Dynamic melting detection

Abstract

The present disclosure relates to methods and systems suitable for simultaneous amplification of many possible targets and then melting detection based on whether any amplification of any target nucleic acid is detected, wherein melting parameters are limited by a melting temperature range characteristic of the target nucleic acid from which the amplification is detected. According to the invention, the resolution ratio realized by nucleic acid melting at a slow and stable heating rate between the annealing temperature and the denaturation temperature is maintained; however, this is achieved faster by using a faster heating rate at a temperature at which the reaction is expected not to produce a melting feature and a slower heating rate at a temperature at which the reaction is expected to produce a melting feature.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 374,350, filed on September 1, 2022, the entire content of which is incorporated herein by reference. Background Art

[0003] 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 can result in a variety of clinical manifestations. Common overt manifestations include fever, pneumonia, meningitis, diarrhea, and bloody diarrhea. While the physical manifestations suggest some pathogens as etiological agents and rule out others, there remain many possible etiological agents, and a definitive diagnosis often requires many assays. Traditional microbiological techniques for diagnosing pathogens can take days or weeks, often delaying appropriate treatment courses.

[0004] In recent years, polymerase chain reaction (PCR) has become the method of choice for rapid diagnosis of infectious agents. PCR can be a rapid, sensitive, and specific tool for diagnosing infectious diseases. The challenge in using PCR as the primary diagnostic means is the diversity of possible pathogenic organisms and the low levels of organisms present in some pathological specimens. Running large panels of PCR assays (one assay for each possible pathogenic organism, most of which are expected to be negative) is often impractical. The problem worsens when the pathogen nucleic acid is at low concentration and large amounts of sample are required to collect sufficient reaction templates. In some cases, there is not enough sample to assay all possible etiological agents. One solution is to run "multiplex PCR," in which multiple targets are assayed simultaneously on a sample in a single reaction. Although multiplex PCR has proven valuable in some systems, there are drawbacks regarding the robustness of high-level multiplex reactions and the difficulty in clearly analyzing multiple products. To address these issues, the assay can subsequently be divided into multiple secondary PCRs. Nesting secondary reactions within the primary product often increases robustness. However, this further operation can be expensive and can lead to contamination or other problems.

[0005] (BioFire Diagnostics, Inc., Salt Lake City, Utah) is a user-friendly, highly multiplexed PCR system developed for the diagnostic market. The single-sample instrument receives a diagnostic "bag" that integrates sample preparation and nested multiplex PCR. The integrated sample preparation provides ease of use, while the highly multiplexed PCR provides PCR sensitivity and the ability to simultaneously assay many organisms (e.g., up to 30 or more different organisms and / or molecular markers). The system is well-suited for pathogen identification where several different pathogens exhibit similar clinical symptoms. Currently available diagnostic panels include a respiratory panel for upper respiratory infections, a blood culture panel for bloodstream infections, a gastrointestinal panel for GI infections, a meningitis / encephalitis panel for central nervous system infections, a pneumonia panel for lower respiratory infections, and a bone and joint panel for bone and joint infections. Other panels are in development.

[0006] PCR can be conceptually divided into three reactions, each of which is typically assumed to occur over time at each of three temperatures. This "equilibrium paradigm" of PCR is easy to understand in terms of the three reactions (denaturation, annealing, and extension) that occur at three temperatures for three time periods in each cycle. However, this equilibrium paradigm does not fit well with physical reality. Instantaneous temperature changes do not occur; it takes time to change the sample temperature, and the temperature may not be uniform throughout the sample, especially when using larger volumes. In addition, the individual reaction rates vary with temperature, and once primer annealing occurs, polymerase extension follows immediately. More accurately, especially for rapid PCR, is a kinetic paradigm where the reaction rates and temperature are always changing. As long as the product is denatured and the primer anneals, there is no need to maintain a constant temperature during PCR. Under the kinetic paradigm of PCR, product denaturation, primer annealing, and polymerase extension may overlap in time, and their rates vary continuously with temperature. Under the equilibrium paradigm, the cycle is defined by three temperatures, each maintained for a period of time, while the kinetic paradigm requires transition rates and target temperatures.

[0007] When PCR first became popular in the late 1980s, the process was slow. A typical protocol was to denature for one minute at 94°C, anneal for two minutes at 55°C, and extend for three minutes at 72°C. When including the time for temperature transitions between them, a typical 8-minute cycle resulted in 30 cycles being completed in four hours. Twenty-five percent of the cycle time was spent on temperature transitions. As the cycle speed increased, the proportion of time spent on temperature transitions also increased, and the kinetic paradigm became increasingly important. During rapid-cycle PCR, the temperature typically changes. For rapid-cycle PCR of short products (<100 bp), 100% of the time may be spent on temperature transitions, and no hold time is required. For rapid-cycle PCR of longer products, a temperature hold at the optimal extension temperature may be included.

[0008] One way to shorten the cycle time is to introduce changes to the PCR protocol to reduce the temperature cycling requirements. Over the years, systems have become faster and the kinetic requirements for denaturation, annealing, and extension have become clearer. Shortening the cycle time, reducing the three-step cycle (denaturation, annealing, and extension) to two steps (denaturation and a combined annealing / extension step), increasing the primer length, shortening the product length, etc. can shorten the PCR reaction time while maintaining high diagnostic accuracy. Even with protocol changes, many diagnostic PCR reactions ultimately still require a confirmatory melt detection step. DNA has a melting temperature range specific to its length and A-T / G-C composition. Probe-free DNA melt analysis typically relies on the fact that a DNA-binding dye fluoresces strongly in the presence of double-stranded DNA and weakly or not at all in the presence of single-stranded DNA. Thus, for a given amplification reaction, the expected product will have an expected melting temperature range characteristic of that product and, therefore, melt analysis can be used to confirm that the expected product was produced in the amplification reaction.

[0009] However, as PCR has become faster, the time spent on melting has become an increasingly large proportion of the run time. One possible solution is to increase the temperature more quickly during melting, but it has been found that melting curves generated by faster temperature increases often result in reduced sensitivity to amplicon differences. It is desirable to generate a melting curve using a faster temperature increase while maintaining sensitivity to amplicon differences. There is a need in the art for a method that maintains the melting resolution achieved by slower temperature increase rates while also shortening the time required for melt analysis. SUMMARY OF THE INVENTION

[0010] The present disclosure relates to methods and systems suitable for simultaneously amplifying many possible targets and then performing melt detection based on whether any amplification of any target nucleic acid is detected, wherein the melt parameters are limited by the melting temperature range characteristic of the amplified target nucleic acid detected. Traditionally, high-resolution DNA melting has been performed using a fixed temperature increase rate that covers the entire temperature range over which the reaction product is expected to melt. The present invention maintains the resolution achieved by slower temperature increase rates but does so in a shorter time by using a faster melting temperature increase rate in the temperature range where the reaction is not expected to produce melting characteristics and a slower melting temperature increase rate in the temperature range where the reaction is expected to produce melting characteristics. These variable melting temperature increase ranges and rates are defined by the predicted or experimentally determined melting temperature of the amplicon for which amplification was actually detected, so there is no need to spend time slowly increasing the temperature over a large temperature window for all possible targets.

[0011] For example, an assay (e.g., a panel including multiple assays for multiple pathogens) can be designed to amplify and detect 20 or more targets. If in a particular use case, only one or two targets are present in the sample, the melting parameters can be set dynamically based on the predicted or experimentally determined melting temperature (referred to herein as the region of interest or ROI) of the target amplicons present, rather than having a large melting range and a slow melting ramp rate to capture the melting of all possible targets. For example, as will be explained in more detail below, the melting temperature can be rapidly increased (greater than 4 °C / sec, e.g., 6 - 20 °C / sec) in a first temperature range where melting of the detected target amplicons is not expected, then slowly increased (e.g., less than 4 °C / sec, 0.01 - 2 °C / sec, 1 - 2 °C / sec) through the ROI, and then rapidly increased again (greater than 4 °C / sec, e.g., 12 - 20 °C / sec) after the ROI until the denaturation temperature. This "fast - slow - fast" ramp protocol can save time while maintaining the resolution of traditional slow melting. If amplification of different target amplicons is detected in more than one well, then this "fast - slow - fast" protocol can be suitably used if the amplification of different target amplicons is detected at the same or nearly the same time and if the melting ranges of the target amplicons sufficiently overlap. In another aspect, if the amplification of different target amplicons is detected at the same or nearly the same time and there is sufficient separation between the regions of interest in the melting between two organisms suspected to be present in the sample, then a modified "fast - slow - fast - slow - fast" protocol can be suitably used. In one aspect, the systems described herein can be suitably designed to determine which melting protocol (e.g., "fast - slow - fast" vs. "fast - slow - fast - slow - fast") is fastest and most efficient in the case where amplification of different target amplicons is detected at the same or nearly the same time.

[0012] The inventions described herein can suitably include: performing a melting detection after a fixed number of PCR cycles if amplification of one or more target nucleic acids is detected (e.g., the fluorescence signal in a sample well rises above a threshold). The inventions described herein can suitably include: performing a melting detection at any point in the reaction if amplification of one or more target nucleic acids is detected. The inventions described herein can suitably include: not performing a melting detection if no amplification of any target nucleic acids is detected.

[0013] The invention described herein suitably may include a method for identifying which of a plurality of target nucleic acids is present in a sample (e.g., from a variety of organisms). The method suitably may include the steps of: providing a sample suspected of containing at least one target organism (e.g., a respiratory sample, a blood sample, a positive blood culture sample, etc.), and 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 the plurality of target nucleic acids. Each target nucleic acid from each different organism and / or molecular marker has a melting temperature range characteristic of that target nucleic acid. The method suitably also may include providing a fluorescent dye that produces an increased fluorescent signal in response to an increase in nucleic acid concentration in the plurality of sample wells, and subjecting the plurality of sample wells simultaneously to amplification conditions for a selected number of cycles. If a sample well exhibits positive nucleic acid amplification during the amplification conditions, the method suitably may include performing a melting detection, wherein the melting detection is defined by the melting temperature range and the melting temperature increase rate characteristic of the target nucleic acid to detect the organism in the sample well.

[0014] The invention described herein suitably may include a method for identifying which of a plurality of target nucleic acids and / or molecular markers is present in a sample. The method suitably may 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 to amplify a plurality of target nucleic acids. Each sample well of the plurality of sample wells suitably may 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 suitably may contain more than one pair of primers for amplifying more than one of the plurality of target nucleic acids. Each target nucleic acid suitably may have a characteristic melting temperature range and a melting temperature increase rate. The method suitably also may include providing a fluorescent dye that produces an increased fluorescent signal in response to an increase in nucleic acid concentration in the plurality of sample wells, dispensing the sample among the plurality of sample wells such that each sample well contains a portion of the sample, subjecting the plurality of sample wells simultaneously to amplification conditions, wherein the amplification conditions include repeated thermal cycles, each thermal cycle including a primer annealing step, a primer extension portion, and a nucleic acid denaturation step, and acquiring a fluorescent signal in each of the plurality of sample wells during the thermal cycles. If the fluorescent signal of a sample well meets the condition indicating the presence of the expected DNA target in the sample (as an example, the fluorescent signal in the sample well is greater than a threshold value that is greater than or equal to the detection limit for inferring an increase in the nucleic acid concentration in the sample well), the method suitably may include performing a melting detection, wherein the melting detection is defined by the melting temperature range and the melting temperature increase rate characteristic of the target nucleic acid to detect the organism in the sample well in which an amplification above the threshold is detected.

[0015] The invention described herein suitably may include a method for the fluorescent detection of nucleic acids. The method suitably may 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 provided with primers for amplifying a target nucleic acid from a different one of the plurality of organisms; moving a portion of the sample into each of the plurality of sample wells and subjecting the plurality of sample wells simultaneously to amplification conditions; and acquiring a fluorescent signal in each of the plurality of sample wells during thermal cycling. If the amplitude of the fluorescent signal of a sample well configured to amplify one target nucleic acid is greater than a threshold, which is greater than or equal to the detection limit for inferring an increase in the concentration of nucleic acid in the sample well, the method suitably may include performing a melt detection, wherein the melt detection is defined by the melting temperature range and the melt heating rate characteristic of the target nucleic acid to detect the organism in the sample well in which amplification above the threshold is detected.

[0016] The invention described herein suitably may include a system for detecting which of a plurality of target organisms is present in a sample. The system suitably may include: a container containing a plurality of sample wells, each sample well configured to receive a portion of the sample, nucleic acid primers that specifically amplify a target nucleic acid from one target organism, a fluorescent dye that produces an increased fluorescent signal in response to an increase in the nucleic acid concentration in the plurality of sample wells, and components for amplification, and an instrument configured to subject the portion of the sample in each of the plurality of sample wells simultaneously to amplification conditions and subsequent melt conditions. The instrument suitably may include a detector for detecting a fluorescent signal indicative of amplification from the fluorescent dye. The instrument suitably may be programmed to have thermal cycling parameters for amplifying the nucleic acids in the plurality of sample wells, and to have information regarding each of the target organisms to be amplified in each of the plurality of wells, including melt range information for each target nucleic acid from the target organism. The instrument may suitably also be programmed to monitor the fluorescence in the plurality of wells during the amplification conditions and, if a sample well shows an increase in fluorescence indicative of positive nucleic acid amplification during the amplification conditions, perform a melt detection, wherein the melt detection is defined by the melting temperature range and the melt heating rate of the target nucleic acid amplified in the sample well.

[0017] The description is as follows:

[0018] A1. A method for identifying which of a plurality of target nucleic acids is present in a sample, comprising:

[0019] providing a sample suspected of containing at least one target nucleic acid,

[0020] 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 the plurality of target nucleic acids, wherein each target nucleic acid sequence has a characteristic melting temperature range,

[0021] A fluorescent dye that produces an increased fluorescent signal in response to an increase in the nucleic acid concentration in the plurality of sample wells,

[0022] subjecting the plurality of sample wells simultaneously to amplification conditions for a selected number of cycles,

[0023] determining whether a sample well exhibits positive nucleic acid amplification, as confirmed by an increased fluorescent signal from the sample well during the amplification conditions, and

[0024] in response to determining that a sample well exhibits positive nucleic acid amplification, performing a melting curve assay configured to detect the amplified target nucleic acid in the sample well, wherein the melting curve assay is defined by a melting temperature range characteristic of the target nucleic acid in the sample well.

[0025] A2. The method of item A1, wherein positive nucleic acid amplification is determined by an increase in the fluorescent signal in the sample well above a threshold.

[0026] A3. The method of item 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 elevated above a threshold, mathematical modeling, signal processing, and combinations thereof.

[0027] A4. The method of any one of items A1 - A3, further comprising real - time analysis of the fluorescent signals of the plurality of wells to determine whether amplification has occurred in a sample well, and if it is determined that amplification has occurred in the well, performing a melting curve assay, wherein the temperature range for the melting curve assay is restricted by the known melting temperature range of the target nucleic acid in the well.

[0028] A5. The method of any one of items A1 - A4, further comprising not performing a melting curve assay if no sample well shows positive nucleic acid amplification.

[0029] A6. The method of any one of items A1 - A5, wherein the plurality of sample wells includes one or more control wells, and wherein if only the one or more control wells show positive nucleic acid amplification, no melting curve assay is performed.

[0030] A7. The method of any one of items A1 - A6, wherein the melting curve assay comprises:

[0031] a first heating rate during a first portion of the melting,

[0032] a second heating rate during a second portion of the melting, and

[0033] a third heating rate during a third portion of the melting,

[0034] The second heating rate is slower than the first and third heating rates, and wherein the second part of the melting is defined by a melting temperature range characteristic of the target nucleic acid to detect an organism in a sample well.

[0035] The method of any one of items A1 - A7 of A7.1., wherein the third heating rate is faster than the first heating rate.

[0036] The method of any one of items A1 - A7.1 of A7.2., wherein the second heating rate is in the range of 0.01 to 4 °C / sec or preferably 0.01 to 2 °C / sec.

[0037] The method of any one of items A1 - A7.2 of A7.3., wherein the second heating rate is preferably not greater than 0.5 °C / sec, more preferably not greater than 1 °C / sec, and most preferably less than or equal to 2 °C / sec.

[0038] The method of any one of items A1 - A7.3 of A7.4., wherein the second heating rate is adapted to detect nucleic acid melting characteristics indicative of a genotype, sequence variant, or gene mutation in the target nucleic acid, the genotype, sequence variant, or gene mutation altering one or more of the nucleic acid melting temperature or nucleic acid melting curve shape relative to a target nucleic acid not having the genotype, sequence variant, or gene mutation.

[0039] The method of any one of items A1 - A7.4 of A7.5., further comprising detecting a single nucleotide polymorphism (SNP).

[0040] The method of any one of items A1 - A7.5 of A7.7., further comprising detecting an antimicrobial resistance (AMR) marker.

[0041] The method of any one of items A1 - A7.7 of A7.8., further comprising detecting the presence of an organism via a first melting detection in a first assay, and if the presence of the organism is detected in the first assay, performing a second melting detection in a second assay to detect the presence or absence of the genotype, sequence variant, or gene mutation.

[0042] The method of any one of items A1 - A7.8 of A8., wherein a positive or negative determination in the sample well determines whether to perform a melting detection and determines the temperature range of the second part of the melting.

[0043] The method of any one of items A1 - A8 of A8.1., wherein the temperature range of the second part of the melting is the melting temperature range of the target amplicon, the melting temperature range of the target amplicon + / - 0.5 °C to 10 °C, preferably the melting temperature range of the target amplicon + / - 2 °C to 6 °C.

[0044] The method according to any one of items A1 - A8.1, wherein two or more wells show a fluorescent signal indicating positive amplification.

[0045] A10. The method according to any one of items A1 - A9, further comprising performing a single denaturation detection with a denaturation temperature range for the amplified target nucleic acid in said two or more wells.

[0046] A11. The method according to any one of items A1 - A10, further comprising performing a first denaturation detection with a characteristic first denaturation temperature range for the amplified target nucleic acid in the first well and performing at least a second denaturation detection with a characteristic second denaturation temperature range for the amplified target nucleic acid in the second well.

[0047] A11.1. The method according to any one of items A1 - A11, wherein the denaturation detection comprises one of the following:

[0048] A first heating rate during a first part of denaturation,

[0049] A second heating rate during a second part of denaturation, and

[0050] A third heating rate during a third part of denaturation,

[0051] The second heating rate is slower than the first and third heating rates, and wherein the second part of denaturation is defined by a denaturation temperature range characteristic of the target nucleic acid in the first and second sample wells, or

[0052] A first heating rate during a first part of denaturation,

[0053] A second heating rate during a second part of denaturation,

[0054] A third heating rate during a third part of denaturation,

[0055] A fourth heating rate during a fourth part of denaturation, and

[0056] A fifth heating rate during a fifth part of denaturation,

[0057] The second and fourth heating rates are slower than the first, third, and fifth heating rates, and wherein the second part of denaturation is defined by a denaturation temperature range characteristic of the target nucleic acid in the first sample well, and the fourth part of denaturation is defined by a denaturation temperature range characteristic of the target nucleic acid in the second sample well.

[0058] A11.2. The method according to any one of items A1 - A11.1, wherein the second heating rate is in the range of 0.05 to 4 °C / sec, or the second and fourth heating rates are each in the range of 0.05 to 4 °C / sec.

[0059] A method according to any one of items A1 - A11.2, wherein the second heating rate or the second and fourth heating rates are preferably not greater than 0.5 °C / sec, more preferably not greater than 1 °C / sec, and most preferably not greater than 2 °C / sec.

[0060] A method according to any one of items A1 - A11.3, wherein the selected number of amplification cycles before the melting detection is at least 20.

[0061] A method according to any one of items A1 - A12, wherein the selected number of amplification cycles before the melting detection is specific and determined by the expected concentrations of the multiple target nucleic acids suspected to be present in the sample.

[0062] A method according to any one of items 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 melting detection have occurred.

[0063] A method according to any one of items A1 - A14, wherein the target nucleic acid can be used to identify cell-free DNA, cells, organisms, molecular markers of antimicrobial drug resistance, host response markers, and combinations thereof.

[0064] B1. A method for identifying which of multiple target nucleic acids are present in a sample, comprising:

[0065] Providing a sample suspected of containing at least one target nucleic acid,

[0066] Providing a plurality of sample wells configured to amplify the multiple target nucleic acids, wherein each sample well of the plurality of sample wells contains a primer pair for amplifying one of the multiple target nucleic acids, and wherein each target nucleic acid has a characteristic melting temperature range,

[0067] Providing a fluorescent dye that produces an increased fluorescent signal in response to an increase in the nucleic acid concentration in the plurality of sample wells,

[0068] Allocating the sample among the plurality of sample wells such that each sample well contains a portion of the sample,

[0069] Subjecting the plurality of sample wells simultaneously to amplification conditions, wherein the amplification conditions include repeated thermal cycles, each thermal cycle including a primer annealing step, a primer extension portion, and a nucleic acid denaturation step,

[0070] Acquiring the fluorescent signal in each of the plurality of sample wells during the thermal cycle,

[0071] Determine that the fluorescence signal amplitude of a sample well configured to amplify a target nucleic acid is greater than a threshold value, which is greater than or equal to the detection limit for inferring an increase in the concentration of nucleic acid in the sample well, and

[0072] In response to determining that the fluorescence signal amplitude of the sample is greater than the threshold value, perform a melting detection, where the melting detection is configured to detect the amplified target nucleic acid in the sample well, and where the melting detection is defined by a melting temperature range characteristic of the amplified target nucleic acid in the sample well to detect an organism in the sample well.

[0073] B2. The method of item B1, where the threshold value is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units raised above a threshold value, mathematical modeling, signal processing, and combinations thereof.

[0074] B3. The method of item B1 or B2, which further includes real-time monitoring of the fluorescence signals of the plurality of wells to determine whether amplification has occurred in at least one of the plurality of sample wells, and if it is determined that amplification has occurred in the at least one well, perform a melting detection, where the temperature range for the melting detection is restricted by the known melting temperature range of the target nucleic acid in the at least one well.

[0075] B4. The method of any one of items B1 - B3, where the timing of the melting detection is determined by the fluorescence signal of one or more wells configured to amplify the plurality of target nucleic acids rising above a threshold value.

[0076] B5. The method of any one of items B1 - B4, where the method does not include performing a set number of thermal cycles before performing the melting detection.

[0077] B6. The method of any one of items B1 - B5, which further includes not performing a melting detection if no sample well shows a fluorescence signal above a threshold value.

[0078] B7. The method of any one of items B1 - B6, where the plurality of sample wells includes one or more control wells, and where no melting detection is performed if only the one or more control wells show a fluorescence signal above a threshold value.

[0079] B8. The method of any one of items B1 - B7, where the melting detection includes:

[0080] A first heating rate during a first temperature range of melting,

[0081] A second heating rate during a second temperature range of melting, and

[0082] A third heating rate during a third temperature range of melting,

[0083] The second heating rate is slower than the first and third heating rates, and wherein the second temperature range of the melting is defined by the melting temperature range of the target nucleic acid amplified in the sample wells having a fluorescence signal above a threshold.

[0084] The method of any one of items B1 - B8, wherein the second temperature range of the 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.

[0085] The method of any one of items B1 - B9, wherein melting detection is performed and the temperature range of the second part of the melting is determined.

[0086] The method of any one of items B1 - B10, wherein two or more wells show a fluorescence signal above a threshold.

[0087] The method of any one of items B1 - B11, further comprising performing a single melting detection with a melting temperature range for the target nucleic acid amplified in the two or more wells.

[0088] The method of any one of items B1 - B12, further comprising performing a first melting detection with a characteristic first melting temperature range that melts the target nucleic acid amplified in the first well and performing at least a second melting detection with a characteristic second melting temperature range that melts the target nucleic acid amplified in the second well.

[0089] The method of any one of items B1 - B13, wherein the plurality of wells are configured to amplify target nucleic acid sequences from: organisms present at a high titer, organisms present at a lower titer relative to organisms present at a high titer, and organisms present at a low titer relative to organisms present at a lower titer, and the method further comprises:

[0090] If one or more wells configured to amplify target nucleic acid sequences from organisms present at a high titer show amplification above a threshold within 20 or fewer thermal cycles, then perform a first melting detection,

[0091] If one or more wells configured to amplify target nucleic acid sequences from organisms present at a lower titer show amplification above a threshold within 25 or fewer thermal cycles, then perform a second melting detection, and

[0092] If one or more wells configured to amplify target nucleic acid sequences from organisms present at a low titer show amplification above a threshold within 30 or fewer thermal cycles, then perform a third melting detection.

[0093] A method according to any one of clauses B1 - B14, wherein the number of first thermal cycles is 20 thermal cycles or fewer, the number of second thermal cycles is 25 thermal cycles or fewer, and the number of third thermal cycles is 30 thermal cycles or fewer.

[0094] B15. A method according to any one of clauses B1 - B14.1, further comprising not performing melt curve detection if one or more wells configured to amplify target nucleic acid sequences from organisms present at a high titer show amplification above a threshold in more than 20 thermal cycles.

[0095] B16. A method according to any one of clauses B1 - B15, further comprising not performing melt curve detection if one or more wells configured to amplify target nucleic acid sequences from organisms present at a lower titer show amplification above a threshold in more than 25 thermal cycles.

[0096] B16.1 A method according to any one of clauses B1 - B16, further comprising not performing melt curve detection if one or more wells configured to amplify target nucleic acid sequences from organisms present at a second titer show amplification above a threshold in more than a second number of thermal cycles.

[0097] B17. A method according to any one of clauses B1 - B16.1, further comprising not performing melt curve detection if none of the one or more wells configured to amplify target nucleic acid sequences from organisms present at a high titer, a lower titer, or a low titer show amplification above a threshold.

[0098] B18. A method according to any one of clauses B1 - B17, further comprising performing multiplex amplification of the sample before the dispensing step.

[0099] B19. A method according to any one of clauses B1 - B18, wherein all steps are performed within a single closed system.

[0100] B20. A method according to any one of clauses 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.

[0101] C1. A method for determining the presence of organisms in a sample, comprising:

[0102] providing a sample suspected of containing at least one of a plurality of organisms,

[0103] providing a plurality of sample wells, each sample well provided with primers for amplifying a target nucleic acid from a different one of the plurality of organisms,

[0104] transferring a portion of the sample into each of the plurality of sample wells

[0105] Subjecting the plurality of sample wells to amplification conditions simultaneously,

[0106] Obtaining the fluorescence signal in each of the plurality of sample wells during thermal cycling,

[0107] Determining that the fluorescence signal amplitude of a sample well configured to amplify a target nucleic acid is greater than a threshold value that is greater than or equal to the detection limit for inferring an increase in the concentration of nucleic acid in the sample well,

[0108] In response to determining that the fluorescence signal amplitude of the sample well is greater than the threshold value, performing a denaturation detection in a denaturation temperature window to detect the amplified target nucleic acid in the sample well, wherein the denaturation temperature window is defined by a denaturation temperature range characteristic of the target nucleic acid, and

[0109] In response to performing the denaturation detection, determining the presence of an organism in the sample.

[0110] C2. The method of item C1, further comprising subjecting the plurality of sample wells to amplification conditions for a selected number of cycles, and performing denaturation detection if a sample well shows positive nucleic acid amplification within the selected number of amplification cycles, wherein the denaturation detection is defined by a denaturation temperature range characteristic of the target nucleic acid to detect an organism in the sample well.

[0111] C3. The method of item C2, wherein the selected number of amplification cycles is at least one but 15 or fewer, at least one but 20 or fewer, at least one but 25 or fewer, at least one but 30 or fewer, or at least one but 35 or fewer.

[0112] C4. The method of any one of items C1 - C3, further comprising not performing denaturation detection if no sample well shows positive nucleic acid amplification.

[0113] C5. The method of any one of items C1 - C4, further comprising performing multiplex amplification on the sample simultaneously before the moving step.

[0114] C6. The method of any one of items C1 - C5, wherein all steps are performed within a single closed system.

[0115] D1. A system for detecting which of a plurality of target nucleic acids is present in a sample, the system comprising:

[0116] A container containing a plurality of sample wells, each sample well configured to receive a portion of the sample, nucleic acid primers that specifically amplify a target nucleic acid, a fluorescent dye that produces an increased fluorescence signal in response to an increase in the nucleic acid concentration in the plurality of sample wells, and components for amplification; and

[0117] An instrument configured to subject portions of samples in each of the plurality of sample wells to amplification conditions and subsequent denaturation conditions simultaneously, the instrument including a detector for detecting a fluorescence signal indicative of amplification from a fluorescent dye,

[0118] wherein the instrument is programmed to have target nucleic acids to be amplified in each of the plurality of wells, melting range information for each of the target nucleic acids, and wherein 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 amplification conditions, perform a denaturation detection, wherein the denaturation detection is defined by the melting temperature range of the target nucleic acid amplified in the sample well.

[0119] D2. The system of item 1, wherein the instrument is programmed to perform a set number of amplification cycles before performing the denaturation detection.

[0120] D3. The system of item D1 or D2, wherein the instrument is programmed to perform the denaturation detection at any number of amplification cycles if amplification is detected in a sample well, and wherein the temperature range of the denaturation detection is restricted by the melting range information of the target nucleic acid amplified in the well.

[0121] D4. The system of one of items D1 - D3, wherein positive nucleic acid amplification is indicated by an increase in the fluorescence signal in the sample well to above a threshold.

[0122] D5. The system of one of items D1 - D4, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units elevated above a threshold, mathematical modeling, signal processing, and combinations thereof.

[0123] D6. The system of any one of items D1 - D5, wherein the instrument is further programmed not to perform the denaturation detection if no sample well shows positive nucleic acid amplification within a set number of amplification cycles.

[0124] D7. The system of any one of items D1 - D6, wherein the system is programmed to include a denaturation detection, the denaturation detection including:

[0125] a first heating rate during a first portion of the denaturation,

[0126] a second heating rate during a second portion of the denaturation, and

[0127] a third heating rate during a third portion of the denaturation,

[0128] the second heating rate being slower than the first and third heating rates, and wherein the second portion of the denaturation is defined by the melting range information of the target nucleic acid amplified in the well.

[0129] A system according to any one of clauses D1 - D7, wherein the system is programmed to identify at least one of a target organism, cell-free DNA, a cell, or a molecular marker of antimicrobial drug resistance present in a sample by identifying the corresponding sample wells in which amplification and melt curve detection have occurred.

[0130] A system according to any one of clauses D1 - D8, wherein the amplification is PCR and the components include a polymerase and dNTPs.

[0131] A system according to any one of clauses D1 - D9, wherein the instrument includes a light source configured to emit a light signal towards the plurality of sample wells during screening of the corresponding samples.

[0132] A system according to any one of clauses D1 - D10, wherein the melt curve result is based on the presence or absence of a melt peak within a predetermined temperature range, and a positive result is output if a melt peak is present and a negative result is output if no melt peak is present.

[0133] E1. A system for detecting which of a plurality of target nucleic acids are present in a sample, the system comprising:

[0134] A container containing a plurality of sample wells, each sample well configured to receive a portion of the sample, nucleic acid primers that specifically amplify one target nucleic acid, a fluorescent dye that produces an increased fluorescent signal in response to an increase in nucleic acid concentration in the plurality of sample wells, and components for amplification,

[0135] An instrument configured to subject the portion of the sample in each of the plurality of sample wells simultaneously to amplification conditions and subsequent melt curve conditions, the instrument including a detector for detecting a fluorescent signal indicative of amplification from the fluorescent dye, and

[0136] Programming to perform the method according to any one of clauses A1 - C6,

[0137] wherein the instrument is programmed to have the identity of the target nucleic acid to be amplified in each of the plurality of wells and to have melt range information for each of the target nucleic acids.

[0138] F1. A computer-implemented method for determining the target nucleic acid sequence of a sample, the method comprising:

[0139] Sending control signals to a thermal cycling element by one or more processors to heat a plurality of sample wells to a first temperature and cool the plurality of sample wells to a second temperature through one or more cycles using an initial heating rate, wherein each cycle includes an in-cycle temperature adjustment segment, the plurality of sample wells are each configured to receive a portion of a sample, each sample well contains a target nucleic acid sequence from a different one of a plurality of target nucleic acids, and each target nucleic acid from each different organism has a characteristic melting temperature range;

[0140] Receiving, at the one or more processors, data from an optical system indicative of the amount of fluorescence emitted by those portions of the samples in the plurality of sample wells during the in-cycle temperature adjustment segments of the one or more cycles;

[0141] In response to determining that the amount of fluorescence in at least one of the sample wells exceeds a threshold:

[0142] Determining, by the one or more processors, the amplified target nucleic acid sequences in each of the sample wells that exceed the threshold;

[0143] Determining, by the one or more processors, an adjusted heating rate curve for heating the sample wells based on the characteristic melting temperature range and / or melting heating rate of the target nucleic acid sequences in each of the sample wells that exceed the threshold; and

[0144] Performing, by the one or more processors, melting detection on the portions of the samples, including sending control signals to the thermal cycling element to heat the sample wells to the first temperature using the adjusted heating rate curve for subsequent cycles.

[0145] F2. The method of item F1, wherein the first temperature is a denaturation temperature, the second temperature is an annealing temperature, the characteristic melting temperature range of the sample wells is from an initial melting temperature to a final melting temperature, and the adjusted heating rate curve includes:

[0146] A first heating rate during a proximity temperature range from the annealing temperature to the initial melting temperature;

[0147] A second heating rate during the characteristic melting temperature range from the initial melting temperature to the final melting temperature; and

[0148] A third heating rate during an end temperature range from the final melting temperature to the denaturation temperature,

[0149] wherein the second heating rate is slower than the first and third heating rates.

[0150] F3. The method of item F1 or item F2, wherein the third heating rate is faster than the first heating rate.

[0151] A method according to any one of items F1 - F3, wherein the second heating rate is in the range of 0.05 to 4 °C / sec.

[0152] F5. A method according to any one of items F1 - F4, wherein the first heating rate is greater than 4 °C / sec (e.g., between 6 and 20 °C / sec), the second heating rate is less than 4 °C / sec (e.g., in the range of 0.05 to 4 °C / sec), and the third heating rate is greater than 4 °C / sec (e.g., 12 - 20 °C / sec).

[0153] F5.1. A method according to any one of items F1 - F5, wherein the third heating rate is faster than the first heating rate.

[0154] F5.2. A method according to any one of items F1 - F5.1, wherein the second heating rate is in the range of 0.01 to 4 °C / sec or preferably 0.01 to 2 °C / sec.

[0155] F5.3. A method according to any one of items F1 - F5.2, wherein the second heating rate is preferably not greater than 0.5 °C / sec, more preferably not greater than 1 °C / sec, and most preferably not greater than 2 °C / sec.

[0156] F5.4. A method according to any one of items F1 - F5.3, wherein the second heating rate is suitable for detecting nucleic acid melting characteristics indicative of a genotype, sequence variant, or gene mutation in a target nucleic acid, the genotype, sequence variant, or gene mutation altering one or more of the nucleic acid melting temperature or the shape of the nucleic acid melting curve relative to a target nucleic acid that does not have the genotype, sequence variant, or gene mutation.

[0157] F5.5. A method according to any one of items F1 - F5.4, further comprising detecting a single nucleotide polymorphism (SNP).

[0158] F5.6. A method according to any one of items F1 - F5.5, further comprising detecting an antimicrobial resistance (AMR) marker.

[0159] F5.7. A method according to any one of items F1 - F5.6, further comprising detecting the presence of an organism via a first melting detection in a first assay, and if the presence of the organism is detected in the first assay, performing a second melting detection in a second assay to detect the presence or absence of the genotype, sequence variant, or gene mutation.

[0160] F6. A method according to any one of items 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 elevated above a threshold, mathematical modeling, signal processing, and combinations thereof.

[0161] A method according to any one of clauses F1 - F6, wherein the controller is further configured to:

[0162] Identify the target nucleic acid corresponding to the sample well among the plurality of target nucleic acids based on the denaturation detection.

[0163] A method according to any one of clauses F1 - F7, wherein the controller is further configured to:

[0164] Determine that the fluorescence amounts of the first sample well and the second sample well exceed a threshold;

[0165] Perform a first denaturation detection by using a first adjusted heating rate curve corresponding to a first characteristic denaturation temperature range for denaturing the amplified target nucleic acid in the first sample well; and

[0166] Perform a second denaturation detection by using a second adjusted heating rate curve corresponding to a second characteristic denaturation temperature range for denaturing the amplified target nucleic acid in the second sample well.

[0167] G1. A system for determining the target nucleic acid sequence of a sample, the system comprising:

[0168] A plurality of sample wells each configured to accommodate a portion of the sample, each sample well containing a target nucleic acid sequence from a different one of a plurality of target nucleic acids, wherein each target nucleic acid from each different organism has a characteristic denaturation temperature range;

[0169] An optical system configured to detect the fluorescence amount emitted by the sample;

[0170] A controller configured to:

[0171] Send a control signal to a thermal cycling element to heat the plurality of sample wells to a first temperature and cool the plurality of sample wells to a second temperature using an initial heating rate through one or more cycles, wherein each cycle includes an in - cycle temperature adjustment segment;

[0172] Receive data from the optical system indicating the fluorescence amount emitted by those portions of the sample in the plurality of sample wells during the in - cycle temperature adjustment segments of the one or more cycles;

[0173] In response to determining that the fluorescence amount of at least one of the sample wells exceeds a threshold:

[0174] Determine an adjusted heating rate curve for heating the sample well based on the characteristic denaturation temperature range of the target nucleic acid sequence in the sample well that exceeds the threshold; and

[0175] Performing denaturation detection on the sample of the portion by sending a control signal to the thermal cycling element to heat the sample well to a first temperature using the adjusted heating rate curve for subsequent cycles.

[0176] G2. The system of item G1, wherein the first temperature is the denaturation temperature, the second temperature is the annealing temperature, the characteristic denaturation temperature range of the sample well is from an initial denaturation temperature to a final denaturation temperature, and the adjusted heating rate curve includes:

[0177] A first heating rate during a proximity temperature range from the annealing temperature to the initial denaturation temperature;

[0178] A second heating rate during a characteristic denaturation temperature range from the initial denaturation temperature to the final denaturation temperature; and

[0179] A third heating rate during an end temperature range from the final denaturation temperature to the denaturation temperature,

[0180] wherein the second heating rate is slower than the first and third heating rates.

[0181] G3. The system of item G1 or item G2, wherein the third heating rate is faster than the first heating rate.

[0182] G4. The system of any one of items G1 - G3, wherein the second heating rate is the same as the initial heating rate.

[0183] G5. The system of any one of items G1 - G4, wherein the first heating rate is greater than 4 °C / sec (e.g., 6 - 20 °C / sec), the second heating 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 rate is greater than 4 °C / sec (e.g., 6 - 20 °C / sec).

[0184] G6. The system of any one of items G1 - G5, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units elevated above the threshold, mathematical modeling, signal processing, and combinations thereof.

[0185] G7. The system of any one of items G1 - G6, wherein the controller is further configured to:

[0186] Identify the target nucleic acid corresponding to the sample well among the multiple target nucleic acids based on the denaturation detection.

[0187] G8. The system of any one of items G1 - G7, wherein the controller is further configured to:

[0188] Determine that the fluorescence amounts in the first sample well and the second sample well exceed the threshold;

[0189] Perform a first melting detection by using a first adjusted heating rate curve corresponding to a first characteristic melting temperature range for denaturing the amplified target nucleic acid in the first sample well; and

[0190] Perform a second melting detection by using a second adjusted heating rate curve corresponding to a second characteristic melting temperature range for denaturing the amplified target nucleic acid in the second sample well.

[0191] H1. A computing device for determining a target nucleic acid sequence of a sample, the computing device comprising:

[0192] One or more processors; and

[0193] A non-transitory computer-readable memory coupled to the one or more processors and storing instructions thereon that, when executed by the one or more processors, cause the computing device to:

[0194] Send a control signal to a thermal cycling element to heat a plurality of sample wells to a first temperature and cool the plurality of sample wells to a second temperature using an initial heating rate through one or more cycles, where each cycle includes an in-cycle temperature adjustment segment, the plurality of sample wells are each configured to receive a portion of a sample, each sample well contains a target nucleic acid sequence from a different one of a plurality of target nucleic acids, and each target nucleic acid from each different organism has a characteristic melting temperature range;

[0195] Receive from an optical system data indicating the fluorescence amounts emitted by those portions of the samples in the plurality of sample wells during the in-cycle temperature adjustment segments of the one or more cycles;

[0196] In response to determining that the fluorescence amount in at least one of the sample wells exceeds a threshold:

[0197] Determine an adjusted heating rate curve for heating the sample well based on the characteristic melting temperature range of the target nucleic acid sequence in the sample well that exceeds the threshold; and

[0198] Perform a melting detection on the portion of the sample by sending a control signal to the thermal cycling element to heat the sample well to the first temperature using the adjusted heating rate curve for subsequent cycles.

[0199] H2. The computing device of item H1, wherein 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 heating rate curve includes:

[0200] The first heating rate during a temperature range approaching from the annealing temperature to the initial melting temperature;

[0201] The second heating rate during a characteristic melting temperature range from the initial melting temperature to the final melting temperature; and

[0202] The third heating rate during an end temperature range from the final melting temperature to the denaturation temperature,

[0203] wherein the second heating rate is slower than the first and third heating rates.

[0204] H3. The computing device of item H1 or item H2, wherein the third heating rate is faster than the first heating rate.

[0205] H4. The computing device of any one of items H1-H3, wherein the second heating rate is the same as the initial heating rate.

[0206] H5. The computing device of any one of items H1-H4, wherein the first heating rate is greater than 4 °C / sec (e.g., 6-20 °C / sec), the second heating 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 rate is greater than 4 °C / sec (e.g., 6-20 °C / sec).

[0207] H6. The computing device of any one of items H1-H5, wherein the threshold is one of a crossing point (Cp), Cp plus one or more additional amplification cycles, Ct, relative fluorescence units raised above the threshold, mathematical modeling, signal processing, and combinations thereof.

[0208] H7. The computing device of any one of items H1-H6, wherein the instructions further cause the computing device to:

[0209] Identify the target nucleic acid corresponding to the sample well among the plurality of target nucleic acids based on the melting detection.

[0210] H8. The computing device of any one of items H1-H7, wherein the instructions further cause the computing device to:

[0211] Determine that the fluorescence amounts of a first sample well and a second sample well exceed the threshold;

[0212] Perform a first melting detection by using a first adjusted heating rate curve corresponding to a first characteristic melting temperature range that causes the target nucleic acid amplified in the first sample well to melt; and

[0213] A second melting detection is performed by using a second adjusted heating rate curve corresponding to a second characteristic melting temperature range for melting the amplified target nucleic acid in the second sample well.

[0214] The present disclosure also provides reaction vessels and devices suitably useful for any of the methods described herein.

[0215] Additional features of the invention will become apparent to those skilled in the art by considering the following detailed description of the preferred embodiments which exemplify the presently contemplated best mode of carrying out the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0216] Figure 1 A schematic bag suitably useful in embodiments of the present disclosure is shown.

[0217] Figure 2 Schematic stages of a melting program suitably useful in embodiments of the present disclosure are shown.

[0218] Figure 3A and 3B Examples comparing slow melting with a fixed temperature increase rate and fast melting with a fixed temperature increase rate are shown.

[0219] Figure 4A Examples comparing slow melting with a fixed heating rate and dynamic melting with a variable heating rate are shown, the variable heating rate using a faster heating rate in a temperature range where no melting features are expected to occur in the reaction and a slower heating rate in a temperature range where melting features are expected to occur in the reaction.

[0220] Figure 4B Shows similar to Figure 4A Another example which compares slow melting with a fixed heating rate and dynamic melting with a variable heating rate.

[0221] Figure 5A An example of performing dynamic melting analysis on a target assay is illustrated.

[0222] Figure 5B Shows for Figure 5A an example of performing dynamic melting on a target after a fixed number of amplification cycles.

[0223] Figure 6A An example of performing dynamic melting analysis on two target assays with one melting temperature range is illustrated.

[0224] Figure 6B Shows for Figure 6A an example of performing dynamic melting on two targets after a fixed number of amplification cycles.

[0225] Figure 7A An example of performing a dynamic melting analysis on an analyte after a dynamically determined number of cycles rather than a fixed number of amplification cycles is shown.

[0226] Figure 7B An example is shown Figure 7A of a dynamic melting where melting is performed based on when the analyte shows amplification above a threshold.

[0227] Figure 8A An example of a determination specific melting detection of a dynamic melting for each target is illustrated, and the number of amplification cycles before each melting is dynamically determined based on when each target shows amplification above a threshold.

[0228] Figure 8B Illustrates Figure 8A the melting specific parameters of the first target.

[0229] Figure 8C Illustrates Figure 8A and 8B the dynamic melting results of the first target illustrated in

[0230] Figure 8D Illustrates Figure 8A the melting specific parameters of the second target.

[0231] Figure 8E Illustrates Figure 8A and 8D the dynamic melting results of the second target illustrated in

[0232] Figure 9 A block diagram of an exemplary embodiment of a thermal cycling system according to aspects of the present disclosure is illustrated.

[0233] Figure 10 A flowchart of an example computer-implemented dynamic melting detection method for determining a target nucleic acid sequence of a sample is illustrated.

[0234] Figure 11A An example of a temperature increase curve for a nucleic acid melting experiment is illustrated.

[0235] Figure 11B An example of nucleic acid melting curves (left inset) and derivative melting curves (right inset) of two amplicons with different melting temperatures is illustrated.

[0236] Figure 12 An illustrative melting program is illustrated that compares a standard melting protocol (……), a dynamic melting protocol (–––), and a dynamic "high resolution" protocol (·–·–).

[0237] Figure 13 It schematically shows the Neisseria gonorrhoeae organism assay (●Org) and the separate assays for the mutation-prone regions of the gyrA gene (○S91F and +WT).

[0238] Figure 14A and 14B The melting curves of the wild-type (–––MG-WT) and A2059G (–––2059G) mutant amplicons of the 23S ribosomal RNA gene of Mycoplasma genitalium were compared.

[0239] Figure 15A and 15B The melting curves of the wild-type (–––MG-WT) and A2058G (–··–2058G) mutant amplicons of the 23S ribosomal RNA gene of Mycoplasma genitalium were compared.

[0240] Figure 16A and 16B The melting curves of the wild-type (–––NG-WT) and S91F (……S91F) mutant amplicons of the gyrA gene of Neisseria gonorrhoeae were compared. DETAILED DESCRIPTION

[0241] As used herein, the terms "a", "an", and "the" are defined to mean "one or more" and include plural referents unless the context dictates otherwise. 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, roughly, around, or in the vicinity of. 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. Generally, the term "about" is used herein to modify a numerical value by plus or minus 5% of the stated value. When expressing such ranges, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by use of the antecedent "about", it is to be understood that the particular value forms another embodiment. It should also be understood that each endpoint of each range is significant both in relation to the other endpoint and independently of the other endpoint.

[0242] As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list.

[0243] "Sample" means an animal; a tissue or organ from an animal; a cell (or a cell that is in a subject, taken directly from a subject, or maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; a solution containing one or more molecules (such as a polypeptide or nucleic acid) derived from a cell, cell material, or viral material; or a solution containing a non-naturally occurring nucleic acid, as measured herein. A sample can also be any body fluid or excrement containing cells, cell components, or nucleic acids (such as, but not limited to, blood, urine, feces, saliva, tears, bile, cerebrospinal fluid).

[0244] As used herein, the term "reaction" can refer to a nucleic acid amplification reaction (such as, for example, a PCR reaction) for amplifying a given target nucleic acid. As used herein, the term "assay" can be used to refer to one or more reactions carried out in a single well or container. As used herein, the term "panel" can be used to refer to a collection of assays that can be grouped together to test a set of targets (such as, for example, a panel for testing respiratory pathogens). For example, a panel can be designed to include multiple assays for amplifying and detecting 20 or more targets.

[0245] As used herein, the expression "nucleic acid" refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA, RNA, or a DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, that is capable of hybridizing with a complementary nucleic acid through Watson-Crick base pairing. The nucleic acids of the present invention can also include nucleotide analogs (such as, for example, BrdU) and non-phosphodiester internucleoside linkages (such as, for example, peptide nucleic acid (PNA) or phosphorothioate linkages). In particular, nucleic acids can include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0246] "Probe", "primer", or "oligonucleotide" refers to a single-stranded DNA or RNA molecule having a defined sequence that can base pair with a second DNA or RNA molecule containing a complementary sequence ("target"). The stability of the resulting hybrid depends on the length, GC content, and 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 the target molecule and the stringency of the hybridization conditions. The stringency of hybridization 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 by methods known to those of skill in the art, whether radioactively labeled, fluorescently labeled, or non-radioactively labeled. A dsDNA-binding dye can be used to detect dsDNA. It should be understood that a "primer" is specifically configured to be extended by a polymerase, while a "probe" or "oligonucleotide" may or may not be so configured.

[0247] "dsDNA-binding dye" refers to a dye that emits different fluorescence when binding to double-stranded DNA than when binding to single-stranded DNA or when free in solution, typically emitting stronger fluorescence in the former case. Although dsDNA-binding dyes are mentioned, it should be understood that any suitable dye can be used herein, and some non-limiting exemplary dyes are described in U.S. Patent No. 7,387,887, which is incorporated herein by reference. Other signal-generating substances can also be used to detect nucleic acid amplification and denaturation, such as enzymes, antibodies, etc., as known in the art.

[0248] "Specific hybridization" refers to the physical interaction (i.e., base pairing) of a probe, primer, or oligonucleotide with a substantially complementary nucleic acid (e.g., a sample nucleic acid) under high-stringency conditions and substantially no base pairing with other nucleic acids.

[0249] "High-stringency conditions" refer to conditions that permit hybridization, and the hybridization results can be comparable to those obtained by hybridizing with a DNA probe having a length of at least 40 nucleotides at a temperature of 65°C in a buffer containing 0.5 M NaHPO4 (pH 7.2), 7% SDS, 1 mM EDTA, and 1% BSA (fraction V) or at a temperature of 42°C 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. Other high-stringency hybridization conditions, such as for PCR, Northern, Southern, or in situ hybridization, DNA sequencing, etc., are well known to those skilled in the art of molecular biology.

[0250] Although the amplification method used in the examples herein is PCR, it should be understood that any amplification method using primers may be suitable. Such suitable procedures include polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), rolling circle cascade amplification (CRCA), loop-mediated isothermal DNA amplification (LAMP), isothermal and chimeric primer-initiated nucleic acid amplification (ICAN), target-based helicase-dependent amplification (HDA), transcription-mediated amplification (TMA), etc. Therefore, when using the term PCR, it should be understood to include other alternative amplification methods. For amplification methods without discrete cycles, in the embodiments described herein, reaction time can be used in cases where measurement is made in terms of cycles or Cp, and additional reaction time can be added in cases where additional PCR cycles are added. It should be understood that the protocol may need to be adjusted accordingly.

[0251] The various embodiments disclosed herein use a stand-alone nucleic acid analysis pouch to determine the presence of various biological substances (schematically, antigens and nucleic acid sequences) in a sample, schematically, in a single closed system. Such systems, including the pouch and the instrument for use with the pouch, are disclosed in more detail in U.S. Patent No. 8,394,608, U.S. Patent No. 8,895,295, and U.S. Patent No. 10,464,060, which are incorporated herein by reference. However, it should be understood that such a pouch is merely schematic, and the multiplex PCR reactions discussed herein can be performed in any of a variety of open or closed system sample containers known in the art (including 96-well plates, plates of other configurations, arrays, rotors, etc.) using a variety of amplification systems known in the art. Although the term "sample well" is used herein, the term is intended to cover wells, tubes, and various other reaction vessels as used in these amplification systems. In one embodiment, the pouch is used to assay for multiple pathogens. Schematically, the various steps, including nucleic acid preparation, one large-volume multiplex PCR, one dilution of the amplification product, and a second PCR, and finally optional real-time detection or post-amplification analysis such as melt-curve analysis, can be performed in an optionally disposable pouch. In addition, it should be understood that although the various steps can be performed in the pouch of the present invention, one or more steps can be omitted for certain uses, and the pouch configuration can be changed accordingly.

[0252] Figure 1 A schematic pouch 510 for the present invention is shown. Pouch 510 is similar to FIG. 15 of U.S. Patent No. 8,895,295, which is incorporated by reference, and like items are numbered the same. Fitting 590 is provided with inlet channels 515a to 515l, which also serve as reagent reservoirs. Schematically, the reagents can be lyophilized in fitting 590 and rehydrated before use. Blisters 522, 544, 546, 548, 564, and 566 with their respective channels 538, 543, 552, 553, 562, and 565 are similar to the identically numbered blisters of FIG. 15 of U.S. Patent No. 8,895,295. Figure 1 The second-stage reaction zone 580 of is similar to U.S. Patent No. 8,895,295, but the second-stage wells 582 of the high-density array 581 are arranged in a slightly different pattern. Figure 1 The more circular pattern of the high-density array 581 of eliminates corners and can result in a more uniform filling of the second-stage wells 582. As shown, the high-density array 581 is provided with 102 second-stage wells 582. Pouch 510 is suitable for use in a FilmArray instrument. However, it should be understood that the pouch embodiment is merely schematic.

[0253] The bag 510 can be used in a manner similar to that described in U.S. Patent No. 8,895,295, which is hereby incorporated by reference. A 300 μl mixture containing a sample to be tested (100 μl) and lysis buffer (200 μl) is injected into an injection port (not shown) in the fitting 590 near the inlet channel 515a, and the sample mixture is aspirated into the inlet channel 515a. Water is also injected into a second injection port (not shown) of the fitting 590 adjacent to the inlet channel 515l and is distributed via a channel (not shown) provided in the fitting 590, thereby hydrating up to eleven different reagents, each of which was previously provided in a dry form at the inlet channels 515b to 515l. Schematically, these reagents can include lyophilized PCR reagents, DNA extraction reagents, wash solutions, immunoassay reagents, or other chemical entities. Schematically, the reagents are used 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 Figure 1 the embodiment shown in, all that needs to be injected is the sample solution injected into one injection port and water injected into the other injection port. After injection, the two injection ports can be sealed. For more information on the various configurations of the bag 510 and the fitting 590, see U.S. Patent No. 8,895,295, which is hereby incorporated by reference.

[0254] After injection, the sample moves from the injection channel 515a to the lysis blister 522 via the channel 514. The lysis blister 522 is provided with ceramic beads and is configured for vortexing by impact using a rotating blade or paddle provided within the FilmArray instrument. Once the cells have been lysed sufficiently, the sample moves through the channel 538, the blister 544, and the channel 543 to the blister 546, where the sample is mixed with nucleic acid-binding magnetic beads. The mixture is incubated for an appropriate length of time, schematically, from about 10 seconds to 10 minutes. A retractable magnet located within the FilmArray instrument adjacent to the blister 546 captures the magnetic beads from the solution, forming a precipitate against the inner surface of the blister 546. The liquid is then removed from the blister 546 and returned through the blister 544 and into the blister 522, which now serves as a waste receiver. One or more wash buffers from one or more of the inlet channels 515c to 515e are provided to the blister 546 via the blister 544 and the channel 543. Optionally, the magnet is retracted and the magnetic beads are washed by moving the beads back and forth between the blisters 544 and 546 via the channel 543. Once the magnetic beads have been washed, the magnetic beads are recaptured in the blister 546 by magnet activation, and then the wash solution is moved to the blister 522. The process can be repeated as necessary to wash the lysis buffer and sample debris from the nucleic acid-binding magnetic beads.

[0255] After washing, the elution buffer stored at the injection channel 515f is transferred to the blister 548, and the magnet is retracted. The solution is circulated between the blisters 546 and 548 via the channel 552, breaking up the pellet of magnetic beads in the blister 546 and allowing the captured nucleic acid to dissociate from the beads and enter the solution. The magnet is activated again, thereby capturing the magnetic beads in the blister 546 and transferring the eluted nucleic acid solution into the blister 548.

[0256] The first-stage PCR master mix from the injection channel 515g is mixed with the nucleic acid sample in the blister 548. Optionally, the mixture is mixed by forcing the mixture via the channel 553 between 548 and 564. After several mixing cycles, the solution is contained in the blister 564, where a pellet of the first-stage PCR primers is provided, with at least one set of primers for each target organism, and the first-stage multiplex PCR is performed. If RNA targets are present, an RT step can be performed before or simultaneously with the first-stage multiplex PCR. The first-stage multiplex PCR temperature cycling in the FilmArray instrument is schematically carried out for 15 - 20 cycles, but other levels of amplification may be desired, depending on the needs of the specific application.

[0257] After the first-stage PCR has been carried out for the desired number of cycles, the sample can be diluted, schematically by forcing most of the sample back into the blister 548, leaving only a small amount of the sample in the blister 564, and adding the second-stage PCR master mix from the injection channel 515i. Alternatively, the dilution buffer from 515i can be transferred to the blister 566 and then mixed with the amplified sample in the blister 564 by moving the fluid back and forth between the blisters 564 and 566. If desired, the dilution can be repeated several times using the dilution buffer from the injection channels 515j and 515k, and then the second-stage PCR master mix from the injection channel 515h is added to some or all of the diluted amplified samples. It should be understood that the dilution level can be adjusted by changing the number of dilution steps or by changing the percentage of the sample discarded before mixing with the dilution buffer or the second-stage PCR master mix containing the components for amplification (schematically, polymerase, dNTP, and a suitable buffer), but other components may be suitable, especially for non-PCR amplification methods. If desired, this mixture of the sample and the second-stage PCR master mix can be preheated in the blister 564 before being transferred to the second-stage well 582 for the second-stage amplification. Such preheating can obviate the need for heat-start components (antibodies, chemicals, or others) in the second-stage PCR mixture.

[0258] The illustrative 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 can lack other reaction components, and these components can also be pre-loaded in the second-stage wells 582. Each primer pair can be similar or identical to the first-stage PCR primer pair, or can be nested within the first-stage primer pair. The sample is transferred from the blister 564 to the second-stage wells 582 to complete the PCR reaction mixture. Once the high-density array 581 is filled, the individual second-stage reactions are sealed in their respective second-stage blisters by any number of means, as known in the art. A schematic manner of filling and sealing the high-density array 581 without cross-contamination is discussed in U.S. Patent No. 8,895,295, which is hereby incorporated by reference. Schematically, the various reactions in the wells 582 of the high-density array 581 are thermocycled simultaneously, schematically using one or more Peltier devices, but other means for thermocycling are known in the art.

[0259] The illustrative second-stage PCR master mix contains a dsDNA-binding dye Plus to produce a signal indicative of amplification. However, it should be understood that this dye is merely illustrative, and other signals can be used, including other dsDNA-binding dyes and fluorescent labels, radioactive labels, chemiluminescent labels, enzymatically labeled probes, etc., as known in the art.

[0260] The illustrative FilmArray instrument is programmed to make a positive or negative determination for each second-stage reaction based on post-PCR melting. For the determination to be positive, the melting curve must produce a melting peak (a first derivative maximum or a negative first derivative maximum) within a predetermined temperature range. It should be understood that this method of determining each second-stage reaction is merely illustrative, and the determination can be made using real-time amplification data or by other means as known in the art.

[0261] Example 1

[0262] Design the melting of amplicons at the end of nucleic acid amplification (e.g., PCR) to confirm that the amplification product is in fact the expected or desired product. That is, real-time monitoring of nucleic acid amplification can be used to identify the presence of amplification, but does not inform the user what has been amplified. The amplification product may be the expected product, or it may be a contaminant, the result of non-specific amplification, etc. Since amplicons can be designed to have specific and known melting properties, post-amplification melting can be used to confirm that the correct product has been prepared.

[0263] Many nucleic acid amplification reactions include nucleic acid binding dyes that are capable of incorporating into double-stranded nucleic acids (e.g., dsDNA). The dye is incorporated when double-stranded nucleic acids are generated in the reaction. Many nucleic acid binding dyes are known to be compatible with nucleic acid amplification conditions, bind tightly to double-stranded nucleic acids, and emit strong fluorescence in the presence of double-stranded nucleic acids. When the nucleic acid (e.g., due to high temperature) denatures, the dye is released and, in the presence of single-stranded nucleic acids, the dye emits weak fluorescence or no fluorescence at all. Such nucleic acid binding dyes typically also emit fluorescence in a concentration-dependent manner in the presence of double-stranded nucleic acids, i.e., the more double-stranded nucleic acids in the reaction, the stronger the fluorescence produced. This can be used to monitor in real time the success (or failure) of a nucleic acid amplification reaction by looking for an increase in fluorescence above a threshold. Examples of the "thresholds" used in the embodiments described herein and in the claims to determine when fluorescence increases and when denaturation can occur include, but are not limited to, the crossing point (Cp), Cp plus one or more additional amplification cycles, the crossing threshold (Ct), relative fluorescence units elevated 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 combinations thereof.

[0264] As used herein, "Cp" or "crossing point" refers to the number of PCR cycles or fractional cycles required to obtain a fluorescence signal above a predetermined threshold. For example, the threshold can be a detection level or the point at which the fluorescence measurement of the reaction reaches an intensity above the background fluorescence level. Cp can be determined experimentally based on a manually set threshold, but other methods for determining Cp are known in the art. Other points can also be used, such as using first, second, or nth derivatives, as taught schematically in U.S. Patent No. 6,303,305, which is incorporated herein by reference in its entirety. Other points known in the art can also be used, and in any of the methods discussed herein, any such point can substitute for Cp. The terms "Ct", "crossing threshold", and "Cq" are generally synonymous with the crossing point (Cp) and these terms can be used interchangeably.

[0265] "Relative fluorescence units" (RFU) are defined herein as fluorescence intensity values reported relative to another value. Fluorescence units are a dimensionless term, and thus, the intensity of a fluorescence signal is typically reported relative to another measurement or relative to a reference measurement employed by the instrument. For example, a baseline fluorescence value can be measured and then the RFU can be reported as a multiple of the baseline value, i.e., 2 RFU, 5 RFU, 10 RFU, etc. Examples of suitable baseline values can include, but are not limited to, background fluorescence or any arbitrary value above or below the baseline. In any given experiment or experimental group using RFU, it is important to use a consistent and reproducible value as the baseline.

[0266] As used herein, "Tm" is the temperature at which half of a DNA duplex dissociates into single strands. In an amplification system, Tm is typically measured after amplification, but techniques for measuring Tm during amplification are known.

[0267] The following are two examples of methods for determining whether amplification has occurred and is suitable for triggering melt detection. These and similar methods can be used in any of the methods and systems described and claimed herein.

[0268] In the first example, a set of reaction wells includes wells with all amplification reagents except the amplification primers (blank 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 use the average fluorescence of the blank wells as a baseline and subtract the baseline fluorescence from the target well fluorescence at a given cycle N (e.g., cycle 5). Other methods for determining the baseline fluorescence are known in the art and can be used in the methods described herein. A target can be run in triplicate (i.e., the target can be amplified in three separate target wells). If, for at least one of the three target wells, at least two of the three target wells, or all three target wells, the value (the difference between the target well at cycle N and the average of the blank wells at cycle N) is above a threshold, melt can be triggered. The basis for the method of using blank wells to define the background is to check whether there has been sufficient change in the RFU of the target wells relative to the baseline to determine that amplification has occurred. This method can be used for assays that are expected to amplify and show a detectable Cp within the number of cycles designed for the assay (e.g., within 15 - 30 PCR cycles) if a target is present.

[0269] The second non-limiting example is designed for assays that require higher sensitivity. A similar algorithm to the previous example can be used, but instead of comparing the target wells to the blank wells 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 at earlier cycles (e.g., N - 1, N - 2, N - 3, etc.). The target wells are still first "compared" to the blank wells to subtract the fluorescence of the blank wells (i.e., the background fluorescence). This is still important for normalizing the overall changes in fluorescence that are seen in both the target and blank wells. If, for at least one of the three target wells, at least two of the three target wells, or all three target wells, the increase in the fluorescence value from an earlier cycle (e.g., N - 1) to cycle N is above a threshold, melt can be triggered. The basis for this method is to check whether the RFU is changing fast enough to determine that amplification is occurring. This algorithm can be used for assays where low-level amplification may occur at the end of the cycle (possibly without a typical Cp determination) but can still trigger melt in response to amplification.

[0270] Copies of double-stranded nucleic acids generated during nucleic acid amplification reactions (referred to as products or amplicons) will have unique sequences based on the template being amplified. The amplicon length and sequence / composition (usually, A-T / G-C content) determine the temperature at which the double-stranded DNA will unwind, which is referred to as the melting temperature (Tm) of the amplicon. Products made from different targets will have different sequences and thus different Tm values. Therefore, nucleic acid-binding dyes added to the reaction can also be used for nucleic acid melting analysis. A specific product will have a specific melting signature (i.e., the temperature transition range over which fluorescence decays as the duplex transitions from fully double-stranded to fully single-stranded), and thus nucleic acid melting can be used to confirm the presence of the correct product in a nucleic acid amplification reaction.

[0271] Typical melting experiments are performed by gradually increasing the temperature of the reaction over a range starting from the annealing temperature or below the annealing temperature to 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 and the fluorescence decreases as the nucleic acid-binding dye is released. This will produce a melting curve, see Figure 11B the left inset of, which shows a rapid decrease in fluorescence as the amplicon melts. By plotting the negative first derivative of the melting curve, a melting peak with a specific Tm is generated for each amplicon, as shown in Figure 11B the right inset of.

[0272] Melting analysis can be used to identify specific PCR products. Since the sequence and Tm of an amplicon from a specific target are known and consistent, a specific PCR product can be identified as being copied from that target. Nonspecific PCR products, contaminating products, and analogs with different Tm values can be excluded.

[0273] Compared to a typical increasing melting temperature (such as that shown in Figure 11A ), Figure 2 shows schematic stages of a melting procedure 20 suitably useful in embodiments of the present disclosure. Melting procedure 20 schematically shows the last two cycles 22 of nucleic acid amplification and a subsequent increasing melting temperature 24. The end of amplification can be followed by nucleic acid amplification cycles, followed by an increasing melting temperature, or an intermediate stage of amplification (e.g., after 10 - 15 amplification cycles) can be followed by an increasing melting temperature, followed by additional amplification cycles. Compared to Figure 11AThe exemplary melting temperature ramps shown are different, and the melting temperature increase 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). Suitably, different ramp rates 26, 27, and 28 can be selected such that the instrument rapidly increases the temperature in regions where no melting information is expected during the melting temperature increase 24 (i.e., the nucleic acid duplex is either an un-melted duplex (near region 26) or fully melted (end region 28)), e.g., near region 26 and end region 28, while increasing the temperature more slowly in the region of interest where a melting transition of the relevant amplicon(s) (i.e., from double-stranded to single-stranded) is expected, e.g., range 27. For example, the temperature in ranges 26 and 28 can be rapidly increased (in ranges 26 and 28, greater than 4 °C / sec, e.g., at 4 - 20 °C / sec), while the temperature in range 27 can be increased more slowly (less than 4 °C / sec, e.g., 0.01 - 4 °C / sec, or e.g., 1 - 2 °C / sec). The foregoing melting ramp rates are illustrative and do not limit the disclosure herein. The temperature can be increased much faster than 20 °C / sec (e.g., 100 °C / sec or greater), and some melting can be performed with ramp rates less than 0.01 °C / sec and greater than 4 °C / sec. This novel melting procedure can save a significant amount of time during melting analysis because the temperature can be rapidly increased in portions of the melting cycle where no melting information is expected and then can be slowly increased only in specific windows where melting of the relevant amplicon(s) is expected. This novel melting procedure can also maintain the high resolution of slow-ramp melting because the temperature is slowly increased in a narrow region of interest around the melting transition temperature of the selected amplicon. Although the devices described herein can be designed to assay for the presence of many potential targets (e.g., up to 30 or more organisms and / or molecular markers), a typical assay scenario may not include any positives or may include only one or two positives. The present invention targets the melting temperature ramp to the actually present amplicon rather than slowly increasing the temperature across the entire melting window designed to cover all possible amplicons.

[0274] The different ramp rates shown at 26, 27, and 28 can be suitably and dynamically selected according to the invention described herein. Referring again to Figure 1, each point of the second-stage holes 582 in the second-stage reaction zone 580 has a specific primer pair for amplifying a target nucleic acid. Amplification of some targets can suitably be performed in duplicate or triplicate, so the same primer pair can be spotted at points in more than one second-stage hole 582. Spotting of the second-stage holes 582 is a specific part of the assay design and requires careful planning. Thus, an instrument with instrument software designed to perform assays with the pouch 510 knows the location of each second-stage hole 582 and, for a given assay, knows in advance the identity and details of the target amplicons expected to be amplified in each second-stage hole 582. When the holes in the second-stage reaction zone 580 show fluorescence indicative of successful amplification, the melting characteristics of the target amplicons in that hole are generally known, and the identity of the analyte (e.g., the analyte for detecting an organism in a sample) associated with that hole is also known. This description of the assay design and primer spotting is specific to FilmArray, but one of ordinary skill in the art should understand that the described principles can be applied to any assay using nucleic acid amplification, such as but not limited to tube-based or 96-well plate-based assays. Thus, the rapid temperature rise ranges 26 and 28 and the slow temperature rise range 27 can suitably be selected based on the expected melting temperature range of the specific amplicon in the sample well in which positive amplification has been detected. The start and end points of the slow temperature rise range 27 can suitably be dynamically selected from the melting ranges of the multiple amplicons in the assay based on which assay (i.e., which reactions in the sample wells) in the panel is actually amplification positive.

[0275] Now refer to Figure 3A and 3B , which shows an example comparing slow melting performed at a fixed rate of temperature increase with fast melting performed at a faster fixed rate of temperature increase. Figure 3A and 3B The difference between Figure 3A is that melting is based on amplified fluorescence at 10 RFU ( Figure 3B ) as compared to 4 RFU ( Figure 3A ). In other words, Figure 3B the amplicons in Figure 3A are amplified to a higher concentration than those in Figure 3B ; this difference results in a stronger fluorescence signal in Figure 3A than in Figure 3B . Negative first derivatives of the slow melting are shown at 31 of Figure 11B and 35 of Figure 3A . The negative first derivative curves shown at 31 and 35 are similar to the negative first derivative melting curves shown in 3B . For Figure 11BThe negative first derivative curves shown are for a temperature increase of approximately 2 °C / sec in the range starting at or below the annealing temperature to above the denaturation temperature (e.g., from about 60 °C to 98 °C). This is the standard melting protocol for FilmArray. In each case, the melting peaks shown in the negative derivative curves are distinct. In contrast, Figure 3A and 3B show negative derivative curves 33 and 37 resulting from temperature increase rates of 12 °C / sec shown at 32 and 36. The only difference between negative derivative curves 31 and 35 and negative derivative curves 33 and 37 is the temperature increase rate. As can be seen from negative derivative curves 33 and 37, between curves 31 and 33, the peak flattens and drifts to higher temperature, and compared to negative derivative curve 35, negative derivative curve 37 does not show a peak. These rapid meltings suggest that rapid temperature increase throughout the melting range may not be sufficient to maintain the melting signal compared to standard melting.

[0276] Now refer to Figure 4A and 4B , which show an example comparing slow melting with a fixed temperature increase rate to melting with a variable temperature increase rate that uses a faster rate in temperature ranges where no melting features are expected and a slower rate in temperature ranges where melting features are expected (fast - slow - fast). Figure 4A and 4B differ in the concentration of nucleic acid at melting and thus the intensity of the fluorescence signal: 10 RFU ( Figure 4A ) versus 4 RFU ( Figure 4B ). In other words, Figure 4A the amplicons in Figure 4B were amplified to a higher concentration than those in Figure 4A before melting analysis; this difference results in a stronger fluorescence signal in Figure 4B than in Figure 3A and 3B . These relative RFUs are similar to those shown in Figure 3A - 4B . Although there is a difference in the threshold concentration before melting analysis (i.e., 10 RFU versus 4 RFU), the derivative melting curves for both sets of data are distinct. Figure 3A - 4B The 2 °C / sec melting shown in Figure 4A and 4B is essentially the same. However, Figure 3A and 3B the melting using a modified dynamic (i.e., fast - slow - fast (6 - 12 °C / sec, 2 °C / sec, 12 °C / sec)) melting temperature increase in is distinct, while Figure 3A and 3B the 12 °C / sec melting shown in is not available.

[0277] can produce, for example, such as Figure 4A and 4BThe denatured nucleic acid amplification reactions shown at 43 and 47 may suitably be included within a panel for more than one organism, and thus, the variable heating rate denaturation information (i.e., using a rapid heating rate in a temperature range where no denaturation features are expected and a slower heating rate in a temperature range where denaturation features are expected) may suitably be determined dynamically based on the organisms showing positive amplification in the nucleic acid amplification panel.

[0278] Figure 4A and 4B the denaturation heating at 40 and 44 and the derivative curves 41 and 45 are the same as those Figure 3A and 3B shown at 30 and 34 and 31 and 35 in. These derivative denaturation curves are generated from denaturation data collected at a heating rate of 2 °C / sec and have well-defined peaks. Similarly, the derivative denaturation curves 43 and 47 also have well-defined peaks. However, the denaturation heating 42 and 46 used to generate these derivative denaturation curves include rapid heating similar to those of denaturation heating 32 and 36 for the approaching and ending portions (for the approaching portion, the temperature increases at 6 - 12 °C / sec, and for the ending portion, the temperature increases at 12 °C / sec), which may suitably save a significant amount of time in the denaturation program. To maintain denaturation resolution, the denaturation heating 42 and 46 include a slow heating portion (about 2 °C / sec) around the denaturation transition temperature specific to the detected amplicon. The denaturation heating 42 and 46 include much of the speed of denaturation heating 32 and 36, but different from denaturation heating 32 and 36, the denaturation heating 42 and 46 maintain the high resolution of slow denaturation with its fast, slow, fast heating program. That is, dynamic denaturation has a denaturation resolution similar to that of the industry standard steady heating rate denaturation using a constant heating rate throughout the denaturation program, while potentially saving a significant amount of time compared to such industry standard denaturation.

[0279] The total time savings associated with the novel dynamic melting schemes described herein may suitably depend on factors such as but not limited to the type of scheme used (e.g., performing a fixed number of cycles before melting or melting one or more analytes as soon as amplification above a selected threshold is detected) and how many analytes are positive. If no analytes are detected (i.e., if the assay result is negative for all analytes), it may suitably be possible to report a negative run result without performing any melting steps. Thus, the time for melting detection and melting data processing can be fully deducted from the panel run time. For the FilmArray panel, a negative run without melting can save ~40 seconds of melting 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 meltings will typically be performed. For example, for single- and three-analyte positive assays, the total time savings for FilmArray runs are typically ~30 seconds or ~10 seconds, respectively. Although these savings may not be considered substantial time savings, any time savings that shorten the time required to obtain results are significant as the overall reaction time gets shorter and as testing moves into more patient-proximal settings. The time savings associated with the novel dynamic melting procedures disclosed herein will be discussed in more detail in Example 6 below.

[0280] In addition to the foregoing, the novel dynamic melting procedures described herein may suitably also allow for positive results of some assays to be reported before the thermal cycling is fully complete and for confirmatory results to be provided at the end of the test. That is, dynamic confirmatory melting can be performed and results can be reported as soon as amplification is detected. This again reduces the time the patient has to wait for results and also reduces the time the patient has to spend in the doctor's office. In a singleplex assay (e.g., a COVID-19 assay), dynamic confirmatory melting can be performed as soon as amplification is detected, the assay run can be stopped, and results can be reported immediately.

[0281] In addition, the novel dynamic melting programs described herein may have other advantages. For example, melting parameters can be altered for certain assays. For example, if an amplicon tests positive and the melt includes a relevant mutation (e.g., a mutation related to antimicrobial susceptibility), then the rate of temperature increase in the region of interest (i.e., the region expected to melt) can be slowed (e.g., 0.01-2 °C / sec, 0.1-1 °C / sec, or slower) to facilitate collection of melting data that can show melting shifts, etc., which can indicate the presence or absence of the relevant mutation in the amplicon population. Such "high-resolution" melting and mutation detection techniques are described in more 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 Publication No. 2018 / 0066137, the entire contents of which are incorporated herein by reference. Specific examples of adjustments to the fast-slow-fast dynamic melting program for detecting genetic variants are shown in Example 10 below. However, if the positive detection is related to an amplicon that is not associated with one or more relevant mutations or genetic variants, then the rate of temperature increase in the region of interest can suitably be a more typical value, such as 2 °C / sec - 3 °C / sec. In another example, the novel dynamic melting programs described herein can suitably be used to delay melting detection in a quantitative or semi-quantitative assay until one or more internal quantitative standards have been detected, to ensure that the entire quantitative range of the assay is accounted for. In yet another example, the novel dynamic melting programs described herein can suitably be used to shorten the run time of an assay with a fixed melt after a selected number of cycles. For example, if it is expected that a particular organism will test positive (if any) by melting within the first 15 PCR cycles, then if the wells used to detect the target organism show positive amplification after the first 15 PCR cycles, then the melt can be omitted.

[0282] Example 2

[0283] In this example, a dynamic melting program for a target analyte after a fixed number of amplification cycles is illustrated. In this example, Mycoplasma pneumoniae is the bacterial target, and the unique gene sequence from Mycoplasma pneumoniae is the amplified amplicon and target analyte. It should be noted that the Mycoplasma pneumoniae assay is run in triplicate (e.g., in a high-density array 581), and two out of three wells need to show positive results for the system to determine that the organism is positive. However, in some systems, some assays may also be run in duplicate or singly. It should also be noted that Mycoplasma pneumoniae is merely an illustrative target, and the procedure can suitably be applied to any amplicon in a nucleic acid amplification assay - whether in a singleplex assay or in a multiplex assay or panel.

[0284] In the case where the exemplary analyte (e.g., Mycoplasma pneumoniae) is the only analyte, if the only analyte shows signs of amplification (e.g., fluorescence increases beyond a threshold) during the amplification phase, then melting at the end of amplification will be based on the melting information of the only analyte. If the only analyte does not amplify, then melting can suitably be omitted to save time, and the assay can be reported as negative without confirmatory melting. In the case where the exemplary analyte is one of multiple analytes being tested, post-amplification melting can suitably be based on the one or more analytes that show signs of amplification during the amplification phase. In the illustrated embodiment, the positive analyte is Mycoplasma pneumoniae, but it can be any one or more of the analytes included in the assay. If no analyte in the assay shows positive amplification, then post-amplification melting can be omitted to save time in obtaining the result.

[0285] Now refer to Figure 5A , which illustrates a procedure for determining the melting characteristics of a target analyte. In this embodiment, the target analyte is an amplicon of Mycoplasma pneumoniae, but Figure 5A the procedure illustrated in Figure 5A can suitably be applied to any analyte.

[0286] Analyte Region of Interest (ROI) Mycoplasma pneumoniae [81.5-4,85.8+2]

[0287] Table 1

[0288] As Figure 5AAs contained herein and shown in Table 2 below, the approach segment that can suitably be heated at 6 - 12°C / sec is in the range of 59°C to 77.5°C, the ROI segment that can suitably be heated at 2°C / sec is in the range of 77.5°C to 87.8°C, and the ending segment that can be heated at 12°C / sec is in the range of 87.8°C up to 98°C. For each of the approach segment, the ROI segment, and the ending segment, the fluorescence acquisition rate is 10 acquisitions per second (i.e., 10F / sec), and denaturation is performed at cycle 27 after a fixed number of PCR cycles. Cycle 27 can be the midpoint of amplification or when amplification is complete. These numbers are specifically based on Mycoplasma pneumoniae, but one of ordinary skill in the art should understand that this procedure and principle can be applied to any amplicon.

[0289]

[0290] Table 2

[0291] Figure 5B Illustrates the dynamic denaturation obtained based on these parameters. Figure 5B The left inset of... graphically shows the temperature increase at 54 based on Table 2. Note the difference between the temperature increase of 54 and Figure 3A the stable temperature increase of 30 of..., which are based on the denaturation of the same Mycoplasma pneumoniae amplicon. Figure 5B The right inset of... shows the derivative denaturation curve at 55, which is sharp and clear. Compare the derivative denaturation curve at 55 with Figure 3A the derivative denaturation curve at 31 of.... It can be seen that the dynamic denaturation heating and the stable denaturation heating produce similar high-quality denaturation curves, and the dynamic denaturation achieves this while significantly saving time.

[0292] Example 3

[0293] Figure 6A and 6B Illustrates another example of a dynamic denaturation procedure with two positive amplicons. The dynamic denaturation procedure illustrated herein can suitably be applied to assays with two analytes, or the two analytes illustrated in this example can be part of a larger assay with more than two analytes. If this is an assay with multiple analytes, this example will illustrate the situation where two amplicons show signs of amplification and both amplicons require denaturation confirmation.

[0294] In this example, the analyte amplicons are the Mycoplasma pneumoniae amplicons discussed in detail in Example 2 and the amplicons for the identification of Bordetella pertussis (Bordetella pertussis 2). It should be noted that in this example, the Mycoplasma pneumoniae and Bordetella pertussis 2 assays are run in triplicate, and generally, two out of three wells for each analyte need to show a positive result for the system to determine that the organism is positive. However, in some systems, some assays may also be run in duplicate or individually. Figure 6A Computer simulation and experimental melting data procedures for determining the melting ranges of Mycoplasma pneumoniae and Bordetella pertussis 2 are illustrated. The computer simulation and experimental melting data (insets 60 - 63) for determining the melting temperature range of Mycoplasma pneumoniae are the same as Figure 5A those illustrated in Example 2. Based on these data, it was determined that the Mycoplasma pneumoniae amplicon used in this assay should melt between 81.5 °C and 85.8 °C. For Bordetella pertussis 2, inset 64 illustrates the computer simulation analysis of the melting range of the Bordetella pertussis 2 amplicon (melting at around 90.3 °C), and insets 65 - 67 illustrate the experimental melting data (melting in the range of approximately 89 °C to 91 °C). Based on these data, it was determined that the Bordetella pertussis 2 amplicon used in this assay should melt between 88.3 °C and 92.7 °C (Table 3).

[0295] The region of interest (ROI) for Mycoplasma pneumoniae is 77.5 °C to 87.8 °C, and the ROI for Bordetella pertussis 2 is 84.3 °C to 94.7 °C. Since these ranges overlap, it was decided in this example to combine the melting of these two amplicons into one melting. If two or more targets have close or overlapping ROIs and sufficient amplification of these two or more targets occurs simultaneously, it may be faster to observe two meltings in a single combined ROI than to melt each target separately. Therefore, the region of interest (ROI) for dynamically melting these two amplicons in a single melting is 77.5 °C to 94.7 °C. That is, the overlapping melting range of the two amplicons includes the lower limit range of Mycoplasma pneumoniae (i.e., 81.5 °C - 4 °C) and the upper limit range of Bordetella pertussis 2 (i.e., 92.7 °C + 2 °C). However, it should be understood that this is only illustrative, and the melting data for Mycoplasma pneumoniae and Bordetella pertussis 2 can be collected in separate melting steps. Example 5 below illustrates such an experiment.

[0296] Analyte Region of Interest (ROI) Mycoplasma pneumoniae [81.5-4,85.8+2] Bordetella pertussis 2 [88.3–4,92.7+2]

[0297] Table 3

[0298] As Figure 6AAs contained in the above and Table 4 below, appropriately, the approach segment that can be heated at 6 - 12 °C / sec is in the range of 59 °C to 77.5 °C, the ROI segment that can be heated at 2 °C / sec is in the range of 77.5 °C to 94.7 °C, and the ending segment that can be heated at 12 °C / sec is in the range of 94.7 °C up to 98 °C. For each of the approach segment, the ROI segment, and the ending segment, the fluorescence acquisition rate is 10 acquisitions per second (i.e., 10F / sec), and the denaturation is fixed at cycle 27, and cycle 27 can appropriately be when the amplification is completed.

[0299]

[0300] Table 4

[0301] These numbers are specifically based on Mycoplasma pneumoniae and Bordetella pertussis 2, but those of ordinary skill in the art should understand that this procedure and principle can be applied to any amplicon with overlapping or adjacent denaturation ranges.

[0302] Figure 6B Illustrates the dynamic denaturation obtained based on these parameters. Figure 6B The left inset of... graphically shows a 68 °C temperature increase based on the information shown in Table 4. Figure 6B The right inset of... shows the derivative denaturation curves of Mycoplasma pneumoniae (69) and Bordetella pertussis 2 (70), which are sharp and clear and are similar to the steady-state denaturation curves shown elsewhere in this document (see, for example Figure 11B ).

[0303] Example 4

[0304] Examples 2 and 3 showed the dynamic denaturation obtained after a fixed number of amplification cycles. In this example, a procedure is shown where, as soon as an amplicon shows amplification above a selected threshold (e.g., a crossing point (Cp) threshold, above a selected relative fluorescence unit value, etc.), the denaturation data of the amplicon can appropriately be collected. Compared to the situations shown in Examples 2 and 3, this has the potential to save additional time because as soon as amplification above a given threshold is detected and denaturation is performed, the results can appropriately be reported.

[0305] In this example, Mycoplasma pneumoniae is the amplicon - the same amplicon as used solely in Example 2 and in combination with Bordetella pertussis 2 in Example 3. It should be noted that in this example, the Mycoplasma pneumoniae assay is run in triplicate, and generally, two out of three wells of the analyte need to show a positive result for the system to determine the organism as positive. However, in some systems, some assays may also be run in duplicate or individually. Although Mycoplasma pneumoniae or another single amplicon can be the sole analyte in the assay (i.e., this example suitably can illustrate a singleplex assay), one of ordinary skill in the art will understand that this example can illustrate an assay or assay panel with many potential analytes (i.e., a multiplex assay), where, for example, the Mycoplasma pneumoniae amplicon is the only amplicon to be amplified, or Mycoplasma pneumoniae is the first amplicon detected to be amplified. In any case, the procedures described in this example apply equally to singleplex assays or multiplex panels or assays.

[0306] The information shown in Table 1 above is shown again in Figure 7A . The Mycoplasma pneumoniae amplicon is expected to melt between 81.5 °C and 85.8 °C, and the region of interest (ROI) for dynamic melting is 77.5 °C to 87.8 °C (Table 1). The parameters for dynamic melting are shown in Table 5 below. The information in Table 5 is nearly the same as the information shown above in Table 2.

[0307]

[0308] Table 5

[0309] The parameters for dynamic melting are: approach segment, 59 °C to 77.5 °C (ramping at 6 - 12 °C / Sec); ROI segment, 77.5 °C to 87.8 °C (ramping at 2 °C / Sec); and end segment, 87.8 °C up to 98 °C (ramping at 6 - 12 °C / Sec) (Table 5). The difference in melting between Example 2 / Table 2 and this example is that the amplicon suitably can be melted after amplification above a selected threshold is detected rather than waiting until a fixed number of amplification cycles have occurred. In this example, as detected by fluorescence, amplification exceeded the threshold of 10 RFU by cycle 15 of amplification (see Figure 7A for 71), and melting detection was performed at that point. Although 10 RFU was used as the threshold in this example, it should be understood that other thresholds can also be used (e.g., see Figure 3B and 4B , where fluorescence data of 4 RFU was used). For example, smaller or larger RFU values, crossing points (Cp), Cp plus a selected number of cycles (e.g., 2 additional amplification cycles after detection of Cp), etc. can be used as thresholds.

[0310] Figure 7BThe "fast-slow-fast" dynamic temperature increase curve that causes Mycoplasma pneumoniae to melt is graphically shown at 72. Figure 7B The melting derivative curve shown at 73 is sharp and clear. As in the previous example, the amplification was performed in triplicate.

[0311] In one aspect of the present disclosure, Figure 7B The confirmatory melt shown in the figure may be immediately followed by reporting the assay result of the detected organism (in this case, Mycoplasma pneumoniae) as positive. If the operator is confident that Mycoplasma pneumoniae is the only positive analyte (e.g., this is a single-plex assay, or in a panel-based test it is statistically unlikely to be a double positive), then the amplification reaction may be stopped after this melt step, as appropriate. This may result in a significant time saving, where possible. In another aspect of the disclosure, the assay result of the detected organism may be reported as positive immediately after the melt step and amplification may be continued as appropriate until the amplification is complete (e.g., a fixed number of cycles or until an internal quality control standard shows positive amplification) to confirm that there are no additional positive assays. If there are additional assay positives in this embodiment, then one or more additional melts may be performed as appropriate when the amplification of the assay positive exceeds a given threshold.

[0312] Example 5

[0313] After the previous embodiment, the present embodiment illustrates the dynamic melting program that comprises more than one positive amplification and the test of more than one melting.In the present embodiment, for given melting, the temperature parameter of dynamic melting suitably can be set according to being the assay thing of positive dynamically, and the number of cycles when melting is also set dynamically according to which amplification cycle the assay thing exceeds the threshold concentration of detection and melting.In one aspect, if a certain assay thing has been detected as positive before and melted based on its assay condition, then for subsequent melting parameter, suitably can not consider the temperature range of interest for this assay thing.

[0314] Although two assay positives and two dynamic melts are included in this example, it will be appreciated by those of ordinary skill that the principles illustrated in this example are applicable to panels or tests with more than two positives and more than two melts. Likewise, even if the positives are processed in separate melts due to the fact that the two positives exceed the threshold at different cycles, if, for example, the amplicons exceed the threshold at the same or nearly the same amplification cycles and if the degree of temperature overlap between the two or more amplicons means that time can be saved by performing one dynamic melt instead of two or more melts, then the two or more amplicons may be included in the same melt in appropriate circumstances.

[0315] Reference now Figure 8A, the thermal cycling temperature traces are shown in the upper small figure, where the first dynamic denaturation is performed at 80, and after five additional PCR cycles, the second dynamic denaturation is performed at 81. In the lower small figure, the real-time fluorescence monitoring of the reaction is schematically shown. In the real-time fluorescence trace, it can be seen that the Mycoplasma pneumoniae amplicon exceeds the threshold (in this example, 10 RFU) at cycle 19 (indicated at 82), while the Bordetella pertussis 2 amplicon exceeds the threshold at cycle 24 (as indicated at 83). Although 10 RFU is used as the threshold in this example, it should be understood that other thresholds can also be used (for example, see Figure 3B and 4B , where fluorescence data of 4 RFU was used). For example, smaller or larger RFU values, crossing points (Cp), Cp plus a selected number of cycles (for example, 2 additional amplification cycles after the detection of Cp), etc. can be used as the threshold. As in the previous examples, these analytes are run in triplicate, and as would be expected if the reagent concentrations (such as primers, polymerase, etc.) and template loading concentrations in each assay well are approximately the same, each of the three wells for each amplicon reaches the threshold at approximately the same time.

[0316] Refer to Figure 8B and 8C , which schematically shows the dynamic denaturation program of the Mycoplasma pneumoniae amplicon. This is largely the same as the Mycoplasma pneumoniae amplicon denaturation program schematically shown in Example 5. In Example 5, the "PCR informed" denaturation of the Mycoplasma pneumoniae amplicon was performed at cycle 15. In this example, the Mycoplasma pneumoniae denaturation is performed at cycle 19. Such between-run variability is not unexpected and is adjusted in a novel way in this disclosure. The Mycoplasma pneumoniae amplicon is expected to denature between 81.5 °C and 85.8 °C, and the region of interest (ROI) for dynamic denaturation is 77.5 °C to 87.8 °C (see, for example, Table 1). The parameters for dynamic denaturation are: approach segment, 59 °C to 77.5 °C (heating at 6 - 12 °C / Sec); ROI segment, 77.5 °C to 87.8 °C (heating at 2 °C / Sec); and end segment, 87.8 °C up to 98 °C (heating at 12 °C / Sec). Figure 8C The "fast - slow - fast" dynamic temperature increase curve for denaturing the Mycoplasma pneumoniae amplicon is graphically shown at 84 in Figure 8C . The resulting derivative denaturation curves for this reaction run in triplicate are shown at 85 in Figure 8C . As in the previous examples, the derivative denaturation curves are sharp and clear.

[0317] Now refer to Figure 8D and 8E, which illustrates the dynamic melting program of Bordetella pertussis 2 amplicons. The melting temperature, ROI, and dynamic melting program of the Bordetella pertussis 2 amplicons are almost the same as those illustrated in references Figure 6A and 6B , except that the timing of the melting cycles is "PCR informed" and based on the cycle when the concentration of the Bordetella pertussis 2 amplicon exceeds the threshold at cycle 24. Additionally, in Examples 3 / Figure 6A and 6B , Mycoplasma pneumoniae and Bordetella pertussis 2 amplicons melt in one melting, while in this example, they melt separately.

[0318] The melting range of the Bordetella pertussis 2 amplicon is from 88.3 °C to 92.7 °C, and the ROI of the Bordetella pertussis 2 amplicon is from 84.3 °C to 94.7 °C. The parameters of the dynamic melting are: approach segment, 59 °C to 84.3 °C (heating at 6 - 12 °C / Sec); ROI segment, 84.3 °C to 94.7 °C (heating at 2 °C / Sec); and end segment, 94.7 °C up to 98 °C (heating at 12 °C / Sec). Figure 8E The "fast - slow - fast" dynamic temperature increase curve for melting the Bordetella pertussis 2 amplicon is graphically shown at 86 of Figure 8E . The resulting melting derivative curves for the triplicate reactions are shown at 87 of Figure 8E . As in the previous examples, the derivative melting curves for the triplicate reactions are sharp and clear.

[0319] Example 6

[0320] As illustrated in the examples presented herein, dynamic melting can give the same melting quality as slow steady - state melting, while also providing a time savings over such steady - state melting. Fast steady - state melting (e.g., collecting melting data while heating at 12 °C / sec) is not feasible. This has been demonstrated on high, medium, and low signal melting curves.

[0321] The type of protocol used (fixed cycle or amplification detection informed) and how many analytes are positive determines the time savings. For a negative run (i.e., no assay positives are detected during amplification), the denaturation step can appropriately be completely omitted. For example, in a FilmArray run, the denaturation program takes about 40 seconds. Thus, omitting the denaturation step in the case of a negative run will shorten the time to obtain results by about 40 seconds. Compared to the time required for a typical FilmArray run, the time savings for fixed cycle dynamic denaturation (see, e.g., Examples 2 and 3) is about 30 seconds. Compared to the time required for a typical FilmArray run, the time savings for amplification detection informed dynamic denaturation (see, e.g., Examples 4 and 5) is about 10 seconds. Of course, if results can be reported as soon as an assay positive is detected and denaturation is performed, the time savings for amplification detection informed dynamic denaturation will be even greater. If results are reported mid-run and no additional assay positives are detected during the run, then denaturation at the end of the run is not required and the mid-run report can be considered the final report.

[0322] As PCR becomes faster and the proportion of time spent on denaturation in the run time becomes larger, the dynamic denaturation programs described herein and the time savings they produce become more significant. The dynamic denaturation programs described herein save a significant amount of time by appropriately configuring the instrument to rapidly increase the temperature in regions of increasing denaturation temperature where no denaturation information is expected (e.g., near regions and end regions) and more slowly increase the temperature in regions of interest (ROIs) of the denaturation transition where relevant amplicons are expected to be present, while maintaining high-quality denaturation resolution.

[0323] Example 7

[0324] The FilmArray Blood Culture Identification (BCID) system is designed to rapidly identify a broad range of microbial pathogens directly from blood cultures. The illustrative 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 ≥95%. Commercially available BCID panels are available from BioFire Diagnostics, Inc. The BCID panel and related methods are described in detail in U.S. Patent No. 10,053,726, the entire content of which is incorporated herein by reference. This example describes an adaptation of the method described in U.S. Patent No. 10,053,726, using the dynamic denaturation programs described herein to distinguish true positives from environmental contamination.

[0325] In the FilmArray instrument, following sample preparation, the first stage multiplex PCR mixture can be suitably amplified in the bubble 564 for a selected number of amplification cycles (e.g., 20-30 cycles). After the first stage PCR is completed, the mixture can be appropriately diluted and transferred to each of the second stage wells 582. The second stage PCR reaction can be suitably subjected to amplification conditions (e.g., 63°C 19 seconds to 94°C 0 seconds) for an additional number of cycles. In this illustrative embodiment, melting can be suitably performed after a fixed number of cycles (e.g., after 20, 26, and 32 cycles), or melting can be "PCR-informed" and can be suitably performed when the well shows signs of amplification exceeding a threshold. In either case, melting can be suitably performed using the dynamic melting program described herein, wherein the temperature increase can be suitably defined by the identity of the organism amplicon in one or more wells showing positive amplification. If the melting curve of a given well shows a melting peak (negative first-order derivative of the melting curve) within the predetermined temperature range of each second stage assay, the amplification assay can be suitably determined to be positive.

[0326] In a BCID panel, each organism tested in the panel can be "grouped" based on expected concentration and contamination risk in the sample to minimize false positives and false negatives. The groups of organisms are specified in Table 6 below:

[0327]

[0328] Table 6

[0329] In general, group 1 may suitably contain targets that are present in the sample with the highest titer and also exist as background organisms and have the highest risk of unexpected positives. Group 2 may suitably contain targets that are present in the sample with high titer and have a low presence rate as background organisms and have a medium risk of unexpected positives. Group 3 may suitably contain targets that are present in positive aerobic blood cultures with low titer and have a low or absent presence rate as background organisms and have a low risk of unexpected positives. Although the organisms grouped in the present embodiment are specific to BCID panels and positive aerobic blood cultures, it will be appreciated by those of ordinary skill in the art that the principles illustrated herein may be applied to any sample with a wide range of expected organism concentrations and / or with a risk of false positives and false negatives due to the environment and symbiotic organisms in the sample. Other examples in which this method may be useful include, but are not limited to, stool samples for diagnosing GI infections and cerebrospinal fluid samples for diagnosing central nervous system infections.

[0330] Generally speaking, organisms in Group 1, if present in a sample, can be expected to amplify early (e.g., within the first 10 - 22 amplification cycles). Thus, if they are present, organisms in Group 1 are suitably expected to show signs of amplification above a threshold, e.g., within the first 10 - 22 amplification cycles. If one or more organisms in Group 1 amplify at this early stage and if the melting curve of a given Group 1 well shows a melting peak within the temperature range pre - defined for that organism's amplicon, the assay may be positive. However, because organisms in Group 1 may face the highest false - positive risk (due to environmental contamination or other factors), later signs of amplification (e.g., after 20 - 22 amplification cycles) are suitably regarded as false positives and suitably do not trigger melting detection. That is, if a well of an organism in Group 1 amplifies early (e.g., before amplification cycle 20 - 22), it suitably can trigger dynamic melting (at the time when amplification above a threshold (e.g., 10 RFU or higher) is detected, or after a fixed number of amplification cycles (e.g., 15 - 20 amplification cycles)), and if the melting curve shows a melting peak within the temperature range pre - defined for the organism's amplicon in that well, such amplification can be determined to be positive. In contrast, if a well used for amplification of an amplicon of an organism in Group 1 amplifies late (e.g., at cycle 25), such amplification suitably does not trigger melting because such amplification may be a false positive and may be attributable to, for example, contamination (e.g., environmental contamination). It should be understood that the cycle numbers presented in this example are merely illustrative, and the novel dynamic melting procedure described herein allows for a more nuanced approach than a cycle cut - off value for true positives versus false positives. Factors such as but not limited to the organism titer in the sample and the amount of sample loaded may vary, and thus, the cycle cut - off value may suitably also vary. For example, if all seem to amplify early, the cut - off value between true positives and false positives may also be advanced. Similarly, if all seem to amplify late, the cut - off value between true positives and false positives may also be postponed.

[0331] Generally speaking, organisms in Group 2, if present in the sample, are expected to amplify in the mid-stage of amplification (e.g., after about 18 - 22 cycles but before about 25 - 27 cycles). However, it should be understood that some true positives of Group 2 organisms may amplify earlier (i.e., before about 18 - 22 amplification cycles). If one or more organisms in Group 2 amplify at this mid-stage and if the melting curve of a given Group 2 well shows a melting peak within the temperature range pre-defined for the amplicon of that organism, the assay may be positive. However, because Group 2 organisms still face a certain risk of false positives (due to contamination), later signs of amplification (e.g., after about 27 amplification cycles) may suitably be regarded as false positives and may suitably not trigger melting detection. Although some true positives of Group 2 organisms may appear late in the amplification, looking for true positives in the mid-stage of amplification may suitably capture most true positives while reducing the risk of false positives from environmental contamination. The above description regarding the number of cycles is only illustrative, and a dynamic melting program may suitably provide a more refined approach than a strict cut-off value, which also applies here.

[0332] Generally speaking, organisms in Group 3, if present in the sample, are expected to amplify in the final stage of amplification (e.g., after about 25 - 27 cycles). However, it should be understood that some true positives of Group 3 organisms may amplify earlier (i.e., before about 25 - 27 amplification cycles). If one or more organisms in Group 3 amplify at this final stage of amplification and if the melting curve of a given Group 2 assay well shows a melting peak within the temperature range pre-defined for the amplicon of that organism, the assay may be positive. Since the risk of false positives due to contamination for Group 3 organisms is low, the risk associated with a positive determination even after 32 - 35 amplification cycles is small. The above description regarding the number of cycles is only illustrative, and a dynamic melting program may suitably provide a more refined approach than a strict cut-off value, which also applies here.

[0333] As an illustrative example, let us study Escherichia coli, which belongs to Group 1 organisms (see Table 6). True sepsis positives in Escherichia coli PABC typically grow to high titers (e.g., >10 8cfu / ml), and is expected to be detected by amplification in a schematic BCID panel and before amplification cycle 20, typically before amplification cycle 10. However, Escherichia coli is almost ubiquitous in the environment and can often be introduced into blood samples and ultimately into blood culture bottles, for example, via environmental sources and / or culture medium raw materials. For a schematic BCID panel, it was found that amplification of Escherichia coli later than about amplification cycles 18 - 22 (e.g., before cycle 20) may be due to a certain type of contamination. Assays of Escherichia coli amplified before about amplification cycle 20 can be considered true positives and suitably can trigger melt curve detection. In contrast, Escherichia coli positives later than about cycle 20 can be considered false positives and suitably may not trigger melt curve detection.

[0334] As another illustrative example, let's examine antimicrobial resistance (AMR) genes that can be detected in a schematic BCID panel. Schematic BCID and BCID2 panels are capable of detecting multiple different antimicrobial resistance (AMR) markers and genes. In one example, the schematic BCID panel is capable of detecting the mecA / C genes associated with methicillin resistance in Staphylococcus aureus. In this embodiment, Staphylococcus aureus belongs to group 2 organisms, and true positives from PABC are expected to amplify in the mid - stage of amplification (e.g., around 20 - 25 cycles). If Staphylococcus aureus is detected but the mecA / C gene is not detected simultaneously, then confirmatory melt curve can suitably be performed at a heating rate of ∼2°C / sec within the region of interest (ROI), as shown in the examples presented herein. However, if amplification is detected in the sample well designated for Staphylococcus aureus and in a separate sample well designated for the mecA / C gene (i.e., Staphylococcus aureus and mecA / C are detected simultaneously), then confirmatory melt curve can suitably be performed for both Staphylococcus aureus and mecA / C. Depending on their respective melt curve temperature ranges and the melt curve heating rates required for optimal detection, and whether amplification is detected at the same or nearly the same cycle for both or whether they are separated in time, confirmatory melt curve for Staphylococcus aureus and mecA / C can be performed together or separately. If Staphylococcus aureus is detected and confirmatory melt curve is performed, and then the mecA / C gene is detected at a later cycle, then confirmatory melt curve for mecA / C can be performed. However, if Staphylococcus aureus amplification is not detected, but the mecA / C gene is detected by amplification, then the melt curve for the mecA / C gene can suitably be omitted because these loci must come from other organisms and are not related to methicillin - resistant Staphylococcus aureus. In other words, before amplification of the relevant pathogen is detected, melt curve for AMR genes or markers can suitably not be performed. This can potentially save the time that might otherwise be spent on melt curve analysis and can make AMR detection more robust and conclusive.

[0335] In addition, it should be understood that information obtained for one organism can be used to assist in the positive or negative determination of other organisms, particularly if there is some cross-reactivity between the organisms, or if there is some other relationship such as between a bacterium and an antibiotic resistance gene associated with that bacterium. As can be seen in Table 6 above, Enterococcus ("Entero") and Staphylococcus ("Staph") are both Group 2 organisms, and if present, amplification of each should be detected between approximately 18 - 27 amplification cycles. However, in many known Enterococcus assays, due to the similarity of the target sequences, they cross-react with Staphylococcus, which can result in a late Cp in an Enterococcus true negative sample that is Staphylococcus positive. To reduce the impact of potential cross-reactivity on the Enterococcus assay in such cases where cross-reactivity becomes a problem, if Staphylococcus amplification is detected between approximately 18 - 27 amplification cycles, a positive or negative determination of Staphylococcus can be made by amplification and melting. If Staphylococcus is positive and thus affects the Enterococcus sample, the Enterococcus can be determined based on previous results, illustratively whether amplification of Enterococcus was detected before amplification cycle 20 - 22. If Staphylococcus is negative, the Enterococcus assay will not be affected, and a determination can be made if (if any) Enterococcus amplification is detected between approximately 18 - 27 amplification cycles, or the whatever cycle range optimal for a non-cross-reactive assay can be selected. However, it should be noted that in blood cultures, positive growth is based on the growth of a complex organism of all organisms present, and one or more organisms may be present in amounts lower than that of any single infection. It may be necessary to adjust the grouping and the cycle ranges for determining cross-reactive assays accordingly. By adjusting the grouping and the cycle ranges used for determining cross-reactive assays based on the positive or negative determination from other assays, the cross-reactivity problem for dual infection samples can be accurately determined, illustratively without having to redesign primers to avoid cross-amplification.

[0336] Although the organisms described in this example are classified into three groups and early, mid, and late melting cycle ranges are used for each group in this example, it should be understood that any number of groups and melting cycles can be used, and any cycle can be selected as a melting cycle. In some assays, the discrimination between false positives and false negatives can be achieved using only two groups and / or two melting cycles, while in other assays four or more groups and / or melting cycles may be required. In addition, although this example uses samples from cultures, it should be understood that multiple groups and / or melting cycles may be appropriate for assays using uncultured materials.

[0337] It should be understood that although the above example identified organisms, it should be understood that the same methods and apparatus can be used to identify different target sequences in one or several organisms by amplifying different loci of the organisms.

[0338] Example 8

[0339] Certain embodiments of the present invention may also relate to or include a PCR system configured to make a positive or negative determination from an amplification curve or a melting curve or a combination thereof. Illustrative examples are described in U.S. Patent No. 8,895,295, which is incorporated by reference and used with the pouch 510 or similar embodiments. However, it should be understood that the embodiments described in U.S. Patent No. 8,895,295 are merely illustrative, and other systems may be used in accordance with the present disclosure. For example, referring to FIG. 15, which shows a block diagram of a schematic system 700 including a control element 702, a thermal cycling element 708, and an optical element 710 according to an exemplary aspect of the present disclosure.

[0340] In at least one embodiment, the system may include at least one PCR reaction mixture contained in a sample container 714. In certain embodiments, the sample container 714 may contain a PCR reaction mixture configured to allow and / or effect amplification of a template nucleic acid. Certain illustrative embodiments may also include at least one sample zone or chamber 716 configured to receive at least one sample container 714. The sample container 714 may include a single, strip, plate, or any number of other forms of sample containers, and schematically, may be provided or received by the sample zone or chamber 716.

[0341] One or more embodiments may also include at least one sample temperature control device 718 and / or 720 configured to manipulate and / or regulate the sample temperature. Such sample temperature control devices may be configured to raise, lower, and / or maintain the temperature of the sample. In one example, the sample control device 718 is a heating system and the sample control device 720 is a cooling system. Illustrative sample temperature control devices include (but are not limited to) heating and / or cooling blocks, elements, exchangers, coils, radiators, refrigerators, filaments, Peltier devices, forced blowers, processors, vents, dispensers, compressors, condensers, water baths, ice baths, flames, and / or other combustible or burnable forms of heat, heat packs, cold packs, dry ice, dry ice baths, liquid nitrogen, devices that emit microwaves and / or other waves, cooling means, heating means, other means for manipulating the sample temperature, and / or any other suitable devices configured to raise, lower, and / or maintain the sample temperature.

[0342] The schematic PCR system 700 also includes an optical system 710 configured to detect the amount of fluorescence emitted by the sample 714 (or a portion or reagent thereof). Such an optical system 710 may include one or more fluorescence channels, as is known in the art, and may detect fluorescence from multiple samples simultaneously or individually.

[0343] At least one embodiment of the PCR system may further include a CPU 706 programmed or configured to, illustratively when the optical system 710 acquires a fluorescence signal, operate, control, execute, or otherwise advance the heating system 718 and the cooling system 720 to thermally cycle the PCR reaction mixture. The CPU 706 may then generate an amplification curve, a melting curve, or any combination thereof, which may or may not be printed, displayed on a screen, or otherwise output. Optionally, a positive, negative, or other determination may be based on the amplification and / or melting curve output. Optionally, only the determination is output, illustratively one determination for each target being tested.

[0344] Additional examples of illustrative features, components, elements, and / or components of an illustrative PCR system and / or thermocycler are known in the art and / or described above or in U.S. Patent Publication Nos. 2014 / 0273181 and 2020 / 0319441 and U.S. Patent No. 10,698,190, the entire contents of these patent publications and patents being incorporated herein by reference.

[0345] Example 9

[0346] Figure 10 Illustrates a flowchart of an example dynamic melting detection method 1000 for determining a target nucleic acid sequence of a sample. Method 1000 may be implemented by various components of the PCR system described below with reference to Figure 9 These components include a processor or controller, optical elements, and one or more temperature control devices, such as a thermocycling element comprising a heating system and a cooling system. In some embodiments, method 1000 or portions thereof may be implemented as a set of instructions and stored on a computer-readable memory and executable on one or more processors or controllers.

[0347] A portion of the sample is incorporated into a sample well together with primers for amplifying the target nucleic acid sequence and a fluorescent dye such as a dsDNA-binding dye. The portion of the sample may be incorporated into multiple sample wells, where each sample well contains primers for amplifying different target nucleic acid sequences from different organisms. Each target nucleic acid sequence has a different characteristic melting temperature range. For example, the characteristic melting temperature range of Mycoplasma pneumoniae amplicons is from 77.5 °C to 87.8 °C, while the characteristic melting temperature range of Bordetella pertussis 2 amplicons is from 84.3 °C to 94.7 °C.

[0348] At block 1002, the samples in each sample well are amplified via thermal cycling, which thermal cycling includes at least a two-step PCR protocol. For each of a number of cycles, the PCR protocol can include an in-cycle temperature adjustment segment or denaturation segment, in which the sample well is heated from an annealing temperature to a denaturation temperature and then cooled from the denaturation temperature to the annealing temperature. For each of a number of cycles, the PCR protocol can further include an extension segment, in which the temperature is held constant at an optimal temperature for DNA polymerase activity. The PCR protocol can also omit a specific elongation temperature hold segment. As explained in more detail elsewhere herein, DNA polymerase can be active and can complete primer elongation while the temperature is rising from the annealing temperature to the denaturation temperature. In some embodiments, a processor or controller provides control signals to the thermal cycling element to heat the sample well to the denaturation temperature, cool the sample well to the annealing temperature, and hold the temperature of the sample well constant using an initial heating rate. The initial heating rate can be a fixed heating rate (e.g., 12 °C / sec for each cycle).

[0349] Then, at block 1004, fluorescence data (which indicates the amount of fluorescence emitted by the sample) is collected from the sample at each sample well during the in-cycle temperature adjustment segment of each of N cycles, where N is 1, 2, 3, 4, 5, 6 or more cycles. The fluorescence data can be collected by an optical system (such as optical system 710 described above with reference to Figure 9 and provided to a processor or controller. For example, the optical system can provide light (e.g., from an LED) to the sample at each sample well and can include an optical detector to detect the amount of fluorescence signal generated by the sample at each sample well. In some embodiments, a processor or controller provides control signals to optical system 710 to detect the amount of light scattered by the sample at each sample well during the in-cycle temperature adjustment segment of each of N cycles. Then, the processor or controller can collect the amount of fluorescence and the temperature of the sample (temperature-fluorescence pairs) at a number of time points during each of N cycles. It should be understood that fluorescence is illustrative only, and other ways of measuring and detecting amplification are within the scope of the present disclosure.

[0350] At block 1006, the processor or controller can determine whether the amount of fluorescence at any sample well exceeds a threshold. The threshold can be an amount greater than or equal to a detection limit for inferring an increase in the concentration of nucleic acid in the sample well. The threshold can also be a crossing point (Cp), a selected amount of relative fluorescence units (RFU), Cp plus a predetermined amount of additional amplification cycles, or any suitable threshold.

[0351] If the amount of fluorescence at one of the sample wells exceeds a threshold, the processor or controller may 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 Mycoplasma pneumoniae amplicons, the characteristic melting temperature range may be from 77.5 °C to 87.8 °C. The processor or controller may 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.

[0352] The processor or controller then determines an adjusted heating rate curve for heating the sample well based on the characteristic melting temperature range of the amplified target nucleic acid in the sample well (block 1008). The adjusted heating rate curve may include different heating rates for heating the sample well from the annealing temperature to the denaturation temperature. At least one heating rate in the adjusted heating rate curve may be faster than the initial heating rate to increase the rate of melting at temperatures where the expected reaction would not produce a melting signature. For example, the adjusted heating rate curve may include a first heating rate for a proximity temperature range, a second heating rate for the characteristic melting temperature range, and a third heating rate for an end temperature range. The proximity temperature range may be from the annealing temperature to the initial melting temperature at the start of the characteristic melting temperature range (e.g., 59 °C to 77.5 °C). The characteristic melting temperature range may 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).

[0353] The first heating rate and the third heating rate may be faster than the second heating rate to increase the rate of melting at temperatures where the expected reaction would not produce a melting signature. For example, the first heating rate may be between 6 and 12 °C / sec and the third heating rate may be 12 °C / sec. The second heating rate may be similar to the initial heating rate (e.g., 2 °C / sec) to slow down melting at temperatures where the expected reaction would produce a melting signature.

[0354] Then at block 1010, melting detection is performed using the adjusted heating rate curve. For example, during a subsequent thermal cycle, the processor or controller provides control signals to the thermal cycling element to heat the sample well to the denaturation temperature, cool the sample well to the annealing temperature, and hold the temperature of the sample well constant using the adjusted heating rate curve. A melting curve is then generated based on the fluorescence data detected during the melting detection process. The melting curve is then analyzed to identify the target nucleic acid corresponding to the sample well among a plurality of target nucleic acids. For example, a melting peak may indicate the organism within the sample well. In a specific example, if the sample well contains Mycoplasma pneumoniae amplicons and the melting peak indicates Mycoplasma pneumoniae, the processor or controller may identify that the sample contains Mycoplasma pneumoniae.

[0355] In some embodiments, a processor or controller may determine that the amount of fluorescence at a plurality of sample wells exceeds a threshold, where each sample well contains primers for amplifying different target nucleic acid sequences from different organisms. For example, the processor or controller may determine that the amount of fluorescence at a first sample well having primers for amplifying a first target nucleic acid having a first characteristic melting temperature range exceeds the threshold and that the amount of fluorescence at a second sample well having primers for amplifying a second target nucleic acid having a second characteristic melting temperature range also exceeds the threshold.

[0356] Then, the processor or controller may generate a first adjusted heating rate curve corresponding to the first characteristic melting temperature range for the first sample well and a second adjusted heating rate curve corresponding to the second characteristic melting temperature range for the second sample well. In some embodiments, the processor or controller may use the first adjusted heating rate curve to perform a first melting detection during a first melting cycle. Then, the processor or controller may use the second adjusted heating rate curve to perform a second melting detection during a second melting cycle that may occur after the first melting cycle. For example, the second melting cycle may occur five cycles after the first melting cycle.

[0357] In other embodiments, the processor or controller may combine the first and second adjusted heating rate curves into a combined heating rate curve and may use the combined heating rate curve to perform melting detection. For example, if the first characteristic melting temperature range is from 77.5 °C to 87.8 °C and the second characteristic melting temperature range is from 84.3 °C to 94.7 °C, then the processor or controller may generate a combined characteristic melting temperature range from 77.5 °C to 94.7 °C. Then, the processor or controller may use the melting ROI from 77.5 °C to 94.7 °C to generate the combined heating rate curve.

[0358] Example 10

[0359] In this embodiment, a dynamic melting program for detecting the presence of gene sequence variants is described. Copies of double-stranded nucleic acids (referred to as products or amplicons) generated during a nucleic acid amplification reaction will have a unique sequence based on the template being amplified. The amplicon length and sequence / composition (generally, A-T / G-C content) determine the temperature at which the double-stranded DNA will unwind, which is referred to as the melting temperature (Tm) of the amplicon. Products made from different targets will have different sequences and thus different Tm values, and in many cases, different melting curve shapes. Thus, due to the nature of nucleic acid-binding dyes, nucleic acid-binding dyes added to the amplification reaction can be used for nucleic acid melting analysis, where they fluoresce strongly in the presence of double-stranded nucleic acids and weakly or not at all in the presence of single-stranded nucleic acids. A particular product will have a particular melting signature (i.e., the temperature transition range over which fluorescence decays as the duplex transitions from fully double-stranded to fully single-stranded), and thus nucleic acid melting can be used to confirm the identity of nucleic acid amplification products. Sequence variants will generally melt at a different temperature and / or have a different melting curve shape compared to the wild-type sequence, and thus, Tm shifts and changes in melting shape can be used to detect the presence of sequence variation in amplified nucleic acids.

[0360] In this context, the term "detection" can include detecting and determining known and unknown nucleic acid sequence variations, including but not limited to SNPs, base deletions, base insertions, sequence duplications, rearrangements; inversions, base methylation, the number of short tandem repeats; and in the case of a diploid genome, whether the genome is homozygous or heterozygous for the sequence variation, and the cis / trans positional relationship of two or more sequence variations on the DNA strand (haplotype analysis). Such nucleic acid sequence variations may be associated with many genotypic and phenotypic differences in organisms. Single nucleotide polymorphisms (SNPs) are the most common genetic variations observed to date in humans and other species. In these polymorphisms, there is a difference in only a single base between individuals. Such an alteration may result in a change in the amino acid in a protein, alter the transcription rate, affect mRNA splicing, or have no apparent effect on cellular processes. Sometimes, when the change is silent (e.g., when the encoded amino acid does not change), SNP genotyping may still be valuable if the alteration is associated (correlated) with a unique phenotype caused by another gene alteration. In one specific instance, a nucleic acid sequence variation (e.g., an SNP) may be associated with an antimicrobial resistance (AMR) marker. Such nucleic acid sequence variations may be associated with detectable Tm shifts that can be used for sequence variants. "High-resolution" melting and mutation detection techniques are described in more detail in U.S. Patent No. 9,657,347 (see, for example, Example 19), U.S. Patent No. 9,290,663, and U.S. Patent Publication No. 2018 / 0066137, the entire contents of which are incorporated herein by reference in their entirety.

[0361] In one aspect of the present invention, there is provided a method which requires only standard PCR reagents, primers, and the simple addition of a "saturating" double-stranded (ds) DNA-binding dye prior to PCR. For the purposes of the present invention, a "saturating" dye is a dye that does not significantly inhibit PCR when present at a concentration (schematically about 10 ng / μL) that provides a maximum fluorescence signal for the amount of dsDNA normally generated by PCR in the absence of the dye. Although these dyes are identified by their compatibility with PCR at near-saturating concentrations, it should be understood that these dyes can also be used at much lower concentrations. During or after amplification, these dyes can be used to identify the presence of known and unknown sequence variants in a manner similar to that when using labeled primers, by melt curve analysis. The identification of known and unknown sequence variants can be used in a variety of assays, 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 any differences present in the sequence. A positive answer indicating the presence of a sequence difference does not necessarily reflect the exact nature of the sequence variant or its location on the nucleic acid fragment. The term "genotyping" includes the detection and determination of nucleic acid sequence variants.

[0362] In addition, while PCR is mentioned, 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), Qβ replicase-mediated amplification, isothermal and chimeric primer-initiated nucleic acid amplification (ICAN), transcription-mediated amplification (TMA), etc. Thus, when using the term PCR, it should be understood to include other alternative amplification methods.

[0363] Now refer to Figure 12, which shows a schematic melting program comparing a standard melting protocol (……), a dynamic melting protocol (–––) (such as the dynamic melting protocol described in detail elsewhere in this document), and a dynamic "high-resolution" protocol (·–·–). Each protocol is held at 62 °C for 3 seconds. For the standard protocol, the temperature is then increased to 68 °C at a maximum heating rate of 12 °C / sec. Then, melting is performed at a standard (fixed) heating rate of 2 °C / sec until the temperature reaches 99 °C. For the dynamic protocol, after holding for 3 seconds, the temperature is increased to 76 °C at a heating rate of 12 °C / sec. Then the heating rate is decreased to 2 °C / sec until a temperature of 95 °C, and then it is increased again to 99 °C at 12 °C / sec. For the dynamic HRM protocol, after holding for 3 seconds, the temperature is increased to 76 °C at a heating rate of 12 °C / sec. From 76 °C to 92 °C, the heating rate is decreased to 1 °C / sec, and then it is increased to 99 °C at 12 °C / sec. In this schematic example, the heating rate for the melting portion is 1 °C / sec. Although this is schematic, other "high-resolution" melting heating rates can be used, such as but not limited to 0.01 - 2 °C / sec, 0.1 - 1 °C / sec, preferably not greater than 0.5 °C / sec, more preferably not greater than 1 °C / sec, and most preferably less than or equal to 2 °C / sec. The selection of the heating rate for detecting genetic variations will depend on factors such as the desired melting resolution and the time requirements of the assay. Generally, the inventors of this case found that a heating rate of 0.5 - 1 °C / sec is sufficient in most cases.

[0364] In this schematic example, the time for the standard melting protocol is 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" heating rate in the standard protocol will increase the run time by ~30 seconds. Although in this example, the implementation of the high-resolution dynamic melting program does take approximately 6 seconds more than the conventional dynamic melting program, compared to the standard protocol, the high-resolution dynamic melting can be completed with only 1 second more. Although these are just hypothetical programs, this schematic diagram illustrates the potential time savings of the dynamic high-resolution protocol compared to the standard high-resolution protocol. As with the actual dynamic melting programs described in detail elsewhere in this document, the actual protocol and actual time savings may depend on the melting characteristics of the amplicons being melted. The total run-time cost of performing the high-resolution melting program will depend on the heating rate and the number of individual meltings performed.

[0365] In some aspects of the present invention, high-resolution melting programs can be routinely used to detect certain nucleic acids. However, since high-resolution melting programs may be more time-consuming, in other aspects of the present invention, it may be necessary to use a first assay as a trigger to determine whether melting should be performed in an assay for detecting genetic variations. Figure 13 A schematic example of such a trigger assay is shown in.Figure 13 Shows a Neisseria gonorrhoeae organism assay (●Org) and separate AMR assays for the mutational regions of the gyrA gene (○S91F and +WT). For the left inset, the S91F assay (open circles) is closest to the organism (solid circles), while the WT assay is much later. This indicates that the isolates tested are resistant. In contrast, in the right inset, the assay closest to the organism is the WT assay, indicating that the sample contains wild-type Neisseria gonorrhoeae. Although Figure 13 Cp differences between wild-type and mutant are shown, but in this case, such Cp differences are not a reliable method for identifying wild-type and mutant isolates. In one aspect, the gyrA assay is performed to identify mutant or wild-type Neisseria gonorrhoeae (i.e., antimicrobial drug-resistant or antimicrobial drug-susceptible Neisseria gonorrhoeae) only when the Org assay is positive. This general approach can be applied to other assays for detecting genetic variation. Thus, the additional time required for detecting genetic variation (e.g., AMR assays) may be needed only when the organism is positive. Additionally, due to potential cross-reactivity between different related species within an organism family (e.g., Neisseria species), there may be cases where the genetic variation assay gives false positives (the target region may be highly conserved between related organisms and thus the assay may not be selective). If the organism assay (which may be highly selective) is positive, that assay can be used to trigger melting to detect genetic variation. Accordingly, the methods of the invention described herein can include detecting the presence of an organism via a first melting detection in a first assay and, if an 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 gene mutation.

[0366] Now referring to FIGS. 14 - 16, which show illustrative examples of using a modified dynamic high-resolution melting procedure to detect genetic variation in nucleic acids amplified in an experimental FilmArray test pouch. FIGS. 14 - 16 show melting curves of amplified wild-type and mutant nucleic acids from Mycoplasma genitalium (23S) and Neisseria gonorrhoeae (gyrA). The 23S mutations shown in FIGS. 14 (A2059G) and 15 (A2058G) render Mycoplasma genitalium resistant to macrolides, while the S91F mutation of gyrA shown in FIG. 16 renders Neisseria gonorrhoeae resistant to ciprofloxacin. Due to the overuse of antibiotics, these and other antimicrobial drug-resistant mutations are becoming increasingly common in many pathogenic bacteria. Accurately and routinely identifying the presence of antimicrobial drug-resistant markers is crucial for antimicrobial stewardship and appropriate patient care. The methods described herein can be used to accurately and routinely detect and identify many types of genetic variation.

[0367] Figure 14A and14B The melting curves of wild-type (–––MG-WT) and A2059G (–––2059G) mutant amplicons of the 23S ribosomal RNA gene of Mycoplasma genitalium were compared. Figure 14A Composite WT and mutant curves are shown, Figure 14B and separate curves from individual amplification reactions and meltings are shown. In both cases, the melting curves of the WT and mutant are clearly distinguishable. The initial heating rate for melting of the WT and mutant amplicons was 2 °C / sec, from 62 °C to 69 °C, and for the melting temperature range from 69 °C to 94 °C (referred to elsewhere herein as the ROI), the heating rate was decreased to 1 °C / sec, and then for the final temperature from 94 °C to 99 °C, the heating rate was increased to 2 °C / sec. These heating rates are only one example. For example, one of ordinary skill in the art would understand that the initial and final heating rates can be higher than 2 °C / sec (e.g., 4 - 12 °C / sec), and other melting heating rates can be used in the ROI, such as but not limited to 0.01 - 2 °C / sec, 0.1 - 1 °C / sec, preferably not greater than 0.5 °C / sec, more preferably not greater than 1 °C / sec, and most preferably less than or equal to 2 °C / sec. Generally, slower heating rates will improve the resolution of melting, at the cost of longer analysis times. Here, with a melting heating rate of 1 °C / sec, each sample was clearly genotyped.

[0368] Figure 15A and 15B The melting curves of (–––MG-WT) and A2058G (–··–2058G) mutant amplicons of the 23S ribosomal RNA gene of Mycoplasma genitalium were compared. Figure 15A and 15B with Figure 14A and 14B similar. Figure 15A Composite WT and mutant curves are shown, Figure 15B and separate curves from individual amplification reactions and meltings are shown. In both cases, the melting curves of the WT and mutant are clearly distinguishable. Figure 15A and 15B The melting parameters of Figure 14A and 14B are the same as those of Figure 14A and 14B As with Figure 14A and 14B one of ordinary skill in the art would understand that the heating parameters can be varied to heat faster or slower and / or to increase or decrease the resolution of melting. Here, with a melting heating rate of 1 °C / sec, each sample was clearly genotyped.

[0369] Figure 16A and 16BThe melting curves of wild-type (–––NG-WT) and S91F (……S91F) mutant amplicons of the gyrA gene of Neisseria gonorrhoeae were compared. Figure 16A Composite WT and mutant curves are shown, Figure 16B and separate curves from individual amplification reactions and melting are shown. In both cases, the melting curves of the WT and mutant are clearly distinguishable. The initial heating rate for melting of the WT and mutant amplicons was 2 °C / sec, ramping from 69 °C to 83.8 °C. For the melting temperature range from 83.8 °C to 91.8 °C (referred to elsewhere herein as the ROI), the heating rate was decreased to 1 °C / sec, and then for the final temperature from 91.8 °C to 99 °C, the heating rate was increased to 2 °C / sec. As Figure 14A - 15B would be understood by one of ordinary skill in the art, the heating parameters can be varied to make the heating faster or slower and / or to increase or decrease the resolution of the melting. Here, with a melting heating rate of 1 °C / sec, each sample was unambiguously genotyped.

[0370] The examples shown in FIGS. 14 - 16 illustrate cases where a dynamic melting protocol can be used to distinguish wild-type melting from mutant melting. The fact that this can be done in a FilmArray test pouch without a significant increase in assay time is a significant advance in the art. While FIGS. 14 - 16 illustrate assays performed in a FilmArray test pouch, one of ordinary skill in the art would understand that these protocols can be adapted for use with nucleic acid amplification and melting systems. Additionally, the examples shown in FIGS. 14 - 16 have relatively small differences in temperature and shape during melting between the wild-type and mutant, but these differences can be clearly resolved using the dynamic melting procedures described herein. This shows that the dynamic melting procedures described herein can be routinely used to detect amplified nucleic acids containing mutations.

[0371] 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 appended claims.

Claims

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

2. The method according to claim 1, wherein positive nucleic acid amplification is determined by the fluorescent signal in the sample well increasing above a threshold.

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

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

5. The method according to any one of claims 1-4, the method further comprising not performing the melting detection if no sample well shows positive nucleic acid amplification.

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

7. The method according to any one of claims 1-4, wherein the melting detection comprises: a first heating rate during a first portion of the melting, a second heating rate during a second portion of the melting, and a third heating rate during a third portion of the melting, the second heating rate being slower than the first and third heating rates, and wherein the second portion of the melting is defined by the melting temperature range characteristic of the target nucleic acid to detect the organism in the sample well.

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

9. The method according to claim 7, wherein the third heating rate is faster than the first heating rate.

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

11. The method according to claim 10, wherein the second heating rate is preferably not greater than 0.5 °C / sec, more preferably not greater than 1 °C / sec, and most preferably less than or equal to 2 °C / sec.

12. The method according to claim 10 or 11, wherein the second heating rate is suitable for detecting nucleic acid melting characteristics indicative of a genotype, sequence variant, or gene mutation in a target nucleic acid, the genotype, sequence variant, or gene mutation altering one or more of the nucleic acid melting temperature or the shape of the nucleic acid melting curve relative to a target nucleic acid that does not have the genotype, sequence variant, or gene mutation.

13. The method according to claim 12, further comprising detecting a single nucleotide polymorphism (SNP).

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

15. The method according to claim 12, further comprising detecting the presence of an organism via a first melting detection in a first assay, and if the presence of the organism is detected in the first assay, performing a second melting detection in a second assay to detect the presence or absence of the genotype, sequence variant, or gene mutation.

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

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

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

19. The method according to claim 18, further comprising performing one melting detection with a melting temperature range for the target nucleic acid amplified in the two or more wells.

20. The method according to claim 18, wherein the method further comprises performing a first melting detection with a characteristic first melting temperature range for melting the amplified target nucleic acid in the first well and performing at least a second melting detection with a characteristic second melting temperature range for melting the amplified target nucleic acid in the second well.

21. The method according to claim 20, wherein the melting detection comprises one of the following: a first heating rate during a first part of the melting, a second heating rate during a second part of the melting, and a third heating rate during a third part of the melting, wherein the second heating rate is slower than the first and third heating rates, and wherein the second part of the melting is defined by the melting temperature range characteristic of the target nucleic acid in the first and second sample wells, or a first heating rate during a first part of the melting, a second heating rate during a second part of the melting, a third heating rate during a third part of the melting, a fourth heating rate during a fourth part of the melting, and a fifth heating rate during a fifth part of the melting, wherein the second and fourth heating rates are slower than the first, third, and fifth heating rates, and wherein the second part of the melting is defined by the melting temperature range characteristic of the target nucleic acid in the first sample well, and the fourth part of the melting is defined by the melting temperature range characteristic of the target nucleic acid in the second sample well.

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

23. The method according to claim 22, wherein the second heating rate or the second and fourth heating rates are preferably not greater than 0.5 °C / sec, more preferably not greater than 1 °C / sec, and most preferably not greater than 2 °C / sec.

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

25. The method according to any one of claims 1-23, wherein the selected number of amplification cycles before the melting detection is assay-specific and determined by the expected concentration of the plurality of target nucleic acids suspected to be present in the sample.

26. The method according to any one of claims 1-25, wherein the method further comprises identifying at least one target organism present in the sample by identifying at least one corresponding sample well in which amplification and melting detection have occurred.

27. The method according to any one of claims 1-26, 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. A method for identifying which of a plurality of target nucleic acids is present in a sample, comprising: providing the sample suspected of containing at least one of the plurality of target nucleic acids, Providing a plurality of sample wells configured to amplify the plurality of target nucleic acids, wherein each of the plurality of sample wells contains a primer pair for amplifying one of the plurality of target nucleic acids, and wherein each target nucleic acid has a characteristic melting temperature range, Providing a fluorescent dye that produces an increased fluorescent signal in response to an increase in nucleic acid concentration in the plurality of sample wells, Dispensing the sample among the plurality of sample wells such that each sample well contains a portion of the sample, Subjecting the plurality of sample wells simultaneously to amplification conditions, wherein the amplification conditions include repeated thermal cycles, each thermal cycle including a primer annealing step, a primer extension portion, and a nucleic acid denaturation step, Acquiring the fluorescent signal in each of the plurality of sample wells during the thermal cycling, Determining that the fluorescent signal amplitude of a sample well configured to amplify one target nucleic acid is greater than a threshold that is greater than or equal to the detection limit for inferring an increase in nucleic acid concentration in the sample well, and In response to determining that the fluorescent signal amplitude of the sample is greater than the threshold, performing a melt detection, the melt detection being configured to detect the amplified target nucleic acid in the sample well, wherein the melt detection is defined by the melting temperature range characteristic of the amplified target nucleic acid in the sample well to detect an organism in the sample well.

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

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

31. The method according to any one of claims 28 - 30, wherein the timing of the melt detection is determined by the fluorescent signal of one or more wells configured to amplify the plurality of target nucleic acids rising above the threshold.

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

33. The method according to any one of claims 28 - 32, the method further comprising not performing the melt detection if no sample well shows a fluorescent signal above the threshold.

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

35. The method according to any one of claims 28 - 32, wherein the melt detection comprises: A first rate of temperature increase during a first temperature range of the melt, A second rate of temperature increase during a second temperature range of the melt, and The third heating rate during the third temperature range of the denaturation wherein the second heating rate is slower than the first and third heating rates, and wherein the second temperature range of the denaturation is defined by the denaturation temperature range of the target nucleic acid amplified in the sample wells having a fluorescence signal above the threshold.

36. The method according to claim 35, wherein the second temperature range of the denaturation is + / - 10 °C of the denaturation temperature range of the target nucleic acid, + / - 8 °C of the denaturation temperature range of the target nucleic acid, + / - 6 °C of the denaturation temperature range of the target nucleic acid, or + / - 4 °C of the denaturation temperature range of the target nucleic acid.

37. The method according to claim 35, wherein denaturation detection is performed and the temperature range of the second part of the denaturation is determined.

38. The method according to claim 28, wherein two or more wells show a fluorescence signal above the threshold.

39. The method according to claim 38, further comprising performing one denaturation detection with a denaturation temperature range for the target nucleic acid amplified in the two or more wells.

40. The method according to claim 38, further comprising performing a first denaturation detection with a characteristic first denaturation temperature range that denatures the target nucleic acid amplified in the first well and performing at least a second denaturation detection with a characteristic second denaturation temperature range that denatures the target nucleic acid amplified in the second well.

41. The method according to any one of claims 28-40, wherein the plurality of wells are configured to amplify target nucleic acid sequences from: an organism present at a first titer, an organism present at a second titer lower than the first titer, and an organism present at a third titer lower than the first and second titers, and the method further comprises: performing a first denaturation detection if one or more wells configured to amplify a target nucleic acid sequence from an organism present at the first titer show amplification above the threshold within a first number of thermal cycles, performing a second denaturation detection if one or more wells configured to amplify a target nucleic acid sequence from an organism present at the second titer show amplification above the threshold within a second number of thermal cycles greater than the first number of thermal cycles, and performing a third denaturation detection if one or more wells configured to amplify a target nucleic acid sequence from an organism present at the third titer show amplification above the threshold within a third number of thermal cycles greater than the first and second numbers of thermal cycles.

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

43. The method according to claim 41, further comprising not performing denaturation detection if the one or more wells configured to amplify a target nucleic acid sequence from an organism present at the first titer show amplification above the threshold in more than the first number of thermal cycles.

44. The method according to claim 41, the method further comprising not performing a melting detection if one or more of the wells configured to amplify a target nucleic acid sequence from an organism present at the second titer exhibit amplification above the threshold in more than the second number of thermal cycles.

45. The method according to claim 41, the method further comprising not performing a melting detection if one or more of the wells configured to amplify a target nucleic acid sequence from an organism present at the third titer exhibit amplification above the threshold in more than the third number of thermal cycles.

46. The method according to any one of claims 41-45, the method further comprising not performing a melting detection if none of the one or more wells configured to amplify a target nucleic acid sequence from an organism present at the first titer, the second titer, or the third titer exhibit amplification above the threshold.

47. The method according to claim 28, the method further comprising performing multiplex amplification on the sample before the dispensing step.

48. The method according to claim 47, wherein all steps are performed within a single closed system.

49. A method for determining the presence of an organism in a sample, the method comprising: providing a sample suspected of containing at least one of a plurality of organisms, providing a plurality of sample wells, each sample well provided with primers for amplifying a target nucleic acid from a different one of the plurality of organisms, transferring a portion of the sample into each of the plurality of sample wells, subjecting the plurality of sample wells simultaneously to amplification conditions, acquiring a fluorescence signal in each of the plurality of sample wells during thermal cycling, determining that the fluorescence signal amplitude of a sample well configured to amplify one target nucleic acid is greater than a threshold, the threshold being greater than or equal to the detection limit for inferring an increase in the concentration of nucleic acid in the sample well, in response to determining that the fluorescence signal amplitude of the sample well is greater than the threshold, performing a melting detection in 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 in response to performing the melting detection, determining the presence of the organism in the sample.

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

51. The method according to claim 50, wherein the selected number of amplification cycles is at least one but 15 or fewer, at least one but 20 or fewer, at least one but 25 or fewer, at least one but 30 or fewer, or at least one but 35 or fewer.

52. The method according to any one of claims 49-51, the method further comprising not performing a melting detection if no sample well exhibits positive nucleic acid amplification.

53. The method according to claim 49, the method further comprising simultaneously multiplex amplifying the sample before the moving step.

54. The method according to claim 53, wherein all steps are performed within a single closed system.

55. A system for detecting which of a plurality of target organisms are present in a sample, the system comprising: A container comprising a plurality of sample wells, each sample well configured to receive a portion of the sample, nucleic acid primers that specifically amplify a target nucleic acid from one target organism, a fluorescent dye that produces an increased fluorescent signal in response to an increase in nucleic acid concentration in the plurality of sample wells, and components for amplification, and An instrument configured to subject the portion of the sample in each of the plurality of sample wells to amplification conditions and subsequent denaturation conditions simultaneously, the instrument including a detector for detecting a fluorescent signal indicative of amplification from the fluorescent dye, wherein the instrument is programmed to have the target organisms to be amplified in each of the plurality of wells, melting range information for each of the target nucleic acids from the target organisms, and wherein the instrument is programmed to monitor fluorescence in the plurality of wells during the amplification conditions and, if a sample well shows an increase in fluorescence indicative of positive nucleic acid amplification during the amplification conditions, perform a denaturation detection, wherein the denaturation detection is defined by the melting temperature range of the target nucleic acid amplified in the sample well.

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

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

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

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

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

61. The system according to any one of claims 55-60, wherein the system is programmed to include a denaturation detection, the denaturation detection including: A first heating rate during a first portion of the denaturation, A second heating rate during a second portion of the denaturation, and A third heating rate during a third portion of the denaturation, the second heating rate being slower than the first and third heating rates, and wherein the second portion of the denaturation is defined by the melting range information of the target nucleic acid of the target organism amplified in the well.

62. The system according to any one of claims 55 - 61, wherein the system is programmed to identify at least one target organism present in the sample by identifying the corresponding sample wells in which amplification and melting curve detection have occurred.

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

64. The system according to any one of claims 55 - 63, wherein the instrument includes a light source configured to emit an optical signal towards the plurality of sample wells during screening of the corresponding samples.

65. The system according to any one of claims 55 - 64, wherein the melting curve result is based on the presence or absence of a melting peak within a predetermined temperature range, and a positive result is output if the melting peak is present and a negative result is output if the melting peak is absent.

66. A system for detecting which of a plurality of target organisms is present in a sample, the system comprising: a container containing a plurality of sample wells, each sample well configured to receive a portion of the sample, nucleic acid primers that specifically amplify a target nucleic acid from one target organism, a fluorescent dye that produces an increased fluorescent signal in response to an increase in nucleic acid concentration in the plurality of sample wells, and components for amplification; an instrument configured to subject the portion of the sample in each of the plurality of sample wells simultaneously to amplification conditions and subsequent melting conditions, the instrument including a detector for detecting a fluorescent signal indicative of amplification from the fluorescent dye, and programming that performs the method according to any one of claims 1 - 54, wherein the instrument is programmed to have the identity of the target organism to be amplified in each of the plurality of wells and to have information on the melting range for each of the target nucleic acids from the target organisms.

67. A computer - implemented method for determining the target nucleic acid sequence of a sample, the method comprising: sending, by one or more processors, control signals to a thermal cycling element to heat a plurality of sample wells to a first temperature and cool the plurality of sample wells to a second temperature using an initial heating rate through one or more cycles, wherein each cycle includes an in - cycle temperature adjustment segment, the plurality of sample wells each configured to receive a portion of the sample, each sample well containing a target nucleic acid sequence from a different one of a plurality of target nucleic acids, and each target nucleic acid having a characteristic melting temperature range; receiving, at the one or more processors, data indicative of the amount of fluorescence emitted by those portions of the sample in the plurality of sample wells during the in - cycle temperature adjustment segments of the one or more cycles from an optical system; in response to determining that the amount of fluorescence in at least one of the sample wells exceeds a threshold: determining, by the one or more processors, the target nucleic acid sequence amplified in each of the sample wells that exceed the threshold; determining, by the one or more processors, an adjusted heating rate curve for heating the sample wells based on the characteristic melting temperature range and / or melting rate of temperature increase of the target nucleic acid sequence in each of the sample wells that exceed the threshold; and Unwinding detection of the sample of the portion by the one or more processors includes sending a control signal to a thermal cycling element to heat the sample well to the first temperature using the adjusted heating rate curve for subsequent cycles.

68. The method according to claim 67, wherein the first temperature is a denaturation temperature, the second temperature is an annealing temperature, the characteristic unwinding temperature range of the sample well is from an initial unwinding temperature to a final unwinding temperature, and the adjusted heating rate curve includes: a first heating rate during a proximity temperature range from the annealing temperature to the initial unwinding temperature; a second heating rate during a characteristic unwinding temperature range from the initial unwinding temperature to the final unwinding temperature; and a third heating rate during an ending temperature range from the final unwinding temperature to the denaturation temperature, wherein the second heating rate is slower than the first and third heating rates.

69. The method according to claim 68, wherein the third heating rate is faster than the first heating rate.

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

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

72. The method according to claim 71, wherein the third heating rate is faster than the first heating rate.

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

74. The method according to claim 73, wherein the second heating rate is preferably not greater than 0.5 °C / sec, more preferably not greater than 1 °C / sec, and most preferably not greater than 2 °C / sec.

75. The method according to claim 73 or 74, wherein the second heating rate is adapted to detect nucleic acid unwinding characteristics indicative of a genotype, sequence variant, or gene mutation in a target nucleic acid, the genotype, sequence variant, or gene mutation changing one or more of the nucleic acid unwinding temperature or nucleic acid unwinding curve shape relative to a target nucleic acid not having the genotype, sequence variant, or gene mutation.

76. The method according to claim 75, the method further comprising detecting a single nucleotide polymorphism (SNP).

77. The method according to claim 75, the method further comprising detecting an antimicrobial resistance (AMR) marker.

78. The method according to claim 75, further comprising detecting the presence of an organism via a first melting detection in a first assay, and if the presence of the organism is detected in the first assay, performing a second melting detection in a second assay to detect the presence or absence of the genotype, sequence variant, or gene mutation.

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

80. The method according to any one of claims 67-79, wherein the controller is further configured to: Identify the target nucleic acid corresponding to the sample well among the plurality of target nucleic acids based on the melting detection.

81. The method according to any one of claims 67-80, wherein the controller is further configured to: Determine that the fluorescence amounts of a first sample well and a second sample well exceed the threshold; Perform a first melting detection by using a first adjusted heating rate curve corresponding to a first characteristic melting temperature range for melting the amplified target nucleic acid in the first sample well; and Perform a second melting detection by using a second adjusted heating rate curve corresponding to a second characteristic melting temperature range for melting the amplified target nucleic acid in the second sample well.

82. A system for determining the target nucleic acid sequence of a sample, the system comprising: A plurality of sample wells each configured to receive a portion of the sample, each sample well containing a target nucleic acid sequence from a different one of a plurality of target nucleic acids, wherein each target nucleic acid has a characteristic melting temperature range; An optical system configured to detect the amount of fluorescence emitted by the sample; A controller configured to: Send control signals to a thermal cycling element to heat the plurality of sample wells to a first temperature and cool the plurality of sample wells to a second temperature using an initial heating rate through one or more cycles, wherein each cycle includes an in-cycle temperature adjustment segment; Receive from the optical system data indicating the amount of fluorescence emitted by those portions of the sample in the plurality of sample wells during the in-cycle temperature adjustment segment of the one or more cycles; In response to determining that the fluorescence amount of at least one of the sample wells exceeds a threshold: Determine an adjusted heating rate curve for heating the sample well based on the characteristic melting temperature range of the target nucleic acid sequence in the sample well that exceeds the threshold; And Perform a melting detection on the portion of the sample by sending control signals to the thermal cycling element to heat the sample well to the first temperature using the adjusted heating rate curve for subsequent cycles.

83. The system according to claim 82, wherein 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 heating rate curve includes: A first heating rate during a proximity temperature range from the annealing temperature to the initial melting temperature; A second heating rate during a characteristic melting temperature range from an initial melting temperature to a final melting temperature; and a third heating rate during an end temperature range from the final melting temperature to a denaturation temperature, wherein the second heating rate is slower than the first and third heating rates.

84. The system according to claim 83, wherein the third heating rate is faster than the first heating rate.

85. The system according to claim 83 or claim 84, wherein the second heating rate is the same as the initial heating rate.

86. The system according to any one of claims 82-85, wherein the first heating rate is greater than 4 °C / sec (e.g., 6-20 °C / sec), the second heating 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 rate is greater than 4 °C / sec (e.g., 6-20 °C / sec).

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

88. The system according to any one of claims 82-87, wherein the controller is further configured to: Identify the target nucleic acid corresponding to the sample well among the plurality of target nucleic acids based on the melting detection.

89. The system according to any one of claims 82-88, wherein the controller is further configured to: Determine that the fluorescence amounts in a first sample well and a second sample well exceed the threshold; Perform a first melting detection by using a first adjusted heating rate curve corresponding to a first characteristic melting temperature range that causes the target nucleic acid amplified in the first sample well to melt; and Perform a second melting detection by using a second adjusted heating rate curve corresponding to a second characteristic melting temperature range that causes the target nucleic acid amplified in the second sample well to melt.

90. A computing device for determining the target nucleic acid sequence of a sample, the computing device comprising: One or more processors; and A non-transitory computer-readable memory coupled to the one or more processors and storing instructions thereon, which when executed by the one or more processors, cause the computing device to: Send a control signal to a thermal cycling element to heat a plurality of sample wells to a first temperature and cool the plurality of sample wells to a second temperature using an initial heating rate through one or more cycles, wherein each cycle includes an in-cycle temperature adjustment segment, each of the plurality of sample wells is configured to receive a portion of the sample, each sample well contains a target nucleic acid sequence from a different one of a plurality of target nucleic acids, and each target nucleic acid has a characteristic melting temperature range; Receive data from an optical system indicating the amount of fluorescence emitted by those portions of the sample in the plurality of sample wells during the in-cycle temperature adjustment segment of the one or more cycles; In response to determining that the fluorescence amount in at least one of the sample wells exceeds a threshold: Determine an adjusted heating rate curve for heating the sample well based on a characteristic melting temperature range of a target nucleic acid sequence in the sample well that exceeds the threshold; and Perform melting detection on the portion of the sample by sending a control signal to the thermal cycling element to heat the sample well to a first temperature using the adjusted heating rate curve for subsequent cycles.

91. The computing device according to claim 90, wherein 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 heating rate curve includes: A first heating rate during a proximity temperature range from the annealing temperature to the initial melting temperature; A second heating rate during the characteristic melting temperature range from the initial melting temperature to the final melting temperature; and A third heating rate during an end temperature range from the final melting temperature to the denaturation temperature, wherein the second heating rate is slower than the first and third heating rates.

92. The computing device according to claim 91, wherein the third heating rate is faster than the first heating rate.

93. The computing device according to claim 91 or claim 92, wherein the second heating rate is the same as the initial heating rate.

94. The computing device according to any one of claims 90-93, wherein the first heating rate is greater than 4 °C / sec (e.g., 6-20 °C / sec), the second heating 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 rate is greater than 4 °C / sec (e.g., 6-20 °C / sec).

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

96. The computing device according to any one of claims 90-95, wherein the instructions further cause the computing device to: Identify the target nucleic acid corresponding to the sample well among the plurality of target nucleic acids based on the melting detection.

97. The computing device according to any one of claims 90-96, wherein the instructions further cause the computing device to: Determine that the fluorescence amounts of a first sample well and a second sample well exceed the threshold; Perform a first melting detection by using a first adjusted heating rate curve corresponding to a first characteristic melting temperature range that causes the amplified target nucleic acid in the first sample well to melt; and Perform a second melting detection by using a second adjusted heating rate curve corresponding to a second characteristic melting temperature range that causes the amplified target nucleic acid in the second sample well to melt.

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