Dynamic Melting Detection

Abstract

The present disclosure relates to methods and systems suitable for simultaneously amplifying multiple potential targets and then performing melt detection based on whether amplification of the target nucleic acids is detected, where the melting parameters are limited by the melting temperature range characteristic of the target nucleic acids whose amplification is detected. The invention maintains the resolution achieved by nucleic acid melting using a slow, steady ramp rate between annealing and denaturation temperatures, but does so more quickly by using faster ramp rates at temperatures where no melt signature is expected from the reaction and slower ramp rates at temperatures where a melt signature is expected from the reaction.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 374,350, filed September 1, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] In the United States, Canada, and Western Europe, infectious diseases account for approximately 7% of human mortality, while in developing regions, infectious diseases account for more than 40% of human mortality. Infectious diseases cause a variety of clinical symptoms. Common manifestations include fever, pneumonia, meningitis, diarrhea, and bloody stools. While physical symptoms suggest some pathogens and exclude others as causative agents, a variety of potential causative agents remains, and a definitive diagnosis often requires the performance of various assays. Traditional microbiological techniques for diagnosing pathogens can take days to weeks, often delaying the appropriate course of treatment.

[0003] In recent years, polymerase chain reaction (PCR) has become the method of choice for the rapid diagnosis of infectious agents. PCR can be a rapid, sensitive, and specific tool for diagnosing infectious diseases. Challenges in using PCR as a primary diagnostic tool include the high diversity of potential causative organisms and the low levels of organisms present in some pathological specimens. It is often impractical to run a large panel of PCR assays, one for each potential causative organism, most of which are expected to be negative. This problem is exacerbated when pathogen nucleic acids are present in low concentrations, requiring large sample volumes to collect sufficient reaction templates. In some cases, there may not be enough sample to assay all possible pathogens. A solution is to perform "multiplex PCR," in which a sample is simultaneously assayed for multiple targets in a single reaction. While multiplex PCR has proven useful in some systems, drawbacks exist regarding the robustness of high-level multiplex reactions and the difficulty of clearly analyzing multiple products. To overcome these challenges, the assay can be subsequently split into multiple secondary PCRs. Nesting secondary reactions within the primary product often improves robustness. However, this additional handling can be expensive and may result in contamination or other problems.

[0004] The FilmArray® (BioFire Diagnostics, Salt Lake City, UT) is a user-friendly, highly multiplexed PCR system developed for the diagnostic market. The single-sample instrument accepts diagnostic "pouches" that integrate sample preparation and nested multiplex PCR. Integrated sample preparation provides ease of use, while highly multiplexed PCR provides both the sensitivity of PCR and the ability to simultaneously test for many organisms (e.g., up to 30 or more different organisms and / or molecular markers). This system is suitable for pathogen identification when many different pathogens all present with similar clinical symptoms. Currently available diagnostic panels include a respiratory panel for upper respiratory tract infections, a blood culture panel for bloodstream infections, a gastrointestinal panel for gastrointestinal infections, a meningitis / encephalitis panel for central nervous system infections, a pneumonia panel for lower respiratory tract infections, and a bone and joint panel for bone and joint infections. Other panels are in development.

[0005] PCR can be conceptually divided into three reactions, each typically assumed to occur at three temperatures over time. This "equilibrium framework" of PCR is easily understood in terms of three reactions (denaturation, annealing, and extension) occurring at three temperatures over three time periods in each cycle. However, this equilibrium framework does not closely match physical reality. Temperature changes do not occur instantaneously; changing sample temperature takes time, and temperature may not be uniform throughout the sample, especially when large volumes are used. Furthermore, individual reaction rates vary with temperature; once primer annealing occurs, polymerase extension immediately follows. A dynamic framework, in which reaction rates and temperature are constantly changing, is more accurate, especially for fast PCR. Maintaining a constant temperature during PCR is not necessary as long as product denaturation and primer annealing occur. In the dynamic framework of PCR, product denaturation, primer annealing, and polymerase extension can overlap in time, and their rates change continuously with temperature. Under the equilibrium framework, a cycle is defined by three temperatures, each held for a certain time, whereas the dynamic framework requires transition rates and target temperatures.

[0006] When PCR first became popular in the late 1980s, the process was slow. A typical protocol involved 1 minute of denaturation at 94°C, 2 minutes of annealing at 55°C, and 3 minutes of extension at 72°C. Including transition times between temperatures, an 8-minute cycle was typical, with 30 cycles completed in 4 hours. 25% of the cycle time was spent in temperature transitions. As the cycle rate increased, the percentage of time spent in temperature transitions also increased, making the dynamic framework increasingly important. During fast-cycle PCR, the temperature is typically changing. In fast-cycle PCR of short products (<100 bps), 100% of the time can be spent in temperature transitions, and no hold period is necessary. In fast-cycle PCR of long products, a temperature hold at the optimal extension temperature can be included.

[0007] One way to reduce cycle time is to introduce variations in PCR protocols to relax temperature cycling requirements. Over the years, systems have become faster, and the kinetic requirements for denaturation, annealing, and extension have become more clearly defined. Shorter cycle times, shortening three-step cycles (denaturation, annealing, and extension) to two steps (combined denaturation and annealing / extension steps), longer primer lengths, and shorter product lengths can reduce PCR reaction times while maintaining high diagnostic accuracy. Despite protocol variations, many diagnostic PCR reactions are completed with a confirmatory melt detection step. DNA has a melting temperature range specific to its length and AT / GC composition. Probe-free DNA melting analysis generally relies on the fact that DNA-binding dyes fluoresce strongly in the presence of double-stranded DNA and fluoresce weakly or not at all in the presence of single-stranded DNA. Therefore, for a given amplification reaction, the expected product has a specific expected melting temperature range, and melting analysis can be used to confirm that the expected product was produced in the amplification reaction.

[0008] However, as PCR speed increases, the time spent on melting increasingly takes up a large portion of the run time.One possible solution is to increase or decrease the temperature faster during melting, but it has been found that the melting curves generated by faster temperature increases often have reduced sensitivity to differences in amplicons.It would be desirable to generate melting curves using faster temperature increases while maintaining sensitivity to differences in amplicons.There is a need in the art to maintain the melting resolution achieved by slower temperature increases and decreases, while also shortening the time required for melting analysis. Summary of the Invention

[0009] The present disclosure relates to methods and systems suitable for simultaneously amplifying many potential targets and then performing melt detection based on whether any amplification is detected for any target nucleic acid, with melting parameters limited by the melting temperature range specific to the target nucleic acid for which amplification is detected. High-resolution DNA melting has traditionally been performed using a fixed temperature ramp rate that encompasses the entire temperature range in which reaction products are expected to melt. This invention achieves this in a shorter time while maintaining the resolution achieved with slower temperature ramp rates by using faster melting rates in temperature ranges where no melting signature is expected from the reaction and slower melting rates in temperature ranges where a melting signature is expected from the reaction. These variable melting ranges and rates are defined by the predicted or experimentally determined melting temperatures of amplicons for which amplification is actually detected, so time is not wasted slowly ramping through a wide temperature range for all possible targets.

[0010] For example, a test (e.g., a panel including many assays for many pathogens) may be designed to amplify and detect 20 or more targets. In certain use cases, when only one or two targets are present in a sample, instead of having one large melting range and one slow melt ramp rate designed to capture the melting of all possible targets, melting parameters can be dynamically set based on the predicted or experimentally determined melting temperatures of the target amplicons present (referred to herein as a range of interest, or ROI). For example, as described in more detail herein below, the melting temperature can be ramped rapidly (greater than 4°C / sec, e.g., 6-20°C / sec) through a first temperature range where the detected target amplicons are not expected to melt, ramped slowly (e.g., less than 4°C / sec, 0.01-2°C / sec, 1-2°C / sec) through the ROI, and then ramped again rapidly (greater than 4°C / sec, e.g., 12-20°C / sec) after the ROI to the denaturation temperature. This fast-slow-fast lift-and-low protocol can save time while maintaining the resolution of traditional slow melting. When amplifications of different target amplicons are detected in more than one well, this "fast-slow-fast" protocol can be appropriately used if the amplifications of the different target amplicons are detected simultaneously or near-simultaneously and the melting ranges of the target amplicons overlap sufficiently. In another embodiment, a modified "fast-slow-fast-slow-fast" protocol can be appropriately used if the amplifications of different target amplicons are detected simultaneously or near-simultaneously and the melting regions of interest between two organisms suspected to be present in the sample are sufficiently separated. In one embodiment, the systems described herein can be appropriately designed to determine which is the fastest and most efficient melting protocol (e.g., "fast-slow-fast" vs. "fast-slow-fast-fast-slow-fast") in situations where amplifications of different target amplicons are detected simultaneously or near-simultaneously.

[0011] The invention described herein may suitably include performing melt detection after a fixed number of PCR cycles if amplification is detected for one or more target nucleic acids (e.g., if the fluorescent signal in the sample well rises above a threshold). The invention described herein may suitably include performing melt detection at any point in the reaction if amplification is detected for one or more target nucleic acids. The invention described herein may suitably include not performing melt detection if amplification is not detected for any target nucleic acids.

[0012] The invention described herein may suitably include a method for identifying which of multiple target nucleic acids is present in a sample (e.g., from multiple organisms). The method may suitably include providing a sample (e.g., a respiratory sample, a blood sample, a positive blood culture sample, etc.) suspected of containing at least one target organism and providing multiple sample wells, each sample well being provided with a portion of the sample and primers for amplifying a target nucleic acid sequence from a different one of the multiple target nucleic acids. Each target nucleic acid from each different organism and / or molecular marker has a melting temperature range specific to that target nucleic acid. The method may suitably further include providing a fluorescent dye that generates a fluorescent signal that increases with increasing concentration of nucleic acid in the multiple sample wells, and simultaneously subjecting the multiple sample wells to amplification conditions for a selected number of cycles. If the sample wells exhibit positive nucleic acid amplification during the amplification conditions, the method may suitably include performing melt detection, the melt detection being defined by a melting temperature range and melting rate specific to the target nucleic acid for detecting the organism in the sample well.

[0013] The invention described herein may suitably include a method for identifying which of a plurality of target nucleic acids and / or molecular markers are present in a sample. The method may suitably include the steps of providing a sample suspected of containing at least one target organism or molecular marker of a plurality of organisms and / or molecular markers, and providing a plurality of sample wells configured for amplification of the plurality of target nucleic acids. Each sample well of the plurality of sample wells may suitably contain a primer pair for amplifying one of the plurality of target nucleic acids. In some embodiments, each sample well of the plurality of sample wells may suitably contain more than one primer pair for amplifying more than one of the plurality of target nucleic acids. Each target nucleic acid may suitably have a unique melting temperature range and melting rate. The method may suitably further include providing a fluorescent dye that generates a fluorescent signal that increases in response to an increase in the concentration of nucleic acid in the plurality of sample wells, distributing the sample among the plurality of sample wells such that each sample well contains a portion of the sample, simultaneously subjecting the plurality of sample wells to amplification conditions, the amplification conditions including repeated thermal cycles each including a primer annealing step, a primer extension portion, and a nucleic acid denaturation step, and acquiring the fluorescent signal from each of the plurality of sample wells during the thermal cycles. If the fluorescent signal from the sample well satisfies a condition indicating the presence of the expected DNA target in the sample (e.g., the fluorescent signal from the sample well is greater than a threshold value, the threshold value being equal to or greater than a detection limit for concluding that the concentration of nucleic acid in the sample well is increased), the method may suitably include performing melt detection, the melt detection being defined by a melting temperature range and melting rate characteristic of the target nucleic acid for detecting organisms in the sample wells in which amplification above the threshold is detected.

[0014] The invention described herein may suitably include a method for fluorescent detection of nucleic acids. The method may suitably include the steps of providing a sample suspected of containing at least one of a plurality of organisms, providing a plurality of sample wells, each sample well being provided with a primer for amplifying a target nucleic acid from a different one of the plurality of organisms, transferring a portion of the sample to each of the plurality of sample wells, simultaneously subjecting the plurality of sample wells to amplification conditions, and acquiring a fluorescent signal from each of the plurality of sample wells during thermal cycling. If the amplitude of the fluorescent signal from a sample well configured for amplification of one target nucleic acid is greater than a threshold value, the threshold value being equal to or greater than a detection limit for concluding an elevated concentration of the nucleic acid in the sample well, the method may suitably include the step of performing melt detection, the melt detection being defined by a melting temperature range and melting rate characteristic of the target nucleic acid for detecting the organism in the sample well in which amplification above the threshold is detected.

[0015] The invention described herein may suitably include a system for detecting which of multiple target organisms are present in a sample. The system may suitably include a container including multiple sample wells, each sample well configured to receive a portion of the sample, nucleic acid primers specific for amplifying one target nucleic acid from one target organism, a fluorescent dye that generates a fluorescent signal that increases with increasing concentration of nucleic acid in the multiple sample wells, and amplification components, and an instrument configured to simultaneously subject the portion of the sample in each of the multiple sample wells to amplification conditions and then melting conditions. The instrument may suitably include a detector for detecting the fluorescent signal from the fluorescent dye that indicates amplification. The instrument may suitably be programmed with thermal cycling parameters for amplification of the nucleic acids in the multiple sample wells and information regarding the target organisms to be amplified in each of the multiple wells, including melting range information for each target nucleic acid from the target organisms. The instrument may suitably be further programmed to monitor the fluorescence of the plurality of wells during the amplification conditions, and to perform melt detection if the sample well exhibits an increase in fluorescence during the amplification conditions indicative of positive nucleic acid amplification, wherein the melt detection is defined by the melting temperature range and melt ramp rate of the target nucleic acid amplified in the sample well.

[0016] Scope of disclosure:

[0017] A1. A method for identifying which of multiple target nucleic acids are present in a sample, comprising: providing a sample suspected of containing at least one target nucleic acid; providing a plurality of sample wells, each sample well provided with a portion of the sample and primers for amplifying a target nucleic acid sequence from a different one of a plurality of target nucleic acids, each target nucleic acid sequence having a unique melting temperature range; providing a fluorescent dye that produces a fluorescent signal that increases in response to increasing concentrations of nucleic acid in the plurality of sample wells; simultaneously subjecting a plurality of sample wells to amplification conditions for a selected number of cycles; determining whether the sample wells exhibit positive nucleic acid amplification as evidenced by an increasing fluorescent signal from the sample wells during amplification conditions; and responsive to determining that the sample well exhibits positive nucleic acid amplification, performing a melt detection configured to detect the amplified target nucleic acid in the sample well, the melt detection being defined by a melting temperature range characteristic of the target nucleic acid in the sample well. A method comprising:

[0018] A2. The method of paragraph A1, wherein positive nucleic acid amplification is determined by a fluorescent signal in the sample well rising above a threshold value.

[0019] A3. The method of paragraph A1 or A2, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units rise above the threshold, mathematical modeling, signal processing, and combinations thereof.

[0020] A4. The method of any one of A1-A3, further comprising the steps of analyzing the fluorescent signals of multiple wells in real time to determine whether amplification has occurred in the sample wells, and performing melting detection if it is determined that amplification has occurred in the wells, wherein the temperature range of melting detection is limited by the known melting temperature range of the target nucleic acid in the wells.

[0021] A5. The method of any one of paragraphs A1-A4, including not performing melt detection if the sample wells do not show positive nucleic acid amplification.

[0022] A6. The method of any one of paragraphs A1-A5, wherein the plurality of sample wells includes one or more control wells, and melting detection is not performed if only one or more control wells show positive nucleic acid amplification.

[0023] A7. Melting detection a first lift rate during a first portion of the melting; a second elevation rate during a second portion of the melting; and a third lift rate during the third portion of the melting wherein the second rate is slower than the first rate and the third rate, and the second portion of the melting is defined by a melting temperature range specific to a target nucleic acid for detection of an organism in the sample well.

[0024] A7.1. The method of any one of paragraphs A1-A7, wherein the third lift speed is faster than the first lift speed.

[0025] A7.2. The method of any one of paragraphs A1-A7.1, wherein the second heating / cooling rate is in the range of 0.01 to 4°C / sec, or preferably 0.01 to 2°C / sec.

[0026] A7.3. The method of any one of paragraphs A1-A7.2, wherein the second heating / cooling rate is preferably 0.5°C / sec or less, more preferably 1°C / sec or less, and most preferably 2°C / sec or less.

[0027] A7.4. The method of any one of sections A1-A7.3, wherein the second elevation rate is adapted to detect a nucleic acid melting signature indicative of a genotype, sequence variant, or genetic mutation in the target nucleic acid that alters one or more of the nucleic acid melting temperature or the shape of the nucleic acid melting curve compared to the target nucleic acid without the genotype, sequence variant, or genetic mutation.

[0028] A7.5. The method of any one of sections A1-A7.4, further comprising detecting single nucleotide polymorphisms (SNPs).

[0029] A7.7. The method of any one of sections A1-A7.5, further comprising detecting an antimicrobial resistance (AMR) marker.

[0030] A7.8. The method of any one of paragraphs A1-A7.7, further comprising detecting the presence of the organism by a first melt detection in a first assay, and, if the first assay detects the presence of the organism, performing a second melt detection in a second assay to detect the presence or absence of a genotype, sequence variant, or genetic mutation.

[0031] A8. The method of any one of paragraphs A1-A7.8, wherein a positive or negative result in the sample well determines whether melting detection is performed and determines the temperature range of the second part of the melting.

[0032] A8.1. The method of any one of paragraphs A1-A8, wherein the temperature range of the second part of the melting is the melting temperature range of the target amplicon, ±0.5°C to 10°C of the melting temperature range of the target amplicon, preferably ±2°C to 6°C of the melting temperature range of the target amplicon.

[0033] A9. The method of any one of sections A1-A8.1, wherein two or more wells show a fluorescent signal indicative of positive amplification.

[0034] A10. The method of any one of paragraphs A1-A9, further comprising performing a single melting detection in one melting temperature range on the amplified target nucleic acids in two or more wells.

[0035] A11. The method of any one of paragraphs A1-A10, further comprising performing a first melting detection in a first melting temperature range specific to the melting of the amplified target nucleic acid in the first well, and performing at least a second melting detection in a second melting temperature range specific to the melting of the amplified target nucleic acid in the second well.

[0036] A11.1. Melting detection is a first lift speed during a first portion of the melting; a second elevation rate during a second portion of the melting; and a third lift rate during the third portion of the melting wherein the second rate is slower than the first rate and the third rate, and the second portion of the melting is defined by a melting temperature range characteristic of the target nucleic acid in the first sample well and the second sample well; or a first lift rate during a first portion of the melting; a second lift rate during a second portion of the melting; a third lift rate during a third portion of the melting; a fourth elevation rate during a fourth portion of the melting; and A fifth lift rate during the fifth portion of the melting wherein the second rate and the fourth rate are slower than the first rate, the third rate, and the fifth rate, and wherein the second portion of the melting is defined by a melting temperature range specific to the target nucleic acid in the first sample well and the fourth portion of the melting is defined by a melting temperature range specific to the target nucleic acid in the second sample well.

[0037] A11.2. The method of any one of paragraphs A1-A11.1, wherein the second rate of change is in the range of 0.05 to 4°C / sec, or the second rate of change and the fourth rate of change are each in the range of 0.05 to 4°C / sec.

[0038] A11.3. The method of any one of paragraphs A1-A11.2, wherein the second rate or the second rate and the fourth rate are preferably 0.5°C / sec or less, more preferably 1°C / sec or less, and most preferably 2°C / sec or less.

[0039] A12. The method of any one of sections A1-A11.3, wherein the selected number of cycles of amplification before melt detection is at least 20.

[0040] A13. The method of any one of paragraphs A1-A12, wherein the selected number of cycles of amplification prior to melt detection is assay specific and determined by the expected concentrations of multiple target nucleic acids suspected to be present in the sample.

[0041] A14. The method of any one of paragraphs A1-A13, further comprising identifying at least one target organism present in the sample by identifying at least one corresponding sample well in which amplification and melt detection occurred.

[0042] A15. The method of any one of paragraphs A1-A14, wherein the target nucleic acid can be used to identify cell-free DNA, cells, organisms, molecular markers of antimicrobial resistance, host response markers, and combinations thereof.

[0043] B1. A method for identifying which of a plurality of target nucleic acids is present in a sample, comprising: providing a sample suspected of containing at least one target nucleic acid; providing a plurality of sample wells configured for amplification of a plurality of target nucleic acids, each sample well of the plurality of sample wells containing a primer pair for amplifying one of the plurality of target nucleic acids, each target nucleic acid having a unique melting temperature range; providing a fluorescent dye that produces a fluorescent signal that increases in response to increasing concentrations of nucleic acid in the plurality of sample wells; distributing the sample among a plurality of sample wells such that each sample well contains a portion of the sample; simultaneously subjecting a plurality of sample wells to amplification conditions, the amplification conditions comprising repeated thermal cycles, each comprising a primer annealing step, a primer extension portion, and a nucleic acid denaturation step; acquiring a fluorescent signal in each of the plurality of sample wells during thermal cycling; determining that the amplitude of the fluorescent signal of a sample well configured for amplification of one target nucleic acid is greater than a threshold value, the threshold value being equal to or greater than a detection limit for concluding an elevated concentration of the nucleic acid in the sample well; and In response to determining that the amplitude of the fluorescent signal of the sample is greater than a threshold, performing melt detection configured to detect the amplified target nucleic acid in the sample well, the melt detection being defined by a melting temperature range characteristic of the amplified target nucleic acid in the sample well for detection of the organism in the sample well. A method comprising:

[0044] B2. The method of paragraph B1, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units rise above the threshold, mathematical modeling, signal processing, and combinations thereof.

[0045] B3. The method of paragraph B1 or B2, further comprising the steps of monitoring the fluorescent signals of the plurality of wells in real time to determine whether amplification has occurred in at least one well of the plurality of sample wells, and performing melting detection if it is determined that amplification has occurred in at least one well, wherein the temperature range of melting detection is limited by the known melting temperature range of the target nucleic acid in at least one well.

[0046] B4. The method of any one of paragraphs B1-B3, wherein the timing of melt detection is determined by the fluorescent signal of one or more wells configured for amplification of multiple target nucleic acids rising above a threshold.

[0047] B5. The method of any one of paragraphs B1-B4, which does not include performing a set number of thermal cycles before performing melt detection.

[0048] B6. The method of any one of paragraphs B1-B5, further comprising not performing melt detection if no sample wells exhibit a fluorescent signal above the threshold.

[0049] B7. The method of any one of paragraphs B1-B6, wherein the plurality of sample wells includes one or more control wells, and melting detection is not performed if only one or more control wells exhibit a fluorescent signal above a threshold.

[0050] B8. Melting detection a first rate of rise and fall through a first temperature range of melting; a second rate of rise and fall during a second temperature range of melting; and a third rate of rise and fall during a third temperature range of melting; wherein the second rate is slower than the first rate and the third rate, and the second temperature range of melting is defined by the melting temperature range of the amplified target nucleic acid in the sample wells having a fluorescent signal greater than a threshold.

[0051] B9. The method of any one of paragraphs B1-B8, wherein the second temperature range for melting is ±10°C of the melting temperature range of the target nucleic acid, ±8°C of the melting temperature range of the target nucleic acid, ±6°C of the melting temperature range of the target nucleic acid, or ±4°C of the melting temperature range of the target nucleic acid.

[0052] B10. The method of any one of paragraphs B1-B9, wherein melt detection is performed and the temperature range of the second portion of the melt is determined.

[0053] B11. The method of any one of paragraphs B1-B10, wherein two or more wells exhibit a fluorescent signal above the threshold.

[0054] B12. The method of any one of paragraphs B1-B11, further comprising performing a single melt detection run at a single melting temperature range on the amplified target nucleic acid in two or more wells.

[0055] B13. The method of any one of paragraphs B1-B12, further comprising performing a first melting detection in a first melting temperature range specific to the melting of the target nucleic acid amplified in the first well, and performing at least a second melting detection in a second melting temperature range specific to the melting of the target nucleic acid amplified in the second well.

[0056] B14. A plurality of wells are configured for amplification of target nucleic acid sequences from organisms present in high titers, organisms present in lower titers than organisms present in high titers, and organisms present in lower titers than organisms present in lower titers; performing a first melt detection if one or more wells configured for amplification of a target nucleic acid sequence from an organism present in high titer show amplification above a threshold within 20 or fewer thermal cycles; performing a second melt detection if one or more wells configured for amplification of target nucleic acid sequences from organisms present in lower titers show amplification above a threshold within 25 or fewer thermal cycles; and performing a third melt detection if one or more wells configured for amplification of a target nucleic acid sequence from an organism present at low titer exhibit amplification above a threshold within 30 or fewer thermal cycles. The method of any one of paragraphs B1-B13, further comprising:

[0057] B14.1. The method of any one of paragraphs B1-B14, wherein the first number of thermal cycles is 20 or less thermal cycles, the second number of thermal cycles is 25 or less thermal cycles, and the third number of thermal cycles is 30 or less thermal cycles.

[0058] B15. The method of any one of paragraphs B1-B14.1, further comprising not performing melt detection if one or more wells configured for amplification of a target nucleic acid sequence from an organism present in high titer show amplification above a threshold for more than 20 thermal cycles.

[0059] B16. The method of any one of paragraphs B1-B15, further comprising not performing melt detection if one or more wells configured for amplification of a target nucleic acid sequence from an organism present in lower titer show amplification above the threshold for more than 25 thermal cycles.

[0060] B16.1 The method of any one of paragraphs B1-B16, further comprising not performing melt detection if one or more wells configured for amplification of target nucleic acid sequences from organisms present at a second titer show amplification above a threshold value for more than a second number of thermal cycles.

[0061] B17. The method of any one of paragraphs B1-B16.1, further comprising not performing melting detection if none of the wells or wells configured for amplification of target nucleic acid sequences from organisms present at high titer, lower titer, or low titer show amplification above the threshold.

[0062] B18. The method of any one of paragraphs B1-B17, further comprising subjecting the sample to multiplex amplification prior to the partitioning step.

[0063] B19. The method of any one of paragraphs B1-B18, wherein all steps are performed in a single closed system.

[0064] B20. The method of any one of paragraphs B1-B19, wherein the target nucleic acid can be used to identify cell-free DNA, cells, organisms, molecular markers of antimicrobial resistance, host response markers, and combinations thereof.

[0065] C1. A method for determining the presence of an organism in a sample, comprising: providing a sample suspected of containing at least one of a plurality of organisms; providing a plurality of sample wells, each sample well being provided with a primer for amplifying a target nucleic acid from a different one of a plurality of organisms; transferring a portion of the sample to each of a plurality of sample wells; simultaneously subjecting a plurality of sample wells to amplification conditions; acquiring a fluorescent signal in each of a plurality of sample wells during thermal cycling; determining that the amplitude of the fluorescent signal of a sample well configured for amplification of one target nucleic acid is greater than a threshold value, the threshold value being equal to or greater than a detection limit for concluding an elevated concentration of the nucleic acid in the sample well; responsive to determining that the amplitude of the fluorescent signal in the sample well is greater than a threshold, performing melt detection at a melting temperature window to detect the amplified target nucleic acid in the sample well, wherein the melting temperature window is defined by a melting temperature range characteristic of the target nucleic acid; and determining the presence of organisms in the sample in response to performing melt detection; A method comprising:

[0066] C2. The method of paragraph C1, further comprising the steps of subjecting a plurality of sample wells to amplification conditions for a selected number of cycles, and if the sample wells show positive nucleic acid amplification within the selected number of amplification cycles, performing melt detection, wherein the melt detection is defined by a melting temperature range characteristic of the target nucleic acid for detection of the organism in the sample well.

[0067] C3. The method of paragraph C2, wherein the selected number of cycles of amplification is at least one but not more than 15, at least one but not more than 20, at least one but not more than 25, at least one but not more than 30, or at least one but not more than 35.

[0068] C4. The method of any one of paragraphs C1-C3, further comprising not performing melt detection if no sample wells show positive nucleic acid amplification.

[0069] C5. The method of any one of paragraphs C1-C4, further comprising simultaneously subjecting the samples to multiplex amplification prior to the transfer step.

[0070] C6. The method of any one of paragraphs C1-C5, wherein all steps are performed in a single closed system.

[0071] D1. A system for detecting which of a plurality of target nucleic acids is present in a sample, comprising: a container including a plurality of sample wells, each sample well configured to contain a portion of the sample, nucleic acid primers specific for amplification of one target nucleic acid, a fluorescent dye that produces a fluorescent signal that increases with increasing concentration of nucleic acid in the plurality of sample wells, and amplification components; An instrument configured to simultaneously subject a portion of a sample in each of a plurality of sample wells to amplification conditions and then to melting conditions, the instrument including a detector for detecting a fluorescent signal from a fluorescent dye indicative of amplification. wherein the instrument is programmed with a target nucleic acid to be amplified in each of a plurality of wells, melting range information for each target nucleic acid, and wherein the instrument is programmed to monitor fluorescence of the plurality of wells during amplification conditions and perform melt detection if a sample well exhibits an increase in fluorescence indicative of positive nucleic acid amplification during the amplification conditions, wherein the melt detection is defined by the melting temperature range of the target nucleic acid to be amplified in the sample well.

[0072] D2. The system of paragraph D1, wherein the instrument is programmed to perform a set number of amplification cycles before performing melt detection.

[0073] D3. A system described in paragraphs D1 or D2, wherein the instrument is programmed to perform melt detection at any number of amplification cycles if amplification is detected in the sample well, and the temperature range of melt detection is limited by the melting range information of the target nucleic acid amplified in the well.

[0074] D4. The system of any one of paragraphs D1-D3, wherein positive nucleic acid amplification is indicated by a fluorescent signal in the sample well rising above a threshold.

[0075] D5. The system of any one of paragraphs D1-D4, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units rise above the threshold, mathematical modeling, signal processing, and combinations thereof.

[0076] D6. A system described in any one of paragraphs D1-D5, wherein the instrument is further programmed to not perform melting detection if no sample wells show positive nucleic acid amplification within a set number of amplification cycles.

[0077] D7. a first lift rate during a first portion of the melting; a second elevation rate during a second portion of the melting; and a third lift rate during the third portion of the melting and programmed to include melt detection, wherein the second rate is slower than the first rate and the third rate, and the second portion of the melt is defined by melting range information of the target nucleic acid amplified in the well.

[0078] D8. A system described in any one of paragraphs D1-D7, programmed to identify at least one of a target organism, cell-free DNA, cells, or molecular markers of antimicrobial resistance present in a sample by identifying at least one corresponding sample well in which amplification and melting detection occurred.

[0079] D9. The system of any one of paragraphs D1-D8, wherein the amplification is PCR and the components include a polymerase and dNTPs.

[0080] D10. The system of any one of paragraphs D1-D9, wherein the device includes a light source configured to emit a light signal toward the plurality of sample wells during screening of each sample.

[0081] D11. A system described in any one of paragraphs D1-D10, wherein the melting result is based on the presence or absence of a melting peak within a predetermined temperature range, the presence of a melting peak outputting a positive result and the absence of a melting peak outputting a negative result.

[0082] E1. A system for detecting which of a plurality of target nucleic acids is present in a sample, comprising: a container comprising a plurality of sample wells, each sample well configured to contain a portion of the sample, nucleic acid primers specific for amplification of one target nucleic acid, a fluorescent dye that produces a fluorescent signal that increases with increasing concentration of nucleic acid in the plurality of sample wells, and amplification components; an instrument configured to simultaneously subject a portion of the sample in each of a plurality of sample wells to amplification conditions and then to melting conditions, the instrument including a detector for detecting a fluorescent signal from the fluorescent dye indicative of amplification; and Programming for carrying out the method of any one of paragraphs A1-C6 Including, A system in which the instrument is programmed with the identity of a target nucleic acid to be amplified in each of a plurality of wells and melting range information for each of the target nucleic acids.

[0083] F1. A computer-implemented method for evaluating a sample for a target nucleic acid sequence, comprising: sending, by the one or more processors, control signals to a thermocycling element to heat a plurality of sample wells to a first temperature using an initial ramp rate and cool the plurality of sample wells to a second temperature through one or more cycles, each cycle including an intra-cycle temperature adjustment segment, each of the plurality of sample wells configured to receive a portion of a sample, each sample well containing a target nucleic acid sequence from a different one of a plurality of target nucleic acids, each target nucleic acid from each different organism having a unique melting temperature range; receiving, at one or more processors from the optical system, data indicative of an amount of fluorescence emitted from a portion of the samples in the plurality of sample wells during an intra-cycle temperature adjustment segment of one or more cycles; In response to determining that the amount of fluorescence for at least one of the sample wells exceeds a threshold value, determining, by one or more processors, the target nucleic acid sequence amplified in each of the sample wells that exceeds a threshold; determining, by the one or more processors, an adjusted heating rate profile for heating the sample wells based on the melting temperature range and / or melting rate characteristic of the target nucleic acid sequence in each of the sample wells that exceeds the threshold; and performing melt detection on a portion of the sample, including sending, by the one or more processors, control signals to the thermocycling element to heat the sample well to the first temperature using the adjusted ramp rate profile for subsequent cycles. A method comprising:

[0084] F2. The first temperature is a denaturation temperature, the second temperature is an annealing temperature, the characteristic melting temperature range of the sample well is from the initial melting temperature to the final melting temperature, and the adjusted ramp rate profile is: a first ramp rate during the approach temperature range from the annealing temperature to the incipient melting temperature; a second ramp rate through a specific melting temperature range from the initial melting temperature to the final melting temperature; and a third ramp rate during the final temperature range from the final melting temperature to the denaturation temperature; wherein the second lift speed is slower than the first lift speed and the third lift speed.

[0085] F3. The method of paragraph F1 or F2, wherein the third lift speed is faster than the first lift speed.

[0086] F4. The method according to any one of F1 to F3, wherein the second temperature increase / decrease rate is in the range of 0.05 to 4°C / sec.

[0087] F5. The method according to any one of items F1-F4, wherein the first heating / cooling rate is greater than 4°C / sec (e.g., between 6°C / sec and 20°C / sec), the second heating / cooling rate is less than 4°C / sec (e.g., in the range of 0.05 to 4°C / sec), and the third heating / cooling rate is greater than 4°C / sec (e.g., 12 to 20°C / sec).

[0088] F5.1. The method of any one of paragraphs F1-F5, wherein the third lift speed is faster than the first lift speed.

[0089] F5.2. The method of any one of paragraphs F1-F5.1, wherein the second heating / cooling rate is in the range of 0.01 to 4°C / sec, or preferably 0.01 to 2°C / sec.

[0090] F5.3. The method of any one of paragraphs F1-F5.2, wherein the second heating / cooling rate is preferably 0.5°C / sec or less, more preferably 1°C / sec or less, and most preferably 2°C / sec or less.

[0091] F5.4. The method of any one of paragraphs F1-F5.3, wherein the second elevation rate is adapted to detect a nucleic acid melting signature indicative of a genotype, sequence variant, or genetic mutation in the target nucleic acid that alters one or more of the nucleic acid melting temperature or the shape of the nucleic acid melting curve compared to the target nucleic acid without the genotype, sequence variant, or genetic mutation.

[0092] F5.5. The method of any one of paragraphs F1-F5.4, further comprising detecting single nucleotide polymorphisms (SNPs).

[0093] F5.6. The method of any one of sections F1-F5.5, further comprising detecting an antimicrobial resistance (AMR) marker.

[0094] F5.7. The method of any one of paragraphs F1-F5.6, further comprising detecting the presence of the organism by a first melt detection in a first assay, and, if the first assay detects the presence of the organism, performing a second melt detection in a second assay to detect the presence or absence of a genotype, sequence variant, or genetic mutation.

[0095] F6. The method of any one of paragraphs F1-F5.7, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units rise above a threshold, mathematical modeling, signal processing, and combinations thereof.

[0096] F7. The controller and identifying a target nucleic acid among a plurality of target nucleic acids corresponding to the sample well based on melt detection. The method of any one of paragraphs F1-F6, further comprising:

[0097] F8. The controller determining that the amount of fluorescence in the first sample well and the second sample well exceeds a threshold; performing a first melt detection by using a first adjusted ramp rate profile corresponding to a first characteristic melting temperature range for melting the amplified target nucleic acid in the first sample well; and and performing a second melting detection by using a second adjusted ramp rate profile corresponding to a second unique melting temperature range for melting the amplified target nucleic acid in the second sample well. The method of any one of paragraphs F1-F7, further comprising:

[0098] G1. A system for determining a sample for a target nucleic acid sequence, comprising: a plurality of sample wells, each configured to receive a portion of a sample, each sample well containing a target nucleic acid sequence from a different one of a plurality of target nucleic acids, each target nucleic acid from each different organism having a distinctive melting temperature range; an optical system configured to detect the amount of fluorescence emitted from the sample; a controller, sending control signals to a thermocycling element to heat the plurality of sample wells to a first temperature using an initial ramp rate and cool the plurality of sample wells to a second temperature through one or more cycles, each cycle including an intra-cycle temperature adjustment segment; receiving data from the optical system during an intra-cycle temperature adjustment segment of one or more cycles that indicates an amount of fluorescence emitted by a portion of the samples in the plurality of sample wells; In response to determining that the amount of fluorescence for at least one of the sample wells exceeds a threshold value, determining a tailored ramp rate profile for heating the sample well based on a melting temperature range characteristic of the target nucleic acid sequence in the sample well above a threshold; and Sending a control signal to the thermocycling element to perform melt detection on a portion of the sample by heating the sample well to a first temperature using the adjusted ramp rate profile for the next cycle. Controller configured as A system equipped with

[0099] G2. The first temperature is a denaturation temperature, the second temperature is an annealing temperature, the characteristic melting temperature range of the sample well is from the initial melting temperature to the final melting temperature, and the adjusted ramp rate profile is a first ramp rate during the approach temperature range from the annealing temperature to the incipient melting temperature; a second ramp rate through a specific melting temperature range from the initial melting temperature to the final melting temperature; and a third ramp rate during the final temperature range from the final melting temperature to the denaturation temperature; wherein the second lift speed is slower than the first lift speed and the third lift speed.

[0100] G3. The system of paragraph G1 or G2, wherein the third lift speed is faster than the first lift speed.

[0101] G4. The system of any one of paragraphs G1-G3, wherein the second lift speed is the same as the initial lift speed.

[0102] G5. A system described in any one of items G1-G4, wherein the first heating / cooling rate is greater than 4°C / sec (e.g., 6-20°C / sec), the second heating / cooling rate is less than 4°C / sec (e.g., in the range of 0.01-4°C / sec or 1-2°C / sec), and the third heating / cooling rate is greater than 4°C / sec (e.g., 6-20°C / sec).

[0103] G6. The system of any one of paragraphs G1-G5, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units rise above the threshold, mathematical modeling, signal processing, and combinations thereof.

[0104] G7. The controller and identifying a target nucleic acid among a plurality of target nucleic acids corresponding to the sample well based on melt detection. The system of any one of paragraphs G1-G6, further comprising:

[0105] G8. The controller determining that the amount of fluorescence in the first sample well and the second sample well exceeds a threshold; performing a first melting detection using a first adjusted ramp rate profile corresponding to a first characteristic melting temperature range for melting the amplified target nucleic acid in the first sample well; and and performing a second melting detection using a second adjusted ramp rate profile corresponding to a second unique melting temperature range for melting the amplified target nucleic acid in the second sample well. The system of any one of paragraphs G1-G7 further comprising:

[0106] H1. A computer device for evaluating a sample for a target nucleic acid sequence, comprising: one or more processors; and a non-transitory computer-readable memory coupled to one or more processors and configured to execute the non-transitory computer-readable memory when executed by the one or more processors; sending control signals to a thermocycling element to heat a plurality of sample wells to a first temperature using an initial ramp rate and cool the plurality of sample wells to a second temperature through one or more cycles, each cycle including an intra-cycle temperature adjustment segment, wherein each of the plurality of sample wells is configured to receive a portion of a sample, each sample well containing a target nucleic acid sequence from a different one of a plurality of target nucleic acids, each target nucleic acid from each different organism having a distinctive melting temperature range; receiving data from the optical system indicative of an amount of fluorescence emitted by a portion of the samples in the plurality of sample wells during an intra-cycle temperature adjustment segment of one or more cycles; In response to determining that the amount of fluorescence in at least one of the sample wells exceeds a threshold value, determining a tailored ramp rate profile for heating the sample well based on the characteristic melting temperature range of the target nucleic acid sequence in the sample well above a threshold; and performing melt detection on a portion of the sample by sending a control signal to the thermocycling element to heat the sample well to a first temperature using the adjusted ramp rate profile for the next cycle. a non-transitory computer-readable memory storing therein instructions for causing a A computer device comprising:

[0107] H2. The first temperature is a denaturation temperature, the second temperature is an annealing temperature, the characteristic melting temperature range of the sample well is from an initial melting temperature to a final melting temperature, and the adjusted ramp rate profile is: a first ramp rate during the approach temperature range from the annealing temperature to the incipient melting temperature; a second ramp rate through a specific melting temperature range from the initial melting temperature to the final melting temperature; and a third ramp rate during the final temperature range from the final melting temperature to the denaturation temperature; wherein the second lift speed is slower than the first lift speed and the third lift speed.

[0108] H3. The computing device of paragraph H1 or H2, wherein the third lift speed is faster than the first lift speed.

[0109] H4. The computer device of any one of paragraphs H1-H3, wherein the second lift speed is the same as the initial lift speed.

[0110] H5. The computer device described in any one of items H1-H4, wherein the first heating / cooling rate is greater than 4°C / sec (e.g., 6-20°C / sec), the second heating / cooling rate is less than 4°C / sec (e.g., in the range of 0.01-4°C / sec or 1-2°C / sec), and the third heating / cooling rate is greater than 4°C / sec (e.g., 6-20°C / sec).

[0111] H6. The computer device of any one of paragraphs H1-H5, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units rise above a threshold, mathematical modeling, signal processing, and combinations thereof.

[0112] H7. Instructions are given to a computer device and identifying a target nucleic acid among a plurality of target nucleic acids corresponding to the sample wells based on melt detection. The computer device of any one of paragraphs H1-H6, further comprising:

[0113] H8. Instructions are given to a computer device determining that the amount of fluorescence in the first sample well and the second sample well exceeds a threshold; performing a first melt detection by using a first adjusted ramp rate profile corresponding to a first characteristic melting temperature range for melting the amplified target nucleic acid in the first sample well; and performing a second melting detection by using a second adjusted ramp rate profile corresponding to a second unique melting temperature range for melting the amplified target nucleic acid in a second sample well; The computer device of any one of paragraphs H1-H7, further comprising:

[0114] Reaction vessels and apparatus that may be suitably used in any of the methods described herein are also provided in this disclosure.

[0115] Additional features of the present invention will become apparent to those skilled in the art upon consideration of the following detailed description of the preferred embodiment, which illustrates the best mode currently known for carrying out the invention. [Brief explanation of the drawings]

[0116] [Figure 1] FIG. 1 shows an exemplary pouch that may be suitably used in embodiments of the present disclosure.

[0117] [Figure 2] FIG. 2 illustrates exemplary steps in a thawing procedure that may be suitably used in embodiments of the present disclosure.

[0118] [Figure 3A] 3A and 3B show an example comparing slow melting at a fixed temperature ramp rate with fast melting at a fixed temperature ramp rate. [Figure 3B] (the above)

[0119] [Figure 4A] FIG. 4A shows an example comparing slow melting at a fixed rate with dynamic melting at a variable rate, using faster rates in temperature ranges where no melting signature is expected from the reaction and slower rates in temperature ranges where a melting signature is expected from the reaction.

[0120] [Figure 4B] FIG. 4B shows another example similar to FIG. 4A, comparing slow melting at a fixed lift rate with dynamic melting at a variable lift rate.

[0121] [Figure 5A] FIG. 5A shows an example of a kinetic melting analysis for one assay target.

[0122] [Figure 5B] FIG. 5B shows an example of dynamic melting after a fixed number of cycles of amplification on the target of FIG. 5A.

[0123] [Figure 6A] FIG. 6A shows an example of a kinetic melting analysis of two assay targets over one melting temperature range.

[0124] [Figure 6B] FIG. 6B shows an example of dynamic melting after a fixed number of cycles of amplification for the two targets of FIG. 6A.

[0125] [Figure 7A] FIG. 7A shows an example of a kinetic melting analysis on one analyte after amplification for a dynamically determined number of cycles rather than a fixed number of cycles.

[0126] [Figure 7B] FIG. 7B shows a dynamic melting example of FIG. 7A, where melting is performed based on the time point at which the analyte shows amplification above a threshold.

[0127] [Figure 8A]FIG. 8A illustrates an example of assay-specific melt detection, with one dynamic melt for each target, where the number of amplification cycles prior to each melt is dynamically determined based on the time at which each target shows amplification above a threshold.

[0128] [Figure 8B] FIG. 8B illustrates the melting specific parameters of the first target of FIG. 8A.

[0129] [Figure 8C] FIG. 8C illustrates the results of dynamic melting of the first target illustrated in FIGS. 8A and 8B.

[0130] [Figure 8D] FIG. 8D illustrates the melting specific parameters of the second target of FIG. 8A.

[0131] [Figure 8E] FIG. 8E illustrates the results of dynamic melting of the second target illustrated in FIGS. 8A and 8D.

[0132] [Figure 9] FIG. 9 illustrates a block diagram of an exemplary embodiment of a thermal cycling system according to aspects of the present disclosure.

[0133] [Figure 10] FIG. 10 illustrates a flow diagram of an embodiment of a computer-implemented dynamic melting detection method for assessing samples for target nucleic acid sequences.

[0134] [Figures 11A-11B] FIG. 11A illustrates an example of a temperature ramp profile for a nucleic acid melting experiment.

[0135] FIG. 11B illustrates an example of nucleic acid melting curves (left panel) and derivative melting curves (right panel) for two amplicons with different melting temperatures.

[0136] [Figure 12] FIG. 12 illustrates a schematic melting procedure comparing a standard melting protocol (····), a dynamic melting protocol (--), and a dynamic "high resolution" protocol (·-·-).

[0137] [Figure 13] FIG. 13 illustrates a N. gonorrhoeae bioassay (● Org) and another assay targeting a mutation-prone region of the gyrA gene (○ S91F and +WT).

[0138] [Figures 14A-14B] 14A and 14B compare the melting curves of wild-type (--MG-WT) and A2059G (- - - 2059G) mutant amplicons of the 23S ribosomal RNA gene of M. genitalium.

[0139] [Figures 15A-15B] Figures 15A and 15B compare the melting curves of the wild-type (-MG-WT) and A2058G (-··-2058G) mutant amplicons of the 23S ribosomal RNA gene of M. genitalium.

[0140] [Figures 16A-16B] 16A and 16B compare the melting curves of the wild-type (--NG-WT) and S91F (- - - S91F) mutant amplicons of the gyrA gene of Neisseria gonorrhoeae. DETAILED DESCRIPTION OF THE INVENTION

[0141] As used herein, the terms "a," "an," and "the" are defined to mean one or more, and include the plural unless the context is inappropriate. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. The term "about" is used herein to mean approximately, in the region of, roughly, or near. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a difference of 5%. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, using the antecedent "about," it is understood that the particular value forms another embodiment. It is further understood that the endpoints of each range are significant both in relation to the other endpoint, and independently of the other endpoint.

[0142] As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list.

[0143] "Sample" means an animal; an animal tissue or organ; a cell (either a subject's internal cells, cells taken directly from a subject, or cells maintained in culture or a cultured cell line); a cell lysate (or lysate fraction) or cell extract; a solution containing one or more molecules (e.g., polypeptides or nucleic acids) derived from cells, cellular material, or viral material; or a solution containing non-naturally occurring nucleic acids to be analyzed as described herein. A sample can be any bodily fluid or excreta that contains cells, cellular components, or nucleic acids (e.g., but not limited to, blood, urine, stool, saliva, tears, bile, cerebrospinal fluid).

[0144] As used herein, the term "reaction" may refer to a nucleic acid amplification reaction (e.g., a PCR reaction) used to amplify a given target nucleic acid. As used herein, the term "assay" may be used to refer to one or more reactions in a single well or container. As used herein, the term "panel" may be used to refer to a collection of assays that may be grouped together to test a group of targets (e.g., a panel testing for respiratory pathogens). For example, a panel may be designed to include many assays that amplify and detect 20 or more targets.

[0145] As used herein, the term "nucleic acid" refers to a natural or synthetic oligonucleotide or polynucleotide, whether DNA, RNA, or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, capable of hybridizing to a complementary nucleic acid via Watson-Crick base pairing. Nucleic acids of the present invention may also include nucleotide analogs (e.g., BrdU) and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids may include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0146] "Probe," "primer," or "oligonucleotide" refers to a single-stranded DNA or RNA molecule of defined sequence that can base-pair with a second DNA or RNA molecule ("target") containing a complementary sequence. The stability of the resulting hybrid depends on the length, GC content, and the degree of base-pairing that occurs. The degree of base-pairing is affected by parameters such as the degree of complementarity between the probe and target molecule and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to those of skill in the art. Probes, primers, and oligonucleotides can be detectably labeled, either radioactively, fluorescently, or non-radioactively, by methods well known to those of skill in the art. dsDNA-binding dyes can be used to detect dsDNA. While a "primer" is specifically configured to be extended by a polymerase, it is understood that a "probe" or "oligonucleotide" may or may not be so configured.

[0147] By "dsDNA binding dye" is meant a dye that fluoresces differently when bound to double-stranded DNA than when bound to single-stranded DNA or when free in solution, typically by emitting more fluorescence. Although dsDNA binding dyes are referred to herein, it is understood that any suitable dye can be used, and some non-limiting exemplary dyes are described in U.S. Patent No. 7,387,887, which is incorporated herein by reference. Other signal-producing agents, such as, for example, enzymes, antibodies, etc., can also be used to detect nucleic acid amplification and melting, as known in the art.

[0148] By "specifically hybridize" is meant that a probe, primer, or oligonucleotide recognizes and physically interacts (i.e., base pairs) with a substantially complementary nucleic acid (e.g., a sample nucleic acid) under high stringency conditions, but does not substantially base pair with other nucleic acids.

[0149] "High stringency conditions" refers to conditions that allow hybridization comparable to that obtained using a DNA probe at least 40 nucleotides in length in a buffer containing 0.5 M NaHPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (fraction V) at a temperature of 65°C, or in a buffer containing 48% formamide, 4.8x SSC, 0.2 M Tris-Cl, pH 7.6, 1x Denhardt's solution, 10% dextran sulfate, and 0.1% SDS at a temperature of 42°C. Other conditions for high stringency hybridization, such as PCR, Northern, Southern, or in situ hybridization, DNA sequencing, etc., are well known to those skilled in the art of molecular biology.

[0150] While PCR is the amplification method used in the examples herein, it is understood that any amplification method using primers may be suitable. Such suitable methods include polymerase chain reaction (PCR); strand displacement amplification (SDA); nucleic acid sequence-based amplification (NASBA); cascade rolling circle amplification (CRCA); loop-mediated isothermal amplification of DNA (LAMP); isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN); target-based helicase-dependent amplification (HDA); transcription-mediated amplification (TMA), and the like. Thus, when the term PCR is used, it should be understood to include other alternative amplification methods. In amplification methods without separate cycles, reaction times measured in cycles or Cp may be used, and additional reaction times may be added, in the embodiments described herein, where additional PCR cycles are added. It is understood that protocols may need to be adjusted accordingly.

[0151] In various embodiments disclosed herein, self-contained nucleic acid analysis pouches are used to examine samples for the presence of various biological substances, such as antigens and nucleic acid sequences, in a single, closed system. Such systems, including pouches and instruments for use with the pouches, are disclosed in more detail in U.S. Pat. Nos. 8,394,608; 8,895,295; and 10,464,060, which are incorporated herein by reference. However, such pouches are exemplary only, and it is understood that the multiple PCR reactions discussed herein can be performed in any of a variety of open or closed sample containers known in the art, including 96-well plates, plates of other configurations, arrays, carousels, and the like, using a variety of amplification systems known in the art. While the term "sample well" is used herein, this term is meant to encompass wells, tubes, and various other reaction vessels used in these amplification systems. In one embodiment, the pouches are used to test for multiple pathogens. Illustratively, various steps can be performed in the optionally disposable pouch, including nucleic acid preparation, primary large-volume multiplex PCR, dilution of the primary amplification products, and secondary PCR culminating in optional real-time detection or post-amplification analysis such as melting curve analysis. Further, while various steps can be performed in the pouch of the present invention, it is understood that for a given application one or more of the steps can be omitted and the pouch configuration can be modified accordingly.

[0152] FIG. 1 shows an exemplary pouch 510 for use in the present invention. Pouch 510 is similar to FIG. 15 of U.S. Pat. No. 8,895,295, previously incorporated by reference, and like parts are numbered the same. Mounting portion 590 is provided with receiving channels 515a-515l, which also function as reagent reservoirs. Illustratively, reagents can be lyophilized within mounting portion 590 and rehydrated prior to use. Blisters 522, 544, 546, 548, 564, and 566, with their respective channels 538, 543, 552, 553, 562, and 565, are similar to the corresponding blisters in FIG. 15 of U.S. Pat. No. 8,895,295. Second-stage reaction zone 580 in FIG. 1 is similar to that in U.S. Pat. No. 8,895,295, although the second-stage wells 582 in high-density array 581 are arranged in a slightly different pattern. The more circular pattern of the high density array 581 of Figure 1 eliminates corners and may result in more uniform filling of the second-stage wells 582. As shown, the high density array 581 is provided with 102 second-stage wells 582. The pouch 510 is suitable for use with a FilmArray machine. However, it should be understood that this pouch embodiment is for illustrative purposes only.

[0153] Pouch 510 may be used in a manner similar to that described in U.S. Patent No. 8,895,295, previously incorporated by reference. A 300 μl mixture containing the sample to be tested (100 μl) and lysis buffer (200 μl) is injected into an injection port (not shown) in fitting 590 adjacent to receiving channel 515 a, drawing the sample mixture into receiving channel 515 a. Water is also injected into a second injection port (not shown) in fitting 590 adjacent to receiving channel 515 l and distributed through channels (not shown) in fitting 590, thereby hydrating up to 11 reagents already provided in dry form in receiving channels 515 b through 515 l, respectively. These reagents may include, for example, lyophilized PCR reagents, DNA extraction reagents, wash solutions, immunoassay reagents, or other chemicals. Illustratively, the reagents are for nucleic acid extraction, first-stage multiplex PCR, dilution of the multiplex reaction, and preparation of second-stage PCR reagents, as well as control reactions. In the embodiment shown in FIG. 1, all that needs to be injected is the sample solution through one injection port and water through the other injection port. After injection, the two injection ports can be sealed. For further details on various configurations of pouch 510 and fitting 590, see U.S. Pat. No. 8,895,295, previously incorporated by reference.

[0154] After injection, the sample travels from injection channel 515a through channel 514 to lysis blister 522. Lysis blister 522 is equipped with ceramic beads and is configured for vortexing by impact using a rotating blade or paddle provided within the FilmArray machine. Once the cells are sufficiently lysed, the sample travels through channel 538, blister 544, and channel 543 to blister 546, where it is mixed with nucleic acid-binding magnetic beads. The mixture is incubated for a suitable period of time, illustratively about 10 seconds to 10 minutes. A retractable magnet located within the FilmArray machine adjacent to blister 546 captures the magnetic beads from the solution and forms a pellet against the inner surface of blister 546. The liquid then travels out of blister 546, back through blister 544, and into blister 522, which is now used as a waste container. One or more wash buffers from one or more injection channels 515c-515e are supplied to blister 546 via blister 544 and channel 543. Optionally, the magnet is retracted and the magnetic beads are washed by transferring the beads back and forth between blister 544 and blister 546 via channel 543. Once the magnetic beads have been washed, activation of the magnet recaptures the magnetic beads in blister 546, after which the wash solution is transferred to blister 522. This process can be repeated as necessary to wash lysis buffer and sample debris from the nucleic acid-bound magnetic beads.

[0155] After washing, elution buffer stored in injection channel 515f is moved into blister 548, and the magnet is retracted. Solution is circulated between blister 546 and blister 548 via channel 552, breaking up the pellet of magnetic beads in blister 546 and dissociating the captured nucleic acids from the beads into solution. The magnet is again activated, capturing the magnetic beads in blister 546, and the eluted nucleic acid solution moves into blister 548.

[0156] First-stage PCR master mix from injection channel 515g is mixed with the nucleic acid sample in blister 548. Optionally, the mixture is mixed by forcing it between 548 and 564 via channel 553. After several cycles of mixing, the solution is deposited in blister 564, where a pellet of first-stage PCR primers is provided, at least one set of primers for each target organism, and first-stage multiplex PCR is performed. If an RNA target is present, an RT step can be performed before or simultaneously with the first-stage multiplex PCR. First-stage multiplex PCR temperature cycling in the FilmArray machine is illustratively performed for 15-20 cycles, although other levels of amplification may be desired depending on the requirements of a particular application.

[0157] After the first-stage PCR has proceeded for the desired number of cycles, the sample can be diluted, illustratively by forcing most of the sample back into blister 548, leaving only a small amount in blister 564, and adding second-stage PCR master mix from injection channel 515i. Alternatively, dilution buffer from 515i can be moved into blister 566 and mixed with the amplified sample in blister 564 by moving fluid back and forth between blister 564 and blister 566. If desired, the dilution can be repeated several times by using dilution buffer from injection channels 515j and 515k and then adding second-stage PCR master mix from injection channel 515h to some or all of the diluted amplified sample. It will be appreciated that the dilution level can be adjusted by varying the number of dilution steps or by varying the proportion of sample that is discarded before mixing with dilution buffer or second-stage PCR master mix containing amplification components (illustratively polymerase, dNTPs, and an appropriate buffer, although other components may be suitable, particularly for non-PCR amplification methods). If desired, this mixture of sample and second-stage PCR master mix may be preheated in blister 564 before being transferred to second-stage well 582 for second-stage amplification. Such preheating may eliminate the need for hot start components (antibodies, chemicals, or other) in the second-stage PCR mixture.

[0158] An exemplary second-stage PCR master mix is ​​incomplete, lacking primer pairs, and each of the 102 second-stage wells 582 is pre-loaded with a specific PCR primer pair. If desired, the second-stage PCR master mix may lack other reaction components, which may also be pre-loaded into the second-stage wells 582. Each primer pair may be similar or identical to a first-stage PCR primer pair, or may be nested within a first-stage primer pair. The PCR reaction mixture is completed when the sample is transferred from blister 564 to second-stage wells 582. Once high-density array 581 is filled, the individual second-stage reactions are sealed within their respective second-stage blisters by any number of means known in the art. An exemplary method for filling and sealing high-density array 581 without cross-contamination is described in U.S. Pat. No. 8,895,295, previously incorporated by reference. Illustratively, the various reactions in wells 582 of high density array 581 are simultaneously thermally cycled, illustratively using one or more Peltier elements, although other means of thermal cycling are known in the art.

[0159] An exemplary second-stage PCR master mix includes the dsDNA binding dye LCGreen® Plus, which generates a signal indicative of amplification, but it is understood that this dye is exemplary only and that other signals can be used, such as other dsDNA binding dyes and fluorescently, radioactively, chemiluminescently, enzyme-labeled probes, etc., as known in the art.

[0160] An exemplary FilmArray machine is programmed to make a positive or negative call for each second-stage reaction based on the melting after PCR. A positive call requires that the melting curve produce a melting peak (maximum first derivative or maximum negative first derivative) within a predefined temperature range. This method of calling each second-stage reaction is for illustrative purposes only, and it is understood that calls can be made using real-time amplification data or by other means known in the art.

[0161] Example 1

[0162] Amplicon melting upon completion of nucleic acid amplification (e.g., PCR) is designed to confirm that the amplification product is in fact the intended or desired product. That is, real-time monitoring of nucleic acid amplification can be used to identify the presence of amplification, but does not tell the user what was amplified. The amplification product may be the intended product, or it may be a product of contamination, the result of non-specific amplification, etc. Because amplicons can be designed to have specific, known melting characteristics, post-amplification melting can be used to confirm that the correct product was generated.

[0163] Many nucleic acid amplification reactions include nucleic acid-binding dyes that can be incorporated into double-stranded nucleic acids (e.g., dsDNA). The dyes are incorporated into double-stranded nucleic acids as they are produced in the reaction. Many nucleic acid-binding dyes are compatible with nucleic acid amplification conditions, bind tightly to double-stranded nucleic acids, and are known to fluoresce strongly in the presence of double-stranded nucleic acids. When the nucleic acid is denatured (e.g., by high temperature), the dye is released, and the dye fluoresces weakly or not at all in the presence of single-stranded nucleic acids. Such nucleic acid-binding dyes also typically fluoresce in a concentration-dependent manner in the presence of double-stranded nucleic acids; that is, the more double-stranded nucleic acids there are in the reaction, the greater the fluorescence. This can be used to monitor the success (or failure) of nucleic acid amplification reactions in real time by monitoring whether the fluorescence increases above a threshold. Examples of "thresholds" used in the embodiments and claims described herein to determine when fluorescence has increased and melting can be performed include, but are not limited to, a crossing point (Cp), Cp plus one or more additional amplification cycles, a crossing threshold (Ct), relative fluorescence units rising above a threshold, relative fluorescence units at a selected value above a threshold (e.g., 2 RFU, 5 RFU, 10 RFU, etc.), mathematical modeling, signal processing, and any combination thereof.

[0164] As used herein, "Cp" or "crossing point" refers to the number of PCR cycles or fractional cycles required to obtain a fluorescent signal that exceeds a predetermined threshold. For example, the threshold can be the detection level or point at which the measured fluorescence for the reaction reaches an intensity above the background level of fluorescence. While Cp can be determined experimentally based on a manually set threshold, other methods for determining Cp are known in the art. For example, other points can be used, such as using first, second, or nth derivatives, as taught in U.S. Pat. No. 6,303,305, the entire contents of which are incorporated herein by reference. As known in the art, other points can also be used, and any such point can be used in place of Cp in any of the methods discussed herein. The terms "Ct," "crossing threshold," and "Cq" are generally synonymous with crossing point (Cp), and these terms can be used interchangeably.

[0165] A "relative fluorescence unit" (RFU) is defined herein as a fluorescence intensity value reported with reference to another value. Fluorescence units are a dimensionless term, and therefore, the intensity of a fluorescent signal is typically reported relative to another measurement or reference measurement obtained by an instrument. For example, a baseline fluorescence value may be measured, and the RFU may then be reported as a multiple of the baseline value; i.e., 2 RFU, 5 RFU, 10 RFU, etc. Suitable examples of baseline values ​​may include, but are not limited to, background fluorescence or any value above or below the baseline. For any given experiment or series of experiments using RFU, it is important to use a consistent and reproducible value as the baseline.

[0166] As used herein, "Tm" is the temperature at which one half of a DNA duplex dissociates into single strands. In amplification systems, Tm is typically measured after amplification, although techniques for measuring Tm during amplification are also known.

[0167] Below are two examples of methods for determining whether amplification has occurred and whether it is appropriate to trigger melt detection. These methods and similar methods can be used with any of the methods and systems disclosed and claimed herein.

[0168] In a first example, the set of reaction wells includes wells with all amplification reagents except for the amplification primers (empty wells) and wells with all amplification reagents, including the amplification primers and a nucleic acid template that can be amplified by the reaction (target wells). In a non-limiting example, the method can treat the average fluorescence of the empty wells as a baseline and subtract the baseline fluorescence from the fluorescence of the target wells at a given cycle N (e.g., cycle 5). Other methods for determining baseline fluorescence are known in the art and can be used with the methods described herein. Targets can be run in triplicate (i.e., targets can be amplified in three separate target wells). Melting can be triggered if the value (the difference between the target well at cycle N and the average of the empty wells at cycle N) exceeds a threshold value in at least one of the three target wells, at least two of the three target wells, or all three target wells. The basis of the method of defining background using empty wells is to determine whether the RFU of the target wells changes sufficiently compared to the baseline to determine that amplification has occurred. This method can be used for assays where the target, if present, is expected to amplify and exhibit detectable Cp within the number of cycles designed for the assay (e.g., within 15–30 cycles of PCR).

[0169] A second non-limiting example is designed for assays requiring greater sensitivity. A similar algorithm to the previous example can be used, but instead of comparing a target well to an empty well to detect whether amplification has occurred, at least one target well (e.g., three target wells) can be compared to itself at cycle N and earlier cycles (e.g., N-1, N-2, N-3, etc.). The target wells are still first "compared" to the empty wells to subtract the fluorescence of the empty wells (i.e., background fluorescence). This is still important for normalizing the overall change in fluorescence that will be seen in both the target and empty wells. Melting can be triggered if the increase in fluorescence value from an earlier cycle (e.g., N-1) to cycle N exceeds a threshold value in at least one of the three target wells, at least two of the three target wells, or all three target wells. The basis of this method is to determine whether the RFU is changing rapidly enough to determine that amplification is occurring. This algorithm can be used for assays where low level amplification can occur at the end of the cycle (perhaps without a typical Cp determination), but melting can still be determined in response to amplification.

[0170] During a nucleic acid amplification reaction, double-stranded nucleic acid copies (called products or amplicons) are generated, each with a unique sequence based on the amplified template. The length and sequence / composition (typically AT / GC content) of the amplicon determine the temperature at which the double-stranded DNA melts and dissociates, known as the amplicon's melting temperature (Tm). Products generated from different targets have different sequences and therefore different Tms. Therefore, nucleic acid-binding dyes added to the reaction can also be used for nucleic acid melting analysis. Nucleic acid melting can be used to confirm the presence of the correct product in a nucleic acid amplification reaction, as specific products have specific melting signatures (i.e., the temperature transition range over which fluorescence decays as the duplex transitions from fully double-stranded to fully single-stranded).

[0171] A typical melting experiment is performed by gradually increasing the reaction temperature, starting at or below the annealing temperature and ranging above the denaturation temperature (e.g., from about 60°C to 98°C). See, for example, Figure 11A. When the temperature reaches the Tm of the amplicon, the amplicon denatures, liberating the nucleic acid-binding dye and decreasing fluorescence. This generates the melting curve seen in the left panel of Figure 11B, which demonstrates the rapid decrease in fluorescence upon amplicon melting. Melting peaks with specific Tm, shown in the right panel of Figure 11B, are generated for each amplicon by plotting the negative first derivative of the melting curve.

[0172] Melting analysis can be used to identify specific PCR products. Because the sequence and Tm of the amplicon from a particular target are known and consistent, specific PCR products can be identified as copies of that target. Nonspecific PCR products with different Tms, contaminants, etc., can be excluded.

[0173] In contrast to a typical melting temperature ramp, such as that shown in FIG. 11A, FIG. 2 illustrates exemplary steps in a melting procedure 20 that may be suitably used in embodiments of the present disclosure. The melting procedure 20 illustratively illustrates the final two cycles 22 of nucleic acid amplification followed by a melting temperature ramp 24. The nucleic acid amplification cycles may occur at the end of amplification, followed by a melting temperature ramp, or the melting temperature ramp may occur midway through amplification (e.g., after 10-15 cycles of amplification), followed by additional amplification cycles. Unlike the typical melting temperature ramp shown in FIG. 11A, the melting temperature ramp 24 includes different ramp rates 26, 27, and 28 between a low annealing temperature and a high denaturation temperature (e.g., between about 60°C and about 98°C). The different ramp rates 26, 27, and 28 can be suitably selected so that the instrument rapidly increases the temperature in regions of melting temperature ramp 24 (e.g., approach region 26 and end region 28) where no melting information is expected (i.e., the nucleic acid duplex is either unmelted (approach region 26) or completely melted (end region 28)), and more slowly increases the temperature in regions of interest (e.g., region 27) where the melting transition (i.e., double-stranded to single-stranded transition) of the associated amplicon(s) is expected. For example, the temperature in regions 26 and 28 can increase rapidly (greater than 4°C / sec, e.g., 4-20°C / sec, in ranges 26 and 28), while the temperature in region 27 can increase more slowly (less than 4°C / sec, e.g., 0.01-4°C / sec, or e.g., 1-2°C / sec). The aforementioned melt ramp rates are exemplary and not limiting of the present disclosure. It is also possible to ramp the temperature much faster than 20° / sec (e.g., 100° / sec or faster), with some melts being performed at ramp rates of less than 0.01° / sec and greater than 4° / sec. This new melting procedure can save considerable time during melting analysis by rapidly ramping the temperature in portions of the melting cycle where no melting information is expected, and then slowly ramping the temperature only in specific regions where the relevant amplicons are expected to subsequently melt. This new melting procedure also maintains high resolution for slower melts because it ramps the temperature slowly in a narrow region of interest around the melting transition temperature of the selected amplicon.While the devices described herein can be designed to test for the presence of many potential targets (e.g., up to 30 or more biological and / or molecular markers), a typical test case will likely include zero positives or only one or two positives. The present invention targets melting elevation to the amplicons actually present, rather than slowly elevating throughout a melting zone designed to encompass all possible amplicons.

[0174] The different lifting and lowering speeds shown at 26, 27, and 28 may be dynamically selected as appropriate in accordance with the invention described herein. Referring again to FIG. 1 , second-stage wells 582 in second-stage reaction zone 580 are each spotted with a specific primer pair for amplification of a single target nucleic acid. Amplification of some targets may suitably be performed in duplicate or triplicate, with the same primer pair spotted in more than one second-stage well 582. The spots of second-stage wells 582 are a specific part of the assay design and are carefully mapped. Thus, an instrument equipped with instrument software designed to perform tests in pouch 510 knows the location of each second-stage well 582 and, for a given assay, knows in advance the identity and details of the target amplicon expected to be amplified in each second-stage well 582. When a well in second-stage reaction zone 580 exhibits fluorescence indicating successful amplification, the melting characteristics of the target amplicon in that well are typically known, as is the identity of the assay associated with that well (e.g., an assay for detecting an organism in a sample). While this assay design and primer spotting description is specific to FilmArray, one skilled in the art will understand that the principles described can be applied to any test using nucleic acid amplification, including, but not limited to, tube-based or 96-well plate-based tests. Thus, the rapidly ramping ranges 26 and 28 and the slowly ramping temperature range 27 can be appropriately selected based on the expected melting temperature range of the particular amplicon in the sample well where positive amplification was detected. The start and end points of the slowly ramping range 27 can be dynamically selected from the many amplicon melting ranges within the assay, depending on which assays in the panel (i.e., which reactions in the sample wells) are actually positive for amplification.

[0175] Referring now to Figures 3A and 3B, an example is shown comparing slow melting at a fixed temperature ramp rate with fast melting at a more rapid fixed temperature ramp rate. The difference between Figures 3A and 3B is that the melting is based on amplified fluorescence at 10 RFU (Figure 3A) and 4 RFU (Figure 3B). In other words, the amplicon in Figure 3A is amplified to a higher concentration than the amplicon in Figure 3B; this difference results in a stronger fluorescent signal in Figure 3A compared to Figure 3B. The negative first derivative of the slow melt is shown at 31 in Figure 3A and 35 in Figure 3B. The negative first derivative curves shown at 31 and 35 are similar to the negative first derivative melting curves shown in Figure 11B. In the negative first derivative curves shown at 31 and 35 in Figures 3A, 3B, and 11B, the temperature was ramped at approximately 2°C / sec starting at or below the annealing temperature and continuing through a range above the denaturation temperature (e.g., from about 60°C to 98°C). This is the standard melting procedure for FilmArray. In both cases, the melting peaks shown in the negative derivative curves are clear and unambiguous. In contrast, Figures 3A and 3B show negative derivative curves 33 and 37 generated from a 12°C / sec ramp rate shown at 32 and 36. The only difference between negative derivative curves 31 and 35 and negative derivative curves 33 and 37 is the ramp rate. As can be seen from negative derivative curves 33 and 37, the peak between curves 31 and 33 is flattened and shifted to higher temperatures, and negative derivative curve 37 does not exhibit a peak compared to negative derivative curve 35. These fast melts, compared to standard melts, indicate that fast ramping across the melting range may not be sufficient to preserve the melting signal.

[0176] Referring now to Figures 4A and 4B, an example is shown comparing a fixed-rate slow melt with a variable-rate melt (fast-slow-fast), using a faster rate in a temperature range where no melting signature is expected and a slower rate in a temperature range where a melting signature is expected. The difference between Figures 4A and 4B is the concentration of nucleic acid at the time of melting, and thus the intensity of the fluorescent signal: 10 RFU (Figure 4A) and 4 RFU (Figure 4B). In other words, the amplicon in Figure 4A was amplified to a higher concentration than in Figure 4B before melting analysis; this difference results in a stronger fluorescent signal in Figure 4A compared to Figure 4B. These relative RFUs are similar to those shown in Figures 3A and 3B. Despite the difference in threshold concentration before melting analysis (i.e., 10 RFU and 4 RFU), the derivative melting curves for both datasets are clear and unambiguous. The 2°C / sec melts shown in Figures 3A-4B are essentially identical, but while the melts using the modified dynamic (i.e., fast-slow-fast (6-12°C / sec, 2°C / sec, 12°C / sec)) melt ramps in Figures 4A and 4B are clear, the 12°C / sec melts shown in Figures 3A and 3B are not usable.

[0177] Nucleic acid amplification reactions capable of producing melts such as those shown at 43 and 47 in Figures 4A and 4B can suitably be included in a panel for more than one organism, and thus variable ramp rate melting information (i.e., fast ramp rates in temperature ranges where no melting signature is expected and slow ramp rates in temperature ranges where a melting signature is expected) can suitably be dynamically determined based on biological assays within the nucleic acid amplification panel that show positive amplification.

[0178] Melting curves 40 and 44 and derivative curves 41 and 45 in Figures 4A and 4B are the same as those shown in Figures 3A and 3B (30 and 34 and 31 and 35, respectively). These derivative melting curves were generated from melting data collected at a rate of 2°C / s and have clear, unambiguous peaks. Similarly, derivative melting curves 43 and 47 also have clear, unambiguous peaks. However, melting curves 42 and 46 used to generate these derivative melting curves include high-speed approaches and terminations, such as melting curves 32 and 36 (the temperature was increased at 6–12°C / s for the approach and 12°C / s for the termination), which advantageously saves considerable time during the melting procedure. To maintain melting resolution, melting curves 42 and 46 include a slower ramp (approximately 2°C / s) near the melting transition temperature specific to the amplicon being detected. Melt lifts 42 and 46 include many of the speeds of melt lifts 32 and 36, but unlike melt lifts 32 and 36, melt lifts 42 and 46 maintain the high resolution of slow melting in a fast, slow, fast lift sequence. That is, dynamic melting can have similar melt resolution to industry-standard constant lift rate melting with a consistent lift rate throughout the melting sequence, while potentially saving significant time over such industry-standard melting.

[0179] The total time savings associated with the novel dynamic melting protocols described herein may suitably depend on factors such as, but not limited to, the type of protocol used (e.g., performing a fixed number of cycles before melting versus performing melting for one or more analytes immediately upon detection of amplification above a selected threshold) and the number of positive analytes. If no analytes are detected (i.e., if the assay results are negative for all analytes), a negative run may suitably be reported without performing a melting step. Thus, the time for melt detection and melt data processing may be deducted entirely from the panel run time. For FilmArray panels, a negative run without melting may save approximately 40 seconds of melt time. If one or more analytes are detected (i.e., if amplification is detected in one or more sample wells associated with one or more assays), one or more melts are typically performed. For example, for single- and triple-analyte positive assays, the total time saved in a FilmArray test is typically about 30 seconds or about 10 seconds, respectively. While this may not be considered a large time savings, as reaction times decrease overall and testing moves closer to the patient, the time savings in reducing time to results becomes significant. The time savings associated with the novel dynamic melting procedure disclosed herein are discussed in more detail in Example 6 below.

[0180] In addition to the above, the novel kinetic melting procedure described herein suitably allows positive results to be reported for some assays before thermal cycling is fully completed, providing confirmatory results at the end of the test. That is, kinetic confirmatory melting can be performed and results reported as soon as amplification is detected. Again, this reduces the time patients must wait for results and the time they must spend in a doctor's office. For singleplex assays (e.g., COVID-19 assays), kinetic confirmatory melting can be performed as soon as amplification is detected, allowing assay run to be stopped and results to be reported immediately.

[0181] Additionally, the novel dynamic melting procedures described herein may have other advantages. For example, melting parameters can be modified for a given assay. For example, if there is a positive detection for an amplicon and the melt contains a relevant mutation (e.g., a mutation associated with antimicrobial susceptibility), the temperature ramp rate in the region of interest (i.e., the region where melting is expected) can be slowed (e.g., 0.01-2°C / sec, 0.1-1°C / sec, or slower) to facilitate collection of melting data that may indicate melting shifts, etc., that may indicate the presence or absence of the relevant mutation in the amplicon population. Techniques for such "high-resolution" melting and mutation detection are described in more detail in U.S. Pat. No. 9,657,347 (see, e.g., Example 19), U.S. Pat. No. 9,290,663, and U.S. Patent Application Publication No. 2018 / 0066137, which are incorporated herein by reference in their entireties. A specific example demonstrating the adaptation of a fast-slow-fast dynamic melting procedure for the detection of genetic mutations is provided herein below in Example 10. However, if a positive detection is associated with an amplicon unrelated to one or more relevant mutations or genetic variations, the ramp rate in the region of interest may suitably be more typical, such as 2°C / s to 3°C / s. In another example, the novel kinetic melting procedures described herein may suitably be used to delay melt detection in a quantitative or semi-quantitative assay until one or more internal quantification standards are detected, ensuring that the full quantitative range of the assay is considered. In yet another example, the novel kinetic melting procedures described herein may suitably be used to shorten the run time of an assay with a fixed melt after a selected number of cycles. For example, if a true positive for a certain organism is expected to be detected by melting within the first 15 cycles of PCR, melting may be omitted if the wells for detecting that target organism show positive amplification after the first 15 cycles of PCR.

[0182] Example 2

[0183] This example illustrates a dynamic melting procedure for one target analyte after a fixed number of amplification cycles. In this example, Mycoplasma pneumoniae is the bacterial target, and a unique gene sequence of M. pneumoniae is the amplified amplicon and target analyte. Note that M. pneumoniae assays are run in triplicate (e.g., on a high-density array 581), and two of the three wells must show positive results for the system to call the organism positive. However, some systems may allow some assays to be run in duplicate or singly. Also note that M. pneumoniae is merely an exemplary target, and this procedure can be suitably applied to any amplicon in a nucleic acid amplification test, either a singleplex assay or a multiplex test or panel, as appropriate.

[0184] In situations where an exemplary analyte (e.g., M. pneumoniae) is the only analyte, if the only analyte shows evidence of amplification (e.g., an increase in fluorescence above a threshold) during the amplification stage, the melt at the end of amplification is performed based on the melt information of the only analyte. If the only analyte does not amplify, the melt can suitably be omitted to save time, and the assay can be reported as negative without a confirmatory melt. In situations where the exemplary analyte is one of multiple analytes being tested, the post-amplification melt can suitably be performed based on the analyte showing evidence of amplification during the amplification stage. In the illustrated example, the positive analyte is M. pneumoniae, but it can be any one or more analytes included in the assay. If none of the analytes in the assay show positive amplification, the post-amplification melt can be omitted to save time to results.

[0185] Referring now to Figure 5A, a procedure for determining the melting characteristics of a target analyte is illustrated. In this example, the target analyte is an M. pneumoniae amplicon; however, the procedure illustrated in Figure 5A can be suitably applied to any analyte. Figure 5A illustrates the determination of the melting range of an M. pneumoniae amplicon based on both "in silico" analysis (panel 50) and experimentally collected melting data (panels 51-53). Based on these data, it was determined that the M. pneumoniae amplicon used in this test should melt between 81.5°C and 85.8°C (Table 1). Also based on these data, the dynamic melting range of interest (ROI) was determined to be 81.5°C - 4°C and 85.8°C + 2°C (Table 1). That is, the M. pneumoniae amplicons used in this test are expected to melt between 81.5°C and 85.8°C, although a margin may be appropriately added to the expected melting range to ensure that the start and end of melting are within the slow ramp period and to account for instrument-dependent factors such as, but not limited to, delays between the fast ramp approach and the slow ramp ROI, variations in the instrument's temperature monitoring system, and differences between heater temperature and reaction fluid temperature. As shown in Figure 5A of TIFF2025529216000002.tif31170 and Table 2 below, the approach temperature range is 59°C to 77.5°C, which can be appropriately ramped at 6-12°C / s; the ROI temperature range is 77.5°C to 87.8°C, which can be appropriately ramped at 2°C / s; and the end temperature range is 87.8°C to 98°C, which can be ramped at 12°C / s. For the approach, ROI, and end temperatures, the fluorescence acquisition rate is 10 acquisitions per second (i.e., 10 F / s), and melting is performed after a fixed number of PCR cycles at cycle 27, which can be the midpoint or the end of amplification. While these values ​​are based specifically on M. pneumoniae, one skilled in the art will understand that the procedures and principles can be applied to any amplicon. TIFF2025529216000003.tif43170

[0186] Figure 5B illustrates the dynamic melting curve obtained based on these parameters. The left panel of Figure 5B graphically depicts the temperature ramp 54 based on Table 2. Note the difference between the temperature ramp 54 and the steady-state ramp 30 in Figure 3A, which are based on melting the same M. pneumoniae amplicon. The right panel of Figure 5B shows the derivative melting curve for 55, which is sharp and distinct. Compare the derivative melting curve for 55 with the derivative melting curve for 31 in Figure 3A. As can be seen, both the dynamic and steady-state melting curves produce similar high-quality melting curves, but the dynamic melting does so with significant time savings.

[0187] Example 3

[0188] 6A and 6B illustrate another example of a dynamic melting procedure using two positive amplicons. The dynamic melting procedure illustrated here is suitably applicable to tests involving two analytes, or the two analytes illustrated in this example may be part of a larger test involving more than two analytes. If this were a test involving multiple analytes, this example illustrates a situation where two amplicons show evidence of amplification and both amplicons require melt confirmation.

[0189] In this example, the analyte amplicons are the M. pneumoniae amplicon discussed in detail in Example 2 and the amplicon for the identification of Bordetella pertussis (B. pertussis 2). Note that in this example, the M. pneumoniae and B. pertussis 2 assays were run in triplicate, and typically, two of the three wells for each analyte must show positive results for the system to call that organism positive. However, in some systems, it may be possible to run some assays in duplicate or alone. The in silico and experimental melting data procedures for determining the melting ranges of M. pneumoniae and B. pertussis 2 are illustrated in Figure 6A. The in silico and experimental melting data (panels 60-63) used to determine the melting temperature range of M. pneumoniae are the same as those shown in Figure 5A and Example 2. Based on these data, it was determined that the M. pneumoniae amplicon used in this test should melt between 81.5 °C and 85.8 °C. For B. pertussis 2, panel 64 shows the in silico analysis of the melting range of the B. pertussis 2 amplicon (melting at approximately 90.3 °C), and panels 65-67 show the experimental melting data (melting in the range between approximately 89 °C and 91 °C). Based on these data, it was determined that the B. pertussis 2 amplicon used in this test should melt between 88.3 °C and 92.7 °C (Table 3).

[0190] The melting range of interest (ROI) for M. pneumoniae is 77.5°C to 87.8°C, and the ROI for B. pertussis 2 is 84.3°C to 94.7°C. Because these ranges overlap, we decided to combine the melting of the two amplicons into a single melt in this example. When two or more targets have close or overlapping ROIs and are sufficiently amplified simultaneously, it may be faster to observe both melts in a single combined ROI than to perform separate melting runs for each target. Therefore, the range of interest (ROI) for dynamic melting of these two amplicons in a single melt is 77.5°C to 94.7°C. This means that the overlapping melting ranges of the two amplicons include the lower range of M. pneumoniae (i.e., 81.5°C - 4°C) and the upper range of B. pertussis 2 (i.e., 92.7°C + 2°C). However, it will be appreciated that this is merely an example and that melting data for M. pneumoniae and B. pertussis 2 can be collected in separate melting steps. Example 5 below illustrates such an experiment. TIFF2025529216000004.tif35170

[0191] As shown in Figure 6A and included in Table 4 below, the approach range is 59°C to 77.5°C, which may be suitably ramped at 6-12°C / s, the ROI range is 77.5°C to 94.7°C, which may be ramped at 2°C / s, and the end range is 94.7°C to 98°C, which may be ramped at 12°C / s. In each case of approach, ROI, and end, the fluorescence acquisition rate is 10 acquisitions per second (i.e., 10 F / s), and the melt is fixed at cycle 27, which may suitably be the completion of amplification. TIFF2025529216000005.tif41170 These values ​​are based specifically on M. pneumoniae and B. pertussis2, but one skilled in the art will understand that the procedures and principles can be applied to any amplicon with overlapping or similar melting ranges.

[0192] Figure 6B illustrates the kinetic melting obtained based on these parameters. The left panel of Figure 6B graphically depicts the temperature ramp 68 based on the information provided in Table 4. The right panel of Figure 6B shows the differential melting curves for M. pneumoniae (69) and B. pertussis 2 (70), which are sharp and distinct and similar to the steady-state melting curves presented elsewhere herein (see, e.g., Figure 11B).

[0193] Example 4

[0194] Examples 2 and 3 show kinetic melts taken after a fixed number of amplification cycles. This example demonstrates a procedure that allows for the proper collection of amplicon melt data as soon as the amplicon shows amplification above a selected threshold (e.g., a crossing point (Cp) threshold, above a selected relative fluorescence unit value, etc.). This potentially saves even more time than shown in Examples 2 and 3, as amplification above a given threshold can be detected and results can be properly reported as soon as the melt is performed.

[0195] In this example, M. pneumoniae is the amplicon, the same amplicon used exclusively in Example 2, combined with B. pertussis 2 in Example 3. Note that in this example, the M. pneumoniae assay is run in triplicate, and typically, two out of three wells of an analyte must show positive results for the system to call that organism positive. However, in some systems, some assays may be run in duplicate or singly. While M. pneumoniae or another single amplicon may be the only analyte in the assay (i.e., this example may suitably illustrate a singleplex assay), one of skill in the art will understand that this example may illustrate a test or assay panel that includes many potential analytes (i.e., a multiplex test), for example, where the M. pneumoniae amplicon is the only amplicon that amplifies, or where M. pneumoniae is the amplicon whose amplification is first detected. In either case, the procedures described in this example are equally applicable to singleplex assays or multiplex panels or tests.

[0196] The information presented in Table 1 above is reproduced in Figure 7A. The M. pneumoniae amplicon is expected to melt between 81.5°C and 85.8°C, with a dynamic melting range of interest (ROI) of 77.5°C to 87.8°C (Table 1). The dynamic melting parameters are presented in Table 5 below. The information in Table 5 is nearly identical to the information presented in Table 2 above. The dynamic melting parameters were: approach, 59°C to 77.5°C (6–12°C / s ramp), ROI, 77.5°C to 87.8°C (2°C / s ramp), and end, 87.8°C to 98°C (6–12°C / s ramp) (Table 5). The difference between the melting in Example 2 / Table 2 and this example is that amplicons can be properly melted after detecting amplification above a selected threshold, rather than waiting until after a fixed number of amplification cycles. In this example, amplification detected by fluorescence exceeded a threshold of 10 RFU by amplification cycle 15 (see 71 in Figure 7A), at which point melt detection was performed. While 10 RFU was used as the threshold in this case, it will be understood that other thresholds can also be used (e.g., see Figures 3B and 4B, where 4 RFU fluorescence data was used). For example, a lower or higher RFU value, a crossing point (Cp), Cp plus a selected number of cycles (eg, two additional amplification cycles after Cp is detected), etc. can be used as the threshold.

[0197] The "fast-slow-fast" dynamic temperature ramp profile for melting M. pneumoniae is shown graphically in Figure 7B at 72. The melting derivative curves, shown in Figure 7B at 73, are sharp and clear. As in the previous example, amplifications were performed in triplicate.

[0198] In one embodiment of the present disclosure, immediately after the confirmatory melt illustrated in FIG. 7B , the assay result may be reported as positive for the detected organism (in this case, M. pneumoniae). If the operator is confident that M. pneumoniae is the only positive analyte (e.g., this is a singleplex assay or double positives are statistically unlikely in a panel-based test), the amplification reaction may suitably be stopped after the melt step. If this is possible, this can save considerable time. In another embodiment of the present disclosure, the assay result may be reported as positive for the detected organism immediately after the melt step, and amplification may suitably be continued until completion of amplification (e.g., a fixed number of cycles or until internal quality control criteria indicate positive amplification) to ensure that no additional positive detections occur. If there are additional assay positives in this example, one or more additional melts may be suitably performed if the amplification of the assay positives exceeds a given threshold.

[0199] Example 5

[0200] Following on from the previous example, this example illustrates a dynamic melting procedure for a test involving more than one positive amplification and more than one melt. In this example, the temperature parameters of the dynamic melting can suitably be dynamically set depending on the assay being positive for a given melt, and the cycle at which melting is performed is also dynamically set depending on the amplification cycle at which the assay exceeds the threshold concentration for detection and melting. In one aspect, if an assay has previously been detected as positive and a melt was performed based on those assay conditions, the temperature range of interest for that assay may not be considered in subsequent melting parameters.

[0201] While this example includes two assay positives and two kinetic melts, one of skill in the art will understand that the principles illustrated in this example may be applicable to panels or tests that include more than two positives and more than two melts. Similarly, even if two positives are processed in separate melts due to the fact that the positives cross the threshold at different cycles, in appropriate scenarios, for example, two or more amplicons may be appropriately included in the same melt if the amplicons cross the threshold at the same or nearly the same amplification cycle and the degree of temperature overlap between the two or more amplicons allows time to be saved by performing a single kinetic melt rather than two or more melts.

[0202] Referring now to FIG. 8A, the top panel shows a thermal cycling temperature trace, with a first kinetic melt at 80 and a second kinetic melt at 81 after five additional cycles of PCR. The bottom panel illustrates real-time fluorescence monitoring of the reaction. The real-time fluorescence trace shows that the M. pneumoniae amplicon crossed the threshold (10 RFU in this case) at cycle 19 (indicated at 82), and the B. pertussis 2 amplicon crossed the threshold at cycle 24, as indicated at 83. While 10 RFU was used as the threshold in this example, it will be appreciated that other thresholds can be used (e.g., see FIGS. 3B and 4B, which used 4 RFU fluorescence data). For example, thresholds can be used that are smaller or larger than the RFU value, the crossing point (Cp), Cp plus a selected cycle number (e.g., two additional amplification cycles after Cp is detected), etc. As in the previous examples, these analyses were run in triplicate, and each of the three wells for each amplicon reached threshold at approximately the same time, as would be expected if the reagent concentrations (e.g., primers, polymerase, etc.) and template loading concentrations were approximately the same in each assay well.

[0203] 8B and 8C, a dynamic melting procedure for the M. pneumoniae amplicon is illustrated. This is nearly identical to the M. pneumoniae amplicon melting procedure illustrated in Example 5. In Example 5, the "PCR-informed" melting of the M. pneumoniae amplicon was performed at cycle 15. In this example, the M. pneumoniae melting was performed at cycle 19. Such run-to-run variation is not unexpected and is novelly accommodated in the present disclosure. The M. pneumoniae amplicon is expected to melt between 81.5°C and 85.8°C, with a dynamic melting range of interest (ROI) of 77.5°C to 87.8°C (see, e.g., Table 1). The dynamic melting parameters were: approach, 59°C to 77.5°C (ramp 6-12°C / sec); ROI, 77.5°C to 87.8°C (ramp 2°C / sec); and end, 87.8°C to 98°C (ramp 12°C / sec). The "fast-slow-fast" dynamic temperature ramp profile for melting the M. pneumoniae amplicon is shown graphically in Figure 8C at 84. The melting derivative curves obtained for triplicate reactions are shown in Figure 8C at 85. As with the previous example, the derivative melting curves are sharp and distinct.

[0204] 8D and 8E, a dynamic melting procedure for the B. pertussis 2 amplicon is illustrated. The melting temperatures, ROIs, and dynamic melting procedure for this B. pertussis 2 amplicon are substantially the same as those illustrated with reference to FIGS. 6A and 6B, except that the timing of the melting cycles is "PCR-informed" and is based on the cycle at which the B. pertussis 2 amplicon concentration crossed a threshold at cycle 24. In addition, whereas in Example 3 / FIGS. 6A and 6B, the M. pneumoniae and B. pertussis 2 amplicons were melted in a single melt, in this example they are melted separately.

[0205] The melting range for the B. pertussis 2 amplicon is 88.3°C to 92.7°C, and the ROI for the B. pertussis 2 amplicon is 84.3°C to 94.7°C. The dynamic melting parameters were: approach, 59°C to 84.3°C (ramp 6-12°C / s); ROI, 84.3°C to 94.7°C (ramp 2°C / s); and end, 94.7°C to 98°C (ramp 12°C / s). The "fast-slow-fast" dynamic temperature ramp profile for melting the B. pertussis 2 amplicon is shown graphically in Figure 8E at 86. The melting derivative curves obtained for triplicate reactions are shown in Figure 8E at 87. As with the previous example, the derivative melting curves for triplicate reactions are sharp and distinct.

[0206] Example 6

[0207] As illustrated in the examples presented herein, dynamic melting can be more time-efficient than slow, steady-state melting while providing the same melt quality. Fast, steady-state melting (e.g., collecting melting data while ramping at 12°C / sec) is not feasible. This is demonstrated for high-, medium-, and low-signal melting curves.

[0208] The type of protocol used (fixed cycle or amplification-detection information) and the number of positive analytes determine the time savings. In the case of a negative run (i.e., no assay positivity is detected during amplification), the melting step can suitably be omitted entirely. For example, in a FilmArray test, the melting procedure takes approximately 40 seconds. Therefore, omitting the melting step in the case of a negative run reduces the time to result by approximately 40 seconds. The time savings with fixed-cycle dynamic melting (see, e.g., Examples 2 and 3) is approximately 30 seconds relative to the time required for a typical FilmArray test. The time savings with detection-information-based dynamic melting (see, e.g., Examples 4 and 5) is approximately 10 seconds relative to the time required for a typical FilmArray test. Of course, the time savings with detection-information-based dynamic melting can be greater if the results are reported as soon as an assay positivity is detected and melting is performed. If results are reported during the run and no additional assay positivity is detected during the run, no end-of-test melting is required, and the interim report can be treated as the final report.

[0209] As PCR speeds increase and the time spent melting takes up an increasingly large portion of run time, the dynamic melting procedure described herein and the resulting time savings become more significant. The dynamic melting procedure described herein saves significant time while maintaining high-quality melting resolution by appropriately configuring the instrument to rapidly increase or decrease the temperature in regions of the melting temperature rise where no melting information is expected (e.g., approach and end regions) and more slowly in regions of interest (ROIs) where melting transitions of relevant amplicons are expected.

[0210] Example 7

[0211] The FilmArray Blood Culture Identification (BCID) system is designed to provide rapid identification of a wide range of microbial pathogens directly from blood cultures. An exemplary BCID panel detects the most common bacteria and yeasts isolated from positive aerobic blood cultures (PABC), as well as selected antibiotic resistance genes, with a sensitivity of over 95%. Commercially available BCID panels are available from BioFire Diagnostics. BCID panels and related methods are described in detail in U.S. Pat. No. 10,053,726, which is incorporated herein by reference in its entirety. This example describes the adaptation of the method described in U.S. Pat. No. 10,053,726 to distinguish true positives from environmental contamination using the dynamic melting procedure described herein.

[0212] Within the FilmArray machine, following sample preparation, the first-stage multiplex PCR mixture may be suitably amplified in blister 564 for a selected number of amplification cycles (e.g., 20-30 cycles). After first-stage PCR is completed, the mixture may be suitably diluted and transferred to each of the second-stage wells 582. The second-stage PCR reaction is suitably subjected to amplification conditions (e.g., 63°C for 19 seconds to 94°C for 0 seconds) for an additional number of cycles. In this illustrative example, melting may suitably be performed after a fixed number of cycles (e.g., after cycles 20, 26, and 32), or melting may be "PCR informative," suitably performed when a well shows evidence of amplification above a threshold. In either case, melting may suitably be performed using the kinetic melting procedure described herein, with temperature ramping suitably defined by the identity of the biological amplicon in the wells showing positive amplification. An amplification assay may suitably be determined to be positive if the melting curve for a given well shows a melting peak (the negative first derivative of the melting curve) within the predefined temperature range of each second-stage assay.

[0213] In a BCID panel, each organism tested in the panel can be "classified" according to its expected concentration in the sample and contamination risk to minimize both false positives and false negatives. Organisms are assigned in Table 6 as follows: In general, bin 1 may suitably contain targets present at the highest titer in the sample, present as background organisms, and present with the highest risk of unexpected positives. bin 2 may suitably contain targets present at high titer in the sample, present with low background organisms, and present with a moderate risk of unexpected positives. bin 3 may suitably contain targets present at low titer in the positive aerobic blood culture, present with low or no background organisms, and present with a low risk of unexpected positives. While the organisms binned in this example are specific to the BCID panel and positive aerobic blood cultures, one skilled in the art will understand that the principles exemplified herein can be applied to any sample with a wide range of expected organism concentrations and / or risk of false positives and false negatives due to environmental and commensal organisms in the sample. Other examples where this approach may be useful include, but are not limited to, stool samples for the diagnosis of gastrointestinal infections and cerebrospinal fluid for the diagnosis of central nervous system infections.

[0214] Generally, organisms in bin 1, if present in a sample, may be expected to amplify early (e.g., within the first 10-22 cycles of amplification). Thus, if present, bin 1 organisms may be expected to show evidence of amplification above a threshold, suitably within, for example, the first 10-22 cycles of amplification. If one or more organisms in bin 1 amplify at this early stage, and the melt curve for a given bin 1 well shows a melt peak within a predefined temperature range for the organism amplicon, the assay may be positive. However, because bin 1 organisms may be at the highest risk of false positives (due to environmental contamination or other factors), later evidence of amplification (e.g., after 20-22 cycles of amplification) may appropriately be considered a false positive and may not appropriately trigger melt detection. That is, if a bin 1 biological well amplifies early (e.g., before amplification cycles 20-22), it may appropriately trigger dynamic melting (upon detection of amplification above a threshold (e.g., 10 RFU or greater) or after a fixed number of amplification cycles (e.g., 15-20 amplification cycles)), and such amplification may be determined as positive if the melting curve shows a melt peak within a predefined temperature range for the biological amplicon in that well. In contrast, if a bin 1 biological amplicon amplification well amplifies later (e.g., at cycle 25), such amplification may not appropriately trigger melting because it may be a false positive, e.g., due to contamination (e.g., environmental contamination). It will be understood that the cycle numbers presented in this example are merely exemplary, and that the novel dynamic melting procedure described herein allows for a nuanced approach to true-positive and false-positive cycle cutoffs. Factors such as, but not limited to, the biological titer in the sample and the amount of sample loaded may vary, and therefore, the cycle cutoffs may also vary accordingly. For example, if everything seems to be amplifying early, the cutoff between true and false positives can be moved earlier, and similarly, if everything seems to be amplifying late, the cutoff between true and false positives can be moved later.

[0215] Generally, organisms in bin 2, if present in the sample, may be expected to amplify at the midpoint of amplification (e.g., after about 18-22 cycles, before about 25-27 cycles). However, it will be understood that some true positives for bin 2 organisms may amplify earlier (i.e., before about 18-22 cycles of amplification). If one or more organisms in bin 2 amplify at this midpoint stage and the melt curve for a given bin 2 well shows a melt peak in a predefined temperature range for the organism amplicon, the assay may be positive. However, because bin 2 organisms still pose some risk of false positives due to contamination, later evidence of amplification (e.g., after about 27 cycles of amplification) may appropriately be considered a false positive and may not appropriately trigger melt detection. While some true positives for bin 2 organisms may appear later in amplification, looking for true positives at the midpoint of amplification suitably captures most true positives while reducing the risk of false positives due to environmental contamination. The above caveat applies here as the cycle numbers are merely exemplary and that dynamic melting procedures may suitably provide a more nuanced approach than strict cutoffs.

[0216] In general, organisms in bin 3, if present in the sample, can be expected to amplify during the final stages of amplification (e.g., after approximately 25-27 cycles). However, it will be understood that some true positives for bin 3 organisms may amplify earlier (i.e., before approximately 25-27 cycles of amplification). If one or more organisms in bin 3 amplify during this final stage of amplification and the melt curve for a given bin 2 assay well exhibits a melt peak within a predefined temperature range for the organism amplicon, the assay may be positive. Because bin 3 organisms have a low risk of false positives due to contamination, there is little risk of a positive reading even after 32-35 cycles of amplification. The cycle numbers are merely exemplary, and the above consideration that a dynamic melting procedure can appropriately provide a more nuanced approach than a strict cutoff still applies here.

[0217] As an illustrative example, consider the bin 1 organism, E. coli (see Table 6). True sepsis-positive E. coli in PABC typically show high titers (e.g., >108 cfu / ml) and would be expected to be detectable by amplification with the exemplary BCID panel before amplification cycle 20, often before amplification cycle 10. However, E. coli is nearly ubiquitous in the environment and can be introduced, for example, into blood samples via environmental sources and / or media raw materials and ultimately into blood culture bottles. For the exemplary BCID panel, E. coli amplification after approximately amplification cycles 18-22 (e.g., before cycle 20) has been found to be likely due to some form of contamination. Assays for E. coli that amplify before approximately amplification cycle 20 can be treated as true positives and may appropriately trigger melt detection. In contrast, E. coli positives after approximately cycle 20 can be treated as false positives and may not appropriately trigger melt detection.

[0218] Another illustrative example considers antimicrobial resistance (AMR) genes that can be detected with the exemplary BCID panel. The exemplary BCID and BCID2 panels can detect many different antimicrobial resistance (AMR) markers and genes. In one example, the exemplary BCID panel can detect the mecA / C gene, which is associated with methicillin resistance in Staphylococcus aureus. In this example, S. aureus is a Bin2 organism, and true positives from PABC can be expected to amplify at the midpoint of amplification (e.g., approximately cycles 20-25). If S. aureus is detected without co-detection of the mecA / C genes, a confirmatory thaw can be appropriately performed in the region of interest (ROI) at a ramp rate of approximately 2°C / sec, as shown in the examples presented here. However, if amplification is detected in a sample well designated for S. aureus and another well designated for the mecA / C genes (i.e., co-detection of S. aureus and mecA / C), a confirmatory thaw can be appropriately performed for both S. aureus and mecA / C. Confirmatory melting of S. aureus and mecA / C may be performed together or separately, depending on the respective melting temperature ranges and melting ramp rates required for optimal detection and whether amplification is detected in the same or nearly the same cycle for the two species, or whether they are separated in time. If S. aureus is detected, confirmatory melting is performed, and the mecA / C gene is detected in a subsequent cycle, confirmatory melting of mecA / C may be performed. However, if S. aureus amplification is not detected but the mecA / C gene is detected by amplification, melting of the mecA / C gene may be omitted because that locus is from a different organism and should not contribute to methicillin-resistant S. aureus. In other words, melting of AMR genes or markers may not be performed until amplification of the relevant pathogen is detected. This potentially saves time that might otherwise be spent on melting analysis and can make AMR detection more robust and confirmatory.

[0219] Additionally, it is understood that information obtained about one organism can be used to determine whether another organism is positive or negative, especially if there is some cross-reactivity between the organisms or some other relationship, such as between the bacteria and their associated antibiotic resistance genes. In Table 6 above, it can be seen that Enterococcus ("Entero") and Staphylococcus ("Staph") are both Bin 2 organisms, and if present, their respective amplifications should be detected between approximately 18 and 27 cycles of amplification. However, many known assays for Entero exhibit cross-reactivity with Staph due to target sequence similarity, potentially resulting in delayed Staph-positive Cp in a true-negative Entero sample. To reduce the impact of potential cross-reactivity in the Entero assay in situations where cross-reactivity is an issue, Staph can be determined positive or negative by amplification and melting if Staph amplification is detected between approximately 18 and 27 cycles of amplification. If Staph is positive for an Entero sample, for example, if Entero amplification is detected before amplification cycles 20 and 22, Entero can be determined based on the earlier results. If Staph is negative, the Entero assay is unaffected and a call can be made if Entero amplification, if any, is detected between approximately 18 and 27 cycles, or within the cycle range selected as optimized for that assay without cross-reactivity. However, in blood cultures, positive growth is based on the combined growth of all organisms present, and it is important to note that one or more organisms may be present in lower amounts than either from a single infection. The binning and cycle range at which the cross-reactivity assay is called may need to be adjusted accordingly. By adjusting the binning and cycle range used to call the cross-reactivity assay based on positive or negative calls from other assays, cross-reactivity issues from dually infected samples can be accurately called for, typically eliminating the need to redesign primers to avoid cross-amplification.

[0220] While the organisms described in this example were grouped into three bins, and this example used early, middle, and late melt cycle ranges for each bin, it is understood that any number of bins and melt cycles can be used, and any cycles can be selected as melt cycles. In some assays, separation of false positives from false negatives may be achieved with only two bins and / or two melt cycles, while other assays may require four or more bins and / or melt cycles. Furthermore, while the examples used samples from culture, it is understood that multiple bins and / or melt cycles may be appropriate for assays using non-cultured material.

[0221] Although the above examples identify organisms, it is understood that the same methods and devices can be used to identify different target sequences within one or several organisms by amplifying different genetic loci of the organism.

[0222] Example 8

[0223] Certain embodiments of the present invention may also involve or include a PCR system configured to make a positive or negative determination from an amplification curve or melting curve, or a combination thereof. An exemplary example is described in U.S. Pat. No. 8,895,295, previously incorporated by reference, and used in pouch 510 or similar embodiments. However, it is understood that the embodiment described in U.S. Pat. No. 8,895,295 is merely exemplary, and other systems may be used in accordance with this disclosure. For example, referring to FIG. 15, a block diagram of an exemplary system 700 including a control element 702, a thermocycling element 708, and an optical element 710 according to an exemplary embodiment of the present disclosure is shown.

[0224] In at least one embodiment, the system can include at least one PCR reaction mixture contained in a sample vessel 714. In certain embodiments, the sample vessel 714 can include a PCR reaction mixture configured to allow for and / or perform amplification of a template nucleic acid. Certain exemplary embodiments can also include at least one sample block or chamber 716 configured to contain at least one sample vessel 714. The sample vessel 714 can include any plurality of sample vessels in individual, strip, plate, or other formats, and can illustratively be provided as or contained by the sample block or chamber 716.

[0225] One or more embodiments may also include at least one sample temperature controller 718 and / or 720 configured to manipulate and / or adjust the temperature of the sample. Such a sample temperature controller may be configured to increase, decrease, and / or maintain the temperature of the sample. In one example, the sample controller 718 is a heating system and the sample controller 720 is a cooling system. Exemplary sample temperature controllers include (but are not limited to) heating and / or cooling blocks, elements, exchangers, coils, radiators, refrigerators, filaments, Peltier elements, forced air blowers, handlers, vents, distributors, compressors, condensers, water baths, ice baths, flames and / or other combustible or flammable forms of heat, hot packs, cold packs, dry ice, dry ice baths, liquid nitrogen, microwave and / or other wave emitting devices, cooling means, heating means, means for otherwise manipulating the temperature of the sample, and / or any other suitable device configured to increase, decrease, and / or maintain the temperature of the sample.

[0226] The exemplary PCR system 700 also includes an optical system 710 configured to detect the amount of fluorescence emitted from (or portions or reagents of) a sample 714. Such an optical system 710 may include one or more fluorescence channels, as known in the art, and may detect fluorescence from multiple samples simultaneously or individually.

[0227] At least one embodiment of the PCR system may further include a CPU 706 that is programmed or configured to operate, control, execute, or otherwise advance a heating system 718 and a cooling system 720 to thermally cycle the PCR reaction mixture, illustratively while an optical system 710 collects the fluorescent signal. The CPU 706 may then generate an amplification curve, a melt curve, or any combination, which may or may not be printed, displayed on a screen, or otherwise output. Optionally, a positive, negative, or other determination may be output based on the amplification curve and / or the melt curve. Optionally, only a determination is output, illustratively one determination for each target tested.

[0228] Further examples of exemplary features, components, elements, and / or parts of exemplary PCR systems and / or thermal cyclers (thermocyclers) are known in the art and / or described above or in U.S. Patent Application Publication Nos. 2014 / 0273181 and 2020 / 0319441 and U.S. Patent No. 10,698,190, which are incorporated herein by reference in their entireties.

[0229] Example 9

[0230] Figure 10 shows a flow diagram of an exemplary dynamic melting detection method 1000 for determining a sample for a target nucleic acid sequence. Method 1000 can be performed by various components of a PCR system, including one or more temperature control devices, such as a processor or controller, optical elements, and thermocycling elements, including heating and cooling systems, as described below with reference to Figure 9. In some embodiments, method 1000, or portions thereof, can be embodied in a series of instructions, stored in a computer-readable memory, and executable by one or more processors or controllers.

[0231] A portion of the sample is placed in a sample well along with primers for amplifying a target nucleic acid sequence and a fluorescent dye, such as a dsDNA-binding dye. Portions of the sample can be placed in multiple sample wells, each containing primers for amplifying a different target nucleic acid sequence from a different organism. Each target nucleic acid sequence has a different, characteristic melting temperature range. For example, the characteristic melting temperature range for the M. pneumoniae amplicon is 77.5°C to 87.8°C, while the characteristic melting temperature range for the B. pertussis 2 amplicon is 84.3°C to 94.7°C.

[0232] In block 1002, the sample in each sample well is amplified by thermal cycling, including at least a two-stage PCR protocol. The PCR protocol may include an intra-cycle temperature adjustment segment or denaturation segment, in which the sample well is heated from an annealing temperature to a denaturation temperature and cooled from the denaturation temperature to the annealing temperature for each of several cycles. The PCR protocol may also include an extension segment, in which the temperature is held constant at an optimal temperature for DNA polymerase activity for each of several cycles. The PCR protocol may also omit a specific extension temperature hold. As described in more detail elsewhere herein, the DNA polymerase may be active and complete primer extension during the temperature increase from the annealing temperature to the denaturation temperature. In some embodiments, the processor or controller provides control signals to the thermocycling elements to heat the sample well to the denaturation temperature using an initial ramp rate, cool the sample well to the annealing temperature, and hold the temperature of the sample well constant. The initial ramp rate may be a fixed ramp rate (e.g., 12°C / sec for each cycle).

[0233] Next, in block 1004, fluorescence data (indicating the amount of fluorescence emitted by the sample) is collected from the sample in each sample well during the intra-cycle temperature adjustment segment for each of the N cycles (where N is 1, 2, 3, 4, 5, 6, or more cycles). The fluorescence data may be collected by an optical system, such as optical system 710 described above with reference to FIG. 9, and provided to a processor or controller. For example, the optical system may provide light (e.g., from an LED) to the sample in each sample well and may include a photodetector that detects the amount of fluorescent signal produced by the sample in each sample well. In some embodiments, the processor or controller provides a control signal to optical system 710 to detect the amount of light scattered by the sample in each sample well during the intra-cycle temperature adjustment segment for each of the N cycles. The processor or controller may then collect the amount of fluorescence along with the temperature of the sample (temperature, fluorescence pair) at several points during each of the N cycles. It is understood that fluorescence is merely exemplary, and other methods of measuring and detecting amplification are within the scope of this disclosure.

[0234] In block 1006, the processor or controller may determine whether the amount of fluorescence in any of the sample wells exceeds a threshold. The threshold may be an amount at or above the detection limit for concluding that the concentration of nucleic acid in the sample well is elevated. The threshold may also be a crossing point (Cp), a selected number of relative fluorescence units (RFU), Cp plus a predetermined number of additional amplification cycles, or any suitable threshold.

[0235] If the amount of fluorescence in one of the sample wells exceeds a threshold, the processor or controller can identify the target nucleic acid sequence of the sample well and the characteristic melting temperature range of the target nucleic acid. For example, if the sample well contains an M. pneumoniae amplicon, the characteristic melting temperature range can be 77.5°C to 87.8°C. The processor or controller can be programmed with information about each sample well in the assay, including, but not limited to, the identity of the target in each well and the characteristic melting temperature range of each target.

[0236] The processor or controller then determines an adjusted ramp rate profile for heating the sample wells based on the characteristic melting temperature range of the target nucleic acid being amplified in the sample wells (block 1008). The adjusted ramp rate profile can include different ramp rates for heating the sample wells from the annealing temperature to the denaturation temperature. At least one of the ramp rates in the adjusted ramp rate profile can be faster than the initial ramp rate to increase the melting rate at temperatures where no melting signature is expected from the reaction. For example, the adjusted ramp rate profile can include a first ramp rate for an approach temperature range, a second ramp rate for a characteristic melting temperature range, and a third ramp rate for an end temperature range. The approach temperature range can be from the annealing temperature at the beginning of the characteristic melting temperature range to the initial melting temperature (e.g., 59°C to 77.5°C). The characteristic melting temperature range can be from the initial melting temperature to the final melting temperature (e.g., 77.5°C to 87.8°C). The characteristic melting temperature range may also be referred to as the melting ROI. The end temperature range may be from the final melting temperature to the denaturation temperature (e.g., 87.8°C to 98°C).

[0237] The first and third rates can be faster than the second rate to increase the melting rate at temperatures where no melting signature is expected from the reaction. For example, the first rate can be between 6°C / sec and 12°C / sec, and the third rate can be 12°C / sec. The second rate can be similar to the initial rate (e.g., 2°C / sec) to slow the melting at temperatures where a melting signature is expected from the reaction.

[0238] Then, in block 1010, melt detection is performed using the adjusted ramp rate profile. For example, during subsequent thermal cycles, the processor or controller provides control signals to the thermocycling elements to heat the sample well to a denaturation temperature using the adjusted ramp rate profile, cool the sample well to an annealing temperature, and maintain a constant temperature in the sample well. A melt curve is then generated based on the fluorescence data detected during melt detection. The melt curve is then analyzed to identify a target nucleic acid among multiple target nucleic acids corresponding to the sample well. For example, a melt peak may indicate an organism in the sample well. In a specific example, if the sample well contains an M. pneumoniae amplicon and the melt peak is indicative of M. pneumoniae, the processor or controller can identify that the sample contains M. pneumoniae.

[0239] In some embodiments, the processor or controller can determine that the amount of fluorescence in multiple sample wells exceeds a threshold, each sample well containing primers for amplifying a different target nucleic acid sequence from a different organism. For example, the processor or controller can determine that the amount of fluorescence in a first sample well containing primers for amplifying a first target nucleic acid having a first characteristic melting temperature range exceeds a threshold, and that the amount of fluorescence in a second sample well containing primers for amplifying a second target nucleic acid having a second characteristic melting temperature range also exceeds a threshold.

[0240] The processor or controller can then generate a first adjusted elevation rate profile for the first sample well corresponding to the first unique melting temperature range and a second adjusted elevation rate profile for the second sample well corresponding to the second unique melting temperature range. In some embodiments, the processor or controller can perform a first melt detection using the first adjusted elevation rate profile in the first melt cycle. The processor or controller can then perform a second melt detection using the second adjusted elevation rate profile in a second melt cycle, which may occur after the first melt cycle. For example, the second melt cycle may occur five cycles after the first melt cycle.

[0241] In other embodiments, the processor or controller may combine the first adjusted elevation velocity profile and the second adjusted elevation velocity profile into a composite elevation velocity profile and perform melt detection using the composite elevation velocity profile. For example, if the first characteristic melting temperature range is 77.5°C to 87.8°C and the second characteristic melting temperature range is 84.3°C to 94.7°C, the processor or controller may generate a composite characteristic melting temperature range of 77.5°C to 94.7°C. The processor or controller may then generate a composite elevation velocity profile using a melting ROI of 77.5°C to 94.7°C.

[0242] Example 10

[0243] This example describes a dynamic melting procedure for detecting the presence of genetic sequence variants. Double-stranded nucleic acid copies (called products or amplicons) generated during a nucleic acid amplification reaction have unique sequences based on the amplified template. The length and sequence / composition (typically, AT / GC content) of the amplicon determine the temperature at which the double-stranded DNA melts and dissociates, known as the amplicon's melting temperature (Tm). Products generated from different targets have different sequences and, therefore, different Tms, and often have different melting curve shapes. Thus, nucleic acid-binding dyes added to the amplification reaction can be used for nucleic acid melting analysis due to their property of fluorescing strongly in the presence of double-stranded nucleic acid and fluorescing weakly or not at all in the presence of single-stranded nucleic acid. Nucleic acid melting can be used to confirm the identity of nucleic acid amplification products because specific products have unique melting signatures (i.e., the temperature transition range over which fluorescence decays as the duplex transitions from fully double-stranded to fully single-stranded). Because sequence variants typically melt at different temperatures and / or have different melting curve shapes than the wild-type sequence, Tm shifts and melting curve changes can be used to detect the presence of sequence mutations in the amplified nucleic acid.

[0244] In this context, the term "detecting" can include the detection and determination of known and unknown nucleic acid sequence variations, including, but not limited to, SNPs, base deletions, base insertions, sequence duplications, rearrangements, inversions, base methylations, and the number of short tandem repeats; and, in the case of diploid genomes, whether the genome is homozygous or heterozygous for a sequence variation, as well as the cis / trans location of two or more sequence variations on a DNA strand (haplotyping). Such nucleic acid sequence variations can be associated with many genotypic and phenotypic differences in organisms. Single nucleotide polymorphisms (SNPs) are the most common genetic variations observed in humans and other species. These polymorphisms involve differences in only a single base between individuals. This variation can cause an amino acid change in a protein, alter the rate of transcription, affect mRNA splicing, or have no apparent effect on cellular processes. Often, even when a variation is silent (e.g., does not change the amino acid it encodes), SNP genotyping can still be useful if the variation is linked (associated) with a unique phenotype caused by another genetic variation. In certain examples, nucleic acid sequence variations (e.g., SNPs) may be associated with antimicrobial resistance (AMR) markers. Such nucleic acid sequence variations may be associated with detectable Tm shifts that can be used for sequence variants. "High-resolution" melting and techniques for detecting variations are described in detail in U.S. Patent No. 9,657,347 (see, e.g., Example 19), U.S. Patent No. 9,290,663, and U.S. Patent Application Publication No. 2018 / 0066137, the entire contents of which are incorporated herein by reference.

[0245] In one aspect of the present invention, a method is provided that requires only standard PCR reagents, primers, and the simple addition of a "saturating" double-stranded (ds) DNA-binding dye prior to PCR. For purposes of this invention, a "saturating" dye is a dye that does not significantly inhibit PCR when present at a concentration that provides a maximum fluorescent signal relative to the amount of dsDNA typically generated by PCR in the absence of the dye, e.g., approximately 10 ng / μL. While the dyes are identified by their compatibility with PCR near saturating concentrations, it is understood that these dyes can be used at much lower concentrations. These dyes can be used during or after amplification to identify the presence of known and unknown sequence mutations by melting curve analysis, similar to the use of labeled primers. Identifying known and unknown sequence mutations can be used in a variety of analyses, including mutation scanning and SNP genotyping. The term "scanning" refers to the process of comparing a nucleic acid fragment with a reference nucleic acid fragment to detect the presence of any sequence differences. A positive result indicating the presence of a sequence difference may not necessarily reflect the exact nature of the sequence mutation or its location on the nucleic acid fragment. The term "genotyping" includes the detection and determination of nucleic acid sequence mutations.

[0246] Furthermore, although reference is made to PCR, other amplification methods may also be compatible with the dyes of the present invention. Such suitable procedures include strand displacement amplification (SDA); nucleic acid sequence-based amplification (NASBA); cascade rolling circle amplification (CRCA), Q-beta replicase-mediated amplification; isothermal chimeric primer nucleic acid amplification (ICAN); transcription-mediated amplification (TMA), etc. Thus, when the term PCR is used, it should be understood to include other alternative amplification methods.

[0247] Referring now to FIG. 12, a schematic melting procedure is shown comparing a standard melting protocol (····), a dynamic melting protocol such as the dynamic melting protocol described in detail elsewhere herein (--), and a dynamic "high-resolution" protocol (·-·-). Each procedure involves a 3-second hold at 62°C. For the standard protocol, the temperature is then increased to 68°C at a maximum rate of 12°C / s. Melting is then carried out at a standard (fixed) rate of 2°C / s to 99°C. For the dynamic protocol, after a 3-second hold, the temperature is increased to 76°C at a rate of 12°C / s. The rate is then reduced to 2°C / s to a temperature of 95°C, after which it is increased again at 12°C / s to 99°C. For the dynamic HRM protocol, after a 3-second hold, the temperature is increased to 76°C at a rate of 12°C / s. The heating rate is ramped down from 76°C to 92°C at 1°C / sec, then ramped up to 99°C at 12°C / sec. In this exemplary embodiment, the melting rate is 1°C / sec. While this is exemplary, other "high-resolution" melting rates can be used, including, but not limited to, 0.01-2°C / sec, 0.1-1°C / sec, preferably 0.5°C / sec or less, more preferably 1°C / sec or less, and most preferably 2°C / sec or less. The selection of the heating rate for detecting genetic variants depends on factors such as the required melting resolution and the time requirements of the assay. In general, the inventors in this example have found that heating rates of 0.5-1°C / sec are sufficient in most cases.

[0248] In this illustrative example, the standard melting protocol takes 20 seconds, the dynamic protocol can be completed in 15 seconds, and the dynamic HRM protocol can be completed in 21 seconds. Implementing a slower "high-resolution" lift rate in the standard protocol adds approximately 30 seconds to the run time. In this example, implementing the high-resolution dynamic melting procedure takes approximately 6 seconds longer than the regular dynamic melting procedure, but the high-resolution dynamic melt can be performed at a cost of only 1 second compared to the standard protocol. While these are hypothetical procedures, this outline illustrates the potential time savings of the dynamic high-resolution protocol versus the standard high-resolution protocol. As with the actual dynamic melting procedures detailed elsewhere herein, the actual protocol and actual time savings may vary depending on the melting characteristics of the amplicons being melted. The total run-time cost for performing the high-resolution melting procedure varies depending on the lift rate and the number of individual melts performed.

[0249] In some embodiments of the invention, high-resolution melting procedures may be routinely used to detect a given nucleic acid. However, because high-resolution melting procedures can be more time-consuming, in other embodiments of the invention, it may be desirable to use a first assay as a trigger to determine whether melting should be performed in an assay to detect genetic variants. An exemplary example of such a trigger assay is shown in Figure 13. Figure 13 shows a N. gonorrhoeae bioassay (● Org) and another AMR assay targeting a mutation-prone region of the gyrA gene (○ S91F and +WT). In the left panel, the S91F assay (open dot) is closest to the organism (solid dot), while the WT assay is much later. This indicates that the isolate tested is resistant. In contrast, in the right panel, the assay closest to the organism is the WT assay, indicating that the sample contains wild-type N. gonorrhoeae. While Figure 13 shows differences in Cp between wild-type and mutant, in this case, such differences in Cp are not a reliable way to identify wild-type and mutant isolates. In one aspect, a gyrA assay to identify mutant or wild-type gonococci (i.e., antibiotic-resistant or antibiotic-susceptible gonococci) may be performed only if the Org assay is positive. This general approach can also be applied to other assays for detecting genetic mutations. In this way, the additional time required for detecting genetic mutations (e.g., AMR assays) may only be required if the organism is positive. In addition, false positives may occur in assays for genetic mutations due to potential cross-reactivity between different related species within an organism family (e.g., Neisseria species) (the target region may be highly conserved among related organisms, and therefore the assay may not be selective). If a biological assay is positive, which can be highly selective, it may be used to trigger melting of genetic mutations. Thus, the methods of the invention described herein may include detecting the presence of an organism by a first melting detection in a first assay, and, if the organism is detected to be present in the first assay, performing a second melting detection in a second assay to detect the presence or absence of a genotype, sequence variant, or genetic mutation.

[0250] Referring now to Figures 14-16, an example is shown using a modified dynamic high-resolution melting procedure to detect genetic variants in nucleic acids amplified in experimental FilmArray test pouches. Figures 14-16 show melting curves for wild-type and mutant nucleic acids amplified from M. genitalium (23S) and Neisseria gonorrhoeae (gyrA). The 23S mutation shown in Figure 14 (A2059G) and Figure 15 (A2058G) confers macrolide resistance to M. genitalium, and the S91F mutation in gyrA shown in Figure 16 confers ciprofloxacin resistance to Neisseria gonorrhoeae. These and other antibiotic resistance mutations are becoming more common in many pathogenic bacteria due to antibiotic overuse. Accurate and routine identification of the presence of antibiotic resistance markers is essential for antibiotic stewardship and appropriate patient care. The methods described herein can be used to accurately and routinely detect and identify many types of genetic variants.

[0251] Figures 14A and 14B compare melting curves for wild-type (-MG-WT) and A2059G (- - - 2059G) mutant amplicons of the M. genitalium 23S ribosomal RNA gene. Figure 14A shows the composite WT and mutant curves, while Figure 14B shows individual curves from individual amplification reactions and melts. In both cases, the WT and mutant melting curves are clearly distinguishable. WT and mutant amplicons were melted at an initial ramp rate of 2°C / s from 62°C to 69°C. The ramp rate was then reduced to 1°C / s for the melting temperature range from 69°C to 94°C (referred to elsewhere in this specification as the ROI). The ramp rate was then increased to 2°C / s to a final temperature of 94°C to 99°C. These ramp rates are merely exemplary. For example, those skilled in the art will understand that the initial and final heating and cooling rates can be higher than 2°C / sec (e.g., 4-12°C / sec), and that other melting heating and cooling rates can be used in the ROI, including, but not limited to, 0.01-2°C / sec, 0.1-1°C / sec, preferably 0.5°C / sec or less, more preferably 1°C / sec or less, and most preferably 2°C / sec or less. Generally, the slower the heating and cooling rate, the higher the melt resolution but the longer the analysis time. Here, each sample was unambiguously genotyped at a melting heating and cooling rate of 1°C / sec.

[0252] Figures 15A and 15B compare melting curves for wild-type (-MG-WT) and A2058G (-··-2058G) mutant amplicons of the M. genitalium 23S ribosomal RNA gene. Figures 15A and 15B are similar to Figures 14A and 14B. Figure 15A shows the composite WT and mutant curves, while Figure 15B shows individual curves from individual amplification reactions and melts. In both cases, the melting curves for the WT and mutants are clearly distinguishable. The melting parameters in Figures 15A and 15B are the same as those in Figures 14A and 14B. As with Figures 14A and 14B, one skilled in the art would understand that the ramp parameters can be modified to speed up or slow down the ramp and / or increase or decrease the melt resolution. Here, each sample was unambiguously genotyped at a melt ramp rate of 1°C / sec.

[0253] Figures 16A and 16B compare melting curves for wild-type (-NG-WT) and S91F (- - - - S91F) mutant amplicons of the gonococcal gyrA gene. Figure 16A shows the composite curve for WT and mutant, while Figure 16B shows the individual curves from individual amplification reactions and melts. In both cases, the melting curves for WT and mutant are clearly distinguishable. WT and mutant amplicons were melted at an initial ramp rate of 2°C / s from 69°C to 83.8°C, slowed to 1°C / s for the melting temperature range from 83.8°C to 91.8°C (referred to elsewhere herein as the ROI), and then ramped to 2°C / s from 91.8°C to a final temperature of 99°C. As with Figures 14A-15B, one skilled in the art will understand that ramp parameters can be modified to speed up or slow down the ramp and / or increase or decrease the resolution of the melt. Here, each sample was unambiguously genotyped at a melting rate of 1°C / s.

[0254] The examples shown in Figures 14-16 illustrate situations in which dynamic melting protocols can be used to distinguish between wild-type and mutant melts. The ability to do this in a FilmArray test pouch without significantly increasing assay time represents a significant advance in the art. Also, while Figures 14-16 illustrate assays performed in a FilmArray test pouch, those skilled in the art will understand that these protocols can be essentially adapted to any nucleic acid amplification and melting system. Additionally, while the examples shown in Figures 14-16 show relatively small differences in melting temperature and shape between wild-type and mutant samples, the differences can be clearly resolved using the dynamic melting procedures described herein. This suggests that the dynamic melting procedures described herein can be routinely used to detect amplified nucleic acids containing mutations.

[0255] Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention, as set forth and defined in the following claims.

Claims

1. 1. A method for identifying which of a plurality of target nucleic acids is present in a sample, comprising: providing a sample suspected of containing at least one of a plurality of target nucleic acids; providing a plurality of sample wells, each sample well being provided with a portion of the sample and primers for amplifying a target nucleic acid sequence from a different one of a plurality of target nucleic acids, each target nucleic acid sequence having a melting temperature range unique to that target nucleic acid; providing a fluorescent dye that produces a fluorescent signal that increases in response to increasing concentrations of nucleic acid in the plurality of sample wells; simultaneously subjecting a plurality of sample wells to amplification conditions for a selected number of cycles; determining whether the sample wells exhibit positive nucleic acid amplification as evidenced by an increasing fluorescent signal from the sample wells during amplification conditions; and responsive to determining that the sample well exhibits positive nucleic acid amplification, performing a melt detection configured to detect the amplified target nucleic acid in the sample well, the melt detection being defined by a melting temperature range characteristic of the target nucleic acid in the sample well. A method comprising:

2. 10. The method of claim 1, wherein positive nucleic acid amplification is determined by a fluorescent signal in the sample well rising above a threshold value.

3. 3. The method of claim 2, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units rise above a threshold, mathematical modeling, signal processing, and combinations thereof.

4. 4. The method of claim 2 or 3, further comprising the steps of analyzing the fluorescent signals of multiple wells in real time to determine whether amplification has occurred in the sample wells, and performing melting detection if it is determined that amplification has occurred in the wells, wherein the temperature range of the melting detection is limited by the known melting temperature range of the nucleic acid of the target organism in the wells.

5. 5. The method of claim 1, further comprising not performing melt detection if the sample well does not show positive nucleic acid amplification.

6. 6. The method of claim 5, wherein the plurality of sample wells includes one or more control wells, and melting detection is not performed if only the one or more control wells show positive nucleic acid amplification.

7. Melting detection a first lift rate during a first portion of the melting; a second elevation rate during a second portion of the melting; and a third lift rate during the third portion of the melting 5. The method of claim 1, wherein the second rate of elevation is slower than the first rate of elevation and the third rate of elevation, and the second portion of the melting is defined by a melting temperature range specific to a target nucleic acid for detection of an organism in the sample well.

8. 8. The method of claim 7, wherein the first ramp rate is greater than 4°C / s, preferably in the range between 4°C / s and 20°C / s, more preferably in the range between 6°C / s and 20°C / s, the second ramp rate is less than 4°C / s, preferably in the range between 0.01°C / s and 4°C / s, and the third ramp rate is greater than 4°C / s, preferably in the range between 4°C / s and 20°C / s, more preferably in the range from 12°C / s to 20°C / s.

9. The method of claim 7 , wherein the third lift speed is faster than the first lift speed.

10. 10. The method according to claim 8 or 9, wherein the second ramp rate is in the range of 0.01 to 4°C / s, or preferably 0.01 to 2°C / s.

11. 11. The method of claim 10, wherein the second ramp rate is preferably 0.5°C / sec or less, more preferably 1°C / sec or less, and most preferably 2°C / sec or less.

12. 12. The method of claim 10 or 11, wherein the second rate of elevation is adapted to detect a nucleic acid melting signature indicative of a genotype, sequence variant, or genetic mutation in the target nucleic acid that alters one or more of the nucleic acid melting temperature or the shape of the nucleic acid melting curve compared to the target nucleic acid without the genotype, sequence variant, or genetic mutation.

13. 13. The method of claim 12, further comprising detecting single nucleotide polymorphisms (SNPs).

14. 13. The method of claim 12, further comprising detecting an antimicrobial resistance (AMR) marker.

15. 13. The method of claim 12, further comprising detecting the presence of the organism by a first melt detection in a first assay, and if the presence of the organism is detected in the first assay, performing a second melt detection in a second assay to detect the presence or absence of a genotype, sequence variant, or genetic mutation.

16. The method of claim 7, wherein whether to perform melting detection is determined by a positive or negative determination in the sample well, and if positive nucleic acid amplification is detected in the sample well, the temperature range of the second part of melting is determined by the melting temperature range of the target amplicon expected for the sample well.

17. 17. The method of claim 16, wherein the temperature range of the second part of the melting is the melting temperature range of the target amplicon, ±0.5°C to 10°C of the melting temperature range of the target amplicon, preferably ±2°C to 6°C of the melting temperature range of the target amplicon.

18. The method of claim 1, wherein two or more wells exhibit a fluorescent signal indicative of positive amplification.

19. 20. The method of claim 18, further comprising performing a single melt detection at a single melting temperature range on the amplified target nucleic acids in two or more wells.

20. 20. The method of claim 18, further comprising the steps of: performing a first melt detection in a first melting temperature range characteristic of melting of the target nucleic acid amplified in the first well; and performing at least a second melt detection in a second melting temperature range characteristic of melting of the target nucleic acid amplified in the second well.

21. Melting detection a first lift rate during a first portion of the melting; a second elevation rate during a second portion of the melting; and a third lift rate during the third portion of the melting wherein the second rate is slower than the first rate and the third rate, and the second portion of the melting is defined by a melting temperature range characteristic of the target nucleic acid in the first sample well and the second sample well; or a first lift rate during a first portion of the melting; a second lift rate during a second portion of the melting; a third lift rate during a third portion of the melting; a fourth elevation rate during a fourth portion of the melting; and A fifth lift rate during the fifth portion of the melt wherein the second rate and the fourth rate are slower than the first, third, and fifth rates, the second portion of the melting is defined by a melting temperature range specific to the target nucleic acid in the first sample well, and the fourth portion of the melting is defined by a melting temperature range specific to the target nucleic acid in the second sample well.

22. 22. The method of claim 21, wherein the second rate is in the range of 0.05 to 4°C / sec, or the second rate and the fourth rate are each in the range of 0.05 to 4°C / sec.

23. 23. The method of claim 22, wherein the second rate or the second rate and the fourth rate is preferably 0.5°C / sec or less, more preferably 1°C / sec or less, and most preferably 2°C / sec or less.

24. 24. The method of any one of claims 1 to 23, wherein the selected number of cycles of amplification before melt detection is at least 20.

25. 24. The method of any one of claims 1 to 23, wherein the selected number of cycles of amplification prior to melt detection is assay specific and determined by the expected concentrations of multiple target nucleic acids suspected to be present in the sample.

26. 26. The method of any one of claims 1 to 25, further comprising identifying at least one target organism present in the sample by identifying at least one corresponding sample well in which amplification and melt detection occurred.

27. 27. The method of any one of claims 1 to 26, wherein the target nucleic acid can be used to identify cell-free DNA, cells, organisms, molecular markers of antimicrobial resistance, host response markers, and combinations thereof.

28. 1. A method for identifying which of a plurality of target nucleic acids is present in a sample, comprising: providing a sample suspected of containing at least one target nucleic acid of a plurality of target nucleic acids; providing a plurality of sample wells configured for amplification of a plurality of target nucleic acids, each sample well of the plurality of sample wells containing a primer pair for amplifying one of the plurality of target nucleic acids, each target nucleic acid having a unique melting temperature range; providing a fluorescent dye that produces a fluorescent signal that increases in response to increasing concentrations of nucleic acid in the plurality of sample wells; distributing the sample among a plurality of sample wells such that each sample well contains a portion of the sample; simultaneously subjecting a plurality of sample wells to amplification conditions, the amplification conditions including repeated thermal cycles, each including a primer annealing step, a primer extension portion, and a nucleic acid denaturation step; acquiring a fluorescent signal in each of a plurality of sample wells during thermal cycling; determining that the amplitude of the fluorescent signal of a sample well configured for amplification of one target nucleic acid is greater than a threshold value, the threshold value being equal to or greater than a detection limit for concluding an elevated concentration of the nucleic acid in the sample well; and In response to determining that the amplitude of the fluorescent signal of the sample is greater than a threshold, performing melt detection configured to detect the amplified target nucleic acid in the sample well, the melt detection being defined by a melting temperature range characteristic of the amplified target nucleic acid in the sample well for detection of the organism in the sample well. A method comprising:

29. 29. The method of claim 28, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units rise above a threshold, mathematical modeling, signal processing, and combinations thereof.

30. The method of claim 28 or 29, further comprising the steps of monitoring the fluorescent signals of the multiple wells in real time to determine whether amplification has occurred in at least one well of the multiple sample wells, and performing melting detection if it is determined that amplification has occurred in at least one well, wherein the temperature range of melting detection is determined based on the known melting temperature range of the nucleic acid of the target organism in at least one well in which amplification has been determined to have occurred.

31. 31. The method of any one of claims 28 to 30, wherein the timing of melting detection is determined by the fluorescent signal of one or more wells configured for amplification of multiple target nucleic acids rising above a threshold.

32. 32. The method of any one of claims 28 to 31, which does not include performing a set number of thermal cycles before performing melt detection.

33. 33. The method of any one of claims 28 to 32, further comprising not performing melting detection if no sample wells exhibit a fluorescent signal above the threshold.

34. 34. The method of claim 33, wherein the plurality of sample wells includes one or more control wells, and melting detection is not performed if only one or more control wells exhibit a fluorescent signal above the threshold.

35. Melting detection a first rate of rise and fall through a first temperature range of melting; a second rate of rise and fall during a second temperature range of melting; and a third rate of rise and fall during a third temperature range of melting; 33. The method of any one of claims 28 to 32, comprising: wherein the second rate of rise and fall is slower than the first rate of rise and fall and the third rate of rise and fall; and wherein the second temperature range of melting is defined by the melting temperature range of the target nucleic acid amplified in the sample wells having a fluorescent signal greater than a threshold.

36. 36. The method of claim 35, wherein the second temperature range of melting is ±10°C of the melting temperature range of the target nucleic acid, ±8°C of the melting temperature range of the target nucleic acid, ±6°C of the melting temperature range of the target nucleic acid, or ±4°C of the melting temperature range of the target nucleic acid.

37. 36. The method of claim 35, wherein melt detection is performed and a temperature range of the second portion of the melt is determined.

38. 29. The method of claim 28, wherein two or more wells exhibit a fluorescent signal above the threshold.

39. 39. The method of claim 38, further comprising performing a single melt detection at a single melting temperature range on the amplified target nucleic acids in two or more wells.

40. 39. The method of claim 38, further comprising the steps of: performing a first melt detection in a first melting temperature range characteristic of melting of the target nucleic acid amplified in the first well; and performing at least a second melt detection in a second melting temperature range characteristic of melting of the target nucleic acid amplified in the second well.

41. the plurality of wells are configured for amplification of target nucleic acid sequences from organisms present at a first titer, organisms present at a second titer less than the first titer, and organisms present at a third titer less than the first titer and the second titer; performing a first melt detection if one or more wells configured for amplification of target nucleic acid sequences from organisms present at a first titer exhibit amplification above a threshold within a first number of thermal cycles; performing a second melt detection if one or more wells configured for amplification of target nucleic acid sequences from organisms present at a second titer exhibit amplification above a threshold within a second number of thermal cycles that is greater than the first number of thermal cycles; and performing a third melt detection if one or more wells configured for amplification of target nucleic acid sequences from organisms present at a third titer exhibit amplification above a threshold within a third number of thermal cycles that is greater than the first number of thermal cycles and the second number of thermal cycles.

41. The method of any one of claims 28 to 40, further comprising:

42. 42. The method of claim 41, wherein the first number of thermal cycles is 20 or less thermal cycles, the second number of thermal cycles is 25 or less thermal cycles, and the third number of thermal cycles is 30 or less thermal cycles.

43. 42. The method of claim 41, further comprising not performing melt detection if one or more wells configured for amplification of target nucleic acid sequences from organisms present at a first titer show amplification above a threshold for more than a first number of thermal cycles.

44. 42. The method of claim 41, further comprising not performing melt detection if one or more wells configured for amplification of target nucleic acid sequences from organisms present at a second titer show amplification above a threshold value for more than a second number of thermal cycles.

45. 42. The method of claim 41, further comprising not performing melt detection if one or more wells configured for amplification of target nucleic acid sequences from organisms present at a third titer show amplification above the threshold for more than a third number of thermal cycles.

46. 46. ​​The method of any one of claims 41 to 45, further comprising not performing melting detection if none of the one or more wells configured for amplification of target nucleic acid sequences from organisms present at the first titer, the second titer, or the third titer show amplification above the threshold.

47. 30. The method of claim 28, further comprising subjecting the sample to multiplex amplification prior to the partitioning step.

48. 48. The method of claim 47, wherein all steps are performed in a single closed system.

49. 1. A method for determining the presence of an organism in a sample, comprising: providing a sample suspected of containing at least one of a plurality of organisms; providing a plurality of sample wells, each sample well being provided with a primer for amplifying a target nucleic acid from a different one of a plurality of organisms; transferring a portion of the sample to each of a plurality of sample wells; simultaneously subjecting a plurality of sample wells to amplification conditions; acquiring a fluorescent signal in each of a plurality of sample wells during thermal cycling; determining that the amplitude of the fluorescent signal of a sample well configured for amplification of one target nucleic acid is greater than a threshold value, the threshold value being equal to or greater than a detection limit for concluding an elevated concentration of the nucleic acid in the sample well; responsive to determining that the amplitude of the fluorescent signal in the sample well is greater than a threshold, performing melt detection at a melting temperature window to detect the amplified target nucleic acid in the sample well, the melting temperature window being defined by a melting temperature range characteristic of the target nucleic acid; and determining the presence of the organism in the sample in response to performing the melting detection. A method comprising:

50. 50. The method of claim 49, further comprising the steps of subjecting a plurality of sample wells to amplification conditions for a selected number of cycles, and if the sample wells show positive nucleic acid amplification within the selected number of amplification cycles, performing melt detection, wherein the melt detection is defined by a melting temperature range characteristic of a target nucleic acid for detection of the organism in the sample well.

51. 51. The method of claim 50, wherein the selected number of cycles of amplification is at least one but not more than 15, at least one but not more than 20, at least one but not more than 25, at least one but not more than 30, or at least one but not more than 35.

52. 52. The method of any one of claims 49 to 51, further comprising not performing melt detection if no sample wells show positive nucleic acid amplification.

53. 50. The method of claim 49, further comprising simultaneously subjecting the samples to multiplex amplification prior to the transferring step.

54. 54. The method of claim 53, wherein all steps are performed in a single closed system.

55. 1. A system for detecting which of a plurality of target organisms are present in a sample, comprising: a container including a plurality of sample wells, each sample well configured to contain a portion of the sample, nucleic acid primers specific for amplification of one target nucleic acid from one target organism, a fluorescent dye that produces a fluorescent signal that increases with increasing concentration of nucleic acid in the plurality of sample wells, and amplification components; An instrument configured to simultaneously subject a portion of a sample in each of a plurality of sample wells to amplification conditions and then to melting conditions, the instrument including a detector for detecting a fluorescent signal from a fluorescent dye indicative of amplification. Equipped with A system in which the instrument is programmed with melting range information for a target organism to be amplified in each of a plurality of wells, and for each target nucleic acid from the target organism, and the instrument is programmed to monitor fluorescence in the plurality of wells during amplification conditions, and if a sample well shows an increase in fluorescence indicative of positive nucleic acid amplification during the amplification conditions, perform melt detection, the melt detection being defined by the melting temperature range of the target nucleic acid to be amplified in the sample well.

56. 56. The system of claim 55, wherein the instrument is programmed to perform a set number of amplification cycles before performing melt detection.

57. The system of claim 55 or 56, wherein the instrument is programmed to perform melting detection at any number of amplification cycles when amplification is detected in the sample well, and the temperature range of melting detection is limited by melting range information of the target nucleic acid of the target organism amplified in the well.

58. 58. The system of any one of claims 55 to 57, wherein positive nucleic acid amplification is indicated by a fluorescent signal in the sample well rising above a threshold value.

59. 59. The system of claim 58, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units rise above a threshold, mathematical modeling, signal processing, and combinations thereof.

60. 60. The system of any one of claims 55 to 59, wherein the instrument is further programmed to not perform melting detection if no sample wells show positive nucleic acid amplification within a set number of amplification cycles.

61. a first lift rate during a first portion of the melting; a second elevation rate during a second portion of the melting; and a third lift rate during the third portion of the melting 61. The system of any one of claims 55 to 60, wherein the second rate of elevation is slower than the first rate of elevation and the third rate of elevation, and wherein the second portion of the melting comprises melting detection defined by melting range information of a target nucleic acid of a target organism amplified in the well.

62. 62. The system of any one of claims 55 to 61, programmed to identify at least one target organism present in a sample by identifying at least one corresponding sample well in which amplification and melting detection occurred.

63. 63. The system of any one of claims 55 to 62, wherein the amplification is PCR and the components include a polymerase and dNTPs.

64. 64. The system of any one of claims 55 to 63, wherein the instrument comprises a light source configured to emit a light signal toward the plurality of sample wells during screening of each sample.

65. 65. The system of any one of claims 55 to 64, wherein the melting result is based on the presence or absence of a melting peak within a predetermined temperature range, the presence of a melting peak outputting a positive result and the absence of a melting peak outputting a negative result.

66. 1. A system for detecting which of a plurality of target organisms are present in a sample, comprising: a container comprising a plurality of sample wells, each sample well configured to contain a portion of the sample, nucleic acid primers specific for amplification of one target nucleic acid from one target organism, a fluorescent dye that produces a fluorescent signal that increases with increasing concentration of nucleic acid in the plurality of sample wells, and amplification components; an instrument configured to simultaneously subject a portion of the sample in each of a plurality of sample wells to amplification conditions and then to melting conditions, the instrument including a detector for detecting a fluorescent signal from the fluorescent dye indicative of amplification; and Programming for carrying out the method of any one of claims 1 to 54. Including, A system in which the instrument is programmed with the identity of a target organism to be amplified in each of a plurality of wells and melting range information for each of the target nucleic acids from the target organism.

67. 1. A computer-implemented method for evaluating a sample for a target nucleic acid sequence, comprising: sending, by the one or more processors, control signals to a thermocycling element to heat a plurality of sample wells to a first temperature using an initial ramp rate and cool the plurality of sample wells to a second temperature through one or more cycles, each cycle including an intra-cycle temperature adjustment segment, each of the plurality of sample wells configured to receive a portion of a sample, each sample well containing a target nucleic acid sequence from a different one of a plurality of target nucleic acids, each target nucleic acid having a distinctive melting temperature range; receiving, with one or more processors from the optical system, data indicative of an amount of fluorescence emitted from a portion of the samples in the plurality of sample wells during an intra-cycle temperature adjustment segment of one or more cycles; In response to determining that the amount of fluorescence for at least one of the sample wells exceeds a threshold value, determining, by one or more processors, the target nucleic acid sequence amplified in each of the sample wells that exceeds a threshold; determining, by the one or more processors, an adjusted heating rate profile for heating the sample wells based on the melting temperature range and / or melting rate characteristic of the target nucleic acid sequence in each of the sample wells that exceeds the threshold; and performing melt detection on a portion of the sample, including sending, by the one or more processors, control signals to the thermocycling element to heat the sample well to a first temperature using the adjusted ramp rate profile for subsequent cycles. A method comprising:

68. the first temperature is a denaturation temperature, the second temperature is an annealing temperature, the characteristic melting temperature range of the sample well is from an initial melting temperature to a final melting temperature, and the adjusted ramp rate profile is: a first ramp rate during the approach temperature range from the annealing temperature to the incipient melting temperature; a second ramp rate through a specific melting temperature range from the initial melting temperature to the final melting temperature; and a third ramp rate during the final temperature range from the final melting temperature to the denaturation temperature; 68. The method of claim 67, comprising:

69. 69. The method of claim 68, wherein the third lift speed is faster than the first lift speed.

70. 70. The method of claim 68 or claim 69, wherein the second ramp rate is in the range of 0.05 to 4°C / sec.

71. 71. The method of any one of claims 68 to 70, wherein the first ramp rate is greater than 4°C / s, preferably in the range of between 4°C / s and 20°C / s, more preferably in the range of between 6°C / s and 20°C / s, the second ramp rate is less than 4°C / s, preferably in the range of between 0.01°C / s and 4°C / s, and the third ramp rate is greater than 4°C / s, preferably in the range of between 4°C / s and 20°C / s, more preferably in the range of 12°C / s to 20°C / s.

72. 72. The method of claim 71, wherein the third lift speed is faster than the first lift speed.

73. 73. A method according to claim 71 or claim 72, wherein the second ramp rate is in the range of 0.01 to 4°C / sec, or preferably 0.01 to 2°C / sec.

74. 74. The method of claim 73, wherein the second ramp rate is preferably 0.5°C / sec or less, more preferably 1°C / sec or less, and most preferably 2°C / sec or less.

75. 75. The method of Claim 73 or 74, wherein the second elevation rate is adapted to detect a nucleic acid melting signature indicative of a genotype, sequence variant, or genetic mutation in the target nucleic acid that alters one or more of the nucleic acid melting temperature or the shape of the nucleic acid melting curve compared to the target nucleic acid without the genotype, sequence variant, or genetic mutation.

76. 76. The method of claim 75, further comprising detecting single nucleotide polymorphisms (SNPs).

77. 76. The method of claim 75, further comprising detecting an antimicrobial resistance (AMR) marker.

78. 76. The method of claim 75, further comprising detecting the presence of the organism by a first melt detection in a first assay, and if the organism is detected as present in the first assay, performing a second melt detection in a second assay to detect the presence or absence of a genotype, sequence variant, or genetic mutation.

79. 79. The method of any one of claims 67 to 78, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units rise above a threshold, mathematical modeling, signal processing, and combinations thereof.

80. The controller and identifying a target nucleic acid among a plurality of target nucleic acids corresponding to the sample well based on melt detection.

80. The method of any one of claims 67 to 79, further comprising:

81. The controller determining that the amount of fluorescence in the first sample well and the second sample well exceeds a threshold; performing a first melting detection by using a first adjusted ramp rate profile corresponding to a first unique melting temperature range for melting the amplified target nucleic acid in the first sample well; and and performing a second melting detection by using a second adjusted ramp rate profile corresponding to a second unique melting temperature range for melting the amplified target nucleic acid in the second sample well.

81. The method of any one of claims 67 to 80, further comprising:

82. 1. A system for determining a sample for a target nucleic acid sequence, comprising: a plurality of sample wells, each configured to receive a portion of a sample, each sample well containing a target nucleic acid sequence from a different one of a plurality of target nucleic acids, each target nucleic acid having a distinctive melting temperature range; an optical system configured to detect the amount of fluorescence emitted from the sample; a controller, sending control signals to a thermocycling element to heat the plurality of sample wells to a first temperature using an initial ramp rate and cool the plurality of sample wells to a second temperature through one or more cycles, each cycle including an in-cycle temperature adjustment segment; receiving data from the optical system during an intra-cycle temperature adjustment segment of one or more cycles that indicates an amount of fluorescence emitted by a portion of the samples in the plurality of sample wells; In response to determining that the amount of fluorescence for at least one of the sample wells exceeds a threshold value, determining a tailored ramp rate profile for heating the sample well based on a melting temperature range characteristic of the target nucleic acid sequence in the sample well that exceeds a threshold; and Sending a control signal to the thermocycling element to perform melt detection on a portion of the sample by heating the sample well to a first temperature using the adjusted ramp rate profile for the next cycle. Controller configured as A system equipped with

83. The first temperature is a denaturation temperature, the second temperature is an annealing temperature, the characteristic melting temperature range of the sample well is from the initial melting temperature to the final melting temperature, and the adjusted ramp rate profile is a first ramp rate during the approach temperature range from the annealing temperature to the incipient melting temperature; a second ramp rate through a specific melting temperature range from the initial melting temperature to the final melting temperature; and a third ramp rate during the final temperature range from the final melting temperature to the denaturation temperature; 83. The system of claim 82, comprising:

84. 84. The system of claim 83, wherein the third lift speed is faster than the first lift speed.

85. 85. The system of claim 83 or claim 84, wherein the second lift speed is the same as the initial lift speed.

86. 86. The system of any one of claims 82 to 85, wherein the first heating / cooling rate is greater than 4°C / s (e.g., 6-20°C / s), the second heating / cooling rate is less than 4°C / s (e.g., in the range of 0.01-4°C / s or 1-2°C / s), and the third heating / cooling rate is greater than 4°C / s (e.g., 6-20°C / s).

87. 87. The system of any one of claims 82 to 86, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units rise above the threshold, mathematical modeling, signal processing, and combinations thereof.

88. The controller and identifying a target nucleic acid among a plurality of target nucleic acids corresponding to the sample well based on melt detection.

88. The system of any one of claims 82 to 87, further comprising:

89. The controller determining that the amount of fluorescence in the first sample well and the second sample well exceeds a threshold; performing a first melt detection by using a first adjusted ramp rate profile corresponding to a first unique melting temperature range for melting the amplified target nucleic acid in the first sample well; and and performing a second melting detection by using a second adjusted ramp rate profile corresponding to a second unique melting temperature range for melting the amplified target nucleic acid in the second sample well.

89. The system of any one of claims 82 to 88, further comprising:

90. 1. A computer apparatus for evaluating a sample for a target nucleic acid sequence, comprising: one or more processors; and a non-transitory computer-readable memory coupled to one or more processors and configured to execute the non-transitory computer-readable memory when executed by the one or more processors; sending control signals to a thermocycling element to heat a plurality of sample wells to a first temperature using an initial ramp rate and cool the plurality of sample wells to a second temperature through one or more cycles, each cycle including an intra-cycle temperature adjustment segment, wherein each of the plurality of sample wells is configured to receive a portion of a sample, each sample well containing a target nucleic acid sequence from a different one of a plurality of target nucleic acids, each target nucleic acid having a distinctive melting temperature range; receiving data from the optical system indicative of an amount of fluorescence emitted by a portion of the samples in the plurality of sample wells during an intra-cycle temperature adjustment segment of one or more cycles; In response to determining that the amount of fluorescence in at least one of the sample wells exceeds a threshold value, determining a tailored ramp rate profile for heating the sample well based on the characteristic melting temperature range of the target nucleic acid sequence in the sample well above a threshold; and performing melt detection on a portion of the sample by sending a control signal to the thermocycling element to heat the sample well to a first temperature using the adjusted ramp rate profile for the next cycle. a non-transitory computer-readable memory storing therein instructions for causing a A computer device comprising:

91. the first temperature is a denaturation temperature, the second temperature is an annealing temperature, the characteristic melting temperature range of the sample well is from an initial melting temperature to a final melting temperature, and the adjusted ramp rate profile is: a first ramp rate during the approach temperature range from the annealing temperature to the incipient melting temperature; a second ramp rate through a specific melting temperature range from the initial melting temperature to the final melting temperature; and a third ramp rate during the final temperature range from the final melting temperature to the denaturation temperature; 91. The computing device of claim 90, comprising:

92. 92. The computer device of claim 91, wherein the third lift speed is faster than the first lift speed.

93. 93. The computer device of claim 91 or claim 92, wherein the second lift speed is the same as the initial lift speed.

94. 94. The computer device of any one of claims 90 to 93, wherein the first heating / cooling rate is greater than 4°C / sec (e.g., 6 to 20°C / sec), the second heating / cooling rate is less than 4°C / sec (e.g., in the range of 0.01 to 4°C / sec or 1 to 2°C / sec), and the third heating / cooling rate is greater than 4°C / sec (e.g., 6 to 20°C / sec).

95. 95. The computer device of any one of claims 90 to 94, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units rise above a threshold, mathematical modeling, signal processing, and combinations thereof.

96. The instructions are sent to the computer device, and identifying a target nucleic acid among a plurality of target nucleic acids corresponding to the sample wells based on melt detection.

96. A computer apparatus according to any one of claims 90 to 95, further configured to:

97. The instructions are sent to the computer device, determining that the amount of fluorescence in the first sample well and the second sample well exceeds a threshold; performing a first melt detection by using a first adjusted ramp rate profile corresponding to a first unique melting temperature range for melting the amplified target nucleic acid in the first sample well; and performing a second melting detection by using a second adjusted ramp rate profile corresponding to a second unique melting temperature range for melting the amplified target nucleic acid in a second sample well; 97. A computer apparatus according to any one of claims 90 to 96, further configured to:

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