Control of the nucleic acid amplification process

Abstract

A method, system, or computer-readable medium for monitoring the introduction of one or more liquids into a reaction chamber. Abnormalities in the volume and composition of buffers may occur during the preparation of the reaction mixture and / or may be caused by errors in the function of the nucleic acid amplification system. An exemplary method includes determining fluorescence-based values ​​from a fluorophore-containing nucleic acid amplification reaction mixture and determining whether those values ​​meet a predetermined threshold and / or range or indicate an abnormality. The methods disclosed herein may depend on the effect of the presence or concentration of nucleic acids, nucleotides, and / or fluorophores on the fluorescence properties of the reaction mixture.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to European Patent Application No. 23315134.9, filed on April 28, 2023, which is incorporated herein by reference in its entirety for all purposes.

[0002] Field This disclosure relates to the field of nucleic acid amplification. More particularly, the disclosure relates to methods, materials, devices, and systems for monitoring a nucleic acid amplification reaction mixture with respect to the introduction of one or more liquids into a reaction chamber.

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 or ligase, either separately or in combination, to generate multiple copies of a target nucleic acid sequence in a primer extension reaction that incorporates nucleotides or by ligation of adjacent probes complementary to the target nucleic acid sequence. In such reactions, each template generates more copies, and the copies themselves can serve as templates. (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, as well as 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, the 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. The target nucleic acid sequence can be subjected to denaturation, annealing, and extension conditions until a sufficient amount of amplicons is present in the reaction mixture to make the target nucleic acid sequence detectable or quantifiable. In the initial stages of PCR, the amplification is exponential.

[0004] Nucleic acid amplification reactions depend on the precision of the buffer composition of the reaction mixture. Amplification of target nucleic acids is primarily carried out by enzymatic methods using one or more polymerases, such as DNA polymerase, reverse transcriptase, and / or RNA polymerase. These enzymes are sensitive to aspects of the buffer composition, such as salt type, salt concentration, and pH. Nucleic acid amplification reactions generally maintain the functionality of the enzymes using buffers such as Tris, magnesium sources such as MgSO4 or MgCl2, and monovalent cation salts such as KCl.

[0005] Abnormalities in the volume and composition of the buffer solution may occur during the preparation of the reaction mixture and / or be caused by errors in the function of the nucleic acid amplification system. Such abnormalities can lead to unreliable or inconsistent results, and even complete failure of the nucleic acid amplification reaction. Existing methods cannot indicate whether the nucleic acid amplification reaction was successful or failed until the reaction run is complete. The absence of amplification is an obvious indicator of failure, but it does not generally indicate the reason for the failure. This specification discloses materials, methods, and systems for detecting abnormalities related to the detection of abnormalities in the liquid filling of the reaction chamber. The methods disclosed herein may depend on the effect of the presence or concentration of nucleic acids, nucleotides, and / or fluorophores on the fluorescence properties 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 the introduction of one or more liquids into a reaction chamber, a) Obtain a first fluorescence measurement from the reaction chamber. b) After step a), introducing or attempting to introduce a first liquid from among one or more liquids into the reaction chamber. c) After step b), obtain a second fluorescence measurement from the reaction chamber. d) Determining a first value from a first fluorescence measurement and a second fluorescence measurement, wherein the first value indicates the degree of change in fluorescence in the reaction chamber resulting from step b), and e) A method comprising comparing a first value with a first predetermined threshold or range, wherein an anomaly is detected if the first value does not meet the first predetermined threshold or if the first value is outside the first predetermined range.

[0008] Embodiment 2 is a system comprising a docking station configured to receive a container having a reaction chamber, a fluorometer configured to measure fluorescence within the reaction chamber, and a processor operably coupled to the fluorometer and memory, The memory, when executed by the processor, contains instructions that cause the system to perform a method of monitoring the introduction of one or more liquids into the reaction chamber, the method being: a) Obtain a first fluorescence measurement from the reaction chamber. b) After step a), introducing or attempting to introduce a first liquid from among one or more liquids into the reaction chamber. c) After step b), obtain a second fluorescence measurement from the reaction chamber. d) Determining a first value from a first fluorescence measurement and a second fluorescence measurement, wherein the first value indicates the degree of change in fluorescence in the reaction chamber resulting from step b), and e) A system that includes comparing a first value with a first predetermined threshold or range, wherein an anomaly is detected if the first value does not meet the first predetermined threshold or if the first value is outside the first predetermined range.

[0009] Embodiment 3 is a computer-readable medium, When executed by the system's processor, the instruction includes a method for causing the system to monitor the introduction of one or more liquids into the reaction chamber, the method being: a) Obtain a first fluorescence measurement from the reaction chamber. b) After step a), introducing or attempting to introduce a first liquid from among one or more liquids into the reaction chamber. c) After step b), obtain a second fluorescence measurement from the amplification reaction chamber. d) Determining a first value from a first fluorescence measurement and a second fluorescence measurement, wherein the first value indicates the degree of change in fluorescence in the reaction chamber resulting from step b), and e) A computer-readable medium that includes comparing a first value with a first predetermined threshold or range, wherein an anomaly is detected if the first value does not meet the first predetermined threshold or if the first value is outside the first predetermined range.

[0010] Embodiment 4 is a method, system, or computer-readable medium of any of the preceding embodiments, wherein introducing or attempting to introduce the first liquid into the reaction chamber involves pumping or attempting to pump the first liquid into the reaction chamber via a tip, the tip being a needle tip or a pipette tip, if necessary.

[0011] Embodiment 5 is a method, system, or computer-readable medium of any of the preceding embodiments in which the container is a multi-chamber receptacle, or the reaction chamber is housed within a multi-chamber receptacle.

[0012] Embodiment 6 is a method, system, or computer-readable medium of any of the preceding embodiments, in which the first value is determined as the ratio of a second fluorescence measurement to a first fluorescence measurement.

[0013] Embodiment 7 is a method, system, or computer-readable medium of any one of the prior embodiments in which the first fluorescence measurement is the mean or median of a plurality of individual measurements obtained in step a).

[0014] Embodiment 8 is any one of the methods, systems, or computer-readable media of the preceding embodiments, wherein the second fluorescence measurement value is the average or median of a plurality of individual measurement values obtained in step c).

[0015] Embodiment 9 is any one of the methods, systems, or computer-readable media of the preceding embodiments, wherein the nucleic acid amplification reaction is initiated in the reaction chamber after step b) and before step c).

[0016] Embodiment 10 is the method, system, or computer-readable media of the immediately preceding embodiment, wherein the initiation of the nucleic acid amplification reaction includes initiating a denaturation step or a reverse transcription step.

[0017] Embodiment 11 is the method, system, or computer-readable media of the immediately preceding embodiment, wherein the second fluorescence measurement value is obtained during the denaturation step or the reverse transcription step.

[0018] Embodiment 12 is any one of the methods, systems, or computer-readable media of the preceding embodiments, wherein the reaction chamber is housed within a microfluidic cartridge.

[0019] Embodiment 13 is such that the microfluidic cartridge includes a plurality of functional areas including a sample preparation area, a nucleic acid amplification area, and a waste area, a central distribution hub, and a fluid network of pumps, a plurality of valves, and microchannels connecting the functional areas to the hub, and the pumps, the plurality of valves, and the fluid network of microchannels can drive the movement of fluid from a first functional area through the central distribution hub to a second functional area among the plurality of functional areas, and the reaction chamber is the nucleic acid amplification area or within the nucleic acid amplification area, which is the method, system, or computer-readable media of the immediately preceding embodiment.

[0020] Embodiment 14 is the method, system, or computer-readable medium of the immediately preceding embodiment, where when successfully introduced in step b), the first liquid is placed in the nucleic acid amplification area during step c).

[0021] Embodiment 15 is the method, system, or computer-readable medium of the immediately preceding embodiment, where the first liquid is placed in the nucleic acid amplification area during step c).

[0022] Embodiment 16 is the method, system, or computer-readable medium of any one of the preceding embodiments, where the first fluorescence measurement value is obtained from each of a plurality of reaction chambers in step a).

[0023] Embodiment 17 is the method, system, or computer-readable medium of the immediately preceding embodiment, where step b) includes introducing or attempting to introduce the first liquid into each of a plurality of reaction chambers in step b).

[0024] Embodiment 18 is the method, system, or computer-readable medium of the immediately preceding embodiment, where step c) includes obtaining a second fluorescence measurement value from each of a plurality of reaction chambers in step c).

[0025] Embodiment 19 is the method, system, or computer-readable medium of the immediately preceding embodiment, where step d) includes determining a first value from each of a plurality of first fluorescence measurement values and each of a plurality of second fluorescence measurement values, and each first value indicates the degree of change in fluorescence within the corresponding reaction chamber resulting from step b).

[0026] Embodiment २० is the method, system, or computer-readable medium of the immediately preceding embodiment, where step e) includes comparing each first value with a first predetermined threshold or range, and an abnormality is detected when any first value does not meet the first predetermined threshold or is outside the first predetermined range.

[0027] Embodiment 21 is any one of the methods, systems, or computer-readable media of Embodiments 16 to 20, wherein multiple reaction chambers are housed in a multiwell plate or multiple tubes.

[0028] Embodiment 22 is a method, system, or computer-readable medium of any one of the preceding embodiments, wherein the first liquid comprises a fluorophore.

[0029] Embodiment 23 is a method, system, or computer-readable medium of the preceding embodiment, wherein a fluorophore is associated with an oligonucleotide probe, and optionally the oligonucleotide probe further comprises a quencher.

[0030] Embodiment 24 is a method, system, or computer-readable medium of any one of Embodiments 1 to 21, wherein the first liquid does not contain fluorophores associated with oligonucleotides, the intrinsic fluorescence of one or more reagents or samples is measured, and optionally one or more reagents include dNTPs and / or one or more primers.

[0031] Embodiment 25 is a method in which, after step c), i) Introducing or attempting to introduce a second liquid into the reaction chamber, ii) After step i), obtain a third fluorescence measurement from the reaction chamber. iii) Determining a second value from a second fluorescence measurement and a third fluorescence measurement, wherein the second value indicates the degree of change in fluorescence in the reaction chamber that occurs in step i), and iv) A method, system, or computer-readable medium of any of the prior embodiments, which includes comparing a second value to a second predetermined threshold or range, wherein an anomaly is detected if the second value does not meet the second predetermined threshold or if the second value is outside the second predetermined range.

[0032] Embodiment 26 is the method, system, or computer-readable medium of the preceding embodiment, wherein the second liquid comprises one or more nucleic acid amplification reagents or samples different from one or more nucleic acid amplification reagents or samples in the first liquid.

[0033] Embodiment 27 is the method, system, or computer-readable medium of Embodiment 25 or 26, wherein the second value is determined as the ratio of the third fluorescence measurement to the second fluorescence measurement.

[0034] Embodiment 28 is any one of the methods, systems, or computer-readable media of Embodiments 25 to 27, wherein the third fluorescence measurement is the mean or median of a plurality of individual measurements obtained in step ii).

[0035] Embodiment 29 is any one of the methods, systems, or computer-readable media of Embodiments 25 to 28, wherein the nucleic acid amplification reaction is initiated after step i) and before step ii).

[0036] Embodiment 30 is a method, system, or computer-readable medium of the preceding embodiment in which the initiation of a nucleic acid amplification reaction includes initiating a denaturation step or a reverse transcription step.

[0037] Embodiment 31 is the method, system, or computer-readable medium of the preceding embodiment, wherein the third fluorescence measurement is obtained during the denaturation step or the reverse transcription step.

[0038] Embodiment 32 is any one of the methods, systems, or computer-readable media of Embodiments 25 to 31, wherein the first liquid and the second liquid together form a nucleic acid amplification reaction mixture.

[0039] Embodiment 33 is the method, system, or computer-readable medium of the preceding embodiment, wherein the nucleic acid amplification reaction mixture is a thermally cycled nucleic acid amplification reaction mixture.

[0040] Embodiment 34 is a method, system, or computer-readable medium of Embodiment 32 or 33, in which the nucleic acid amplification reaction mixture is contained within a microfluidic cartridge.

[0041] Embodiment 35 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.

[0042] Embodiment 36 is the method, system, or computer-readable medium of the preceding embodiment, wherein the nucleic acid amplification reaction mixture is placed in the nucleic acid amplification area during step ii).

[0043] Embodiment 37 is one of the methods of Embodiments 25 to 36, wherein the second liquid is introduced into the reaction chamber and no abnormality is detected in step iii).

[0044] Embodiment 38 is one of the methods of Embodiments 25 to 36, wherein the second liquid is not introduced into the reaction chamber and an abnormality is detected in step iii).

[0045] Embodiment 39 is one of the methods of Embodiments 25 to 36, wherein step i) involves introducing a second liquid in a first volume into the reaction chamber, but the second liquid in a second volume is introduced into the reaction chamber, the second volume being smaller than the first volume, and an abnormality is detected in step iv).

[0046] Embodiment 40 is one of the methods of Embodiments 25 to 36, wherein step i) attempts to introduce a first volume of a second liquid into the reaction chamber, and the first volume within an acceptable range of 10%, 5%, 2%, 1%, or 0.5% is introduced into the reaction chamber, and no abnormality is detected in step iii).

[0047] Embodiment 41 is a method, system, or computer-readable medium of any of the preceding embodiments in which the abnormality is a defect in liquid handling.

[0048] Embodiment 42 is one of the methods of Embodiments 1 and 4-41, wherein the first liquid is introduced into the reaction chamber and no abnormality is detected in step e).

[0049] Embodiment 43 is one of Embodiments 1 and 4-41, wherein the first liquid is not introduced into the reaction chamber and an abnormality is detected in step e).

[0050] Embodiment 44 is one of Embodiments 1 and 4-41, wherein step b) involves attempting to introduce a first volume of the first liquid into the reaction chamber, but a second volume of the first liquid is introduced into the reaction chamber, the second volume being smaller than the first volume, and an abnormality is detected in step e).

[0051] Embodiment 45 is one of Embodiments 1 and 4-41, wherein step b) involves introducing a first volume of a first liquid into the reaction chamber, the first volume being introduced into the reaction chamber within an acceptable range of 10%, 5%, 2%, 1%, or 0.5%, and no abnormality is detected in step e).

[0052] Embodiment 46 is one of the systems of Embodiments 2, 4 to 36, and 41, configured to not expose the reaction chamber to nucleic acid amplification conditions if an abnormality is detected, and / or to expose the reaction chamber to nucleic acid amplification conditions only if no abnormality is detected.

[0053] Embodiment 47 is a system or computer-readable medium of any one of Embodiments 2-36, 41, and 45, further comprising subjecting the reaction chamber to nucleic acid amplification conditions only when no abnormality is detected.

[0054] Embodiment 48 is the system or computer-readable medium of Embodiment 45 or 46, wherein the nucleic acid amplification conditions include thermal cycling.

[0055] Embodiment 49 is a system or computer-readable medium of any one of Embodiments 2-36, 41, and 46-48, further comprising interrupting the nucleic acid amplification reaction if an abnormality is detected.

[0056] 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.

[0057] 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]

[0058] Brief explanation of the drawing [Figure 1] Figure 1 is an illustrative graph of fluorescence over time during monitoring of liquid handling in a reaction chamber. It shows the times of the first and second attempts to introduce the first and second liquids into the reaction chamber (see arrows). Fluorescence measurements were obtained before the first attempt to introduce the liquid and after each of the first and second attempts (see horizontal bars). Relative fluorescence units (signal; RFU; y-axis) are plotted against time (milliseconds on the x-axis).

[0059] [Figure 2]Figure 2 is an exemplary histogram of the fluorescence ratio before and after attempts to introduce liquid into the reaction chamber for multiple trials, as described in Example 1. For each of these chambers, the histogram shows the number of conditions (y-axis) against the after / before fluorescence ratio (x-axis) obtained after the second injection. Ranges of values ​​typical for chambers with normal liquid handling conditions ("normal"), no injection, and incorrect fluid composition are shown.

[0060] [Figure 3] Figure 3 shows exemplary fluorescence signals from multiple qPCR amplification reactions with or without various liquid handling anomalies (e.g., correct liquid filling). Arrows indicate the times corresponding to the first and second attempts to introduce the first and second liquids into the reaction chamber. Fluorescence measurements are obtained before the first attempt to introduce the liquid (during step 1) and after each of the first and second attempts to introduce the liquid (during step 2 and step 3, respectively). Relative fluorescence units (signal; RFU; y-axis) are plotted against the measurements (once every 200 ms, x-axis).

[0061] [Figure 4] Figure 4 is an illustrative two-dimensional plot of data from reactions with or without anomalies, such as missing injection steps, plotted as shown in Figure 3, with the ratio of median fluorescence measurements from steps 3 and 1 to the ratio of median fluorescence measurements from steps 2 and 1 (x-axis) (y-axis).

[0062] [Figure 5] Figure 5 is a two-dimensional plot shown in Figure 4, marked with exemplary thresholds (thr1, thr2, and thr3) used to detect and / or identify anomalies in liquid filling.

[0063] [Figure 6]Figure 6 is an exemplary two-dimensional plot of data from reactions with various anomalies, such as missing injection steps, plotted as the ratio (y-axis) of the median fluorescence measurements from steps 2 and 3 to the number of runs (x-axis), as shown in Figure 3. The two-dimensional map is marked with alternative exemplary thresholds (thr1, thr2, and thr3) used to detect and / or identify anomalies in liquid filling.

[0064] [Figure 7] Figure 7 shows an exemplary nucleic acid amplification system 700 with a microfluidic cartridge 1001 loaded into a docking station 703. System 700 includes a heat sink 705, a fluorometer 706, a press 701, and a press / heater 704 used to interact with and measure the sample loaded in cartridge 1001.

[0065] [Figure 8] Figure 8 is a side view of the nucleic acid amplification system 700, including the docking station 703 of Figure 7. The side view shows an optical fiber 803 extending from the cartridge 1001 to the light source 707 (e.g., one or more LEDs) of the fluorometer 706, and a hole 804 formed in an aluminum block 802 for guiding the fluorescence from the cartridge 1001 to a photodetector 801 used to perform fluorescence measurements, the photodetector 801 being a component of the fluorometer 706.

[0066] [Figure 9]Figure 9 shows a cross-sectional view of the nucleic acid amplification system 700, including the docking station 703 and microfluidic cartridge 1001 shown in Figures 7 and 8. The cross-sectional view shows internal components such as a thermal cycler 908 including a thermal block 905, a Peltier module, and a heat sink 907, an array detector 911, and a rotary valve system 901 including a piston 902, a ball 903, and a cam 904 useful for moving and analyzing the liquid in different functional areas of the cartridge 1001. The system 700 also includes a clamp 909 for securing a detection tip (not shown) and an illuminator 910.

[0067] [Figure 10-1]Figures 10A–10D show various diagrams of an exemplary microfluidic cartridge 1001. Figure 10A shows a top perspective view of a microfluidic cartridge 1001 containing multiple chambers for holding samples, reagents, or other liquids. Figure 10B shows a bottom perspective view of an exemplary microfluidic cartridge 1001 showing a fluid network of microchannels 1006 connecting the various chambers to other areas within the cartridge 1001. For example, chambers 1002, 1003, 1004, and 1005 may be sample chamber 1002, metering chamber 1003, waste chamber 1004, and PCR chamber 1005. Figure 10C shows a bottom perspective view of a fully constructed microfluidic cartridge 1001, including a bottom cover 1013 and a microarray slide 1014, as shown in Figure 10D, which include a sample preparation area 1011, a nucleic acid amplification area 1010, and a nucleic acid analysis area 1009. The nucleic acid amplification area 1010 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 1006, which are shown in detail in Figure 10B. The sample preparation area 1011 contains liquids in adjacent chambers. The chambers within the sample preparation area 1011 are arranged around a central distribution hub 1008. 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 1010 is in close proximity to the thermal cycler 908 in Figure 9. Similarly, when docked to the system, the nucleic acid analysis area 1009 is in close proximity to the array detector 911, as shown in Figure 9. Figure 10D shows an exploded view of a fully constructed exemplary microfluidic cartridge 1001, which includes a cartridge body 1012, a vent cap 1023 for covering the sample after input, a sample filter 1024, and components for the cartridge 1001 to interact with the system's mechanical components to move and combine the liquid in the sample preparation area 1011, including a stopper 1018, a nucleic acid binding matrix column 1019, a protective cover 1021, a blocker 1022, and a blocker ring 1025.The exploded view also shows how the microarray slide 1014 is attached to the cartridge body 1012 via the microarray adhesive tape 1015 in the nucleic acid analysis area 1009. The cartridge 1001 also includes polypropylene (PP) coverings 1013, 1016 on the bottom and top of the cartridge, and optionally includes a pre-printed sticker 1017. [Figure 10-2] Same as above. [Figure 10-3] Same as above.

[0068] [Figure 11] Figure 11 shows an exploded view of an exemplary microfluidic cartridge 1001 and plunger 1101, which works to unlock the blocker 1022 and interact with the stopper 1018 to move and combine the liquid in the microfluidic cartridge 1001. [Modes for carrying out the invention]

[0069] 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.

[0070] "Liquid handling" refers to the aspiration, transfer, and dispensing of liquids. "Liquid filling" refers to the filling of a reaction chamber with liquid via liquid handling. Manual or automatic liquid handling methods may be used. Abnormalities in the liquid filling of the chamber may result from errors in liquid handling.

[0071] "Liquid handling defects" refer to the aspiration, movement, and dispensing of liquids that result in deviations from the intended volume or composition within the reaction chamber after an attempt has been made to introduce the liquid into the reaction chamber.

[0072] "Nucleic acids" and "polynucleotides" refer to multimeric compounds containing two or more covalently bonded nucleosides or nucleoside analogs or base analogs having nitrogen heterocyclic bases, where nucleosides are bonded together by phosphodiester bonds or other bonds to form polynucleotides. Nucleic acids include RNA, DNA, and combinations and analogs thereof such as "peptide nucleic acids" or PNAs (see, e.g., WO95 / 32305) and "locked nucleic acids" (LNAs), where one or more nucleotide monomers have a bicyclic furanose unit locked to RNA mimicking sugar conformation (see, e.g., Vester et al., Biochemistry 43:13233-41, 2004). Nitrogen bases include conventional bases (A, G, C, T, U), purine or pyrimidine base derivatives (e.g., N 4 These may include analogs such as methyl-13-deoxyguanosine, deaza- or aza-purine, deaza- or aza-pyrimidine (see U.S. Patent No. 5,378,825, U.S. Patent No. 6,949,367, and International Publication No. 93 / 13121) (e.g., inosine, 5-methylisocytosine, isoguanine, e.g., The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992; Abraham et al., 2007, BioTechniques 43: 617-24), and / or "decayable" residues (e.g., see U.S. Patent No. 5,585,481).

[0073] 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.

[0074] 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 from its 3' end. The 5' region of the primer may be complementary to the target nucleic acid. If the 5' complementary region contains a promoter sequence, it is called a "promoter-primer."

[0075] As used herein, the term “region” refers to a portion of a nucleic acid, which may be 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 in a promoter-primer).

[0076] "Sample" refers to any composition that may or is suspected to contain the target material. A sample may be a composite mixture of its components. Examples of samples include "biological samples" containing any tissue or material derived from living or dead mammals or organisms, including, for example, feces, blood, plasma, serum, blood cells, saliva, mucus, and cerebrospinal fluid. Samples also include "nucleic acid samples" containing the nucleic acids described above. 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 also include foods containing any material intended for or suitable for consumption, including solids, suspensions, emulsions, gels, and liquids (i.e., gelatin, milk, soups, beverages, ice cream, fruit smoothies, emulsified cheese dips, fruit purees, nut butters, processed and / or textured proteins, as well as bread, fruits, vegetables, and meat). Samples 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.

[0077] The interchangeable terms “oligomer,” “oligo,” and “oligonucleotide” refer to nucleic acids generally having fewer than 1,000 nucleotides (nt), including polymers with a lower limit of about 5 nt residues and an upper limit of about 500–900 nt residues. In some embodiments, oligonucleotides are in a size range with a lower limit of about 12–15 nt and an upper limit of about 50–600 nt, while in other embodiments, they are in a range with a lower limit of about 15–20 nt and an upper limit of about 22–100 nt. Oligonucleotides can perform one or more of a variety of different functions, such as primers and / or promoters, detection probes, and capture oligomers.

[0078] "Amplifying" or "nucleic acid 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 PCT Publications 88 / 01302, 88 / 10315, and 95 / 03430 (TMA), U.S. Patent 4,786,600 (RCA), U.S. Patents 5,427,930, and 5,516,663 (LCR), as well as U.S. Patents 5,422,252, 5,547,861, and U.S. Patent 5,648,211 (SDA). See, for example, Compton, Nature 350:91-92, 1991; Malek et al., Methods Mol. Biol. 28:253-260, 1994 (NASBA). Briefly, PCR amplification uses DNA polymerase, primer pairs, and thermal cycling to synthesize multiple copies of two complementary strands from dsDNA or cDNA (see, for example, U.S. Patents 4,683,195, 4,683,202, and 4,800,159).

[0079] As used herein, the term “real-time amplification” refers to the amplification of a target nucleic acid monitored by real-time detection. Real-time PCR amplification includes, for example, what is commonly called TaqMan® PCR (see, e.g., Holland et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, 1991; and Livak et al., US Pat. No. 6,030,787). TaqMan® PCR is a type of real-time PCR that uses a nucleic acid probe complementary to the internal segment of the target DNA. The probe is labeled with two fluorescent moieties. The emission spectrum of one overlaps with the excitation spectrum of the other, resulting in the “quenching” of the first fluorophore by the second fluorophore.

[0080] 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.”

[0081] As used herein, “nucleic acid amplification system” refers to a device or apparatus that can be used to carry out nucleic acid amplification. In many examples, a nucleic acid amplification system includes a fluorometer that can obtain fluorescence measurements before, during, and after nucleic acid amplification. In many examples, a nucleic acid amplification system includes a temperature controller that can provide or transfer heat to the nucleic acid amplification reaction mixture via one or more heating elements, which may include, for example, a heat block. 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.

[0082] 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.

[0083] "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. The hybridization or amplification conditions are "stringent" and selectively allow 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 to the target sequence intended by standard nucleic acid base pairing (e.g., G:C, A:T, or A:U pairing). "Fully complementary" means a sequence that can hybridize to another sequence by hydrogen bonding between a series of complementary bases, which may contain one or more debasic residues that are complementary or non-complementary at each position in the sequence by standard base pairing. 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.).

[0084] As used herein, “label” or “detection label” refers to a moiety or compound that can detect or generate a detectable signal and is directly or indirectly linked to a molecule such as a probe. Direct labeling may occur via a bond or interaction that links the label to the molecule, including covalent or non-covalent interactions, e.g., hydrogen bonds, hydrophobic and ionic interactions, or the formation of chelates or coordination complexes. Indirect labeling may occur through the use of a crosslinking moiety or “linker,” e.g., a binding pair member, an antibody, or an additional oligomer, which is directly or indirectly labeled and can amplify a detectable signal. Examples of labels include any detectable moiety such as radionuclides, ligands (e.g., biotin, avidin), enzymes or enzyme substrates, reactive groups, or chromophores (e.g., dyes, particles, or beads that impart a detectable color), luminescent compounds (e.g., bioluminescent labels, phosphorescent labels, or chemiluminescent labels), or fluorophores. Common labels used with TaqMan® probes include fluorophores and quenchers. As used herein, “fluorophore” refers to any label whose presence can be detected by its fluorescence properties. As used herein, “quencher” refers to a portion that absorbs at least a portion of the intensity of fluorescence emission. Quenchers include fluorescent quenchers and dark quenchers (sometimes called non-fluorescent quenchers). Dark quenchers are substances that absorb excitation energy from fluorophores and dissipate that energy as heat, while fluorescent quenchers re-emit much of this energy as light. A fluorescent quencher is a portion that can absorb the fluorescence signal emitted from a fluorescence source at a first wavelength, typically a fluorophore, such as, for example, a nucleic acid dye associated with the double-stranded segment of a nucleic acid, and after absorbing sufficient fluorescence energy, the fluorescent quencher can emit fluorescence at a second wavelength characteristic of the quencher, a process called “fluorescence resonance energy transfer” or FRET.Examples of fluorophores include FAM, SYBR® Green, ATTO fluorescent labels, VIC, JOE, NED, Cy3, ROX, Texas Red, and Cy5 dyes (all available from numerous commercial sources). The synthesis and methods for attaching labels to nucleic acids and detecting the labels are known in the art (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2). nd (ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10; see U.S. Patents Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333). More than one label and more than one type of label may be present on a particular probe, or detection may be performed using a mixture of probes in which each probe is labeled with a compound that produces a different detectable signal (see, for example, U.S. Patents Nos. 6,180,340 and 6,350,579).

[0085] 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).

[0086] As used herein, "elution buffer" is a liquid suitable for separating nucleic acids from a solid support.

[0087] 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.

[0088] A "reaction chamber," also called a "reaction space," refers to the space in which the amplification of a target nucleic acid takes place. A reaction chamber contains tubes or wells. Multiple reaction chambers may be arranged parallel or substantially parallel to each other within various containers. Examples of containers that can accommodate multiple reaction chambers include multiwell strips, multiwell plates, microfluidic chips, or microfluidic cartridges.

[0089] As used herein, “fluorescence spectrometer,” “fluorescence detector,” or “fluorescence sensor” refers to an optical detector capable of obtaining fluorescence measurements. Such measurements can be obtained before, during, and after nucleic acid amplification. The fluorescence spectrometer may be a photodiode or a photomultiplier tube. The fluorescence spectrometer receives light (fluorescence) emitted from a sample. Real-time detection of PCR products can be achieved by using a fluorescent dye or probe. The fluorescence signal measured by the fluorescence spectrometer increases with each PCR cycle as more polynucleotide molecules are produced.

[0090] As used herein, the term “relative fluorescence unit” (“RFU”) is a unit of measurement for fluorescence intensity. RFUs vary depending on the characteristics of the detection instrument used for measurement and can be used as a measurement for comparing the relative intensity between a sample and a control.

[0091] 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.

[0092] Where used herein, the term “approximately” refers to a number, whether explicitly stated or not, including integers, fractions, and percentages. When this term precedes a list of numbers or ranges, it modifies all values ​​or ranges. When applied to a measurement, the term includes the exact number modified by this term and the range of values ​​expected to be within experimental error. For example, “approximately 5°C” means “5°C” and a temperature range within experimental error, e.g., 5°C plus or minus (±) 20%, 5°C ± 15%, 5°C ± 10%, or 5°C ± 5%. The term has a similar meaning with respect to other parameters. For example, “approximately 5 minutes” means “5 minutes” and a time range within experimental error, e.g., 5 minutes plus or minus (±) 20%, 5 minutes ± 15%, 5 minutes ± 10%, or 5 minutes ± 5%. In some situations, the percentage of experimental error is implied or evident and does not need to be explicitly stated. In some situations, the percentage of experimental error is explicitly provided. The term "approximately" can be used to modify any measurable quantity, including quantities of time, temperature, volume, mass, weight, length, density, size, percentage, ratio, dose, frequency, pressure, speed, and intensity. In some cases, the term "approximately" may include a number rounded to the nearest significant figure.

[0093] 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.

[0094] 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.

[0095] "Or" is used in an inclusive sense, meaning it is equivalent to "and / or" unless the context clearly indicates otherwise.

[0096] Detailed explanation This specification discloses a method for monitoring the introduction of one or more liquids into a reaction chamber to detect anomalies in the reaction chamber. This method utilizes the fluorescence of a probe and / or reagent having a specific fluorescence, such as a labeled reagent like dNTPs and primers, to detect changes in the volume and / or composition of liquids within the reaction chamber, thereby facilitating the detection of anomalies such as failed attempts to introduce liquids into the chamber or improper liquid filling. Typically, an improper liquid filling event can lead to errors in the final volume and / or composition of the nucleic acid amplification reaction mixture, potentially causing failure of the nucleic acid amplification reaction. Therefore, this method can enable the detection of anomalies within the reaction chamber that could adversely affect the nucleic acid amplification 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 fluorometer, without requiring additional hardware. Alternatively, this method uses existing features of such systems to monitor fluorescence from the reaction chamber before and after one or more attempts to introduce liquids into the reaction chamber, thereby detecting whether there was an anomaly in the liquid filling.

[0097] A. Detection labels and probes Detection labels (e.g., fluorophores) may be used in accordance with this disclosure. The first and / or second liquids of this disclosure, as well as nucleic acid amplification reaction mixtures, may include detection oligomers having detection labels (e.g., designed to hybridize to an amplicon) and / or amplification oligomers, e.g., forward primers and / or reverse primers. Generally, amplification oligomers or detection oligomers having detection labels used in amplification reactions include 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.

[0098] Suitable fluorophores for use as detection labels may include compounds that emit a detectable light signal, such as fluorophores ("fluorescent dye compounds"). More than one label and more than one type of label may be present on a particular probe, or a mixture of probes in which each probe is labeled with a compound that produces a detectable signal may be used (see, for example, U.S. Patents 6,180,340 and 6,350,579). Labels may be attached to the probe by a variety of means, including covalent bonding, chelation, and ionic interactions, but preferably the labels are covalently bonded. Suitable fluorophores are well known in the art and include, for example, CAL Flour® Orange 560, CAL Flour® Red 610, FAM, or ATTO 490LS. In some embodiments, the fluorophores are temperature-sensitive fluorophores. In some embodiments, the temperature-sensitive fluorophores are sulforhodamines. In embodiments including fluorophore-labeled detection probes, each detection probe further includes a quencher. Suitable quenchers are well known in the art and include, for example, BHQ, TAMRA, and DABCLY. In other embodiments, the detection probe includes both a fluorescent label and a quencher, and this combination is particularly useful in fluorescence resonance energy transfer (FRET) assays. Specific variations of such detection probes include, for example, the TaqMan® detection probe (Roche Molecular Diagnostics), “Molecular Beacon” (see, e.g., Tyagi et al., Nature Biotechnol. 16:49-53, 1998; U.S. Patents 5,118,801 and 5,312,728), and “Molecular Torch” (see, e.g., U.S. Patents 6,849,412, 6,835,542, 6,534,274, and 6,361,945). In some embodiments, a fluorophore in a nucleic acid amplification reaction mixture associates with a probe further comprising a quencher.

[0099] B. Reaction mixture In some embodiments, the reaction mixture or first or second liquid introduced into the reaction chamber used in the method or system described herein comprises one or more amplification oligomers for amplifying a target nucleic acid. In some embodiments, the amplification oligomer comprises a detection label. The reaction mixture typically comprises a buffer, a salt solution, suitable nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP), and / or other reagents suitable for carrying out in vitro amplification, such as enzymes (plural) (e.g., DNA polymerase, reverse transcriptase, and RNA polymerase), and the first or second liquid may comprise these, and may comprise test sample components in which an internal control (IC) target nucleic acid may be present. In some embodiments, the reaction mixture is a nucleic acid amplification reaction mixture.

[0100] Various liquids may be used in nucleic acid amplification reaction mixtures used in accordance with this disclosure. In some embodiments, the first or second liquid may contain one or more reagents used in the nucleic acid amplification reaction. Such reagents may include enzymes, master mixes, and other PCR components. In some embodiments, the first or second liquid may contain a sample, such as a nucleic acid sample. In some embodiments, the first liquid may contain a sample, and the second liquid may contain all or substantially all of the reagents necessary for the nucleic acid amplification reaction. In some embodiments, the second liquid may contain a sample, and the first liquid may contain all or substantially all of the reagents necessary for the nucleic acid amplification reaction.

[0101] C. Reaction chamber, sample Various reaction vessels may be used to receive the first and / or second liquids and / or to contain the reaction mixture used in accordance with this disclosure. In some embodiments, the first or second liquid contains a sample, such as a nucleic acid sample. In some embodiments, the reaction mixture contains a sample, such as a nucleic acid sample. The reaction vessel may include one reaction chamber or a plurality of reaction chambers. In some embodiments, the steps of the method disclosed herein are carried out using an empty reaction chamber or using a reaction chamber containing a nucleic acid amplification reaction mixture. In some embodiments, the plurality of fluorophores-containing nucleic acid amplification reaction mixture is contained in a plurality of wells or a plurality of tubes.

[0102] The reaction chamber can be configured as a variety of reaction vessels, each containing multiple reaction chambers. In some embodiments, the reaction vessel includes a reaction chamber containing multiple empty reaction chambers and / or nucleic acid amplification reaction mixtures. In some embodiments, the vessel is a multi-chamber receptacle. In some embodiments, the nucleic acid amplification chambers are housed within the multi-chamber receptacle. Non-limiting examples of vessels include multi-well strips, multi-well plates, microfluidic chips, and microfluidic cartridges. In some embodiments, multiple nucleic acid amplification reaction mixtures are contained in a multi-well plate or multiple tubes.

[0103] In some embodiments, at least one nucleic acid amplification reaction mixture is contained in a multiwell strip, multiwell plate, microfluidic chip, or microfluidic cartridge. In some embodiments, the reaction vessel is a microfluidic cartridge. In some embodiments, the microfluidic cartridge is also referred to as a “lab-on-a-chip” and can carry out a complete nucleic acid analysis of a sample, from sample collection to nucleic acid amplification and reading of the results(s). Exemplary microfluidic cartridges that can be used to carry out the steps of the methods disclosed herein are shown in Figures 10A–D. Figure 10A shows a top perspective view of an exemplary microfluidic cartridge 1001 containing multiple chambers for containing a sample, reagent, or other liquid. Microchannels 1006 connect such chambers to move liquid between different functional areas within the cartridge 1001. Figure 10B shows a bottom perspective view of an exemplary microfluidic cartridge 1001 showing a network of microchannels 1006 connecting various chambers to other areas within the cartridge. Figure 10C shows a bottom perspective view of a fully constructed microfluidic cartridge 1001, including a bottom cover 1013 and a microarray slide 1014 as shown in Figure 10D, which include a sample preparation area 1011, a nucleic acid amplification area 1010, and a nucleic acid analysis area 1009. The nucleic acid amplification area may also constitute a nucleic acid detection area capable of detecting signals from real-time PCR reactions. These functional areas are connected by a series of microchannels 1006, which are shown in detail in Figure 10B. The sample preparation area 1011 contains liquid in an adjacent chamber. The system, e.g., nucleic acid amplification system 700, can be programmed to combine the liquids contained in these chambers in a specific order and in separate volumes. When the exemplary microfluidic cartridge 1001 is docked to the system, the nucleic acid amplification area 1010 is adjacent to the thermal cycler 908, as shown in Figure 9. Similarly, when the exemplary microfluidic cartridge 1001 is docked to the system, the nucleic acid analysis area 1009 is adjacent to the array detector 911, as shown in Figure 9.Figure 10D shows an exploded view of a fully constructed exemplary microfluidic cartridge 1001, which includes a cartridge body 1012, a vent cap 1023 for covering the sample after input, and a sample filter 1024 for removing cell debris after lysis, which is performed either before sample addition and / or in the sample reservoir 1002. The cartridge further includes components for moving and combining the liquid in the sample preparation area 1011 by interacting with the mechanical components of the system, including a stopper 1018, a nucleic acid binding matrix column 1019, a protective cover 1021, a blocker 1022, and a blocker ring 1025. The exploded view in Figure 10D also shows how a microarray slide 1014 is attached to the body 1012 of the microfluidic cartridge 1001 in the nucleic acid analysis area 1009 via a microarray adhesive tape 1015. The cartridge 1001 also includes top and bottom coverings 1016, 1013 of polypropylene (PP), respectively, and a pre-printed sticker 1017, if necessary. Figure 11 shows an exploded view of an exemplary microfluidic cartridge 1001, which includes a plunger 1101 for engaging with a stopper 1018 to move and combine the liquid within the microfluidic cartridge 1001. Microfluidic cartridges have several advantages, including the ability to perform automated operations while consuming small reagent volumes, being inexpensive and disposable. An example of a microfluidic cartridge is also disclosed in U.S. Patent No. 10,654,039.

[0104] In some embodiments, the microfluidic cartridge 1001 includes at least (1) a plurality of functional areas including a sample preparation area 1011, a nucleic acid amplification area 1010, a nucleic acid analysis area 1009, and a waste area 1004; (2) a central distribution hub 1008; and (3) a fluid network of a pump or plunger 1101, a plurality of valves 1026, and microchannels 1006 connecting the functional areas to the central distribution hub 1008. The fluid network of the pump or plunger 1101, the plurality of valves 1026, and microchannels 1006 can drive the movement of fluid from a first functional area through the central distribution hub 1008 to a second functional area among the plurality of functional areas. The nucleic acid amplification reaction may be contained within the nucleic acid amplification area 1010.

[0105] The sample preparation area 1011 may include a network of reservoirs, chambers, or tanks holding multiple liquids or gases of various configurations, as shown in Figure 10A. 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.

[0106] The nucleic acid amplification area 1010 may be adjacent to the sample preparation area 1011, or it may be connected via a microfluidic channel 1006. Such a channel 1006 allows the prepared sample to move through the central distribution hub 1008 to the nucleic acid amplification area 1010 and be subjected to nucleic acid amplification conditions. The nucleic acid amplification area 1010 (see, for example, Figure 10C) may be the location where some or all of the detection steps for real-time PCR are performed. Nucleic acid detection may include fluorescence detection of the amplified nucleic acid.

[0107] The nucleic acid analysis area 1009 may include microarray components or slides, as shown in Figures 10C and 10D. 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.

[0108] In some embodiments in which the nucleic acid amplification reaction mixture is contained in a microfluidic cartridge, the nucleic acid amplification reaction mixture may or may not be placed in the nucleic acid amplification area. In some embodiments, the first liquid may or may not be placed in the nucleic acid amplification area. In these embodiments, if the introduction of the first liquid is successful when an attempt is made to introduce the first liquid into the reaction chamber, the first liquid is placed in the nucleic acid amplification area when obtaining a second fluorescence measurement from the reaction chamber.

[0109] In some embodiments, the second liquid may or may not be placed in the nucleic acid amplification area. In some embodiments, the nucleic acid amplification reaction mixture is placed in the nucleic acid amplification area when obtaining a third fluorescence measurement from the reaction chamber. In these embodiments, if the introduction of the second liquid into the reaction chamber is successful after an attempt, the second liquid is placed in the nucleic acid amplification area when obtaining a third fluorescence measurement from the reaction chamber.

[0110] 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.

[0111] 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.

[0112] Each microchannel may include a central distribution hub end ("hub end") and a functional area end ("area end"). The area end of a microchannel is adjacent to the corresponding functional area, and the hub end is adjacent to the central distribution hub. Each microchannel may also include a valve located near the relevant area end. Thus, a microfluidic cartridge includes multiple valves located at or near the area end of a hub-connected microchannel.

[0113] 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.

[0114] 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.

[0115] D. Method for monitoring abnormalities in the liquid filling of the reaction chamber. In one embodiment, a method for monitoring the introduction of one or more liquids into a reaction chamber includes obtaining a first fluorescence measurement from the reaction chamber, introducing or attempting to introduce a first liquid into the reaction chamber, obtaining a second fluorescence measurement from the reaction chamber, determining a first value from the first and second fluorescence measurements, wherein the first value indicates the degree of change in fluorescence within the reaction chamber resulting from the attempt to introduce the first liquid into the reaction chamber, and comparing the first value with a first predetermined threshold or range, wherein an anomaly is detected if the first value does not meet the first predetermined threshold or if the first value is outside the first predetermined range.

[0116] In one embodiment, a method for monitoring the introduction of one or more liquids into a plurality of reaction chambers includes obtaining a first fluorescence measurement from each of the reaction chambers, introducing or attempting to introduce a first liquid into each of the reaction chambers, obtaining a second fluorescence measurement from each of the plurality of first fluorescence measurements and each of the plurality of second fluorescence measurements, wherein each first value indicates the degree of change in fluorescence in the corresponding reaction chamber resulting from introducing or attempting to introduce a first liquid into each of the reaction chambers, and comparing each of the first values ​​to a first predetermined threshold or range, wherein an anomaly is detected if any one of the first values ​​does not meet the first predetermined threshold or if the first value is outside the first predetermined range.

[0117] In some embodiments, when attempting to introduce a first liquid into the reaction chamber, the attempt may or may not successfully introduce the first liquid into the reaction chamber.

[0118] In some embodiments, the method involves pumping or attempting to pump a first liquid through a tip into a reaction chamber. In some embodiments, the tip is a needle tip or a pipette tip.

[0119] In some embodiments, after attempting to introduce a first liquid, the nucleic acid amplification reaction is initiated in the reaction chamber before obtaining a second fluorescence measurement. In some embodiments, the nucleic acid amplification reaction includes initiating a denaturation step or a reverse transcription step.

[0120] In some embodiments, the first liquid comprises one or more reagents or nucleic acid samples. In some embodiments, the second liquid comprises one or more reagents or nucleic acid samples. In some embodiments, after obtaining a second fluorescence measurement from the reaction chamber, the method further includes i) attempting to introduce the second liquid into the reaction chamber. The second liquid may comprise one or more nucleic acid amplification reagents or nucleic acid samples different from the nucleic acid amplification reagents or nucleic acid samples of the first liquid. After the step of attempting to introduce the second liquid, the method further includes obtaining a third fluorescence measurement from the reaction chamber. The second value may be determined from the second and third fluorescence measurements, and the second value may indicate whether a change in fluorescence was induced by introducing the second liquid into the nucleic acid amplification reaction chamber. The method further includes iii) comparing the second value to a second predetermined threshold or range, wherein an anomaly is detected if the second value does not meet the second predetermined threshold or if the second value is outside the second predetermined range.

[0121] In some embodiments, the first and second liquids together form a nucleic acid amplification mixture. In some embodiments, the nucleic acid amplification mixture is a thermally cycled nucleic acid amplification mixture. In some embodiments, the nucleic acid amplification mixture is 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 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.

[0122] 1. Fluorescence measurement This method involves obtaining fluorescence measurements from a reaction chamber that may contain a nucleic acid amplification reaction mixture or its components, or that may not yet be filled with such a mixture or components. In some embodiments, fluorescence measurements are obtained from multiple reaction chambers. The fluorescence source may be a fluorophore, oligonucleotide probe, dNTP, primer, and / or other reagents in the nucleic acid amplification reaction mixture. Fluorescence may be measured in terms of relative fluorescence units (RFU). The fluorescence measurements described herein differ from fluorescence measurements that may be performed in previous quantitative and / or real-time PCR methods in that the measurements herein are obtained before and / or during the initial stages of nucleic acid amplification and do not reflect the generation of amplicons, but rather reflect the presence and compositional changes of the liquid in the chamber resulting from liquid filling. Furthermore, the measurements are used qualitatively in the sense that they are evaluated in terms of a threshold of 1 or greater to give a qualitative output (such as a determination of whether there is an anomaly). Furthermore, the measurements may be performed during the period before thermal cycling begins. Therefore, this method does not require fluorescence measurements during thermal cycling to detect anomalies.

[0123] In some embodiments, fluorescence measurements may be obtained before introducing or attempting to introduce the first liquid into the reaction chamber. In some embodiments, fluorescence measurements may be obtained when the reaction chamber is empty. In some embodiments, fluorescence measurements may be obtained after attempting to introduce the first liquid into the reaction chamber and, if necessary, before attempting to introduce the second liquid into the reaction chamber. In some embodiments, fluorescence measurements may be obtained after introducing or attempting to introduce the second liquid into the reaction chamber. In some embodiments, fluorescence measurements may be obtained when the reaction chamber contains liquid. In some embodiments, fluorescence measurements are obtained at regular time intervals.

[0124] In some embodiments, the first liquid and / or the second liquid comprises one or more fluorophores. In some embodiments, the fluorophores are associated with an oligonucleotide probe. In some embodiments, the oligonucleotide probe further comprises a quencher.

[0125] In some embodiments, the first liquid and / or the second liquid do not contain fluorophores associated with oligonucleotides. In these embodiments, the intrinsic fluorescence of one or more reagents and / or nucleic acid samples is measured. In some embodiments, one or more reagents include dNTPs and / or one or more primers.

[0126] a) First fluorescence measurement In some embodiments, the first fluorescence measurement is obtained from the reaction chamber. The fluorescence measurement may be obtained using a photodetector, such as a fluorometer, such as a photodiode or photomultiplier tube. In some embodiments, when the first measurement is obtained, the nucleic acid amplification reaction mixture is not yet present in the chamber, or not all components of the nucleic acid amplification reaction mixture are present in the chamber. In some embodiments, the first fluorescence measurement is obtained before the first liquid is introduced into or attempted to be introduced into the reaction chamber.

[0127] In some embodiments, the first fluorescence measurement is the mean or median of several individual measurements obtained from the nucleic acid amplification reaction chamber. In some embodiments, the several individual measurements are obtained over a predetermined period of time.

[0128] In some embodiments, the first fluorescence measurement is obtained from each of the multiple reaction chambers. In some embodiments, the first fluorescence measurement is obtained before or before introducing the first liquid into the multiple reaction chambers. In some embodiments, after the first fluorescence measurement is obtained, the method further includes introducing, or attempting to introduce, the multiple first liquids into the multiple reaction chambers.

[0129] b) Second fluorescence measurement In some embodiments, a second fluorescence measurement is obtained. In some embodiments, the second fluorescence measurement is obtained during the denaturation step or the reverse transcription step. In some embodiments, the second fluorescence measurement is obtained before introducing or attempting to introduce the second liquid into the reaction chamber. In some embodiments, the nucleic acid amplification reaction is initiated after the second fluorescence measurement is obtained.

[0130] In some embodiments, a second fluorescence measurement is obtained after introducing or attempting to introduce the first liquid into the reaction chamber. The first liquid may be a nucleic acid amplification mixture or may contain one or more components of a nucleic acid amplification mixture. Additional components of the mixture may be provided after obtaining the second fluorescence measurement. In some embodiments, the second fluorescence measurement is the mean or median of a plurality of individual measurements obtained from the nucleic acid amplification reaction chamber. In some embodiments, the plurality of measurements are obtained over a predetermined period of time.

[0131] In some embodiments, the second fluorescence measurement is obtained from multiple reaction chambers. In some embodiments, the second fluorescence measurement is obtained after introducing or attempting to introduce the first liquid into multiple reaction chambers. c) Third fluorescence measurement

[0132] In some embodiments, a third fluorescence measurement is obtained. In some embodiments, the nucleic acid amplification reaction is initiated before the third measurement is obtained. In some embodiments, the third fluorescence measurement is obtained during the denaturation or reverse transcription step.

[0133] In some embodiments, the third fluorescence measurement is obtained after introducing or attempting to introduce the second liquid into multiple reaction chambers. In some embodiments, the third fluorescence measurement is the mean or median of multiple individual measurements obtained from the reaction chambers.

[0134] 2. Value as a ratio of fluorescence measurement values a) Determination of the first value as the ratio of the second fluorescence measurement to the first fluorescence measurement. This method involves determining a first value from a first fluorescence measurement and a second fluorescence measurement, the first value representing the degree of change in fluorescence within the nucleic acid amplification reaction chamber resulting from introducing, or attempting to introduce, a first liquid of 1 or more liquids into the reaction chamber. In some embodiments, the degree of change in fluorescence may be 0 or nearly 0 in certain abnormalities, such as the inability to introduce any first liquid of 1 or more liquids into the nucleic acid amplification reaction chamber.

[0135] The first value should reflect whether the fluorescence changed between the first and second fluorescence measurements due to the introduction of the first liquid into the reaction chamber or an attempt to introduce the first liquid, or to what extent the fluorescence changed between the first and second fluorescence measurements due to the introduction of the first liquid into the nucleic acid amplification reaction chamber or an attempt to introduce the first liquid. In some embodiments, the first value is determined as the ratio of the second fluorescence measurement to the first fluorescence measurement.

[0136] In some embodiments, if first and second fluorescence measurements are obtained from each of a plurality of chambers, the method includes determining a plurality of first values ​​from the plurality of first and plurality of second fluorescence measurements. The plurality of first values ​​can indicate, for each of the plurality of chambers, whether a change in fluorescence was induced by introducing the first liquid into the nucleic acid amplification reaction chamber.

[0137] b) Determination of the second value as the ratio of the third fluorescence measurement to the second fluorescence measurement. In some embodiments, the method includes determining a second value from a fluorescence measurement and a third fluorescence measurement. The second value should reflect whether or not fluorescence has changed between the second and third fluorescence measurements due to the introduction of the second liquid into the reaction chamber or an attempt to introduce the second liquid, or to what extent fluorescence has changed between the second and third fluorescence measurements due to the introduction of the second liquid into the reaction chamber or an attempt to introduce the second liquid. In some embodiments, the second value is determined as the ratio of the third fluorescence measurement to the second fluorescence measurement. In some embodiments, the second value may indicate whether the introduction of the second liquid into the nucleic acid amplification reaction chamber caused a change in fluorescence. In some embodiments, the degree of fluorescence change may be 0 or nearly 0 in certain abnormalities, such as the inability to introduce the second liquid (a liquid of 1 or more) into the nucleic acid amplification reaction chamber.

[0138] In some embodiments, if second and third fluorescence measurements are obtained from each of a plurality of chambers, the method includes determining a plurality of second values ​​from the plurality of third fluorescence measurements and the plurality of second fluorescence measurements. In some embodiments, the plurality of second values ​​can indicate, for each of the plurality of chambers, whether a change in fluorescence was induced by introducing a second liquid into the nucleic acid amplification reaction chamber.

[0139] 3. Detection of anomalies This method includes comparing a first value with a first predetermined threshold or range. In some embodiments, an anomaly is detected if the first value does not meet the first predetermined threshold. In some embodiments, an anomaly is detected if the first value is outside the first predetermined range.

[0140] In some embodiments, if a plurality of first values ​​have been determined, the method includes comparing the plurality of first values ​​to a first predetermined threshold or range. In some embodiments, an anomaly is detected if any of the first values ​​does not meet the first predetermined threshold or if any of the first values ​​are outside the first predetermined range.

[0141] In some embodiments, once a second value is determined, the method includes comparing the second value to a second predetermined threshold or range. In some embodiments, an anomaly is detected if the second value does not meet the second predetermined threshold or if the second value is outside the second predetermined range.

[0142] In some embodiments, a predetermined threshold or range may be established from a reference liquid filling run. The reference liquid filling run of the nucleic acid amplification reaction chamber may include several successful and defective liquid handling events in the method described herein. Typically, an abnormal liquid filling event may result in an error in the final volume and / or composition of the nucleic acid amplification reaction mixture, potentially causing failure of the nucleic acid amplification reaction. Thus, the method may enable the detection of anomalies in the nucleic acid amplification reaction chamber that could adversely affect the nucleic acid amplification reaction. The reference run may include liquid handling events performed under different conditions. These conditions may include liquid filling anomalies such as the introduction of bubbles into the nucleic acid amplification reaction chamber, improper filling of the nucleic acid amplification reaction chamber with liquid reagents, and / or filling of the nucleic acid amplification reaction chamber with an inaccurate liquid volume. Inaccurate liquid volumes may include insufficient liquid volume, excess liquid volume, and / or no liquid volume transferred to the nucleic acid amplification reaction chamber. These conditions may include nucleic acid amplification reaction mixtures with one or more reagent changes that cause the reaction to fail. Fluorescence measurements may be obtained at various time points during these reference filling runs. The ratio of these measurements can be used to determine a threshold and / or acceptable range of values. In some embodiments, the threshold or range is referred to as a “predetermined threshold” or “predetermined range” and is used for comparison with values ​​described herein.

[0143] Values ​​within a specific range, or values ​​that meet or do not meet a specific threshold, may indicate a normal or abnormal liquid filling event of 1 or greater. Exemplary thresholds are shown in Figures 5 and 6.

[0144] In some embodiments, after attempting to introduce the first liquid into the nucleic acid amplification reaction chamber, no abnormality is detected when the first value is compared to a first predetermined threshold or range. In some embodiments, after attempting to introduce the first liquid into the nucleic acid amplification reaction chamber, an abnormality is detected when the first value is compared to a first predetermined threshold or range.

[0145] In some embodiments, after attempting to introduce the second liquid into the nucleic acid amplification reaction chamber, no abnormality is detected when the second value is compared to a second predetermined threshold or range. In some embodiments, after attempting to introduce the second liquid into the nucleic acid amplification reaction chamber, an abnormality is detected when the second value is compared to a second predetermined threshold or range.

[0146] 4.Liquid filling abnormality In some embodiments, to detect an anomaly in the liquid filling within the nucleic acid amplification reaction chamber, a first value of the method herein is compared to a first predetermined threshold or range. In some embodiments, a second value of the method herein is compared to a second predetermined threshold or range.

[0147] In some embodiments, an anomaly is detected if any first value does not meet a first predetermined threshold. In some embodiments, an anomaly is detected if any first value is outside a first predetermined range. In some embodiments, an anomaly is detected if any second value does not meet a second predetermined threshold. In some embodiments, an anomaly is detected if any second value is outside a second predetermined range.

[0148] In some embodiments, the abnormality includes defects in liquid handling. In some embodiments, defects in liquid handling may include introducing bubbles, incorrect liquid volume, and / or incorrect liquid reagents (e.g., buffers, salts, and other aqueous solutions) into the nucleic acid amplification reaction chamber, thereby causing abnormalities in the nucleic acid amplification reaction.

[0149] In some embodiments, when attempting to introduce a liquid into a nucleic acid amplification reaction chamber, an incorrect liquid or volume may be used. In such cases, an anomaly will be detected.

[0150] In some embodiments, when attempting to introduce the first liquid into the reaction chamber, the first liquid is introduced. In these embodiments, no abnormality is detected. In some embodiments, when attempting to introduce the first liquid into the reaction chamber, the first liquid is not introduced. In these embodiments, an abnormality is detected.

[0151] In some embodiments, when attempting to introduce a first liquid in a first volume into the reaction chamber, a second volume of the first liquid is introduced instead. In some embodiments, the second volume is smaller than the first volume, and an anomaly is detected.

[0152] In some embodiments, when introducing a first volume of a first liquid into a reaction chamber, the first volume is introduced into the nucleic acid amplification reaction chamber within an acceptable range of 10%, 5%, 2%, 1%, or 0.5% of the first volume. In these embodiments, no abnormalities are detected.

[0153] In some embodiments, when attempting to introduce a second liquid into the reaction chamber, the second liquid is not introduced. In these embodiments, an abnormality is detected. In some embodiments, when attempting to introduce a first volume of the second liquid into the reaction chamber, a second volume of the second liquid is introduced instead. In some embodiments, the second volume is less than the first volume. In these embodiments, an abnormality is detected.

[0154] In some embodiments, when introducing a first volume of a second liquid into the reaction chamber, the first volume is introduced into the reaction chamber within an acceptable range of 10%, 5%, 2%, 1%, or 0.5% of the first volume. In these embodiments, no abnormalities are detected.

[0155] E. Systems and computer-readable media This specification discloses a system. A system means a device or apparatus that can be used to perform, monitor, and / or analyze the filling of a reaction chamber with liquid as described herein. The nucleic acid amplification system 700 provided herein includes (1) a docking station 703 configured to accept a container containing at least one reaction chamber, (2) a fluorometer 706 configured to measure fluorescence within the reaction chamber, and (3) a processor operably coupled to the fluorometer 706 and memory. The memory coupled to the processor includes, when executed by the processor, instructions causing the system to perform a method of monitoring for anomalies in the introduction of one or more liquids into the reaction chamber. The monitoring method includes the steps of: obtaining a first fluorescence measurement from the nucleic acid amplification reaction chamber; introducing or attempting to introduce a first liquid into the reaction chamber; obtaining a second fluorescence measurement from the reaction chamber; determining from the first and second fluorescence measurements a first value indicating the degree of change in fluorescence within the nucleic acid amplification reaction chamber that occurs when the first liquid is attempted to be introduced into the reaction chamber; and comparing the first value to a first predetermined threshold or range. An anomaly is detected when a first value is compared to a first predetermined threshold or range, and the first value does not meet the first predetermined threshold or is outside the first predetermined range. In some embodiments, the nucleic acid amplification system includes instructions, when executed by a processor, that cause the system to perform a method of monitoring or introducing one or more liquids into a reaction chamber as described herein.

[0156] Exemplary nucleic acid amplification systems are shown in Figures 7 to 9. Figure 7 shows an exemplary nucleic acid amplification system 700 with a microfluidic cartridge 1001 loaded into a docking station 703. The exemplary system may include a heat sink 705, a fluorometer 706 including a light source 707 and a fluorescence detector 801, a press 701, and a press / heater 704 used to interact with and measure a sample loaded in a container (e.g., cartridge 1001). Figure 8 is a side view of the exemplary system 700 shown in Figure 7. The side view shows an optical fiber 803 extending from cartridge 1001 to light source 707, a hole 804 formed in an aluminum block 802, and a fluorescence detector 801 for performing fluorescence measurements. Figure 9 shows a cross-sectional view of the nucleic acid amplification system 700 including the docking station 703 and microfluidic cartridge 1001 shown in Figures 7 and 8. The cross-sectional view shows internal components such as a thermal cycler 908 including a thermal block 905, a Peltier module 906, and a heat sink 907, an array detector 911, a piston 902, a ball 903, and a rotary valve system 901 including a cam 904 useful for moving and analyzing liquids in different functional areas of the cartridge 1001, and for subjecting the container (e.g., microfluidic cartridge 1001) to nucleic acid amplification conditions. The system 700 may also include additional internal components such as a clamp 909 for securing a detection tip (not shown), an illuminator 910, and a press / heater 912. An exemplary nucleic acid amplification system is the Novodiag® system (Mobidiag Oy; Espoo, Finland), which may be configured for use with several different cartridges (reaction vessels).

[0157] In some embodiments, the container is a multi-chamber receptacle, or the nucleic acid amplification chamber is housed within a multi-chamber receptacle.

[0158] In some embodiments, the 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 system is programmable to maintain the nucleic acid amplification reaction chamber at a set temperature for different durations.

[0159] In some embodiments, the fluorometer is configured to measure and record the fluorescence of one or more fluorophores within the nucleic acid amplification reaction chamber. If the vessel is located within a system, for example, in a docking station, the fluorometer 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 vessel that is optically in communication with the fluorometer).

[0160] In some embodiments, the system may also include sensors for monitoring and / or estimating the liquid handling in the nucleic acid amplification reaction chamber. In some embodiments, the sensors may be thermal sensors, capacitive sensors, or infrared sensors.

[0161] In some embodiments, if an abnormality is detected, the nucleic acid amplification system provides one or more alarms. Alarms may be provided to indicate liquid filling abnormalities. Examples of alarms include visual and audible notifications. In some embodiments, when an alarm is provided, the nucleic acid amplification system pauses the nucleic acid amplification process, which may include adjusting the heating element to about 4°C and maintaining that temperature for a period of time.

[0162] In some embodiments, the system is configured to prevent the reaction chamber from being subjected to nucleic acid amplification conditions if an abnormality is detected, and / or to only be subjected to nucleic acid amplification conditions if no abnormality is detected.

[0163] Computer-readable media are also disclosed herein. In some embodiments, the computer-readable media includes instructions, when executed by the system's processor, causing the system to perform a method for monitoring the introduction of one or more liquids into a reaction chamber as described herein, for example, the steps of: obtaining a first fluorescence measurement from the reaction chamber; introducing or attempting to introduce a first liquid into the reaction chamber, wherein the first liquid contains one or more reagents or nucleic acid samples; obtaining a second fluorescence measurement from the reaction chamber; determining a first value from the first and second fluorescence measurements, wherein the first value indicates the degree of change in fluorescence within the reaction chamber resulting from obtaining the second fluorescence measurement from the reaction chamber; and comparing the first value to a first predetermined threshold or range, wherein an anomaly is detected if the first value does not meet the first predetermined threshold or if the first value is outside the first predetermined range.

[0164] In some embodiments, instructions for carrying out a method for monitoring the liquid filling of a reaction chamber are executed by the processor of the system described herein.

[0165] In some embodiments, the method includes subjecting the reaction mixture to nucleic acid amplification conditions only if no abnormalities are detected. In some embodiments, the nucleic acid amplification conditions include thermal cycling. In some embodiments, the method includes interrupting the nucleic acid amplification reaction if an abnormality is detected. [Examples]

[0166] Examples This embodiment describes performing liquid filling runs of a nucleic acid amplification chamber under normal conditions and under conditions with liquid filling abnormalities. This set of runs provided a basis for predetermining thresholds and / or ranges for comparing the first or second values ​​in the method described herein.

[0167] 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.

[0168] The reaction mixture was amplified using a Novodiag® system. Attempts were made to introduce the first and second liquids, as indicated by the arrows in Figure 3. Fluorescence measurements were obtained from the nucleic acid amplification reaction chamber at various points during these packing runs, including before and after liquid injection (see Figures 1 and 3, indicated by horizontal bars).

[0169] The ratio of fluorescence measurements obtained before and after liquid injection was plotted to determine the threshold (see Figures 2 and 4-6).

[0170] Values ​​within a specific range, or values ​​that meet or do not meet a specific threshold, may indicate liquid filling events greater than 1, such as run failure, missing liquid injection (a type of failed liquid filling attempt), a dry chamber, and / or standard liquid filling.

[0171] As shown in Figure 5, the threshold can be determined by plotting the ratio of fluorescence measurements obtained before and after the liquid filling attempt. The y-axis represents the ratio of measurements obtained before the first liquid introduction attempt and after the second liquid introduction attempt. The x-axis represents the ratio of measurements obtained after the first liquid introduction attempt and before the first liquid introduction attempt. The threshold is shown by a solid black line and can be determined during the liquid filling run according to user and / or instrument parameters. As shown in Figure 5, values ​​above threshold 2 (thr2; y-axis) and below threshold 1 (thr1; x-axis) indicate a reaction mixture where the second liquid filling attempt failed. Values ​​below threshold 2 (thr2; y-axis) and below threshold 1 (thr1; x-axis) indicate a run failure or a dry chamber (i.e., the liquid filling attempt was unsuccessful). Values ​​above threshold 3 (thr3; y-axis) and above threshold 1 (thr1; x-axis) indicate a reaction mixture where the first and second liquid filling attempts were successful (i.e., standard filling). Values ​​below threshold 3 (thr3; y-axis) and above threshold 1 (thr1; x-axis) indicate a reaction mixture where the first liquid filling attempt failed (i.e., the first injection was missing).

[0172] Alternatively, the threshold can be determined by plotting the ratio of fluorescence measurements obtained after attempting liquid filling. As shown in Figure 6, the y-axis represents the ratio of measurements obtained after attempting to introduce the first liquid and after attempting to introduce the second liquid, and the x-axis represents the number of runs. The threshold is shown by a solid black line and can be determined according to user and / or instrument parameters during a liquid filling run. In this embodiment, a value above threshold 1 (thr1) indicates a failed first liquid filling attempt. A value between threshold 1 (thr1) and threshold 2 (thr2) indicates a failed run or a dry chamber. A value between threshold 2 (thr2) and threshold 3 (thr3) indicates successful first and second liquid filling attempts (i.e., standard filling). A value below threshold 3 (thr3) indicates a failed second liquid filling attempt.

[0173] Results: The fluorescence ratios of multiple runs are plotted in Figures 2 to 6. Most runs with fluid errors have different fluorescence ratios than normal runs. Therefore, this method was effective in detecting fluid filling anomaly errors based on fluorescence data obtained during reference fluid filling runs of nucleic acid amplification chambers under normal conditions and under conditions with fluid filling anomalies in thermal cycle amplification reactions.

[0174] Equal portions 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 the introduction of one or more liquids into a reaction chamber, a) Obtain a first fluorescence measurement value from the reaction chamber, b) After step a), introducing or attempting to introduce a first liquid from among the one or more liquids into the reaction chamber, c) After step b), obtain a second fluorescence measurement from the reaction chamber. d) Determining a first value from the first fluorescence measurement and the second fluorescence measurement, wherein the first value indicates the degree of change in fluorescence in the reaction chamber resulting from step b), and e) A method comprising comparing the first value with a first predetermined threshold or range, wherein an anomaly is detected if the first value does not meet the first predetermined threshold or if the first value is outside the first predetermined range.

2. It is a system, A docking station configured to accept a vessel containing a reaction chamber, A fluorite spectrometer configured to measure fluorescence in the reaction chamber, Furthermore The system comprises a processor operably connected to the aforementioned fluorescence photometer and memory, The memory, when executed by the processor, includes instructions causing the system to perform a method for monitoring the introduction of one or more liquids into the reaction chamber, the method being: a) Obtain a first fluorescence measurement value from the reaction chamber, b) After step a), introducing or attempting to introduce a first liquid from among the one or more liquids into the reaction chamber, c) After step b), obtain a second fluorescence measurement from the reaction chamber. d) Determining a first value from the first fluorescence measurement and the second fluorescence measurement, wherein the first value indicates the degree of change in fluorescence in the reaction chamber resulting from step b), and e) A system that includes comparing the first value with a first predetermined threshold or range, wherein an anomaly is detected if the first value does not meet the first predetermined threshold or if the first value is outside the first predetermined range.

3. Computer-readable medium, When executed by the system's processor, the instruction causes the system to perform a method for monitoring the introduction of one or more liquids into a reaction chamber, the method being: a) Obtain a first fluorescence measurement value from the reaction chamber, b) After step a), introducing or attempting to introduce a first liquid from among the one or more liquids into the reaction chamber, c) After step b), obtain a second fluorescence measurement value from the amplification reaction chamber. d) Determining a first value from the first fluorescence measurement and the second fluorescence measurement, wherein the first value indicates the degree of change in fluorescence in the reaction chamber resulting from step b), and e) A computer-readable medium that includes comparing the first value with a first predetermined threshold or range, wherein an anomaly is detected if the first value does not meet the first predetermined threshold or if the first value is outside the first predetermined range.

4. The method, system, or computer-readable medium according to any one of the preceding claims, wherein introducing or attempting to introduce the first liquid into the reaction chamber includes pumping or attempting to pump the first liquid into the reaction chamber via a tip, wherein the tip is optionally a needle tip or a pipette tip.

5. The method, system, or computer-readable medium according to any one of the preceding claims, wherein the first value is determined as the ratio of the second fluorescence measurement to the first fluorescence measurement.

6. The method, system, or computer-readable medium according to any one of the preceding claims, wherein the first fluorescence measurement is the average or median of a plurality of individual measurements obtained in step a).

7. The method, system, or computer-readable medium according to any one of the preceding claims, wherein the second fluorescence measurement is the mean or median of a plurality of individual measurements obtained in step c).

8. The method, system, or computer-readable medium according to any one of the preceding claims, wherein a nucleic acid amplification reaction is initiated in the reaction chamber after step b) and before step c).

9. The method, system, or computer-readable medium according to the preceding claim, wherein the second fluorescence measurement is obtained during a thermal denaturation step or a reverse transcription step.

10. The method, system, or computer-readable medium according to any one of the preceding claims, wherein the reaction chamber is housed within a microfluidic cartridge.

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