Control of the nucleic acid amplification process

Abstract

Methods, systems, reaction mixtures, kits, apparatus, and systems for monitoring nucleic acid amplification reaction reactions for abnormalities. Deviations of temperature and buffer composition from normal or expected may occur during the preparation of the reaction mixture, under conditions, and / or due to errors in the functionality of the nucleic acid amplification system. Such deviations may lead to unreliable or inconsistent results, and even complete failure of the nucleic acid amplification reaction. Exemplary methods include determining fluorescence-based values ​​from fluorophore-containing nucleic acid amplification reaction mixtures and determining whether those values ​​meet predetermined values ​​or indicate abnormalities.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to European Patent Application No. 23315135.6, filed on April 28, 2023, which is incorporated herein by reference in its entirety for all purposes.

[0002] Field 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 nucleic acid amplification reaction mixtures for abnormalities during the amplification process.

Background Art

[0003] Introduction Nucleic acid amplification reactions are widely used in research and clinical laboratories for detecting genetic disorders and infectious diseases. Nucleic acid amplification reactions utilize enzymes such as polymerase and 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. (Nucleic acid copies are called "amplicons".) In a nucleic acid amplification reaction, the reaction mixture can be subjected to a number of thermal cycles ( "PCR cycles") each including a denaturation step, a primer annealing step and a primer extension step. In the denaturation step, double - stranded DNA template molecules are made single - stranded, in the primer annealing step, primers bind to complementary sequences in the single - stranded DNA template, and in the primer extension step, new DNA strands are formed from the primers. By selecting appropriate temperatures for each step of the PCR cycle, the PCR cycle can be repeated until a sufficient amount of amplicon is present in the reaction mixture to make the target nucleic acid sequence detectable or quantifiable. In the initial stages of PCR, amplification is exponential.

[0004] Nucleic acid amplification reactions depend on the temperature of the reaction mixture and the precision of the buffer composition. Amplification of target nucleic acids is mainly carried out by enzymatic methods using DNA polymerase (for DNA target amplification) or DNA polymerase and reverse transcriptase (for RNA target amplification). These enzymes are sensitive to changes in temperature and reaction conditions, such as pH. To maintain enzyme stability, nucleic acid amplification reactions may contain tris and sulfate compounds (e.g., MgSO4, NH4SO4).

[0005] Deviations of normal or expected temperature and buffer composition may occur during the preparation of the reaction mixture, under conditions, and / or due to errors in the functionality of the nucleic acid amplification system. Such deviations can lead to unreliable or inconsistent results, and even complete failure of the nucleic acid amplification reaction. Typically, whether a nucleic acid amplification reaction is successful or unsuccessful cannot be determined until the reaction is complete, or should be complete, and even then, a control reaction is generally necessary to reliably detect problems with the buffer and / or cycling conditions. The absence of amplification (of the target nucleic acid, if present in the reaction mixture, or of the control) is a clear indicator of failure, but generally not the cause of the failure. Materials, methods, and systems for detecting anomalies associated with such process errors are disclosed herein. The methods disclosed herein may rely on the responsiveness of oligonucleotide primers, fluorophores, and quenchers to changes in the temperature of the reaction mixture. [Overview of the project]

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

[0007] Embodiment 1 is a method for monitoring abnormalities in nucleic acid amplification reaction mixtures, a) To provide a nucleic acid amplification reaction reaction mixture containing fluorophores to a nucleic acid amplification system. b) Adjusting the heating element of the nucleic acid amplification system to a first temperature over a first period, thereby heating the nucleic acid amplification reaction mixture to a first incubation temperature during the first period. c) After step b), the fluorescence from the fluorophores is measured multiple times during the first period, wherein the nucleic acid amplification reaction mixture is not exposed to nucleic acid amplification conditions before or during the first period. d) Determine the following: i) A first value representing the change in the fluorescence signal during the first period, and / or ii) A second value representing the rate of change in fluorescence during the first period, and e) Comparing a first value to a first predetermined threshold or range, and / or comparing a second value to a second predetermined threshold or range, in the following cases: i) The first value does not meet the first predetermined threshold or shows a change in fluorescence outside the first predetermined range, ii) The second value indicates a rate of temperature change that does not satisfy the second predetermined threshold or is outside the second predetermined range. iii) The first value shows a change in fluorescence that does not meet the first predetermined threshold or is outside the first predetermined range, or the second value shows a rate of temperature change that does not meet the second predetermined threshold or is outside the second predetermined range, iv) A method including comparison in which an anomaly is detected when the first value shows a change in fluorescence that does not meet a first predetermined threshold or is outside a first predetermined range, and the second value shows a rate of temperature change that does not meet a second predetermined threshold or is outside a second predetermined range.

[0008] Embodiment 2 is a nucleic acid amplification system comprising: a docking station configured to receive a reaction vessel for containing at least one nucleic acid amplification mixture; a heating element positioned in close proximity to the location occupied by the nucleic acid amplification mixture when the reaction vessel is present in the docking station; a fluorescence detector configured to measure the fluorescence of fluorophores in the nucleic acid mixture; and a processor operably coupled to the heating element and memory. The memory includes instructions, when executed by the processor, that cause the nucleic acid amplification system to perform a method of monitoring the nucleic acid amplification reaction mixture containing a fluorophore for anomalies, and the method is a) Adjusting the heating element to a first temperature over a first period of time, b) After step a), the fluorescence from fluorophores contained in the nucleic acid amplification mixture is measured multiple times during the first period using a fluorescence detector, wherein the first period is the period before the planned nucleic acid amplification. c) Determine the following: i) A first value representing the change in the fluorescence signal during the first period, and / or ii) A second value representing the rate of change in fluorescence during the first period, and d) Comparing a first value to a first predetermined threshold or range, and / or comparing a second value to a second predetermined threshold or range, in the following cases: i) The first value does not meet the first predetermined threshold or shows a change in fluorescence outside the first predetermined range, ii) The second value indicates a rate of temperature change that does not satisfy the second predetermined threshold or is outside the second predetermined range. iii) The first value shows a change in fluorescence that does not meet the first predetermined threshold or is outside the first predetermined range, or the second value shows a rate of temperature change that does not meet the second predetermined threshold or is outside the second predetermined range, iv) A nucleic acid amplification system that includes a comparison in which an anomaly is detected when the first value shows a change in fluorescence that does not meet a first predetermined threshold or is outside the first predetermined range, and the second value shows a rate of temperature change that does not meet a second predetermined threshold or is outside the second predetermined range.

[0009] Embodiment 3 is a computer-readable medium, The method includes an instruction, when executed by the processor of a nucleic acid amplification system, that causes the nucleic acid amplification system to perform a method of monitoring the nucleic acid amplification reaction mixture containing a fluorophore for abnormalities, and the method is a) Adjusting the heating element of the nucleic acid amplification system to a first temperature over a first period of time. b) After step a), the fluorescence from fluorophores contained in the nucleic acid amplification mixture is measured multiple times during the first period using the fluorescence detector of the nucleic acid amplification system, wherein the first period is the period before the scheduled nucleic acid amplification. c) Determine the following: i) A first value representing the change in the fluorescence signal during the first period, and / or ii) A second value representing the rate of change in fluorescence during the first period, and d) Comparing a first value to a first predetermined threshold or range, and / or comparing a second value to a second predetermined threshold or range, in the following cases: i) The first value does not meet the first predetermined threshold or shows a change in fluorescence outside the first predetermined range, ii) The second value indicates a rate of temperature change that does not satisfy the second predetermined threshold or is outside the second predetermined range. iii) The first value shows a change in fluorescence that does not meet the first predetermined threshold or is outside the first predetermined range, or the second value shows a rate of temperature change that does not meet the second predetermined threshold or is outside the second predetermined range, iv) A computer-readable medium including comparing, wherein an anomaly is detected when the first value indicates a change in fluorescence that does not meet a first predetermined threshold or is outside a first predetermined range, and the second value indicates a rate of temperature change that does not meet a second predetermined threshold or is outside a second predetermined range.

[0010] Embodiment 4 is a method, system, or computer-readable medium of any one of the preceding embodiments, wherein the first value is determined by subtracting an early measurement value of fluorescence from a fluorophore from a late measurement value of fluorescence from the fluorophore.

[0011] Embodiment 5 is a method, system, or computer-readable medium of the immediately preceding embodiment, wherein an early measurement value of fluorescence from a fluorophore is obtained in the first half or during the first half of a first period, within 1 minute from the start of the first period, or approximately at the start of the first period.

[0012] Embodiment 6 is a method, system, or computer-readable medium of any one of Embodiments 4 to 5, wherein an early measurement value of fluorescence from a fluorophore is the lowest measurement value of fluorescence from the fluorophore.

[0013] Embodiment 7 is a method, system, or computer-readable medium of any one of Embodiments 4 to 6, wherein a late measurement value of fluorescence from a fluorophore is obtained during the second half of a first period, within 1 minute from the end of the first period, or approximately at the end of the first period.

[0014] Embodiment 8 is a method, system, or computer-readable medium of any one of Embodiments 4 to 7, wherein a late measurement value of fluorescence from a fluorophore is the highest measurement value of fluorescence from the fluorophore.

[0015] Embodiment 9 is a) The second value is determined by dividing a first partial value by a second partial value, b) The first partial value represents the lower area of the quadrilateral region of the plot of fluorescence from the fluorophore against time during the first period, and c) The second partial value represents the upper area of the quadrilateral region, The quadrilateral region is bounded by i) The first and last measured values of fluorescence from the fluorophore during the first period, and the times of the first and last measured values of fluorescence from the fluorophore during the first period, ii) The minimum and maximum measured values of fluorescence from the fluorophore during the first period, and the times of the minimum and maximum measured values of fluorescence from the fluorophore during the first period, iii) The minimum and maximum measured values of fluorescence from the fluorophore during the first period, and the times of the first and last measured values of fluorescence from the fluorophore during the first period, or iv) The first and last measured values of fluorescence from the fluorophore during the first period, and the times of the minimum and maximum measured values of fluorescence from the fluorophore during the first period, and the lower area is equal to the area of the quadrilateral region below the measured values of fluorescence from the fluorophore, and the upper area is equal to the area of the quadrilateral region above the measured values of fluorescence from the fluorophore, which is a method, system, or computer-readable medium according to any one of the preceding embodiments. The lower area is equal to the area of the quadrilateral region below the measured values of fluorescence from the fluorophore, and the upper area is equal to the area of the quadrilateral region above the measured values of fluorescence from the fluorophore, which is a method, system, or computer-readable medium according to any one of the preceding embodiments.

[0016] [[ID=,17]] Embodiment 10 is a method, system, or computer-readable medium according to any one of the preceding embodiments, where the first value represents the change in the Y-axis of the fluorescence signal.

[0017] Embodiment 11 is a method, system, or computer-readable medium according to any one of the preceding embodiments, where the fluorescence changes over time during the first period, and the rate of change of fluorescence during the first period correlates with the rate of change of temperature of the nucleic acid amplification reaction mixture.

[0018] Embodiment 12 is a method, system, or computer-readable medium of any one of the preceding embodiments in which the abnormality is a thermal contact defect and / or a liquid handling defect.

[0019] Embodiment 13 is a method, system, or computer-readable medium of any one of the preceding embodiments, wherein the nucleic acid in the fluorophore-containing nucleic acid amplification reaction mixture is denatured during a first period.

[0020] Embodiment 14 is a method, system, or computer-readable medium of any one of the prior embodiments, wherein the first period is in the range of approximately 2 to 10 minutes, 3 to 9 minutes, 3 to 7 minutes, or 4 to 6 minutes, or approximately 5 minutes.

[0021] Embodiment 15 is a method, system, or computer-readable medium of any one of the prior embodiments, wherein the first temperature is in the range of 90°C to 120°C or 95°C to 115°C, or approximately 110°C.

[0022] Embodiment 16 is a method, system, or computer-readable medium of any of the preceding embodiments, wherein the method further includes adjusting a heating element to a preheating temperature over a preheating period prior to step a).

[0023] Embodiment 17 is a method, system, or computer-readable medium of the preceding embodiment, where the pre-temperature is suitable for reverse transfer.

[0024] Embodiment 18 is the method, system, or computer-readable medium of the preceding embodiment, wherein reverse transcription occurs in a fluorophore-containing nucleic acid amplification reaction mixture during a preliminary period.

[0025] Embodiment 19 is one of the methods, systems, or computer-readable media of Embodiments 16 to 18, wherein the pre-temperature is in the range of 25°C to 50°C, 30°C to 50°C, 35°C to 50°C, 40°C to 50°C, or 42°C to 49°C, or approximately 48°C.

[0026] Embodiment 20 is one of the methods, systems, or computer-readable media of Embodiments 16 to 18, wherein the preparatory period is in the range of 15 to 60 minutes, 20 to 50 minutes, or 25 to 40 minutes, or approximately 30 minutes.

[0027] Embodiment 21 is a method, system, or computer-readable medium of any one of the preceding embodiments in which the fluorophore-containing nucleic acid amplification reaction mixture is a thermal cycling reaction mixture.

[0028] Embodiment 22 is a method, system, or computer-readable medium of any one of the preceding embodiments, wherein the fluorophore-containing nucleic acid amplification reaction mixture is a PCR reaction mixture or an RT-PCR reaction mixture.

[0029] Embodiment 23 is a method, system, or computer-readable medium of any of the preceding embodiments, in which a fluorophore-containing nucleic acid amplification reaction mixture is contained in a microfluidic cartridge.

[0030] Embodiment 24 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 pump, a plurality of valves, and a microchannel fluid network can drive the movement of fluid from a first functional area through a central distribution hub to a second functional area among a plurality of functional areas, as described in the preceding embodiment, method, system, or computer-readable medium.

[0031] Embodiment 25 is the method, system, or computer-readable medium of the preceding embodiment, wherein the fluorophore-containing nucleic acid amplification reaction mixture is placed in the nucleic acid amplification area during a first period.

[0032] Embodiment 26 is a method, system, or computer-readable medium of Embodiment 25, wherein the method further comprises adjusting a heating element to a preheating temperature over a preheating period prior to step a), and the fluorophore-containing nucleic acid amplification reaction mixture is placed in the nucleic acid amplification area during the preheating period.

[0033] Embodiment 27 is a method, system, or computer-readable medium of any one of the prior embodiments, wherein a plurality of fluorophore-containing nucleic acid amplification reaction mixtures are provided to a nucleic acid amplification system in step a).

[0034] Embodiment 28 is the method, system, or computer-readable medium of the preceding embodiment, wherein in step c), fluorescence is measured from each of the fluorophores in a plurality of fluorophore-containing nucleic acid amplification reaction mixtures.

[0035] Embodiment 29 is, a) A first value representing the change in the fluorescence signal during the first period, and / or b) The second value representing the rate of change in fluorescence during the first period is: In step d), the method, system, or computer-readable medium of the preceding embodiment is determined for each of the multiple fluorophore-containing nucleic acid amplification reaction mixtures.

[0036] Embodiment 30 is the method, system, or computer-readable medium of the preceding embodiment, wherein in step e), for each of a plurality of fluorophore-containing nucleic acid amplification reaction mixtures, a first value is compared to a first predetermined threshold or range, and / or a second value is compared to a second predetermined threshold or range.

[0037] Embodiment 31 is any one of the methods, systems, or computer-readable media of Embodiments 27-30, wherein a plurality of fluorophore-containing nucleic acid amplification reaction mixtures are contained in a multiwell plate or a plurality of tubes.

[0038] Embodiment 32 is a method, system, or computer-readable medium of any of the prior embodiments in which an anomaly is detected when a first value shows a change in fluorescence that does not meet a first predetermined threshold or is outside a first predetermined range.

[0039] Embodiment 33 is a method, system, or computer-readable medium of any one of Embodiments 1 to 28 in which an anomaly is detected when the second value shows a temperature change rate that does not meet a second predetermined threshold or is outside a second predetermined range.

[0040] Embodiment 34 is a method, system, or computer-readable medium from any one of Embodiments 1 to 28 in which an anomaly is detected if the first value does not meet a first predetermined threshold or shows a change in fluorescence that is outside the first predetermined range, or if the second value does not meet a second predetermined threshold or shows a rate of temperature change that is outside the second predetermined range.

[0041] Embodiment 35 is a method, system, or computer-readable medium according to any one of Embodiments 1 to 28, in which an anomaly is detected when a first value indicates a change in fluorescence that does not meet a first predetermined threshold or is outside a first predetermined range, and a second value indicates a rate of temperature change that does not meet a second predetermined threshold or is outside a second predetermined range.

[0042] Embodiment 36 is a method, system, or computer-readable medium of any one of the prior embodiments, wherein a fluorophore associates with an oligonucleotide probe, and optionally the oligonucleotide probe further comprises a quencher.

[0043] Embodiment 37 is a method, system, or computer-readable medium of any of the preceding embodiments in which the reverse transcriptase inactivation step is to adjust the heating element to a first temperature over a first period of time.

[0044] Embodiment 38 is a method, system, or computer-readable medium of any of the prior embodiments in which the fluorophore is temperature-sensitive.

[0045] Embodiment 39 is a method, system, or computer-readable medium of any one of the prior embodiments, wherein the fluorophore is a sulforhodamine.

[0046] Embodiment 40 is one of Embodiments 1 or 4-39, wherein an abnormality is detected and the nucleic acid amplification reaction mixture is not subjected to nucleic acid amplification conditions.

[0047] Embodiment 41 is one of Embodiments 1 or 4-39, wherein no abnormalities are detected and the nucleic acid amplification reaction mixture is subjected to nucleic acid amplification conditions.

[0048] Embodiment 42 is a system of any one of Embodiments 2 or 4-39, configured such that if an abnormality is detected, the nucleic acid amplification reaction mixture is not subjected to nucleic acid amplification conditions, and / or only if no abnormality is detected, the nucleic acid amplification reaction mixture is subjected to nucleic acid amplification conditions.

[0049] Embodiment 43 is a system or computer-readable medium of any one of Embodiments 2, 3, 4-39, or 42, further comprising subjecting the nucleic acid amplification reaction mixture to nucleic acid amplification conditions only if no abnormalities are detected.

[0050] Embodiment 44 is a system or computer-readable medium of any one of Embodiments 2, 3, 4-39, or 42-43, further comprising a method of reducing the temperature of a heating element without thermal cycling when an abnormality is detected.

[0051] Additional objectives and benefits are partially described in the following description, partially understood from the description, or acquired through practice. These objectives and benefits will be realized and achieved by the elements and combinations specifically indicated in the attached claims.

[0052] Please understand that both the general description above and the detailed description below are illustrative and descriptive only, and do not limit the scope of the claims. [Brief explanation of the drawing]

[0053] Brief explanation of the drawing [Figure 1] Figure 1 shows an exemplary curve of a single nucleic acid amplification reaction for determining the signal gain and heat ratio from the first period. Relative fluorescence units (RFU; y-axis) are plotted over time (milliseconds; x-axis). The signal gain is the difference between RFU Max and RFU Min over the shown period. The heat ratio is the ratio of the lower area of ​​the quadrilateral region to the upper area of ​​the quadrilateral region.

[0054] [Figure 2] Figure 2 is an illustrative plot of the signal gain (y-axis) versus heat ratio (x-axis) for multiple nucleic acid amplification reactions performed under different conditions, including normal conditions, thermal contact loss, and fluid errors (such as elution buffer omission, master mix buffer omission, and partial valve opening).

[0055] [Figure 3] Figure 3 is an exemplary plot of PCR module target and heater temperature curves recorded during nucleic acid amplification. The curves are split to identify the reverse transcription (RT) step and the RT inactivation phase. The limit of the RT inactivation phase can be determined by the target temperature or heater temperature. This period includes a 4-second delay to allow the temperature to exceed the target temperature of 80°C.

[0056] [Figure 4]Figure 4 shows an exemplary nucleic acid amplification system 400, including a docking station 403 loaded with a microfluidic cartridge 701. The system includes a heat sink 405, a fluorometer 406, a press 401, and a press / heater 404 used to interact with and measure the sample loaded in the cartridge 701.

[0057] [Figure 5] Figure 5 is a side view of the nucleic acid amplification system 400, including the docking station 403 of Figure 4. The side view shows an optical fiber 503 extending from the cartridge 701 to the light source 407 (e.g., one or more LEDs) of the fluorometer 406, and a hole 504 formed in an aluminum block for guiding the fluorescence from the cartridge 701 to a fluorescence detector 501 used to perform fluorescence measurements, the fluorescence detector 501 being a component of the fluorometer 406.

[0058] [Figure 6] Figure 6 shows a cross-sectional view of the nucleic acid amplification system 400, including the docking station 403 and microfluidic cartridge 701 shown in Figures 4 and 5. The cross-sectional view shows internal components such as a thermal cycler 608 including a thermal block 605, a Peltier module 606, and a heat sink 607, an array detector 611, and a rotary valve system 601 including a piston 602, a ball 603, and a cam 604 useful for moving and analyzing the liquid in different functional areas of the cartridge 701. The system also includes a clamp 609 for securing a detection tip (not shown) and an illuminator 610.

[0059] [Figure 7-1]Figures 7A–7D show various diagrams of an exemplary microfluidic cartridge 701. Figure 7A shows a top perspective view of the microfluidic cartridge 701 containing multiple chambers for holding samples, reagents, or other liquids. Figure 7B shows a bottom perspective view of the exemplary microfluidic cartridge 701, showing a fluid network of microchannels 706 connecting the various chambers to other areas within the cartridge 701. For example, chambers 702, 703, 704, and 705 may be the sample chamber 702, the weighing chamber 703, the waste chamber 704, and the PCR chamber 705. Figure 7C shows a bottom perspective view of a fully constructed microfluidic cartridge 701, including a bottom cover 713 and a microarray slide 714, as shown in Figure 7D, which include a sample preparation area 711, a nucleic acid amplification area 710, and a nucleic acid analysis area 709. The nucleic acid amplification area 710 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 706, which are shown in detail in Figure 7B. The sample preparation area 711 contains liquids in adjacent chambers. The chambers within the sample preparation area 711 are arranged around a central distribution hub 708. 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 710 is in close proximity to the thermal cycler 608 in Figure 6. Similarly, when docked to the system, the nucleic acid analysis area 709 is in close proximity to the array detector 611, as shown in Figure 6. Figure 7D shows an exploded view of a fully constructed exemplary microfluidic cartridge 701, which includes a cartridge body 712, a vent cap 723 for covering the sample after input, a sample filter 724, and components for the cartridge 701 to interact with the system's mechanical components to move and combine the liquids in the sample preparation area 711, including a stopper 718, a nucleic acid binding matrix column 719, a protective cover 721, a blocker 722, and a blocker ring 725.The exploded view also shows how the microarray slide 714 is attached to the cartridge body 712 via the microarray adhesive tape 715 in the nucleic acid analysis area 709. The cartridge 701 also includes polypropylene (PP) coverings 713, 716 on the bottom and top of the cartridge, and optionally includes a pre-printed sticker 717. [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0060] [Figure 8] Figure 8 shows an exploded view of an exemplary microfluidic cartridge 701 and plunger 801 of the nucleic acid amplification system 400, where the plunger 801 operates to unlock the blocker 722 and interact with the stopper 718 to move and combine the liquid in the microfluidic cartridge 701. [Modes for carrying out the invention]

[0061] 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.

[0062] "Nucleic acids" and "polynucleotides" refer to polymeric 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., International Publication 95 / 32305) and "locked nucleic acids" (LNAs), where one or more nucleotide monomers have a bicyclic furanose unit locked to RNA that mimics a sugar conformation (see, e.g., Vester et al., Biochemistry 43:13233-41, 2004). Nitrogen bases may be conventional bases (A, G, C, T, U), derivatives of purine or pyrimidine bases (e.g., N4-methyldeoxyguanosine, deaza- or aza-purine, deaza- or aza-pyrimidine, etc., see U.S. Patent No. 5,378,825, U.S. Patent No. 6,949,367, and International Publication No. 93 / 13121), their analogues (e.g., inosine, 5-methylisocytosine, isoguanine, see 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 "debasic" residues (e.g., see U.S. Patent No. 5,585,481).

[0063] 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.

[0064] As used herein, the term “region” refers to a portion of a nucleic acid that is smaller 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).

[0065] A “primer,” “amplifying oligonucleotide,” or “oligonucleotide primer” refers to an oligonucleotide containing a polynucleotide, generally the “target” binding region, 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, for example, from its 3' end. The 5' region of the primer may be non-complementary to the target nucleic acid. If the 5' non-complementary region contains a promoter sequence, it is called a “promoter-primer.” A promoter-primer can function as a primer for a DNA polymerase that extends the promoter-primer from its 3' end, or as a promoter for an RNA polymerase that initiates de novo RNA synthesis at or near the promoter sequence contained in the promoter-primer.

[0066] "Sample" refers to any material that may contain or is suspected of containing target nucleic acids. A sample may be a complex mixture of components. Examples of samples include "biological samples" which include 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 in vitro cell culture components, including, for example, conditioned media resulting from the growth of cells and tissues in culture media. Samples may also include foods which include any material intended for consumption 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.

[0067] The interchangeable terms “oligomer,” “oligo,” and “oligonucleotide” refer to nucleic acids generally having fewer than 1,000 nucleotide (nt) residues, including polymers with a lower limit of about 5 nt residues and an upper limit of about 500 to about 900 nt residues. In some embodiments, oligonucleotides are in a size range with a lower limit of about 12 to about 15 nt and an upper limit of about 50 to about 600 nt, and in other embodiments, they are in a range with a lower limit of about 15 to 20 nt and an upper limit of about 22 to about 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.

[0068] "Amplifying" or "amplification" 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. As used herein, the term "nucleic acid amplification conditions" refers to both the temperature and chemical conditions that enable nucleic acid amplification. Methods for forming reaction mixtures and subjecting these reaction mixtures to conditions suitable for nucleic acid amplification are well-established. 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-limiting 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 International 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), and U.S. Patents 5,422,252, 5,547,861, and 5,648,211 (SDA). See, for example, Compton, Nature 350:91-92, 1991; Malek et al., Methods Mol. Biol. 28:253-260, 1994 (NASBA). PCR is a preferred amplification method and is well known in the art. 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).

[0069] As used herein, the term “real-time amplification” refers to the amplification of a target nucleic acid monitored by real-time detection means. 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 may be labeled with two fluorescent moieties. The emission spectrum of one overlaps with the excitation spectrum of the other, resulting in “quenching” of the first fluorophore by the second fluorophore. In some applications, the fluorescent moieties may be used in combination with a non-fluorescent quencher moiety.

[0070] 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.”

[0071] As used herein, “nucleic acid amplification system” refers to a device or apparatus that may be used to carry out nucleic acid amplification. In many examples, a nucleic acid amplification system includes a temperature controller and may 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. Nucleic acid amplification systems are typically programmable and can maintain a temperature over different time periods. A 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.

[0072] 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.

[0073] "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. "Stringent" hybridization or amplification conditions selectively enable hybridization of highly homologous nucleic acid sequences. Such conditions may include high hybridization temperatures and low concentrations of salt in the buffer. Sequences that hybridize with each other may be fully or partially complementary by standard nucleic acid base pairings (e.g., G:C, A:T, or A:U pairings). "Fully complementary" means a sequence that can hybridize to another sequence by hydrogen bonds 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 pairings, or that contain debasic residues that are not complementary. Sufficiently complementary contiguous 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 being "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. Nucleotide sequences are "perfectly" complementary if a contiguous 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.).

[0074] 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.Exemplary fluorophores include carboxyfluorescein (FAM®), N',N'-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazole-2-ylidene)methyl]-1-phenylquinoline-1-ium-2-yl]-N-propylpropane-1,3-diamine (SYBR® Green), ATTO fluorescent labeling (Atto-Tec; Siegen, Germany), 2'-chloro-7'phenyl-1,4-dichloro-6-carboxyfluorescein (VIC®), 5'-dichloro-dimethoxy-fluorescein dye (JOE®), NED®, cyanine 3 (Cy3), carboxyrhodamine (ROX®), Texaled sulfonyl chloride (Texas Red), CAL Fluor® Orange 560, and CAL Fluor® Red. Contains 610 and Cyanine 5 (Cy5) dye (all available from numerous commercial sources). Exemplary quenchers include Blackberry® Quenchers (BBQ®), ATTO Quencher (Atto-Tec; Siegen, Germany), Black Hole Quenchers (BHQ®), TAMRA®, and 4-((4-(dimethylamino)phenyl)azo)benzoic acid (DABCYL acid) (all available from numerous commercial sources). Synthesis and methods for attaching labels to nucleic acids and detecting those labels are known in the art (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10; 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).

[0075] 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).

[0076] As used herein, "elution buffer" is a liquid suitable for separating nucleic acids from a solid support.

[0077] 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.

[0078] 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.

[0079] As used herein, “fluorescence detector” or “fluorescence sensor” refers to an optical detector from which fluorescence measurements can be obtained. Such measurements can 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 the sample. Real-time detection of PCR products can be achieved by using a fluorescent dye or probe. The fluorescence signal measured by the fluorescence detector increases with each PCR cycle as more polynucleotide molecules are produced.

[0080] 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 means used for measurement and can be used as a measurement for comparing the relative intensity between a sample and a control.

[0081] 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.

[0082] Where used herein, the term “approximately” refers to numerical values, including integers, fractions, and percentages, whether explicitly stated or not. When this term precedes a list of numerical values ​​or ranges, it modifies all values ​​or ranges. When applied to a measurement, the term includes the exact numerical value modified by this term, and the range of values ​​that are expected to be within the experimental error. Unless the context specifically indicates otherwise, “approximately” encompasses a range of plus or minus (±) 10%. For example, “approximately 5°C” means a temperature range of “5°C” and within ±10% of 5°C. The term has a similar meaning with respect to other parameters. For example, “approximately 5 minutes” means a time range of “5 minutes” and within ±10% of 5 minutes. 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.

[0083] 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.

[0084] 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.

[0085] "Or" is used in an inclusive sense, meaning it is equivalent to "and / or" unless the context clearly indicates otherwise.

[0086] Detailed explanation A method for monitoring a nucleic acid amplification reaction mixture for anomalies is disclosed herein. This method utilizes the presence of fluorophores, quenchers (substances whose fluorescence emission can be affected by static or dynamic quenching), and / or reagents having intrinsic fluorescence, such as dNTPs and primers, to detect changes in temperature and buffer composition, thereby detecting abnormal temperature or buffer composition in the nucleic acid amplification reaction mixture. 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 a fluorescence detector, without requiring additional hardware. Alternatively, this method uses existing features of such systems to monitor fluorescence signals for anomaly detection during the nucleic acid amplification reaction.

[0087] A. Detection labels and probes Detection labels may be used in accordance with this disclosure. The nucleic acid amplification reaction mixture of this disclosure may include a detection oligomer having a detection label (e.g., designed to hybridize to an amplicon) and / or amplification oligomers, e.g., 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.

[0088] 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 Fluor® Orange 560, CAL Fluor® 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 fluorophor-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 (see, e.g., U.S. Patent No. 5,723,591), “Molecular Beacon” (see, e.g., Tyagi et al., Nature Biotechnol. 16:49-53, 1998; see, U.S. Patents No. 5,118,801 and 5,312,728), and “Molecular Torch” (see, e.g., U.S. Patents No. 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. The quencher-containing probes include TaqMan® probes, molecular beacons, and molecular torches.

[0089] B. Reaction mixture In some embodiments, the reaction mixture used in the method or system described herein includes one or more amplification oligomers for amplifying a target nucleic acid. In some embodiments, the amplification oligomer includes a detection label. Using the labeled amplification oligomer, a labeled amplicon can be produced that can be detected, for example, by hybridization with an immobilized probe. The reaction mixture typically includes 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 include a test sample component in which an internal control (IC) target nucleic acid may be present.

[0090] C. Reaction vessel, sample Various reaction vessels may be used to house the nucleic acid amplification reaction mixture used in accordance with this disclosure. Non-limiting examples of reaction vessels include tubes, multiwell strips, multiwell plates, microfluidic chips, and microfluidic cartridges. In some embodiments, the nucleic acid amplification reaction mixture containing multiple fluorophores is contained in multiple tubes, multiwell strips, multiwell plates, microfluidic chips, or microfluidic cartridges.

[0091] In some embodiments, the reaction vessel is a microfluidic cartridge. In some embodiments, the microfluidic cartridge (e.g., “lab-on-a-chip”) can perform 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 perform the steps of the methods disclosed herein are shown in Figures 7A–D. Figure 7A shows a top perspective view of an exemplary microfluidic cartridge 701 containing multiple chambers for containing a sample, reagent, or other liquid. Microchannels 706 connect such chambers to move liquids between different functional areas within the cartridge 701. Figure 7B shows a bottom perspective view of an exemplary microfluidic cartridge 701 showing a network of microchannels 706 connecting the various chambers to other areas within the cartridge. Figure 7C shows a bottom perspective view of a fully constructed microfluidic cartridge 701, including a bottom cover 713 and a microarray slide 714, as shown in Figure 7D, which include a sample preparation area 711, a nucleic acid amplification area 710, and a nucleic acid analysis area 709. The nucleic acid amplification area 710 can 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 706, which are shown in detail in Figure 7B. The sample preparation area 711 contains liquid in an adjacent chamber. The system, for example, the nucleic acid amplification system 400, can be programmed to combine the liquids contained in these chambers in a specific order and in separate volumes. When an exemplary microfluidic cartridge 701 is docked to the system 400, the nucleic acid amplification area 710 is adjacent to the thermal cycler 608, as shown in Figure 6. Similarly, when an exemplary microfluidic cartridge 701 is docked to the system 400, the nucleic acid analysis area is adjacent to the array detector 611, as shown in Figure 6.Figure 7D shows an exploded view of a fully constructed exemplary microfluidic cartridge 701, which includes a cartridge body 712, a vent cap 723 for covering the sample after input, and a sample filter 724 for removing cell debris after lysis, which is performed either before sample addition and / or in the sample reservoir 702. The cartridge 701 further includes components for moving and combining the liquid in the sample preparation area 711 by interacting with the mechanical components of the system, including a stopper 718, a nucleic acid binding matrix column 719, a protective cover 721, a blocker 722, and a blocker ring 725. The exploded view in Figure 7D also shows how a microarray slide 714 is attached to the body 712 of the microfluidic cartridge 701 in the nucleic acid analysis area 709 via a microarray adhesive tape 715. The cartridge also includes top and bottom coverings 716, 713 of polypropylene (PP), respectively, and, optionally, a pre-printed sticker 717. Figure 8 shows an exploded view of an exemplary microfluidic cartridge 701, which includes a plunger 801 for engaging with a stopper 718 to move and combine the liquid within the microfluidic cartridge 701. Microfluidic cartridges can have several advantages, including the ability to perform automated operations while consuming small reagent volumes and / or being inexpensive and disposable. An example of a microfluidic cartridge is disclosed in U.S. Patent No. 10,654,039.

[0092] In some embodiments, the microfluidic cartridge 701 includes at least (1) a plurality of functional areas including a sample preparation area 711, a nucleic acid amplification area 710, a nucleic acid analysis area 709, and a waste area 704; (2) a central distribution hub 708; and (3) a fluid network of a pump or plunger 801, a plurality of valves 726, and microchannels 706 connecting the functional areas to the central distribution hub 708. Exemplary microfluidic channels are shown in Figures 7A to 7D. The fluid network of the pump or plunger 801, the plurality of valves 726, and microchannels 706 can drive the movement of fluid from a first functional area through the central distribution hub 708 to a second functional area among the plurality of functional areas. In some embodiments, the nucleic acid amplification reaction is contained within the nucleic acid amplification area 710.

[0093] The sample preparation area 711 may include a network of reservoirs, chambers, or tanks holding multiple liquids or gases of various configurations, as shown in Figure 7A. Such liquids or gases may contain samples and / or reagents commonly used in the art, such as master mixes, washing 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.

[0094] The nucleic acid amplification area 710 may be adjacent to the sample preparation area 711, or it may be connected via a microfluidic channel 706. Such a channel 706 allows the prepared sample to move through the central distribution hub 708 to the nucleic acid amplification area 710 and be subjected to nucleic acid amplification conditions. The nucleic acid amplification area (see, for example, Figure 7C) may be 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.

[0095] The nucleic acid analysis area 709 may include microarray components or slides, as shown in Figures 7C and 7D. 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.

[0096] 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.

[0097] 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.

[0098] 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 at or near the associated area end. Thus, a microfluidic cartridge includes multiple valves located at or near the area ends of hub-connected microchannels. The multiple valves enable the targeted distribution of liquids and gases within the channel network by actively opening and closing the channels and metering the liquids. In some embodiments, the valves are operated in an automated manner by rotating the valves in a previously defined increment. In some embodiments, the valves are operated via valve actuators.

[0099] 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.

[0100] In some embodiments, the fluorophore-containing nucleic acid amplification reaction mixture is placed in the nucleic acid amplification area of ​​a reaction vessel (e.g., a well, a chamber, etc.) during a first period. In some embodiments, the method further includes adjusting the heating element of the nucleic acid system to an initial temperature over a predetermined period, during which the fluorophore-containing nucleic acid amplification reaction mixture is placed in the nucleic acid amplification area. In some embodiments, the nucleic acid amplification reaction mixture can be exposed to an initial temperature for a predetermined period to inactivate the reverse transcriptase after the reverse transcription step used to transcribe RNA into DNA, and then amplified by PCR.

[0101] 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.

[0102] D. Methods for monitoring nucleic acid amplification reaction reactions for abnormalities The methods described herein may include providing a fluorophore-containing nucleic acid amplification reaction mixture. For example, the fluorophore may be provided as a fluorophore-containing nucleic acid (e.g., a labeled detection probe). 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.

[0103] In one embodiment, the method comprises a) providing a fluorophore-containing nucleic acid amplification reaction mixture to a nucleic acid amplification system, and b) adjusting the heating element of the nucleic acid amplification system to a first temperature over a first period, thereby heating the nucleic acid amplification reaction mixture to a first incubation temperature during the first period. The first period may be a high-temperature step before the nucleic acid amplification reaction mixture is exposed to conditions for nucleic acid amplification, such as PCR or qPCR (e.g., thermal cycling). In some embodiments, the first period is a reverse transcriptase inactivation period for inactivating the reverse transcriptase used in RT-PCR. In some embodiments, the nucleic acids in the amplification reaction mixture are denatured during the first period. In some embodiments, the first temperature during this first period is higher than 80°C and is about 80–110°C, about 85–125°C, about 90–120°C, about 95–115°C, about 109°C, or about 110°C. In some embodiments, the first temperature may vary during the first period, for example, being higher in the first part of the first period than in the second part of the first period, which is followed by the first part. In some embodiments, the first temperature is about 95–110°C (e.g., 100–110°C, 105–110°C, or about 109°C) in the first part of the first period and about 80–105°C (e.g., 95–100°C or about 99°C) in the second part of the first period. In some embodiments, the first period is about 20 seconds to about 10 minutes, for example, about 2–10 minutes, about 3–9 minutes, about 3–7 minutes, or about 4–6 minutes. Where applicable, the first and second parts of the first period may each be about half of the first period (e.g., 40–60%). In some embodiments, the first period is approximately 48–52 seconds, which may include a delay of approximately 4 seconds to allow the heating element to exceed 80°C at the start of the first period. In some embodiments, the first period includes a first portion of approximately 20 seconds at approximately 109°C and a second portion of approximately 20 seconds at approximately 99°C.

[0104] In some embodiments, the first period includes a delay of about 1 to 10 seconds to allow the heating element to reach or exceed the target temperature for the first period or the first portion thereof. In some embodiments, the target temperature is about 80°C. An exemplary delay of 4 seconds is shown in Figure 3. In Figure 3, the x-axis shows the individual temperature measurements taken during the indicated amplification steps. The target temperature (Target T°) represents the programmed temperature during each step of the reaction, and the heater temperature (Heater T°) represents the recorded temperature of the heating element over time. The time indicated by the arrow at 48 seconds represents the first period including a 4-second delay. No fluorescence signal is recorded during such a delay.

[0105] In some embodiments, the first period lasts for about 35 seconds to about 60 seconds. In some embodiments, the first period lasts for about 5 seconds to about 50 seconds when the temperature of the first period reaches a plateau. In some embodiments, the first period lasts for about 5 seconds to about 50 seconds when the temperature of the first period exceeds about 80°C.

[0106] In some embodiments, the first period includes a delay sufficient to ensure that the heating element reaches or exceeds the target temperature in the reaction vessel. In some embodiments, the method includes adjusting the heating element to an initial temperature over a predetermined period during the delay. In some embodiments, the method further includes this delay before the fluorophore-containing nucleic acid amplification reaction mixture is subjected to amplification conditions. In some embodiments, the initial temperature is suitable for reverse transcription. In some embodiments, reverse transcription occurs during a predetermined period. In some embodiments, reverse transcription occurs in the fluorophore-containing nucleic acid amplification reaction mixture during a predetermined period. In some embodiments, the initial temperature is about 25–50°C, about 30–50°C, about 35–50°C, about 40–50°C, about 42–49°C, about 35–40°C, about 40–45°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 50°C, or about 55°C. In some embodiments, the predetermined period is approximately 15 to 100 minutes, for example, approximately 20 to 50 minutes.

[0107] 1. Fluorescence measurement The method of this disclosure includes measuring fluorescence from fluorophores present in a nucleic acid amplification reaction mixture. Fluorescence may be measured over the entire first period described in the previous section. Fluorescence may also be measured during the first period, for example, after a delay described in the previous section. A first and / or second value may then be determined from these fluorescence measurements. Fluorescence may be measured in terms of relative fluorescence units (RFU). In some embodiments, if fluorescence is measured during the first period, the nucleic acid amplification reaction mixture has not yet been exposed to nucleic acid amplification conditions, such as those typically seen in PCR or qPCR reactions. The fluorescence measurements described herein differ from fluorescence measurements that may be performed by quantitative and / or real-time PCR methods. This is because the fluorescence measurements in the disclosed method are used qualitatively in that they are evaluated in terms of a threshold or range greater than 1 to give a qualitative output (such as a determination of whether there is an anomaly, depending on whether the fluorescence measurement meets a threshold or falls within a given range). Fluorescence measurements that do not meet the threshold or fall within a predetermined range may indicate defects in the heating element, thermal contact, thermocycler, fluorescence detector, insufficient or incorrect reagents in the reaction mixture, and / or other abnormalities. Furthermore, measurements may be performed during the period before thermal cycling begins, and this method does not require fluorescence measurements during thermal cycling to detect abnormalities.

[0108] In some embodiments, the step of providing a fluorophore-containing nucleic acid amplification reaction mixture for use in a nucleic acid amplification system includes providing a plurality of fluorophore-containing nucleic acid amplification reaction mixtures. These plurality of fluorophore-containing nucleic acid amplification reaction mixtures may be provided in a reaction vessel that allows multiple reactions to be carried out in parallel, while being contained in a single vessel. Such vessels include multiwell strips, multiwell plates, microfluidic chips, and microfluidic cartridges. In some embodiments, the step of measuring fluorescence from fluorophores multiple times during a first period includes measuring fluorescence from each fluorophore in a plurality of fluorophore-containing nucleic acid amplification reaction mixtures contained in a plurality of vessels (e.g., tubes, multiwell strips, multiwell plates, microfluidic chips, or microfluidic cartridges).

[0109] In some embodiments, a single value is determined from the fluorescence measurement. In other embodiments, both the first and second values ​​are determined from the fluorescence measurement.

[0110] In some embodiments, a first value representing the change in fluorescence signal during a first period is determined for each of the multiple fluorophore-containing nucleic acid amplification reaction mixtures. In some embodiments, a second value representing the rate of change in fluorescence during the first period is determined for each of the multiple fluorophore-containing nucleic acid amplification reaction mixtures. In some embodiments, the first and second values ​​are determined for each of the multiple fluorophore-containing nucleic acid amplification reaction mixtures.

[0111] a) Determination of the first value from fluorescence measurement In some embodiments, the first value is a measure of the total change in fluorescence during a first period. In some embodiments, the first value is determined from multiple fluorescence measurements recorded over time. If it is assumed that the fluorescence of the fluorophore increases in response to rising temperature, the change in fluorescence is the total change in fluorescence from the start of the first period when the temperature of the fluorophore-containing nucleic acid amplification reaction mixture is lowest, to the end of the first period when the temperature of the fluorophore-containing nucleic acid amplification reaction mixture is highest. Thus, in some embodiments, this change in fluorescence during the first period is the total increase or total gain of fluorescence. In some embodiments, the first fluorescence measurement is the lowest fluorescence measurement during the first period. In some embodiments, the highest temperature is maintained by a heating element over a period of time, and after the fluorescence signal has plateaued, the last fluorescence measurement is recorded at the end of the first period. In these embodiments, the last fluorescence measurement may be the highest fluorescence measurement during the first period.

[0112] In some embodiments, fluorescence measurements are recorded at regular time intervals. In some embodiments, fluorescence measurements are recorded approximately every 100 milliseconds, every 200 milliseconds, every 300 milliseconds, every 400 milliseconds, or every 500 milliseconds. In some embodiments, fluorescence measurements are recorded over periods such as 1 second to 10 seconds.

[0113] In some embodiments, the first fluorescence measurement is recorded at the start of the first period when the temperature of the heating element reaches the target temperature for the first period. In some embodiments, the first fluorescence measurement is recorded after the heating element reaches or exceeds 80°C.

[0114] In some embodiments, the first measurement is obtained about 1 to 10 seconds before the start of the first period. In some embodiments, the first measurement is obtained about 1 to 10 seconds before the heating element reaches or exceeds 80°C. In some embodiments, the first measurement is obtained about 1 to 10 seconds after the start of the first period. In some embodiments, the first measurement is obtained about 1 to 10 seconds after the heating element reaches or exceeds 80°C.

[0115] In some embodiments, the final measurement is obtained approximately 35 to 60 seconds after the start of the first period. In some embodiments, the final measurement is obtained approximately 5 to 30 seconds after the temperature reaches a temperature plateau during the first period.

[0116] In some embodiments, the first value represents the change in the fluorescence signal measured in relative fluorescence units. In some embodiments, the first value is determined by subtracting the early fluorescence measurement from the late fluorescence measurement. In some embodiments, the early fluorescence measurement is obtained before or during the first half of the first period, within one minute of the start of the first period, or approximately at the start of the first period. In some embodiments, the early fluorescence measurement constitutes the minimum measurable fluorescence from the fluorophore. In some embodiments, the late fluorescence measurement is obtained during the second half of the first period, within one minute of the end of the first period, or approximately at the end of the first period. In some embodiments, the late fluorescence measurement constitutes the maximum measurable fluorescence from the fluorophore. In some embodiments, the difference between the late fluorescence measurement of the first period and the early fluorescence measurement of the first period is called the "contrast".

[0117] b) Determination of the second value from fluorescence measurement In some embodiments, the second value is a heat ratio equal to the area of ​​the lower quadrilateral region divided by the area of ​​the upper quadrilateral region. An exemplary quadrilateral region, along with its upper and lower areas, is shown in Figure 1. During the first period described above, a plot, e.g., a graph, can be created with the quadrilateral region to represent the change in fluorescence over time (i.e., gain). For example, as shown in Figure 1, the unit of time may be shown on the x-axis and the relative fluorescence units (RFU) may be shown on the y-axis. Thus, in some embodiments, the fluorescence signal can be plotted over time to generate a curve. In some embodiments, the quadrilateral region is vertically bounded by early and late fluorescence measurements from the fluorophore during the first period and horizontally bounded by the times of the early and late fluorescence measurements from the fluorophore during the first period. In some embodiments, the quadrilateral region is vertically bounded by the lowest and highest fluorescence measurements from the fluorophore during the first period and horizontally bounded by the times of the lowest and highest fluorescence measurements from the fluorophore during the first period. In some embodiments, the quadrilateral region is vertically bounded by the lowest and highest fluorescence measurements from the fluorophore during a first period, and horizontally bounded by the times of the early and late fluorescence measurements from the fluorophore during the first period.

[0118] In some embodiments, the plot includes a curve representing the change in fluorescence over time. In some embodiments, the plot includes a histogram, and a curve may be created by drawing a line through the RFU data point of each bar in the histogram, and a median filter is applied to smooth the curve. In any embodiment, the curve represents the boundary between (1) the lower area of ​​the quadrilateral region and (2) the upper area of ​​the quadrilateral region. Thus, (1) the lower area can be equal to the area of ​​the quadrilateral region below the fluorescence measurement from the fluorophore, and (2) the upper area can be equal to the area of ​​the quadrilateral region above the fluorescence measurement from the fluorophore. Thus, the surface areas (or two-dimensional spaces) of the lower and upper areas can be defined by the curve and the boundary of the quadrilateral region.

[0119] The shape of the curve during the first period described above can be quantified by determining the ratio of the lower area to the upper area. This surface ratio of the lower area to the upper area may represent a second value. In some embodiments, the lower area is called the first partial value and the upper area is called the second partial value. Thus, the second value can be determined by dividing the first partial value by the second partial value. In some embodiments, the first partial value represents the lower area of ​​the quadrilateral region of the fluorescence plot from the fluorophore over time during the first period. In some embodiments, the second partial value represents the upper area of ​​the quadrilateral region. In some embodiments, the second value represents the rate of change of fluorescence over time, and the rate of change correlates with the rate of change of temperature of the nucleic acid amplification reaction mixture.

[0120] In some embodiments, fluorescence measurements are recorded at regular time intervals. In some embodiments, a curve smoothing function is applied to the measurements recorded at regular time intervals. In these embodiments, the exact areas above and below the curve are not determined. In these embodiments, the lower area, i.e., the first partial value, is the sum of a series of differences between the minimum values ​​represented by the data points along the curve. Also in these embodiments, the upper area, i.e., the second partial value, is the sum of the maximum value from the quadrilateral region minus each minimum value.

[0121] c) Determination of abnormalities by comparing the first and second values ​​with a predetermined value. In some embodiments, first and second values, such as (1) gain at RFU and (2) surface ratio of lower area to upper area (first partial value / second partial value), are used to identify nucleic acid amplification reaction mixtures associated with anomalies. These first and second values ​​may be determined for the nucleic acid amplification reaction mixture of interest. The first and second values ​​may then be compared with first and second values ​​determined from a reference nucleic acid amplification reaction mixture.

[0122] In some embodiments, in the step of comparing a first and / or second value with a first and / or second predetermined threshold or a first and / or second predetermined range, for each of the plurality of fluorophore-containing nucleic acid amplification reaction mixtures, (1) the first value is compared with a first predetermined threshold or range, (2) the second value is compared with a second predetermined threshold or range, or (3) the first value is compared with a first predetermined threshold or range and the second value is compared with a second predetermined threshold.

[0123] A reference run of a nucleic acid amplification reaction may include multiple successful and unsuccessful reactions. The reference run may include reactions performed under different conditions ranging from normal to completely unsuccessful due to fluid anomalies including poor thermal contact, a faulty thermocycler, or insufficient amounts of reagent. Unsuccessful reactions may include nucleic acid amplification reaction mixtures having one or more conditions that caused those reactions to fail. A first and second value may be determined for each of these reference reactions, and in some embodiments, the first and second values ​​are referred to as the first predetermined value and the second predetermined value. In some embodiments, the first predetermined value is a range or threshold. In some embodiments, the second predetermined value is a range or threshold.

[0124] 2. Abnormality To detect anomalies in the nucleic acid amplification reaction mixture, in some embodiments, a first value of the method described herein is compared to a first predetermined threshold or range. In some embodiments, a second value of the method described herein is compared to a second predetermined threshold or range. These comparisons enable the detection of abnormal curve shapes. In some embodiments, if an anomaly is detected, the nucleic acid amplification system provides an alarm. Advantageously, the method and system require no additional reagents compared to existing nucleic acid amplification reactions. Also advantageously, the method can be run on existing nucleic acid amplification systems that can detect fluorescence without modifying their hardware. The method and system collect and utilize fluorescence that is commonly generated but ignored by existing methods. Such fluorescence is used in novel ways to detect anomalies and thus can improve assay throughput and efficiency (e.g., by enabling early termination of assays with anomalies) without requiring additional hardware, reagents, or space.

[0125] In some embodiments, an abnormality is detected in the nucleic acid amplification reaction mixture if the first value does not meet a first predetermined threshold or shows a change in fluorescence that is outside a first predetermined range.

[0126] In some embodiments, an abnormality is detected in the nucleic acid amplification reaction mixture if the second value does not meet a second predetermined threshold or exhibits a temperature change rate that is outside the second predetermined range.

[0127] In some embodiments, an abnormality is detected in the nucleic acid amplification reaction mixture if (1) the first value does not meet a first predetermined threshold or shows a change in fluorescence that is outside the first predetermined range, or (2) the second value does not meet a second predetermined threshold or shows a rate of temperature change that is outside the second predetermined range.

[0128] In some embodiments, an abnormality is detected in the nucleic acid amplification reaction mixture if (1) the first value shows a change in fluorescence that does not meet a first predetermined threshold or is outside a first predetermined range, and (2) the second value shows a rate of temperature change that does not meet a second predetermined threshold or is outside a second predetermined range.

[0129] In some embodiments, an anomaly is detected when the sum of the maximum value from the quadrilateral region minus each minimum value (i.e., a second partial value) is greater than a predetermined limit or threshold for that sum. In some embodiments, an anomaly is detected when a second value (i.e., a heat ratio) is greater than a predetermined limit or threshold for the second value.

[0130] In some embodiments, the abnormality includes a defect in thermal contact. For example, contact between the heating element in the nucleic acid amplification system and the reaction vessel containing the nucleic acid amplification reaction mixture may be insufficient or inadequate. A defect in thermal contact can lead to improper heat transfer to the nucleic acid amplification reaction mixture, thereby causing an abnormality in the nucleic acid amplification reaction.

[0131] In some embodiments, the abnormality includes a defect in the heating element. In some embodiments, the defect in the heating element is a defect in controlling or changing the temperature or in transferring heat to the nucleic acid amplification reaction chamber.

[0132] In some embodiments, the abnormality includes defects in liquid handling. In some cases, errors may occur in liquid handling during the preparation of the nucleic acid amplification reaction mixture. These can include introducing bubbles, incorrect liquid volume, and / or incorrect liquid reagents (e.g., buffers, salts, and other aqueous solutions) into the reaction vessel, thereby causing abnormalities in the nucleic acid amplification reaction. Incorrect liquid volume may include insufficient liquid volume, excessive liquid volume, and / or no liquid volume transferred to the nucleic acid amplification reaction chamber. Incorrect liquid reagents may include nucleic acid amplification reaction mixtures with reagent changes of one or more that cause the reaction to fail.

[0133] E. 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 carry out, monitor, and / or analyze a nucleic acid amplification reaction as described herein. The nucleic acid amplification system may include a docking station configured to receive a reaction vessel for containing at least one nucleic acid amplification mixture, a heating element positioned in close proximity to the location occupied by the nucleic acid amplification mixture when the reaction vessel is present in the docking station, a fluorescence detector configured to measure the fluorescence of fluorophores in the nucleic acid mixture, and a processor operably coupled to the heating element and memory, and a memory containing instructions, when executed by the processor, causing the nucleic acid amplification system to carry out a method of monitoring the nucleic acid amplification reaction mixture, including fluorophores, for anomalies. Features of any exemplary nucleic acid amplification system are identified above. Furthermore, an exemplary commercial system is the Novodiag® system (Mobidiag Oy; Espoo, Finland), which can be used with several different cartridges (reaction vessels). The monitoring method includes adjusting the heating element of the nucleic acid amplification system to a first temperature over a first period, and then measuring the fluorescence from fluorophores contained in the nucleic acid amplification mixture multiple times during the first period using the fluorescence detector of the nucleic acid amplification system. The first period may occur before the planned nucleic acid amplification. The method then includes determining a first value representing the change in the fluorescence signal during the first period, and / or a second value representing the rate of change in fluorescence during the first period, and comparing the first value to a first predetermined threshold or range, and / or comparing the second value to a second predetermined threshold or range.An abnormality is detected if: the first value does not meet a first predetermined threshold or shows a change in fluorescence that is outside the first predetermined range; the second value does not meet a second predetermined threshold or shows a rate of temperature change that is outside the second predetermined range; the first value does not meet a first predetermined threshold or shows a change in fluorescence that is outside the first predetermined range, or the second value does not meet a second predetermined threshold or shows a rate of temperature change that is outside the second predetermined range; or the first value does not meet a first predetermined threshold or shows a change in fluorescence that is outside the first predetermined range, AND the second value does not meet a second predetermined threshold or shows a rate of temperature change that is outside the second predetermined range.

[0134] In some embodiments, the vessel docking station is configured to receive a multi-chamber receptacle, or the nucleic acid amplification chamber is housed within the multi-chamber receptacle. In some embodiments, if the reaction vessel is located within the system, for example, within the docking station, the heating element 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 reaction vessel that is in thermal communication with the heating element).

[0135] 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.

[0136] In some embodiments, the nucleic acid amplification system includes an optical detector (e.g., a fluorophotometer) configured to measure and record the fluorescence of one or more fluorophores in the nucleic acid amplification reaction mixture. The fluorescence measurement and recording may be a single event or multiple events over time.

[0137] In some embodiments, the nucleic acid amplification system may also include sensors for monitoring and / or estimating the temperature or liquid filling of the nucleic acid amplification reaction mixture. In some embodiments, the sensors may be thermal sensors, capacitive sensors, or infrared sensors. The nucleic acid amplification system may include a heating element and a processor operably coupled to memory. The memory includes instructions that cause the system to perform methods for monitoring the nucleic acid amplification reaction mixture for anomalies described herein.

[0138]

[92] In some embodiments, if an abnormality is detected, the nucleic acid amplification system provides one or more alarms. 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 method, which may include adjusting the heating element to about 4°C and holding that temperature for a period of time.

[0139] In some embodiments, the system is configured to detect anomalies including defects in the heater (e.g., a thermal cycler), improper contact between the heater and the reaction vessel (e.g., a microfluidic cartridge), improper passage of reaction reagents or nucleic acid amplification reaction mixtures into the functional area for carrying out nucleic acid amplification reactions (e.g., PCR), or problems with the composition of the reaction mixture.

[0140] In some embodiments, the system is configured to adjust a heating element to an initial temperature over a predetermined period of time before providing a fluorophore-containing nucleic acid amplification reaction mixture within the nucleic acid amplification system. The initial temperature may be suitable for reverse transcription. Reverse transcription may occur in the fluorophore-containing nucleic acid amplification reaction mixture over a predetermined period of time, as described herein.

[0141] In some embodiments, the system is configured to subject the nucleic acid amplification reaction mixture to conditions for thermal cycling under conditions where no abnormalities are detected. Such conditions may include time and temperature conditions for performing PCR or RT-PCR.

[0142] The fluorophore-containing nucleic acid amplification reaction mixture may be contained in a microfluidic cartridge, and such a cartridge may have any of the features described elsewhere in this specification.

[0143] In some embodiments, the system is configured to accept multiple fluorophore-containing nucleic acid amplification reaction mixtures housed in a multiwell plate or multiple tubes. For example, the system may include a receptacle for a multiwell plate or multiple receptacles for tubes.

[0144] In some embodiments, the system is configured to detect an anomaly if a first value indicates a change in fluorescence that does not meet a first predetermined threshold or is outside a first predetermined range. In some embodiments, the system is configured to detect an anomaly if a second value indicates a rate of temperature change that does not meet a second predetermined threshold or is outside a second predetermined range. In some embodiments, the system is configured to detect an anomaly if either the first value indicates a change in fluorescence that does not meet a first predetermined threshold or is outside a first predetermined range, or the second value indicates a rate of temperature change that does not meet a second predetermined threshold or is outside a second predetermined range. In some embodiments, the system is configured to detect an anomaly if the first value indicates a change in fluorescence that does not meet a first predetermined threshold or is outside a first predetermined range, and the second value indicates a rate of temperature change that does not meet a second predetermined threshold or is outside a second predetermined range.

[0145] In some embodiments, the system is configured such that adjusting the heating element to a first temperature over a first period of time constitutes a reverse transcriptase inactivation step.

[0146] In some embodiments, the method includes subjecting the nucleic acid amplification reaction mixture to nucleic acid amplification conditions only if no abnormality is detected. In some embodiments, the nucleic acid amplification conditions include thermal cycling. In some embodiments, the method includes stopping the nucleic acid amplification reaction if an abnormality is detected. In some embodiments, the system is configured to subject the nucleic acid amplification reaction mixture to nucleic acid amplification conditions only if no abnormality is detected. In some embodiments, the system is configured to reduce the temperature of the heating element (for example, to ambient temperature, or to a temperature higher than 0°C but below ambient temperature, e.g., 0.1 to 10°C) without thermal cycling if an abnormality is detected.

[0147] Furthermore, computer-readable media are disclosed herein. In some embodiments, the computer-readable media includes instructions, when executed by the processor of the nucleic acid amplification system, that cause the nucleic acid amplification system to perform a method for monitoring the nucleic acid amplification reaction mixture described herein. The monitoring method includes adjusting the heating element of the nucleic acid amplification system to a first temperature over a first period, and then measuring the fluorescence from fluorophores contained in the nucleic acid amplification mixture multiple times during the first period using the fluorescence detector of the nucleic acid amplification system. The first period may occur before the planned nucleic acid amplification. The method then includes determining a first value representing the change in the fluorescence signal during the first period, and / or a second value representing the rate of change in fluorescence during the first period, and comparing the first value to a first predetermined threshold or range, and / or comparing the second value to a second predetermined threshold or range. An abnormality is detected if: the first value does not meet a first predetermined threshold or shows a change in fluorescence that is outside the first predetermined range; the second value does not meet a second predetermined threshold or shows a rate of temperature change that is outside the second predetermined range; the first value does not meet a first predetermined threshold or shows a change in fluorescence that is outside the first predetermined range, or the second value does not meet a second predetermined threshold or shows a rate of temperature change that is outside the second predetermined range; or the first value does not meet a first predetermined threshold or shows a change in fluorescence that is outside the first predetermined range, AND the second value does not meet a second predetermined threshold or shows a rate of temperature change that is outside the second predetermined range.

[0148] In some embodiments, a computer-readable medium includes instructions for carrying out the method of monitoring the nucleic acid amplification reaction mixture described herein. In some embodiments, these instructions are executed by the processor of the nucleic acid amplification system described herein. [Examples]

[0149] Examples This embodiment describes performing reference runs of a nucleic acid amplification reaction under normal and abnormal conditions. This set of reference runs provides a basis for determining the signal gain and heat ratio of normal and abnormal runs. The objective is to implement additional controls in the nucleic acid amplification reaction run to detect any thermal anomalies from the run data before the completion of the reaction run.

[0150] 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. The first chamber contained 0.6 μM ATTO (LSS signal) with a quencher, 0.4 μM 6-FAM (blue signal) with a quencher, and 0.4 μM Quasar® 670 (red signal) with a quencher. The second chamber contained 1.2 μM ATTO (LSS signal) with a quencher, 0.4 μM 6-FAM (blue signal) with a quencher, and 0.8 μM Quasar® 670 (red signal) with a quencher.

[0151] The reaction mixture was subjected to a protocol including the following steps, performed using a Novodiag® system (Mobodiag Oy; Espoo, Finland): 46℃ 300 seconds 109℃ 24 seconds 99℃ 24 seconds

[0152] Reactions to abnormalities included reactions to poor thermal contact and fluid errors. Thermal errors occurred by adding a layer of adhesive between the heater and the qPCR chamber, which hindered heat transfer. Fluid errors occurred by modifying the protocol to skip one or more injection steps. Further fluid errors occurred by altering valve force and / or position to cause leaks or by preventing valves from opening.

[0153] The system began recording RFUs after the heating element exceeded 80°C (with a +4 second delay). The system stopped recording RFUs at the end of the 109°C period. In this example, data points were recorded every 200 milliseconds for a total of 48 seconds.

[0154] For each run, a curve of the type shown in Figure 1 was created, and a curve smoothing function with a five-point average filtering window was applied. The minimum and maximum values ​​of each smoothed curve were also determined, and the signal gain was calculated as the difference between them. Next, the heat ratio (i.e., upper area / lower area) was determined from each smoothed curve.

[0155] Results: The signal gain and heat ratio of multiple runs are plotted in Figure 2. The distribution of normal runs is indicated and limited by the upper and lower limits of the heat ratio, as well as the upper and lower limits of the signal gain. Most runs with fluid errors have a signal gain and / or heat ratio higher than the upper limit of normal runs. Most runs with poor thermal contact have a heat ratio lower than the lower limit of normal runs. Therefore, this method was effective in detecting both fluid errors and poor thermal contact based on fluorescence data obtained during heating and incubation of nucleic acid reaction mixtures, such as in the initial denaturation step in thermal cycle amplification reactions.

[0156] 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 abnormalities in nucleic acid amplification reaction mixtures, a) To provide a nucleic acid amplification reaction mixture containing fluorophores to a nucleic acid amplification system. b) Adjusting the heating element of the nucleic acid amplification system to a first temperature over a first period of time, thereby heating the nucleic acid amplification reaction mixture to a first incubation temperature during the first period. c) After step b), the fluorescence from the fluorophore is measured multiple times during the first period, wherein the nucleic acid amplification reaction mixture is not exposed to nucleic acid amplification conditions before or during the first period. d) Determine the following: i) A first value representing the change in the fluorescence signal during the first period, and / or ii) A second value representing the rate of change of fluorescence during the first period, and e) Comparing the first value with a first predetermined threshold or range, and / or comparing the second value with a second predetermined threshold or range, in the following cases: i) The first value does not meet the first predetermined threshold or shows a change in fluorescence outside the first predetermined range, ii) The second value indicates a temperature change rate that does not satisfy the second predetermined threshold or is outside the second predetermined range. iii) The first value does not meet the first predetermined threshold or shows a change in fluorescence that is outside the first predetermined range, or the second value does not meet the second predetermined threshold or shows a rate of temperature change that is outside the second predetermined range, iv) A method for detecting an anomaly when the first value shows a change in fluorescence that does not meet the first predetermined threshold or is outside the first predetermined range, and the second value shows a rate of temperature change that does not meet the second predetermined threshold or is outside the second predetermined range.

2. A nucleic acid amplification system, A docking station configured to accept a reaction vessel for containing at least one nucleic acid amplification mixture, A heating element installed in close proximity to the position occupied by the nucleic acid amplification mixture when the reaction vessel is located in the docking station, A fluorescence detector configured to measure the fluorescence of fluorophores in the nucleic acid mixture, and The system comprises a processor operably connected to the heating element and memory, The memory includes an instruction, when executed by the processor, that causes the nucleic acid amplification system to perform a method of monitoring the nucleic acid amplification reaction mixture containing a fluorophore for abnormalities, the method being: a) Adjusting the heating element to a first temperature over a first period of time, b) After step a), the fluorescence from fluorophores contained in the nucleic acid amplification mixture is measured multiple times during the first period using the fluorescence detector, wherein the first period occurs before the planned nucleic acid amplification. c) Determine the following: i) A first value representing the change in the fluorescence signal during the first period, and / or ii) A second value representing the rate of change of fluorescence during the first period, and d) Comparing the first value with a first predetermined threshold or range, and / or comparing the second value with a second predetermined threshold or range, in the following cases: i) The first value does not meet the first predetermined threshold or shows a change in fluorescence outside the first predetermined range, ii) The second value indicates a temperature change rate that does not satisfy the second predetermined threshold or is outside the second predetermined range. iii) The first value does not meet the first predetermined threshold or shows a change in fluorescence that is outside the first predetermined range, or the second value does not meet the second predetermined threshold or shows a rate of temperature change that is outside the second predetermined range, iv) A nucleic acid amplification system that includes detecting an anomaly when the first value shows a change in fluorescence that does not meet the first predetermined threshold or is outside the first predetermined range, and the second value shows a rate of temperature change that does not meet the second predetermined threshold or is outside the second predetermined range.

3. Computer-readable medium, When executed by the processor of a nucleic acid amplification system, the instruction includes a command to cause the nucleic acid amplification system to perform a method of monitoring the nucleic acid amplification reaction mixture containing a fluorophore for abnormalities, and the method is a) Adjusting the heating element of the nucleic acid amplification system to a first temperature over a first period of time, b) After step a), the fluorescence from fluorophores contained in the nucleic acid amplification mixture is measured multiple times during the first period using the fluorescence detector of the nucleic acid amplification system, wherein the first period is the period before the scheduled nucleic acid amplification. c) Determine the following: i) A first value representing the change in the fluorescence signal during the first period, and / or ii) A second value representing the rate of change of fluorescence during the first period, and d) Comparing the first value with a first predetermined threshold or range, and / or comparing the second value with a second predetermined threshold or range, in the following cases: i) The first value does not meet the first predetermined threshold or shows a change in fluorescence outside the first predetermined range, ii) The second value indicates a temperature change rate that does not satisfy the second predetermined threshold or is outside the second predetermined range. iii) The first value does not meet the first predetermined threshold or shows a change in fluorescence that is outside the first predetermined range, or the second value does not meet the second predetermined threshold or shows a rate of temperature change that is outside the second predetermined range, iv) A computer-readable medium that includes comparison, in which an anomaly is detected when the first value shows a change in fluorescence that does not meet the first predetermined threshold or is outside the first predetermined range, and the second value shows a rate of temperature change that does not meet the second predetermined threshold or is outside the second predetermined range.

4. The method, system, or computer-readable medium according to any one of the preceding claims, wherein the first value is determined by subtracting an early measurement of fluorescence from the fluorophore from a late measurement of fluorescence from the fluorophore.

5. The method, system, or computer-readable medium of the preceding claim, wherein the early measurement of fluorescence from the fluorophore is obtained before or during the first half of the first period, within one minute from the start of the first period, or approximately at the start of the first period.

6. The method, system, or computer-readable medium according to any one of claims 4 to 5, wherein the early measurement of fluorescence from the fluorophore is the lowest measurement of fluorescence from the fluorophore.

7. a) The second value is determined by dividing the first partial value by the second partial value, b) The first partial value represents the area of ​​the quadrilateral region of the plot of fluorescence from the fluorophore over time during the first period, and c) The second partial value represents the upper area of ​​the quadrilateral region, The aforementioned quadrilateral region is as follows: i) the first and last measured values ​​of fluorescence from the fluorophore during the first period, and the time of the first and last measured values ​​of fluorescence from the fluorophore during the first period, ii) The minimum and maximum measured values ​​of fluorescence from the fluorophore during the first period, and the time intervals for the minimum and maximum measured values ​​of fluorescence from the fluorophore during the first period. iii) The minimum and maximum measured values ​​of fluorescence from the fluorophore during the first period, and the time of the first and last measured values ​​of fluorescence from the fluorophore during the first period, or iv) Bounded by the first and last measured values ​​of fluorescence from the fluorophore during the first period, and the time of the minimum and maximum measured values ​​of fluorescence from the fluorophore during the first period, The method, system, or computer-readable medium according to any one of the preceding claims, wherein the lower area is equal to the area of ​​the quadrilateral region below the measured value of fluorescence from the fluorophore, and the upper area is equal to the area of ​​the quadrilateral region above the measured value of fluorescence from the fluorophore.

8. The method, system, or computer-readable medium according to any one of the preceding claims, wherein the first value represents a change in the Y axis of a fluorescence signal.

9. The method, system, or computer-readable medium according to any one of the preceding claims, wherein the fluorescence changes over time during the first period, and the rate of change of the fluorescence during the first period correlates with the rate of change of temperature of the nucleic acid amplification reaction mixture.

10. The method, system, or computer-readable medium according to any one of the preceding claims, wherein the abnormality is a thermal contact defect and / or a liquid handling defect.

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