucleotide," "detection oligomer," "probe oligomer," and "detection probe oligomer" are used interchangeably to refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a nucleic acid, such as an amplified nucleic acid, under conditions that facilitate hybridization enabling detection of the target sequence or amplified nucleic acid. Detection can be either direct (e.g., a probe that directly hybridizes to its target sequence) or indirect (e.g., a probe linked to its target via an intermediate molecular structure). The detection probe may be DNA, RNA, its analogues, or combinations thereof (e.g., a DNA / RNA chimera), and may or may not be labeled. The detection probe may further include alternative skeletal linkages, such as a 2'-O-methyl bond. The "target sequence" of a detection probe generally refers to a smaller nucleic acid sequence region within a larger nucleic acid sequence that specifically hybridizes to at least a portion of the probe oligomer by standard base pairing. The detection probe may include target-specific sequences and other sequences that contribute to the probe's three-dimensional conformation (see, for example, U.S. Patents 5,118,801, 5,312,728, 6,849,412, 6,835,542, 6,534,274, and 6,361,945, and U.S. Patent Application Publication 20060068417).

[0055] As used herein, "elution buffer" is a liquid suitable for separating nucleic acids from a solid support.

[0056] As used herein, “Master Mix Buffer,” “Master Mix,” or “Amplification Master Mix Buffer” includes amplification reagents and, if necessary, primers used to amplify the target nucleic acid, but does not include the sample to be amplified.

[0057] An "amplification reaction chamber," also called a "reaction chamber" or "reaction space," refers to the space in which the amplification of a target nucleic acid is carried out. Multiple amplification reaction chambers may be arranged parallel or substantially parallel to each other within various containers. Examples of containers that can accommodate multiple amplification reaction chambers include multiwell strips, multiwell plates, microfluidic chips, or microfluidic cartridges.

[0058] As used herein, the term “relative fluorescence unit” (“RFU”) is a unit of measurement for fluorescence intensity. RFUs vary depending on the characteristics of the detection instrument used for measurement and can be used as a measurement for comparing the relative intensity between a sample and a control.

[0059] As used herein in the context of data analysis, "smoothing" refers to techniques for reducing noise in data by adjusting the measurements to reduce or eliminate noise in the measurements, generally making them closer to the surrounding measurements. Smoothing creates an approximation function that attempts to capture important patterns in the data while excluding noise. Examples of smoothing include moving average / median, local regression (loess), low-pass filters, Savitzky-Golay smoothing filters, and / or Ramer-Douglas-Peucker algorithms.

[0060] As used herein, the term “substantially” may be synonymous with the term “essentially” and indicates that a process, reagent, component, or other element achieves the result or has properties that may result in a slight difference or deviation from the result or properties to which the materially identical but “substantially” applied result or properties are. For example, “substantially avoiding variation” may mean that the variation is about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% or less.

[0061] Where used herein, the term “approximately” refers to a number, whether explicitly stated or not, including integers, fractions, and percentages. When this term precedes a list of numbers or ranges, it modifies all values ​​or ranges. When applied to a measurement, the term includes the exact number modified by this term and the range of values ​​expected to be within experimental error. For example, “approximately 5°C” means “5°C” and a temperature range within experimental error, e.g., 5°C plus or minus (±) 20%, 5°C ± 15%, 5°C ± 10%, or 5°C ± 5%. The term has a similar meaning with respect to other parameters. For example, “approximately 5 minutes” means “5 minutes” and a time range within experimental error, e.g., 5 minutes plus or minus (±) 20%, 5 minutes ± 15%, 5 minutes ± 10%, or 5 minutes ± 5%. In some situations, the percentage of experimental error is implied or evident and does not need to be explicitly stated. In some situations, the percentage of experimental error is explicitly provided. The term "approximately" can be used to modify any measurable quantity, including quantities of time, temperature, volume, mass, weight, length, density, size, percentage, ratio, dose, frequency, pressure, speed, and intensity. In some cases, the term "approximately" may include a number rounded to the nearest significant figure.

[0062] References to numerical ranges in this specification (e.g., "X to Y", "from X to Y", or "between X and Y") include the endpoint defining the range and all values ​​that fall within that range.

[0063] The terms “a,” “an,” and “the” refer to multiple objects unless the context clearly indicates otherwise. For example, as used herein, “a nucleic acid” is understood to refer to one or more nucleic acids. Thus, the terms “a” (or “an”), “one or more,” and “at least one” are interchangeable herein.

[0064] "Or" is used in an inclusive sense, meaning it is equivalent to "and / or" unless the context clearly indicates otherwise.

[0065] Detailed explanation A method for detecting fluorescent signals from nucleic acid amplification reactions for the detection of anomalous fluorescence is disclosed herein. This method utilizes the presence of fluorophores, quenchers (substances that can influence fluorescence emission by static or dynamic quenching), and / or reagents having intrinsic fluorescence, such as dNTPs and primers, to detect anomalous fluorescence while monitoring the fluorescent signal from a nucleic acid amplification reaction. Typically, anomalous fluorescence detection correlates with failures of the light source and / or fluorescence detector. Anomalies can negatively impact the reliability of data collected from the reaction. Importantly, this method can be performed on many existing nucleic acid amplification systems, such as any other system equipped with a real-time nucleic acid amplification system and fluorescence detector, without requiring additional hardware. Alternatively, this method uses existing features of such systems to monitor the fluorescent signal for anomalous detection during a nucleic acid amplification reaction.

[0066] A. Detection labels and probes Detection labels (e.g., fluorophores) may be used in accordance with this disclosure. The nucleic acid amplification reaction mixture used in the methods of this disclosure may include a detection oligomer having a detection label (e.g., designed to hybridize to an amplicon) and / or amplification oligomers, such as forward primers and / or reverse primers. Generally, an amplification oligomer or detection oligomer having a detection label used in an amplification reaction includes at least (1) a region for specifically hybridizing to a region on a target nucleic acid sequence, and (2) a detection label. In some embodiments, the oligomer having a detection label is referred to as a detection probe.

[0067] Suitable fluorophores for use as detection labels may include compounds that emit a detectable light signal, such as fluorophores ("fluorescent dye compounds"). More than one label and more than one type of label may be present on a particular probe, or a mixture of probes in which each probe is labeled with a compound that produces a detectable signal may be used (see, for example, U.S. Patents 6,180,340 and 6,350,579). Labels may be attached to the probe by a variety of means, including covalent bonding, chelation, and ionic interactions, but preferably the labels are covalently bonded. Suitable fluorophores are well known in the art and include, for example, CAL Flour® Orange 560, CAL Flour® Red 610, FAM, or ATTO 490LS. In some embodiments, the fluorophores are temperature-sensitive fluorophores. In some embodiments, the temperature-sensitive fluorophores are sulforhodamines. In embodiments including fluorophore-labeled detection probes, each detection probe further includes a quencher. Suitable quenchers are well known in the art and include, for example, BHQ, TAMRA, and DABCLY. In other embodiments, the detection probe includes both a fluorescent label and a quencher, and this combination is particularly useful in fluorescence resonance energy transfer (FRET) assays. Specific variations of such detection probes include, for example, the TaqMan® detection probe (Roche Molecular Diagnostics), “Molecular Beacon” (see, e.g., Tyagi et al., Nature Biotechnol. 16:49-53, 1998; U.S. Patents 5,118,801 and 5,312,728), and “Molecular Torch” (see, e.g., U.S. Patents 6,849,412, 6,835,542, 6,534,274, and 6,361,945). In some embodiments, a fluorophore in a nucleic acid amplification reaction mixture associates with a probe further comprising a quencher.

[0068] B. Reaction mixture In some embodiments, the reaction mixture used in the method or system described herein comprises one or more amplification oligomers for amplifying a target nucleic acid. In some embodiments, the amplification oligomer comprises a detection label. The reaction mixture typically comprises a buffer, a salt solution, suitable nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP), and / or other reagents suitable for carrying out in vitro amplification, such as enzymes (e.g., DNA polymerase, reverse transcriptase, and RNA polymerase), and may comprise a test sample component in which an internal control (IC) target nucleic acid may be present.

[0069] C. Reaction chamber, sample Various reaction vessels may be used to accommodate the nucleic acid amplification reactions used in accordance with this disclosure. The reaction vessel may contain one reaction chamber or multiple reaction chambers. In some embodiments, the steps of the methods disclosed herein are carried out using a nucleic acid reaction chamber. In some embodiments, the steps of the methods disclosed herein are carried out using an empty nucleic acid reaction chamber or using a nucleic acid reaction chamber containing a nucleic acid amplification reaction mixture. In some embodiments, the nucleic acid amplification reaction mixture containing multiple fluorophores is contained in multiple wells or multiple tubes.

[0070] A nucleic acid reaction chamber can be configured as a variety of reaction vessels, each containing multiple reaction chambers. In some embodiments, the reaction vessel includes multiple empty reaction chambers and / or reaction chambers containing nucleic acid amplification reaction mixtures. In some embodiments, the vessel is a multi-chamber receptacle. In some embodiments, the nucleic acid amplification chambers are housed within the multi-chamber receptacle. Non-limiting examples of vessels include multi-well strips, multi-well plates, microfluidic chips, and microfluidic cartridges. In some embodiments, multiple nucleic acid amplification reaction mixtures are contained in a multi-well plate or multiple tubes.

[0071] In some embodiments, at least one nucleic acid amplification reaction is contained within a multiwell strip, multiwell plate, microfluidic chip, or microfluidic cartridge. In some embodiments, multiple fluorophore-containing nucleic acid amplification reactions are contained within a multiwell plate or multiple tubes. In some embodiments, the reaction vessel is a microfluidic cartridge. In some embodiments, the microfluidic cartridge is also referred to as a “lab-on-a-chip” and can carry out a complete nucleic acid analysis of a sample, from sample collection to nucleic acid amplification and reading of the results(s). Exemplary microfluidic cartridges that can be used to carry out the steps of the methods disclosed herein are shown in Figures 6A–6D. Figure 6A shows a top perspective view of an exemplary microfluidic cartridge 601 containing multiple chambers for containing a sample, reagent, or other liquid. Microchannels 606 connect such chambers to move liquid between different functional areas within the cartridge 601. Figure 6B shows a bottom perspective view of an exemplary microfluidic cartridge 601 showing a network of microchannels 606 connecting the various chambers to other areas within the cartridge 601. Figure 6C shows a bottom perspective view of a fully constructed microfluidic cartridge 601, including a bottom cover 613 and a microarray slide 614 as shown in Figure 6D, which include a sample preparation area 611, a nucleic acid amplification area 610, and a nucleic acid analysis area 609. The nucleic acid amplification area may also constitute a nucleic acid detection area capable of detecting signals from real-time PCR reactions. These functional areas are connected by a series of microchannels 606, which are shown in detail in Figure 6B. The sample preparation area 611 contains liquid in an adjacent chamber. The system, e.g., nucleic acid amplification system 300, can be programmed to combine the liquids contained in these chambers in a specific order and in separate volumes. When the exemplary microfluidic cartridge 601 is docked to the system, the nucleic acid amplification area 610 is adjacent to the thermal cycler 508, as shown in Figure 5.Similarly, when the exemplary microfluidic cartridge 601 is docked to the system, the nucleic acid analysis area 609 is in close proximity to the array detector 511, as shown in Figure 5. Figure 6D shows an exploded view of a fully constructed exemplary microfluidic cartridge 601, which includes the cartridge body 612, a vent cap 623 for covering the sample after input, and a sample filter 624 for removing cell debris after lysis, which is performed either before sample addition and / or in the sample reservoir 602. The cartridge further includes components for moving and combining the liquid in the sample preparation area 611 by interacting with the mechanical components of the system, including a stopper 618, a nucleic acid binding matrix column 619, a protective cover 621, a blocker 622, and a blocker ring 625. The exploded view in Figure 6D also shows how the microarray slide 614 is attached to the body 612 of the microfluidic cartridge 601 in the nucleic acid analysis area 609 via the microarray adhesive tape 615. Cartridge 601 also includes top and bottom coverings 613 and 616 made of polypropylene (PP), respectively, and optionally includes a pre-printed sticker 617. Figure 7 shows an exploded view of an exemplary microfluidic cartridge 601, which includes a plunger 701 for moving and combining the liquid within the microfluidic cartridge 601 by engaging with a stopper 618. Microfluidic cartridges have several advantages, including the ability to perform automated operations while consuming small reagent volumes, being inexpensive and disposable. An example of a microfluidic cartridge is also disclosed in U.S. Patent No. 10,654,039.

[0072] In some embodiments, the microfluidic cartridge 601 includes at least a) a plurality of functional areas including a sample preparation area 611, a nucleic acid amplification area 610, a nucleic acid analysis area 609, and a waste area 604; b) a central distribution hub 608; and c) a fluid network of a pump or plunger 701, a plurality of valves 626, and microchannels 606 connecting the functional areas to the central distribution hub 608. The fluid network of the pump or plunger 701, the plurality of valves 626, and microchannels 606 can drive the movement of fluid from a first functional area through the central distribution hub 608 to a second functional area among the plurality of functional areas. The nucleic acid amplification reaction may be contained within the nucleic acid amplification area 610.

[0073] The sample preparation area 611 may include a network of reservoirs, chambers, or tanks holding multiple liquids or gases of various configurations, as shown in Figure 6A. Such liquids or gases may contain samples and / or reagents commonly used in the art, such as master mixes, wash buffers, elution buffers, hybridization buffers, or gases such as CO2 or O2. The arrangement, distribution, and transfer of such reagents can be customized based on the experimental protocol or amplification system used.

[0074] The nucleic acid amplification area 610 may be adjacent to the sample preparation area 611, or it may be connected via a microfluidic channel 606. Such a channel 606 allows the prepared sample to move through the central distribution hub 608 to the nucleic acid amplification area 610 and be subjected to nucleic acid amplification conditions. The nucleic acid amplification area 610 (see, for example, Figure 6C) may be the location where some or all of the detection steps for real-time PCR are performed. Nucleic acid detection may include fluorescence detection of the amplified nucleic acid.

[0075] The nucleic acid analysis area 609 may include microarray components or slides, as shown in Figures 6C and 6D. An exemplary nucleic acid analysis area in a microfluidic cartridge is disclosed in U.S. Patent No. 10,184,891. Microarray slides can be used to analyze amplified nucleic acids via capture probes. Such microarrays may include an array of many individual fragments of DNA immobilized on a solid support (e.g., a glass slide) that hybridize with complementary target sequences in the organism of interest. Hybridization can be detected using a fluorescent reporter molecule, such as a fluorophore. Including different probe sequences on a single microarray allows for the simultaneous detection of differences between different organisms or between organisms of the same species, enabling symptomatic testing with a high degree of specificity.

[0076] Functional areas may be spaces dedicated to specific operations on a sample. Functional areas of a microfluidic cartridge may be fluidly connected to a central distribution hub by a fluid network of microchannels. Functional areas can be arranged in various ways, and multiple functional areas may be identical or distinct from one another. Examples of functional areas include nucleic acid extraction areas, nucleic acid purification areas, nucleic acid preparation areas, nucleic acid hybridization areas, nucleic acid amplification areas, nucleic acid detection areas, nucleic acid analysis areas, and disposal areas. In some embodiments, the detection area is a biochip.

[0077] A central distribution hub can be connected to a pump and multiple valves, and it can pump and inject fluid from one functional area to another. Therefore, the central distribution hub makes it possible to use only one simple fluid displacement system (typically a pump system) for most of the fluid movement of a microfluidic cartridge in order to displace fluid from one functional area to another and to induce depressurization and pressurization in order to reduce the volume of the microfluidic cartridge.

[0078] Each microchannel may include a central distribution hub end ("hub end") and a functional area end ("area end"). The area end of a microchannel is adjacent to the corresponding functional area, and the hub end is adjacent to the central distribution hub. Each microchannel may also include a valve located near the relevant area end. Thus, a microfluidic cartridge includes multiple valves located at or near the area end of a hub-connected microchannel.

[0079] The valves may be spatially arranged so as to be actuated independently by actuators. In some embodiments, the actuators are external cam-driven actuators, linear motion actuators, rotational motion actuators, linear actuators, or rotational actuators.

[0080] As used herein, the nucleic acid amplification reaction mixture may contain a sample that may contain or is suspected of containing the target nucleic acid of interest. Various sample types and preparations may be used. In some embodiments, the sample may be subjected to mechanical disruption, for example, by bead beating or sonication, before being added to the nucleic acid amplification mixture, thereby making it easier to obtain the target nucleic acid from the sample. In some embodiments, extraction and purification of the target nucleic acid are performed. If a microfluidic cartridge is used, these steps may be performed within the microfluidic cartridge. In some embodiments, the extraction and purification of the target nucleic acid are performed in the sample preparation area of ​​the microfluidic cartridge.

[0081] D. Method for monitoring fluorescent signals from nucleic acid amplification reactions for abnormal fluorescence detection. In one embodiment, a method for monitoring a fluorescence signal from a nucleic acid amplification reaction for abnormal fluorescence detection includes measuring the fluorescence signal from the nucleic acid amplification reaction at multiple time points during the period in which the nucleic acid amplification reaction is occurring, determining multiple fluorescence intensities for each or some of the multiple time points from the measured fluorescence signals, and comparing each of the multiple fluorescence intensities with a predetermined threshold. If the fluorescence intensity falls below the predetermined threshold, abnormal fluorescence detection is detected.

[0082] In some embodiments, the nucleic acid amplification reaction is a thermally cycled nucleic acid amplification reaction. In some embodiments, the thermally cycled nucleic acid amplification reaction is a qPCR reaction. In some embodiments, this period begins after the start of thermal cycling or after the first thermal cycle.

[0083] In some embodiments, the nucleic acid amplification reaction includes a fluorophore. In some embodiments, the fluorophore is associated with an oligonucleotide probe. In some embodiments, the probe further includes a quencher. For example, the fluorophore may be provided as a fluorophore-containing nucleic acid (e.g., a TaqMan® probe or a labeled detection probe such as a molecular beacon or torch). In some embodiments, the fluorophore-containing nucleic acid reaction mixture is a reaction mixture subjected to thermal cycling. In some embodiments, the fluorophore-containing nucleic acid reaction mixture is a PCR reaction mixture or an RT-PCR reaction mixture.

[0084] In some embodiments, the nucleic acid amplification reaction does not involve fluorophores associated with oligonucleotides, and a fluorescence signal is measured, including intrinsic fluorescence from one or more reagents or templates in the nucleic acid amplification reaction. In some embodiments, one or more reagents include dNTPs and / or one or more primers.

[0085] 1. Fluorescence measurement This method involves measuring the fluorescence signal from the nucleic acid amplification reaction at multiple time points during the period in which the nucleic acid amplification reaction is occurring.

[0086] In some embodiments, the fluorescence signal is measured by a fluorescence detector. In some embodiments, the fluorescence detector is a sensor configured to measure fluorescence. In some embodiments, the fluorescence detector includes a photomultiplier tube or a photodiode.

[0087] In some embodiments, the fluorescence signal is measured from multiple nucleic acid amplification reactions.

[0088] 2. Determination of fluorescence intensity This method involves determining multiple fluorescence intensities for each or some of multiple time points from a measured fluorescence signal. In some embodiments, the multiple fluorescence intensities are measured in relative fluorescence units.

[0089] In some embodiments, multiple fluorescence intensities are determined by smoothing fluorescence signals measured at multiple time points. Examples of smoothing include moving average / median, local regression (loess), low-pass filters, Savitzky-Golay smoothing filters, and the Ramer-Douglas-Peucker algorithm. Determining fluorescence intensity using smoothing can provide more accurate detection of anomalous fluorescence, as a single outlier measurement unrelated to equipment failure, such as a light source or detector, is less likely to result in an intensity below a threshold. In some embodiments, smoothing the fluorescence signal involves determining fluorescence intensity using a window of 3 to 21 measured fluorescence signals, e.g., 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21 measured fluorescence signals.

[0090] In some embodiments, multiple fluorescence intensities are determined as a moving average or moving median of fluorescence signals measured at multiple time points.

[0091] In some embodiments, determining multiple fluorescence intensities for each or a portion of a set of time points from the measured fluorescence signals includes determining multiple fluorescence intensities for at least a portion of a set of time points from the measured fluorescence signals for each of a set of nucleic acid amplification reactions.

[0092] E. Detection of anomalies This method involves comparing each of several fluorescence intensities with a predetermined threshold, and detecting abnormal fluorescence if the fluorescence intensity falls below the predetermined threshold.

[0093] In some embodiments, a predetermined threshold can be established from a reference optical run. A reference optical run of a nucleic acid amplification reaction may include optical events that are multiple successes and failures in the method described herein. The reference run may include optical events performed under different conditions. These conditions may include the presence or absence of a sample, the presence or absence of a fluorophore, the presence or absence of LED light, and / or a functional fluorescence detector. Fluorescence measurements taken at various points in time during these reference optical runs may be obtained. Using the distribution of these measurements, a threshold of acceptable values ​​may be determined. For example, the threshold may be selected as a value between (i) the fluorescence intensity observed in the absence of working LED light and / or a functional fluorescence detector and (ii) the fluorescence intensity observed in the presence or absence of a sample (e.g., the correct amount of master mix), but in the presence or absence of both working LED light and a functional fluorescence detector. In some embodiments, the threshold is referred to as the “predetermined threshold” and is used for comparison with the fluorescence intensity in the method described herein.

[0094] Fluorescence intensity that does not meet a predetermined threshold may indicate abnormal fluorescence detection.

[0095] In some embodiments, when fluorescence intensity is determined for multiple nucleic acid amplification reactions, comparing each of the multiple fluorescence intensities to a predetermined threshold includes comparing each of the multiple fluorescence intensities corresponding to the multiple nucleic acid amplification reactions to a predetermined threshold.

[0096] In some embodiments, abnormal fluorescence detection is detected when the fluorescence intensity falls below a predetermined threshold. In some embodiments, the method includes interrupting the nucleic acid amplification reaction after detecting abnormal fluorescence. In some embodiments, the method includes generating an alarm after detecting abnormal fluorescence. In some embodiments, the method includes notifying the user of the abnormal fluorescence detection after detecting it. In some embodiments, a defect in the fluorescence detector is detected when the fluorescence intensity falls below a predetermined threshold.

[0097] In some embodiments, each of the multiple fluorescence intensities exceeds a predetermined threshold, and no abnormal fluorescence is detected.

[0098] F. Nucleic acid amplification systems and computer-readable media A nucleic acid amplification system is disclosed herein. A nucleic acid amplification system means a device or apparatus that can be used to perform, monitor and / or analyze a fluorescence signal in a nucleic acid amplification reaction described herein. A nucleic acid amplification system 300 is provided herein, comprising (1) a docking station 303 configured to receive a vessel 601 having at least one nucleic acid amplification reaction chamber; (2) a fluorescence detector 401 configured to measure fluorescence from a nucleic acid amplification reaction in the nucleic acid reaction chamber; and (3) a processor operably coupled to the fluorescence detector 401 and memory. The memory includes instructions, when executed by the processor, causing the nucleic acid amplification system to perform a method of monitoring a fluorescence signal from a nucleic acid amplification reaction for abnormal fluorescence detection, the method comprising: a) measuring a fluorescence signal from a nucleic acid amplification reaction at several time points over a period of time during which the nucleic acid amplification reaction is occurring; b) determining several fluorescence intensities from the measured fluorescence signals for each or a portion of the several time points; and c) comparing each of the several fluorescence intensities to a predetermined threshold, wherein abnormal fluorescence detection is detected if the fluorescence intensity falls below the predetermined threshold. An exemplary nucleic acid amplification system is the Novodiag® system (Mobidiag Oy; Espoo, Finland), which can be configured for use with multiple different cartridges 601 (reaction vessels).

[0099] In some embodiments, the container is a multi-chamber receptacle, or the nucleic acid amplification chamber is housed within a multi-chamber receptacle.

[0100] In some embodiments, the nucleic acid amplification system includes a temperature controller that can supply or transfer heat to the nucleic acid amplification reaction chamber via one or more heating elements. In some embodiments, the nucleic acid amplification system is programmable to maintain the nucleic acid amplification reaction chamber at a set temperature over different time periods.

[0101] In some embodiments, the fluorescence detector is configured to measure and record the fluorescence of one or more fluorophores within the nucleic acid amplification reaction chamber. If the container is located within the system, for example, in a docking station, the fluorescence detector 401 is positioned close to the location occupied or to be occupied by the nucleic acid amplification reaction mixture (i.e., the functional area of ​​the container that is optically in communication with the fluorescence detector).

[0102] In some embodiments, the nucleic acid amplification system may also include sensors for monitoring and / or estimating the liquid handling in the nucleic acid amplification reaction chamber. In some embodiments, the sensors may be thermal sensors, capacitive sensors, or infrared sensors.

[0103] In some embodiments, if an anomaly is detected, the nucleic acid amplification system provides one or more alarms. Alarms may be provided to indicate liquid filling anomalies. Examples of alarms include visual and audible notifications. In some embodiments, when an alarm is provided, the nucleic acid amplification system pauses the nucleic acid amplification process, which may include lowering the temperature of the heating element to, for example, about 4°C and maintaining the lowered temperature for a period of time.

[0104] In some embodiments, the system is configured to interrupt the nucleic acid amplification reaction if abnormal fluorescence is detected.

[0105] Computer-readable media are also disclosed herein. In some embodiments, the computer-readable media includes instructions, when executed by the processor of the nucleic acid amplification system, to cause the nucleic acid amplification system to perform a method for monitoring a fluorescence signal from a nucleic acid amplification reaction for anomalous fluorescence detection as described herein, for example, a method including: measuring a fluorescence signal from a nucleic acid amplification reaction at several points in time during which the nucleic acid amplification reaction is occurring; determining several fluorescence intensities for each or some of the multiple points in time from the measured fluorescence signals; and comparing each of the multiple fluorescence intensities to a predetermined threshold, wherein anomalous fluorescence detection is detected if the fluorescence intensity falls below the predetermined threshold.

[0106] In some embodiments, a computer-readable medium includes instructions for carrying out a method for monitoring a fluorescence signal from a nucleic acid amplification reaction for anomalous fluorescence detection as described herein. In some embodiments, these instructions are executed by a processor of the nucleic acid amplification system described herein.

[0107] In some embodiments, the method includes stopping the nucleic acid amplification reaction if an abnormality is detected. [Examples]

[0108] Examples This example describes performing reference optical runs of nucleic acid amplification reactions under various conditions, including normal conditions, conditions with fluorescence detection abnormalities, and other abnormal conditions. This set of reference runs provided a basis for pre-determining thresholds for comparison with fluorescence intensity in the methods described herein.

[0109] A reaction mixture was prepared containing Amplidiag® Multiplex PCR Master Mix (Mobidiag Oy; Espoo, Finland), nucleic acid templates, forward and reverse primers, and a fluorescent probe.

[0110] The reaction mixture was amplified using a Novodiag® system. Fluorescence measurements from the nucleic acid amplification reaction chamber were obtained at various points during these reference optical runs, including before and after the LED light source was turned off (see Figure 1).

[0111] Further runs were performed to obtain fluorescence intensity at multiple time points under various conditions (e.g., LED light off, no sample, less reagent, etc.) (see Figure 2). Values ​​within a specific range, or values ​​that meet or do not meet a specific threshold, indicate an optical error or instrument malfunction of 1 or greater. As shown in Figure 2, a threshold of 100 RFU in the blue channel was used as the threshold to indicate abnormal fluorescence detection (in this case, without LED light). Therefore, this method was effective in detecting abnormal fluorescence detection and distinguishing normal fluorescence detection (from both normal and abnormal reactions) based on the fluorescence intensity obtained from nucleic acid amplification reactions.

[0112] Equal parts The foregoing specification is considered sufficient to enable those skilled in the art to carry out the embodiments. The foregoing description and examples detail specific embodiments and illustrate the best mode intended by the inventors. However, it will be understood that, no matter how detailed the foregoing is, these embodiments can be carried out in many ways and should be interpreted in accordance with the appended claims and their equivalents.

Claims

1. A method for monitoring fluorescent signals from nucleic acid amplification reactions for the detection of abnormal fluorescence, a) Measuring the fluorescence signal from the nucleic acid amplification reaction at multiple points in time during the period in which the nucleic acid amplification reaction is occurring. b) Determining multiple fluorescence intensities for each or a portion of the multiple time points from the measured fluorescence signal, c) A method comprising comparing each of the plurality of fluorescence intensities with a predetermined threshold, wherein abnormal fluorescence detection is detected when the fluorescence intensity falls below the predetermined threshold.

2. A nucleic acid amplification system, A docking station configured to accept a container having at least one nucleic acid amplification reaction chamber, A fluorescence detector configured to measure fluorescence from a nucleic acid amplification reaction in the nucleic acid reaction chamber, and The system comprises a processor operably connected to the fluorescence detector and memory, The memory includes instructions, when executed by the processor, that cause the nucleic acid amplification system to perform a method for monitoring a fluorescence signal from a nucleic acid amplification reaction for abnormal fluorescence detection, the method being: a) Measuring the fluorescence signal from the nucleic acid amplification reaction at multiple points in time during the period in which the nucleic acid amplification reaction is occurring. b) Determining multiple fluorescence intensities for each or a portion of the multiple time points from the measured fluorescence signal, and c) A nucleic acid amplification system comprising comparing each of the plurality of fluorescence intensities with a predetermined threshold, wherein abnormal fluorescence detection is detected when the fluorescence intensity falls below the predetermined threshold.

3. Computer-readable medium, When executed by the processor of a nucleic acid amplification system, the instruction includes a method for causing the nucleic acid amplification system to perform a method for monitoring a fluorescence signal from a nucleic acid amplification reaction for abnormal fluorescence detection, wherein the method a) Measuring the fluorescence signal from the nucleic acid amplification reaction at multiple points in time during the period in which the nucleic acid amplification reaction is occurring. b) Determining multiple fluorescence intensities for each or a portion of the multiple time points from the measured fluorescence signal, c) A computer-readable medium that includes comparing each of the plurality of fluorescence intensities with a predetermined threshold, wherein abnormal fluorescence detection is detected when the fluorescence intensity falls below the predetermined threshold.

4. The method, system, or computer-readable medium according to any one of the preceding claims, wherein the nucleic acid amplification reaction is a thermal cycle nucleic acid amplification reaction.

5. The method, system, or computer-readable medium according to any one of the preceding claims, wherein the plurality of fluorescence intensities are determined by smoothing the fluorescence signals measured at the plurality of time points.

6. The method, system, or computer-readable medium according to any one of the preceding claims, wherein the plurality of fluorescence intensities are determined as a moving average or moving median of the fluorescence signals measured at the plurality of time points.

7. The method, system, or computer-readable medium according to any one of the preceding claims, wherein the fluorescence signal is measured by a fluorescence detector, and the fluorescence detector optionally includes a photomultiplier tube or a photodiode.

8. The method, system, or computer-readable medium according to the immediately preceding claim, wherein a defect in the fluorescence detector is detected if the fluorescence intensity falls below a predetermined threshold.

9. The method, system, or computer-readable medium according to any one of the preceding claims, wherein the nucleic acid amplification reaction is contained within a microfluidic cartridge.

10. A method, system, or computer-readable medium according to any one of the preceding claims, wherein a fluorescent signal is measured from a plurality of nucleic acid amplification reactions in step a).

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