Biological sample analysis for mutation detection

Abstract

A method of biological sample analysis includes subjecting a biological sample comprising nucleic acid to an amplification reaction conducted in the presence of a detector probe and a calibrator probe, wherein the detector probe is configured to specifically interact with a first region of a wild type target nucleic acid sequence and emit a first emission signal in response to the interaction, and wherein the calibrator probe is configured to specifically interact with a second region of the wild type target nucleic acid sequence and emit a second emission signal in response to the interaction, the first region and the second region differing from each other. The method further includes calculating a ratio of the change in the first emission signal to the change of the second emission signal at a point in time of the amplification reaction.

Description

PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 BIOLOGICAL SAMPLE ANALYSIS FOR MUTATION DETECTION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 590,062, filed October 13, 2023, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] This disclosure relates to biological sample analysis techniques utilizing probe carrying a detectable label. More specifically, aspects of the disclosure relate to systems and methods for detection of nucleic acids in biological samples by utilizing one or more such probes during an amplification reaction, such as in a quantitative polymerase chain reaction (qPCR) assay. INTRODUCTION

[0003] Nucleic acid detection assays can be carried out by adding a sample that is suspected of including one or more target nucleic acids to a reaction mixture. The reaction mixture can include one or more probes carrying detectable labels each designed to interact with one or more different target nucleic acids and generate an emission signal, via the detectable labels, which corresponds to the amount of target nucleic acid in the reaction mixture. Subjecting the sample and reaction mixture to an amplification reaction results in an amplified product of the target nucleic acid (copies of the target nucleic acid) when present in the starting sample. By detecting the signal from the detectable labels of the probe which interacted with the amplified product, the initial amount of target nucleic acid can be determined.

[0004] Quantitative polymerase chain reaction (qPCR) is a type of amplification reaction used to link a detectable signal to the amount of a target nucleic acid present in a biological sample during multiple cycles of a polymerase chain reaction (PCR). In various implementations, a probe sequence that matches the target nucleic acid sequence, or a portion thereof, is used to carry a detectable label, such as a fluorophore. During the amplification cycles of the PCR reaction, fluorescent signal isPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 detected to determine an amount of amplified product of the target nucleic acid generated.

[0005] Use of probes and amplification reactions, such as in a qPCR assay, can pose challenges in the presence of relatively low concentrations of the target nucleic acid, including when mutation of the target nucleic acid may be present, such as a mutant allele of the wild type. For example, binding efficiency of the probe that is chosen due to its match with a target nucleic acid sequence can be reduced in the presence of mutations of the target nucleic acid due to imperfections of the match. Further, if the probe matches a region of the target nucleic acid that does not include the differing region (subsequence) of the target nucleic acid (due to the mutation), then it may not be possible to determine the existence of mutant sequences in the final amplified product. This may result in skewing the determination of the concentration of the wild type target nucleic acid versus a mutation of the target nucleic acid in the sample.

[0006] There exists a need to improve upon probe-based qPCR assays that permit detection of mutant target nucleic acid sequences and of lower concentrations of target nucleic acids in samples, including detection of mutations and distinguishing of wild-type target nucleic acids from mutations of the target nucleic acid. Moreover, there exists a need to provide reliable signal detection techniques that can distinguish between signals detected during amplification reactions, such as in qPCR assays, so as to provide improved detection at lower concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Various objects, features, characteristics, and / or advantages of embodiments of the present disclosure will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings and the appended claims, all of which form a part of this application. In the Drawings, like reference numerals may be utilized to designate corresponding or similar parts in the various Figures, and the various elements depicted are not necessarily drawn to scale. In the Drawings:PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304

[0008] FIG.1 is a schematic illustration of an amplification plot illustrating aspects relating to the present disclosure.

[0009] FIG.2 is a schematic overview of a biological sample analysis workflow utilizing a target probe and a calibrator probe in accordance with various embodiments.

[0010] FIGs.3A and 3B respectively illustrate amplification curves from amplification data from an example of mutation detection utilizing a biological sample analysis assay employing detector and calibrator probes for Mycoplasma Genitalium (MG) for samples containing the wild type (WT) MG and containing a mutant MG having mutation A-260G on the ParC gene.

[0011] FIG.4 is a schematic illustration of an amplification plot illustrating yet other aspects relating to the present disclosure.

[0012] FIG.5 illustrates amplification curves from amplification data form an example of mutation detection utilizing a biological sample analysis assay employing target probe for Micoplasma Genitalium (MG) for samples containing the wild type (WT) MG and containing a mutant MG having mutation A-260G on the ParC gene.

[0013] FIG.6 is a plot showing amplification curves and illustrating the effect of concentration on the amplification curve profiles.

[0014] FIG.7 is a schematic overview of another biological sample analysis workflow utilizing a target probe in accordance with various embodiments.

[0015] FIG.8 illustrates the relation between max dRn and the cycle at which threshold is reached (Ct) for wild type samples (diamonds) and mutant samples (solid circles) from amplification data, as well as calculated “boundary” data (open circles) and linear equations from an example of mutation detection utilizing a biological sample analysis assay employing detector probe for Mycoplasma Genitalium (MG) for samples containing the wild type (WT) MG and a mutant MG having mutation A-260G on the ParC gene. DETAILED DESCRIPTIONPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304

[0016] The present disclosure contemplates a method of biological sample analysis that includes subjecting a biological sample containing a nucleic acid to an amplification reaction conducted in the presence of a detector probe and a calibrator probe, with the detector probe configured to specifically interact with a first region of a wild type target nucleic acid sequence and emit a first emission signal in response to the interaction, and the calibrator probe configured to specifically interact with a second region of the wild type target nucleic acid sequence and emit a second emission signal in response to the interaction, the first region and the second region differing from each other. The method also includes measuring the first and second emission signals during the amplification reaction; calculating a ratio of the change in the first emission signal to the change of the second emission signal; comparing the ratio to a threshold ratio; and based on the comparing, determining one of a presence or absence of an mutation of the target nucleic acid sequence in the amplified product.

[0017] Implementations of the above method may include one or more of the following aspects in various combinations. The method where the amplification reaction may include a polymerase chain reaction (PCR). The calculating the ratio corresponds to a predetermined point in time of the amplification reaction. The amplification reaction is a PCR amplification reaction and the predetermined point in time is at predetermined number of cycles of the PCR amplification reaction. The predetermined number of cycles is a last cycle of the PCR amplification reaction or a predetermined number of cycles after a cycle threshold value for the calibrator probe is reached, whichever occurs first. Each of the detector probe and the calibrator probe emit differing emission signals in response to interaction with the mutation of the target nucleic acid. The second region of the wild type target nucleic acid may include a region of the wild type target nucleic acid that remains relatively unchanged in the presence of a mutation of the target nucleic acid. The first and second emission signals are fluorescence.

[0018] The present disclosure further contemplates a method of biological sample analysis that includes receiving first data corresponding to a change of an amount of a first nucleic acid subsequence of a wild type target nucleic acid measured over a time period during an amplification reaction on the biological sample, the amplificationPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 reaction configured to produce an amplification product of the wild type target nucleic acid; receiving second data corresponding to a change of an amount of a second nucleic acid subsequence of the wild type target nucleic acid sequence measured over the number of cycles during the amplification reaction; at a predetermined number of cycles, calculating a ratio of the change of the amount of the first nucleic acid subsequence to the change of the amount of the second nucleic acid subsequence. The method also includes comparing the ratio to a predetermined threshold; and based on the comparing, determining one of a presence or absence of a mutation of the target nucleic acid sequence in the biological sample.

[0019] Implementations of this aspect may include one or more of the following in various combinations. The method where the first data may include first emission signal data from a detector probe and the second data may include second emission signal data from a calibrator probe, where the detector probe is configured to specifically interact with the first nucleic acid subsequence and the calibrator probe is configured to specifically interact with the second nucleic acid subsequence. The emission signal is fluorescence. The first emission signal differs from the second emission signal in the presence of a mutation of the target nucleic acid in the sample.

[0020] The present disclosure yet further contemplates a method of biological sample analysis that includes subjecting a biological sample to an amplification reaction including multiple cycles conducted in the presence of a detector probe, with the detector probe configured to specifically interact with a wild type target nucleic acid and emit an emission signal in response to interaction with amplified product of the wild type target nucleic acid; measuring the emission signal during the amplification reaction; based on the measuring, identifying a value corresponding to a cycle of the amplification reaction at which the change in measured emission signal reaches a threshold and identifying a maximum amplitude value associated with the change of emission signal generated during the amplification reaction; comparing the value to a calculated cycle value, with the calculated cycle value is determined by a mathematical function of the maximum; and based on the comparing, determining one of a presence or absence of a mutation of the target nucleic acid sequence in the amplified product.PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304

[0021] Implementations of the above may include one or more of the following in various combinations. The mathematical function is a linear slope intercept equation. The mathematical function is a boundary condition function, where calculated Ct from the mathematical function greater than a predefined value indicates presence of the wild type target nucleic acid and calculated Ct less than the predefined value indicates presence of the mutation of the target nucleic acid.

[0022] The present disclosure also contemplates a method of biological sample analysis that includes determining, for a plurality of concentrations of a sample containing a wild type target nucleic acid, a respective plurality of wild type data points may include pairs of a cycle threshold and maximum change in emission signal from a detector probe generated by an amplification reaction configured to produce amplified product of the wild type target nucleic acid; determining, for a plurality of concentrations of a sample containing a mutation of the target nucleic acid, a respective plurality of mutant data points may include pairs of a cycle threshold and maximum change in emission signal from the detector probe generated by the amplification reaction; using at least some of the mutant data points, generating a mathematical function expressing maximum limit on change in amount of emission signal as a function of cycle threshold; using the mathematical function and the cycle thresholds from the wild type data points, calculating putative maximum limits of change in amount of generated amplified product of the mutation of target nucleic acid; calculating boundary points based on values between the calculated putative maximum limits and the measured target maximum limits corresponding to at least some of the wild type data points; based on the boundary points, determining a boundary mathematical function of cycle threshold and maximum limit of change of nucleic acid sequence in an amplification product.

[0023] Implementations of the above method may include one or more of the following in various combinations. The method may include storing instructions to execute the boundary function on computer readable media on amplification data of an amplification curve obtained from an amplification reaction of a biological sample, the amplification reaction configured to produce amplified product of the wild type target nucleic acid and of the mutation of the target nucleic acid. The method of any may include outputtingPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 information regarding presence of wild type target nucleic acid in the amplified product or presence of the mutation of the target nucleic acid in the amplified product. The amplification reaction is an amplification reaction of a qPCR assay using a detector probe configured to specifically interact with the wild type target nucleic acid. The mathematical functions are determined using a linear regression analysis. The mathematical functions are determined using machine learning models. The mathematical functions are one of linear, exponential, or polynomial.

[0024] The present disclosure yet further contemplates a method of biological sample analysis that includes obtaining emission signal data from an amplification reaction conducted on a biological sample, the amplification reaction configured to produce amplified product of a wild type target nucleic acid. The method also includes identifying from the emission signal data: a cycle of the amplification reaction at which a change in the emission signal data measured over the course of the amplification reaction reaches a threshold, and a maximum value of the change in emission signal data measured over the course of the amplification reaction. The method also includes utilizing a mathematical function expressing cycle as a function of maximum change in emission signal, calculating a cycle value from the identified maximum value. The method also includes comparing the identified cycle to the calculated cycle value; and based on the comparing, outputting one of an indication that the biological sample contains the wild type target nucleic acid or an indication that the biological sample contains the mutation of the target nucleic acid.

[0025] Implementations may include one or more of the following in various combinations. The method where the emission signal data is fluorescence. The emission signal data is from a detector probe used in the amplification reaction and configured to specifically interact with the wild type target nucleic acid. Implementations of the described techniques may include hardware, a method or process, or computer software on a computer-accessible medium

[0026] Other implementations according to the present disclosure include corresponding kits and systems capable of carry out the above methods, computer readable media storing instructions to be executed by processor to carry out the above methods, and / orPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 a system comprising such a processor of a computer device and a memory or computer readable media storing such instructions.

[0027] In the context of a nucleic acid probe and a target nucleic acid, the term “specifically interact” (and similar terms) indicates that the probe is designed to interact with the target nucleic acid to a greater degree than with non-target nucleic acids that may also be present in the reaction mixture. For example, specific interaction may include hybridization (binding) of the probe, in whole or in part, with the corresponding target nucleic acid. The hybridization between the probe and target need not be 100%. For example, functionally effective interaction may be accomplished with probes having homology to their respective target of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or up to 100%.

[0028] In the context of a nucleic acid probe and a target nucleic acid, the term “reduced interaction” , “inefficient interaction” (and similar terms) indicates that the probe that is designed to interact with a target nucleic acid to a greater degree than with non-target nucleic acids that may also be present in the reaction mixture can nonetheless have a level of interaction with the target nucleic acid but that is reduced or inefficient compared to if it specifically interacted. For example, reduced interaction may include hybridization (binding) of the probe, in whole or in part, with the corresponding target nucleic acid but at a reduced rate due to imperfections in the matching of the probe sequence and the actual sequence of the target nucleic acid found in the sample. This can occur for example when the sample contains different alleles of the target nucleic acid, including for example, a mutant rather than a wild type (WT) target nucleic acid in the sample. For example, reduced or inefficient interaction may thus result in a diminished amplification (signal) of the probe relative to that of the amplification (signal) resulting from the interaction with the target nucleic acid (e.g., WT). The diminished amplification can range from 0-80% of that of the WT amplification, for example.

[0029] As used herein with respect to signals, a “detectable signal” has a value significantly above a baseline level, including an emission (e.g., fluorescence) signalPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 that is significantly above a baseline level of emission (e.g., fluorescence). This may be defined by a threshold value, for example, that separates baseline from detectable fluorescence. The threshold value may vary according to particular testing protocols and application needs. In some embodiments (e.g., without a passive reference), the threshold is set at a dRn of about 1,000 to about 30,000, or about 2,000 to about 20,000, or about 3,000 to about 15,000 or about 4,000 to about 6,000, for example, or within a range having endpoints defined by any two of the foregoing values. In some embodiments, such as for example those including passive fluorescence with a signal that does not change with amplification, the threshold is set at a dRn of about 0.01 to 0.5, for example. In some embodiments, the threshold value is some percentage above the baseline level, such as about 5 percent to about 10 percent above the baseline level.

[0030] The “baseline” level of signal, which sometimes is referred to by those of ordinary skill in the art as a “background” level, during an amplification process may be determined according to methods known to those of skill in the art. As a non-limiting example, the baseline level may be determined as the median signal of the amplification cycles before exponential amplification occurs. For example, exponential amplification may be determined when the change in signal from one amplification cycle to the next exceeds a certain percentage indicative of exponential change.

[0031] The terms “detectable signal” and “label signal” are used synonymously herein. For example, a “label signal” is the signal emitted by a label associated with a probe. Multiple different “detectable signals” / “label signals” may contribute to the same total signal.

[0032] The term “target nucleic acid” is used to refer to the nucleic acid of interest in a sample and having a sequence that is the typical expression of a gene sequence. The nucleic acid can be ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In applications relating to mutation detection, the target nucleic acid can be the wild type (WT) expression of the nucleic acid sequence or a mutated version. The nucleic acid can also correspond to specific alleles.PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304

[0033] The term “mutation”, “mutant allele”, “mutant” and variants thereof are used to refer to any modification of the oligonucleotide sequence of the target nucleic acid, but such that the overall sequence can be discerned as a version of the target nucleic acid. Thus, these terms are used interchangeably to refer to versions of the target nucleic acid that differ in a single oligonucleotide base or a region (subsequence) of the oligonucleotide bases of the overall sequence of the wild type target nucleic acid.

[0034] The term “detector probe” and variants thereof is used to refer to a molecule having a nucleic acid sequence that is configured to specifically interact with a specified (e.g., first) region of a target nucleic acid sequence. In various embodiments, the detector probe can carry a label that is configured to emit a label signal. With an amplified product of the target nucleic acid sequence being generated during the course of the amplification reaction, the detectable label signal increases as the specific interaction of the detector probes and amplicons of the target nucleic acid sequence increases, resulting in an increase in an emitted label signal associated with the detector probe.

[0035] The term “calibrator probe” and variants thereof is used to refer to a molecule having a nucleic acid sequence that is configured to specifically interact with a specific (e.g., second) region of a target nucleic acid sequence that differs from (e.g., does not overlap with) the region that a detector probe is configured to specifically interact with. In various embodiments, the calibrator probe can carry a label that is configured to emit a label signal. With an amplified product of the target nucleic acid sequence being generated during the course of the amplification reaction, the calibrator probe increases as the specific interaction of the calibrator probes and amplicons of the target nucleic acid sequence increases, resulting in an increase in an emitted label signal associated with the calibrator probe. To distinguish from the detector probe, the calibrator probe label can have an emission signal that does not overlap within a detection channel for the detector probe label emission signal.

[0036] The terms “determine,” “calculate,” and “estimate” are used synonymously herein. These terms are not intended to imply an exact level of measurement precision.PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 Thus, where a value is “determined,” “calculated,” or “estimated” using the embodiments described herein, it will be understood that such a value may include some degree of inherent error due to factors such as detection instrument tolerances, rounding, chemical reaction variability, and other inherent measurement imperfections known and understood by those of skill in the art.

[0037] In addition, unless otherwise indicated, numbers expressing quantities, constituents, distances, or other measurements used in the specification and claims are to be understood as optionally being modified by the term “about” or its synonyms. When the terms “about,” “approximately,” “substantially,” or the like are used in conjunction with a stated amount, value, or condition, it may be taken to mean an amount, value or condition that deviates by less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of the stated amount, value, or condition. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0038] Any headings and subheadings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims.

[0039] It will also be noted that, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” do not exclude plural referents unless the context clearly dictates otherwise. Thus, for example, an embodiment referencing a singular referent (e.g., “widget”) may also include two or more such referents.

[0040] It will also be appreciated that embodiments described herein may also include properties and / or features (e.g., substances, components, members, elements, parts, and / or portions) described in one or more separate embodiments and are not necessarily limited strictly to the features expressly described for that particular embodiment. Accordingly, the various features of a given embodiment can be combined with and / or incorporated into other embodiments of the present disclosure. Thus, disclosure of certain features relative to a specific embodiment of the present disclosure should not be construed as limiting application or inclusion of said features to thePCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 specific embodiment. Rather, it will be appreciated that other embodiments can also include such features. Overview of Biological Sample Analysis Using Quantitative Polymerase Chain Reaction

[0041] In biological sample analysis using amplification reactions, such as in qPCR assays, the accumulation of amplification product can be measured as the amplification reaction progresses, in real time, with quantification of amplified product occurring along the way, e.g., after each cycle of the amplification reaction. In qPCR, the cycle is a thermal cycle of a PCR amplification reaction. As noted above, real-time detection of amplification product is enabled by the inclusion of a detectable label (e.g., fluorescent molecule) used as part of the reaction mixture that yields increased emission signal (e.g., fluorescence) with an increasing amount of amplified product, which product is the number of copies of the target nucleic acid sequence (amplicon). In some implementations, the detectable label is part of (e.g., carried by) sequence-specific primers and / or probes which are configured to specifically interact with a target nucleic acid of interest so that the amount of amplification product of the target nucleic acid can be determined based on the detection of the label. For example, an emission signal from the detectable label, such as fluorescence for example, is proportional to the total amount of amplicons generated and the change in the emission signal over time is used to calculate the amount of amplicons produced in each cycle.

[0042] Thus, in some implementations of a qPCR assay, a detector probe is used and the change in emission signal amplitude (dRn) (e.g., fluorescence) is measured over the course of the amplification reaction. The detector probe, as noted above, is a nucleic acid molecule with a sequence designed to specifically interact with a target nucleic acid of interest to detect the presence and / or quantity (e.g., concentration) of the target nucleic acid in the biological sample being analyzed. The label signal (e.g., fluorescence emission) is measured during multiple cycles of amplification and if the label signal is detectable over a threshold level of signal, it can be determined that the target nucleic acid is present in the sample being analyzed. If the label signal is not detectable overPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 the threshold level, the target nucleic acid may be determined to not be present in the sample tested.

[0043] However, in some cases, although the label signal is not detectable, a mutation of the target nucleic acid may nevertheless be present in the sample and / or the amount of amplified product of the target nucleic acid may be sufficiently low in concentration so as to not produce signal above the threshold. For example, because nucleic acids are subject to mutation, if a mutant of the target nucleic acid that the detector probe is designed for is present in the sample being analyzed, the detector probe sequence may imperfectly match the mutant form of target nucleic acid sequence that is present due to one or more different nucleotides over a subsequence of the target nucleic acid sequence. An imperfect match can reduce the efficiency with regard to occurrence of binding of the detector probe with the mutation of the target nucleic acid sequence, thereby resulting in a relative reduction of the label signal from the detector probe that would otherwise be detectable. In other words, a reduced or inefficient interaction of the detector probe with the mutant target nucleic acid sequence occurs. This can lead to inaccuracy in the quantification of the target nucleic acid sequence, including both the wild type target nucleic acid and the mutation of the target nucleic acid. Such inaccuracy can include false negatives in situations where the label signal is below the threshold, whether due to inefficient interaction because of the presence of the mutation in the sample or due to low concentration of the mutation and / or the target nucleic acid in the sample. Use of Calibrator Probe and Ct Shift to Detect Mutant Target Nucleic Acid

[0044] In some implementations of biological sample analysis, to assist with the aforementioned issues, another probe (second probe) can be used in addition to the detector probe (first probe). The second probe can have a nucleic acid sequence that is configured to specifically interact with a region (subsequence) of the target nucleic acid that is well-preserved (not anticipated to change) between the target nucleic acid and the mutation of the target nucleic acid. The second probe, also referred to as a calibrator probe, specifically interacts with a well-preserved region (subsequence) of thePCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 target nucleic acid which is less likely to change even in the mutated form as compared to other regions (subsequences) of the target nucleic acid targeted by the detector probe. The calibrator probe emits a second label signal different and distinguishable from the first label signal emitted by the detector probe. By measuring and comparing the respective label signals from the calibrator and detector probes, a determination can be made as to the presence of the mutation of the target nucleic acid, and also quantification of the wild type target nucleic acid and the mutation of the target nucleic acid.

[0045] In particular, in one implementation, use of the calibrator probe involves measuring the first and second label signals and determining that the second label change in signal level of the calibrator probe and the first label change in signal of the detector probe reaches a predefined threshold level. If the second label change in signal is detected to reach the predefined threshold level sooner than a predefined number of cycles as compared to the first label signal, then it is determined that the mutation of the target nucleic acid sequence exists; otherwise, if the first and second label changes in signal reach the predefined threshold level but after the predefined number of cycles, it is determined that only the wild type target nucleic acid exists. If the first and second label changes in signal do not reach the predefined threshold level, then it is determined neither the wild type target nucleic acid nor the mutation of the target nucleic acid exists in the sample.

[0046] The cycle at which the first or second label signal reaches the predefined threshold level is referred to herein as a “cycle threshold (Ct),” and thus the implementation looks for a “Ct shift” between the first and second label signals. In other words, if the respective cycles at which the change in first and second label signals of the first and second (detector and calibrator) probes reach a threshold level differ a Ct shift exists. The degree of Ct shift can thus serve as an indication of the presence of the mutation of the target nucleic acid in the sample because otherwise it would be expected for the change in first and second label signals to reach the predefined threshold level at the same time as they correspond to probes (detector and calibrator) that would each be interacting with the same wild type target nucleic acid. But in thePCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 presence of the mutation of the target nucleic acid sequence, the second (calibrator) label signal would grow faster and reach the Ct sooner than the first (detector) label signal due to the binding of the calibrator probe with the well-preserved subsequence of the allele of the target nucleic acid versus the inefficient (or absence of) interaction of the detector probe with the mutation of the target nucleic acid sequence.

[0047] To facilitate an understanding of the Ct, Ct shift, and related aspects of the present disclosure, FIG.1 is a generic amplification plot that schematically illustrates how the signal traces respectively associated with the calibrator probe and the detector probe differ in the cases of sample containing the wild type target nucleic acid versus a sample that contains the mutation of the target nucleic acid. In FIG.1, the X-axis represents the number of amplification cycles (Cycle) and the Y-axis the measured change in label signal (e.g., fluorescence in embodiments wherein fluorescent labelled probes are utilized) (dRn). The dashed line 100 represents the threshold signal level required for both the calibrator and the detector label signal resulting from amplified product from a biological sample undergoing amplification to be determined to be at a level indicative the presence of the amplified product, and thus the original nucleic acid being amplified in the sample. As described above, the cycle where the measured change in signal crosses the threshold line 100 is referred to as a cycle threshold (Ct) herein, with the “Ct” for the different amplification curves described below indicated on the plot.

[0048] In various embodiments, the threshold signal level noted above is determined by measuring the amount of noise in the signal and may be established using experiments and measuring how much change in dRn can be expected during the amplification (e.g., PCR) reaction when the target nucleic acid is absent. In this manner, a statistical estimation of the maximum value that dRn could reach can be established. The threshold can then be set to something sufficiently higher than that value, such as for example two times or more, so that a dRn signal would not reach that level in the absence of amplification. To address issues with potential contamination and amplification related to the same, in addition to the threshold level, a Ct cutoff cycle can be utilized as a limit on how late in the overall amplification (e.g., PCR) process the CtPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 value can be detected and still be considered as indicative that the target nucleic acid is present. Various embodiments may use a cutoff cycle of about 30-40 cycles, such as for example, 35-37 cycles.

[0049] The amplification curves on the amplification plot generally follow a predictable concentration-dependent shape in the presence of a target nucleic acid in a sample and during an amplification reaction that relies on a detector probe with a detectable label designed to specifically interact with the nucleic acid. In an initial phase of the amplification reaction, the dRn curves are relatively unchanging as the target nucleic acid of interest is amplifying. Once the amplified product reaches a sufficient concentration for the emitted label signal of the detector probe to be detected, the curve undergoes a transient exponential growth phase, as labeled in FIG.1 with respect to generally the upper amplification curve shown. The exponential phase refers to the increasing accumulation of amplification products over the course of amplification, where no factor is limiting. After the exponential phase, the amplification curve transitions to a “non-exponential plateau phase,” which also is labeled in FIG.1. In this latter phase, reaction components (e.g., reagents, etc.) are reduced, thereby limiting the maximum amplitude of the amplification product being generated and corresponding change in emission signal, until the amplification reaction eventually stops. The amplification curves thus follow a sigmoid shape, as reflected by the two different curves illustrated in FIG.1.

[0050] For purposes of explanation, FIG.1 depicts on a single amplification plot two different amplification curves associated with: a first emission signal (e.g., fluorescent signal) from a detector probe (lower curve) and a second emission signal (upper curve) from a calibrator probe in the presence of a mutation of the wild type target nucleic acid in the sample. In the presence of the wild type target nucleic acid in the sample, the corresponding detector amplification curve would be substantially coincident with the calibrator probe curve (not shown in FIG.1).

[0051] The Ct shift technique discussed above thus address some of the issues with qPCR assays for biological sample analysis and quantification of target nucleic acid in the presence of mutant sequences. But such technique can also depend on the first andPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 second label signals (from the detector and calibrator probes) being sufficiently high so as to at least register the Ct of the calibrator and obtain a clear separation between the respective curves in the amplification plot. Stated differently, due to the different rate of increase of the detector and calibrator in presence of a mutant, the higher the threshold for detection, the larger the difference in Ct values between the two so as to discern a clear separation of the Ct shifts. But setting a relatively high threshold level to determine the Ct shift can result in the detector (first) label signal being too low to detect that it has reached the threshold level due to the concentration of the target nucleic acid sequence in the sample is relatively low.

[0052] Further, at relatively low concentrations of the target nucleic acid in the sample, variability of the Ct can be relatively large due to the nonlinear relation between dRn amplitude and Ct value making it difficult to identify an absolute criteria for what is a significant difference in Ct shift to distinguish between a wild type and a mutant sample. For example, if variability is high the Ct shift can range from 2 cycles to 4 cycles or more, which results in a loss of sensibility (i.e. mutants will be detected only when Ct shift value is large).

[0053] With reference to FIG.1, it can be seen that for a low threshold (e.g., TH1 = 5000) the Ct shift given by point 2 to point 1 on the calibrator and detector curves is less than 4 cycles, while for a higher threshold (e.g, TH 2 = 15000) the Ct shift given by point 4 to point 3 on the calibrator and detector curves is 9 cycles. Thus, the threshold has a relatively large impact on the efficacy of the Ct shift as a metric to differentiate between amplification curves that indicate wild type versus mutant target nucleic acid in a sample. If in this case the criteria to identify mutants is that Ct shift must be larger than 4, only for the larger threshold (e.g., TH2) would the correct discrimination be able to be made. But if the threshold is set too high (e.g., 50,000) the calibrator might not reach the threshold before the cutoff cycle value, resulting in the sample being determined as not having an amplification product (i.e., no wild type of mutant target nucleic acid). Thus, the Ct shift methodology requires samples to have large enough concentration so the calibrator signal will always reach the threshold which might be larger than the usual threshold used for presence / absence detection, as the number of cycles of separationPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 may need to be sufficiently large in order to differentiate between the calibrator and detector curves to indicate presence of wild type or mutant target nucleic acid in the sample.

[0054] Various embodiments in accordance with the disclosure provide robust techniques that enable accurate and relatively fast biological sample analysis using amplification assays, such as, e.g., qPCR assays, even in the presence of relatively low concentrations of target nucleic acid sequences of interest in the sample and that can utilize one probe instead of two, reducing the cost of the assays and allowing for higher multiplex capabilities. For example, various embodiments described herein can improve the accuracy of detecting the presence of and quantifying the amount of a mutant target nucleic acid present in a sample, even for relatively low concentrations. Moreover, various embodiments can be implemented in a manner that is somewhat independent of an actual threshold level of signal that might be applied to determine the Ct, thereby making them more robust to implement regardless of instrument variability. Biological Sample Analysis Using Ratio of Calibrator to Detector Probe Emission Signals for Nucleic Acid Detection

[0055] In accordance with one embodiment of the present disclosure, to determine the presence of wild type versus mutant target nucleic acid in a sample, the ratio of the change in label signal of the detector probe to the change in label signal of the calibrator probe is utilized. Using the ratio addresses issues associated with the Ct shift technique discussed above because, given the assumption that the target nucleic acid is in the sample in some form (e.g., wild type target or mutant), it is expected that the calibrator will cross a threshold level of label signal. Given this, if the sample corresponds to the mutant target nucleic acid present, the calibrator probe label signal is expected to amplify significantly more than the detector probe label signal. Consequently, the aforementioned ratio will be small. As a corollary, if the wild type target nucleic acid is present in the sample, the calibrator and detector probe label signals will amplify substantially by a similar amount and thus the ratio will be closer to 1. A threshold ratio (e.g., 0.5) can be used to determine whether the resulting ratio above corresponds to aPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 determination that the wild type target nucleic acid or a mutation of the target nucleic acid is in the sample.

[0056] With reference again to FIG.1, various aspects relating to the use of a ratio of the change in label signal (dRn) of a calibrator probe and detector probe are illustrated. In FIG.1, dRn values labeled “D” for denominator and “N” for numerator are obtained either at the last cycle or at a number of amplification cycles past the Ct associated with the calibrator amplification curve produced from detecting the label signal associated with the calibrator probe. These dRn values are incorporated into a ratio wherein the denominator is always the dRn value, D, for the calibrator probe and the numerator would be the dRn value of the detector probe (N), which values differ for the different amplification curves as discussed above.

[0057] As can be seen graphically in FIG.1, N / D is noticeably lower than 1, for example about 0.3 in the schematic illustration of FIG.1. Thus, by looking at the ratio at a cycle sufficiently past the Ct cycle of the calibrator but before both curves reach saturation, the ratio can be used to determine that the mutant target nucleic acid is in the sample being analyzed, and this is the case whether the sample contains relatively high or low concentrations of the wild type target nucleic acid or mutant target nucleic acid. Depending on the design of the probes, a ratio significantly larger, such as ranging from 0.5 to 2, or for example close to 1 is expected to occur if the wild type target nucleic acid is in the sample.

[0058] As can also be seen by the schematic illustration of FIG.1, the relevant ratio can drop to sufficiently lower than 1 so as to be able to determine the mutant target nucleic acid sequence exists relatively more quickly after the calibrator probe reaches the Ct than the cycle at which the Ct shift is observed. This is particularly evident when comparing the cycle at which the ratio is sufficiently lower than 1 and the Ct shift associated corresponding to the lower amplification curve. With reference to FIG.1, by way of example, it can be seen that if the ratio is computed 2 cycles after the calibrator’s Ct, the dRn ratio can be computed at cycle 31.3 for the higher threshold TH 2, whereas the Ct shift would need to be computed at cycle 38.4.PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304

[0059] Turning now to FIG.2, an example workflow 200 for biological sample analysis in accordance with the present disclosure is illustrated and will now be described. At 202, a biological sample comprising nucleic acid is subjected to an amplification reaction conducted in the presence of reagents comprising a detector probe and a calibrator probe. The amplification reaction can be a reaction associated with a qPCR assay.

[0060] As discussed above, the detector and calibrator probes can be designed to specifically interact with a target nucleic acid such that the calibrator probe is designed to specifically interact with a region (subsequence) of the wild type target nucleic acid that does not significantly change (is well-preserved) even in the presence of a mutation of the target nucleic acid. The detector probe can be designed to specifically interact with a region (subsequence) of the wild type target nucleic acid that does change in the presence of an allele of the target nucleic acid, including, e.g., a mutation of the WT target nucleic acid. The detector and calibrator probes are each associated with (carry or otherwise react with) a detectable label configured to emit label signals that increase as the amplification product with which the respective probes are designed to specifically interact increases.

[0061] During the amplification reaction, a first emission signal associated with the detector probe interacting with amplification product is sensed and a second emission signal associated with the calibrator probe interacting with the amplification product is sensed. At action 204 in workflow 200, the change in label signal (e.g., fluorescence) from the detectable label of the target probe that is emitted as the amplification reaction takes place is determined from the sensed signals. In one example, the first emission signal from the detector probe is measured at predetermined time points (e.g., cycles) during the amplification reaction. At action 206, the change in label signal (e.g., fluorescence) from the detectable label of the calibrator probe that is emitted as the amplification reaction takes place is determined from the sensed signals.

[0062] At action 208, if the second emission signal is indicative of amplifying, the workflow 200 includes calculating a ratio (e.g., N / D) of the change of the first emission signal to the change of the second emission signal at a time after the first emission signal of the calibrator probe crosses the Ct threshold. The time period for calculatingPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 the ratio can be set to a predetermined number of cycles after the Ct threshold is crossed by the first emission signal, which can be predetermined for each type of assay. Alternatively, in one embodiment, the cycle at which the ratio is calculated can be determined by using the negative peak that follows the positive peak, which corresponds to a minimum value after its positive peak and can be found using the second derivative of a smoothed amplification curve of the calibrator probe and considering only those minimum values of the second derivative after the positive peak is identified.

[0063] The ratio calculated at action 208 is compared to a threshold ratio at action 210. At action 212, based on the comparison of the ratio to the threshold ratio, a determination is made with respect to the presence or absence of a mutation of the target nucleic acid in the amplified product, or in other words that amplified product contains the mutant target nucleic acid and thus so does the biological sample. For example, in various embodiments, if the ratio is lower than the threshold ratio, then the amplified product, and thus starting sample, is determined to contain the mutation of target nucleic acid. If the ratio is higher than the threshold, the amplified product, and thus starting sample, is determined to contain the wild type target nucleic acid.

[0064] In some examples, it is assumed that the target nucleic acid is present in the sample so the calibrator probe is expected to provide amplified signal and cross the predefined Ct threshold. In still other examples, the ratio is smaller as the calibrator probe is expected to amplify much more than the detector probe in the presence of mutant target nucleic acid. However, the ratio will be closer to one if the sample does not contain the mutation of the target nucleic acid because the calibrator and detector probe amplification will be about the same. In yet other examples, a threshold ratio can be established to use across assays or individually for each assay to establish whether the resulting ratio (e.g., dRn ratio) obtained corresponds to a sample with a mutation of the target nucleic acid present or with the wild type target nucleic acid.

[0065] By way of nonlimiting, illustrative example, to detect mutation A260G on the PARC gene of Mycoplasma genitalium, the dRn ratio computed 5 cycles after the Ct of a calibrator probe emission signal has been observed to be larger than 0.9 for samplesPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 containing the wild type nucleic acid sequence and less than 0.3 for samples containing different mutations of the wild type nucleic acid sequence. Thus, a threshold in a range of equal or greater than 0.4, such as, for example, 0.5 or 0.6, can be selected and applied for this assay to differentiate samples containing wild type target nucleic acid versus mutant target nucleic acid.

[0066] In a similar manner, optimal thresholds for differing assays could be obtained. Further, normalizing the signals by their brightness or a similar type of normalization, can allow establishment of an absolute threshold that could be applied for a variety of assays. It is expected that samples containing wild type target nucleic acid will produce a significantly larger detector amplification curve than a sample containing a mutant target nucleic acid, but not necessarily equal to the calibrator amplification curve, thus the threshold for dRn ratio can vary from 0.1 to 10 or more depending on the relative amplification of the detector and calibrator curves. Example 1

[0067] Turning to FIGs.3A and 3B, an example of utilizing the above-described technique for detection of a mutation of the target nucleic acid associated with Mycoplasma Genitalium (MG) in a biological sample was conducted. In FIG.3B the MG sequence has a mutation (A260G) on the PARC MG gene and is at a relatively low starting concentration of 5 copies per reaction. In this example, the Ct value associated with the calibrator probe (Ct_c) was identified at 37.2 and the predetermined number of cycles after the Ct_c used was 10 cycles. In accordance with the embodiment described above, the dRn ratio was thus computed on the last cycle (40) of the amplification reaction since Ct_c + 10 cycles would go beyond the last cycle. Therefore, the numerator (N) of the ratio is the value of dRn, taken at the last cycle, associated with the target probe and the denominator is the value of dRn, taken at the last cycle, associated with the calibrator probe (D) and the ratio was 0.2.

[0068] With a sample containing the wild type target nucleic acid of MG, like the one shown in FIG.3A, the ratio is 0.98, and thus there is a difference of a factor of almost 5 that is observed compared to the above ratio associated with the sample containing thePCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 mutant target nucleic acid. Based on these ratios, assays using the ratio technique described above for the MG mutation detection can utilize a ratio threshold of, for example, 0.6 and a ratio below that value can indicate the presence of the mutant MG in the sample.

[0069] Thus, the results of FIGs.3A and 3B demonstrate the robustness of the technique of using the ratio of the emission signal change (e.g., dRn) associated with the detector probe to the emission signal change (e.g., dRn) associated with the calibrator probe to detect mutant target nucleic acid in a sample over a wide range of concentrations, including relatively low concentrations.

[0070] Because the ratio technique described above relies on the relatively highly amplified emission signal associated with the calibrator probe and the relatively mildly amplified emission signal associated with the detector probe in presence of a mutant targe nucleic acid, a reliable output for determining the presence (or absence) of a mutation of the target nucleic acid in the sample results even for samples at low concentrations in which the absolute values of both emission signals are relatively small. Biological Sample Analysis Using Amplification Curve Profiles and Single Target Probe for Nucleic Acid Detection

[0071] As discussed above, with reference to FIG.1, an amplification curve generally follows a sigmoid shape in the presence of the target nucleic acid for which the amplification reaction is producing an amplification product. If there is a large amount of the target nucleic acid in the sample, the exponential phase of amplification will be reached relatively early (at a lower cycle), and the amplitude of the curve (e.g., dRn value) will grow as long as the amplification reagents are still allowing for amplification to occur and amplification product (and thus measured emission signal (e.g., fluorescence) from the probe in the reagents) to increase. On the other hand, if the initial concentration of the target nucleic acid is low, the exponential phase of amplification will occur relatively late (at a higher cycle). And, in some cases, thePCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 amplification process might be cut short at the last cycle, preventing it from achieving higher amplification, including reaching the non-exponential plateau phase.

[0072] For example, for an amplification reaction (e.g., qPCR assay) relying on a detector probe designed to specifically interact with a target nucleic acid in the sample being subjected to the reaction, with the sample containing a relative low concentration of the target nucleic acid, whether it be the wild type target nucleic acid sequence or a mutation of the target nucleic acid sequence, amplification curves may follow those shown in the schematic illustration of FIG.4. More specifically, using the detector probe, a sample containing a relatively low concentration of the target nucleic acid would generate a signal response as reflected in the top amplification curve labeled T in FIG.4, which shows a relatively strong signal response (relatively large amplitude), although not reaching the non-exponential plateau phase of amplification by the last cycle of amplification. In the presence of a sample containing the relatively low concentration of the mutation of the target nucleic acid, the detector probe (designed to specifically interact with the wild type target nucleic acid T) would generate a signal response as reflected by the bottom amplification curve labeled A in FIG.4, which shows a relatively weak signal response (relatively small amplitude) and the exponential phase beginning later than the T curve does and also not reaching the non-exponential plateau phase by the last cycle. In other words, due to the inefficient binding of the detector probe to the mutation of the target nucleic acid, the amplification process produces a proportionally lower emission signal emitted from the detector probe as compared to if the wild type target nucleic acid is present in the sample.

[0073] The response signal and overall shape of the amplification curves produced therefrom can be used to establish a target-specific relationship between the Ct value (i.e., cycle at which a threshold value of amplitude of the curve is crossed) and the maximum amplitude of the amplification curve (e.g., maximum dRn) that can be reached during the amplification cycles. This relationship can in turn be leveraged due to the substantially different relation and spatial discrepancy between the pairs [Ct, max(dRn)] obtained for the wild type target nucleic acid and the mutant target nucleic acid using an amplification reaction that relies on the detector probe, and without thePCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 need for a calibrator probe, assuming the concentration is above the detection level (limit of detection (LoD)). The Ct points (Ct_T, Ct_A) and max(dRn) points (max(dRn)_T, max(dRn)_A) for each of the amplification curves are labeled in FIG.5 for reference, with the threshold dRn (amplification curve amplitude) level to identify the Ct indicated by the horizontal dashed line in FIG.4.

[0074] Thus, for each assay for a particular target nucleic acid, a discrimination region that separates pairs of points [Ct, maximum amplitude] of the amplification curves of the wild type target nucleic acid and mutation of the target nucleic acid can be obtained at different concentrations. From this information, a function F(Ct, maximum amplitude) can be determined, the solution (output) of which indicates that either the wild type target nucleic acid is present or the mutation of the target nucleic acid is present. Recognizing and leveraging the amplification curve shapes and the discrimination region that exists between the curves produced enables an analysis protocol that allows for a single probe type (detector probe) to be used during the amplification reaction, while still permitting an accurate determination of the presence of wild type target nucleic acid or a mutation of the target nucleic acid in the sample. In various embodiments, the maximum amplitude refers to the maximum dRn (change in emission (e.g., fluorescence) signal associated with the relevant probe).

[0075] Moreover, the technique is useful for its ability to distinguish between low amplitudes of amplification (probe emission signal) resulting from the presence of a mutation of the target nucleic acid (reflected by the bottom curve in FIG.4) in the sample versus that resulting from a low concentration of the wild type target nucleic acid in the sample (reflected by the top curve in FIG.4). Other techniques may not distinguish the same and output that the presence of the wild type target nucleic acid exists (e.g., at low concentration) when it is instead mutant target nucleic acid that is present in the sample. One non-limiting application in which this distinction can become significant is in assays detecting bacterial diseases because which particular sequence of nucleic acid exists, (wild type or any of various mutants that, for example, may confer antibiotic resistance), can affect subsequent treatment.PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304

[0076] FIG.5 is an amplification plot with the overlap of actual amplification curves of the detector probe obtained from two qPCR assays in which one sample contained the wild type target nucleic acid of MG PARC gene and the other sample contained the mutant PARC-A260G, with the wild type (WT) and mutant (Mut) curves and Ct values labeled. The curves of FIG.5 demonstrate the same effect of the different shapes and separations of the curves as discussed above with reference to the schematic illustration of FIG.4.

[0077] FIG.6 shows amplification curves corresponding to emission signal from a detector probe for samples wild type (WT) and A260G mutant (Mut) target nucleic acid of MG PARC at different concentrations. As can be seen the low concentration WT sample at 10 copies per reaction (WT 10), can have a similar max dRn than the mutant sample but differ significantly in their Ct values. On the other hand, a sample with relatively high concentration of WT target nucleic acid (WT 105) can have a similar Ct value to the mutant sample, but a significantly higher max dRn. Accordingly, FIG.6 illustrates the differing Ct and max dRn dynamics (shapes of amplification curves) exhibited by the detector probe in the presence of different sample types.

[0078] Turning to FIG.7, an embodiment of a workflow for biological sample analysis that utilizes only detector probe in an amplification reaction (e.g., of a qPCR assay) for detection of wild type or mutant target nucleic acid in a sample is described. In this exemplary workflow, a detector probe is used which is designed to specifically interact with a wild type target nucleic acid and the shape of amplification curves analyzed to determine whether the amplification curves have been generated by the wild type target nucleic acid or by an mutation of the target nucleic acid.

[0079] Referring to FIG.7, a workflow 700 for analysis of a biological sample comprising target nucleic acid includes at action 702 subjecting the biological sample to an amplification reaction conducted in the presence of a detector probe designed to specifically interact with a wild type target nucleic acid. In various embodiments, the detector probe is designed to specifically interact (e.g., bind) to a region (e.g., subsequence) of the wild type target nucleic acid that is anticipated to change in the presence of a mutation of the target nucleic acid. Accordingly, the detector probePCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 experiences reduced interaction (e.g., reduced binding efficiency) when the sample being subject to the amplification reaction at action 702 contains a mutation of the target nucleic acid instead of the wild type target nucleic acid due to differences in the region (e.g., subsequence) with which the detector probe is designed to interact specifically.

[0080] In various embodiments, the amplification reaction is PCR conducted as part of a qPCR assay. The detector probe carries or otherwise causes emission of a label signal, e.g., a fluorescence signal, which increases in response to generation of amplification product of the target nucleic acid due to the interaction of the detector probe with the amplification product. At action 604, based on emission signal from the detector probe measured during the amplification reaction, the change in label signal (e.g., change in fluorescence) from the detectable label of the detector probe that is emitted as the amplification reaction takes place is calculated. The measuring can occur at each cycle during the amplification reaction, as can occur during a qPCR assay, so as to generate an amplification curve from the measuring and based on the calculation of the change of emission signal. As reflected by the different curves of FIG.4, the emission signal from the detector probe would be reduced in the presence of a mutation of the target nucleic acid due to the reduced (inefficient) interaction of the detector probe and the resulting amplified product of the amplification reaction.

[0081] At action 706 of workflow 700, a value corresponding to a cycle at which a threshold of the amount of amplification product, as (e.g., as determined by the change of emission signal determined at action 704) is reached is identified as a cycle threshold (Ct). Further, a value of the maximum amplitude of the amplification curve, which is associated with a maximum amount of amplification product generated during the amplification reaction, is identified. For example, the maximum change of emission signal (e.g., fluorescence) from the detector probe is identified (e.g., max(dRn) with reference to FIG.4).

[0082] At action 708, the Ct value is compared to calculated cycle threshold value, which is determined by a mathematical function of the maximum emission signal amplitude value corresponding to the maximum amount of amplification product. The calculated cycle threshold is determined by a mathematical function of the maximumPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 amplitude value. Based on the comparison, a presence or absence of a mutation or wild type of the target nucleic acid sequence in the amplified product can be determined at action 710.

[0083] In various embodiments, the number of amplification cycles can range from 5-60 cycles. In various embodiments, the mathematical function that is used to provide the determination of target nucleic acid or an allele of the target nucleic acid in the sample subjected to the amplification reaction in the presence of a target probe can be determined as a straight line function that is based on previously run amplification assays at differing concentrations for the target nucleic acid and the allele of the target nucleic acid in the sample, as is further described below. In other embodiments, a more complex decision boundary can be used, determined, for example, using support vector machines or other machine learning models suitable for finding optimal boundaries between classes, for example as described in the following publications: "Fully Convolutional Networks for Semantic Segmentation" by Jonathan Long, Evan Shelhamer, and Trevor Darrell (Published in 2015); "U-Net: Convolutional Networks for Biomedical Image Segmentation" by Olaf Ronneberger, Philipp Fischer, and Thomas Brox (Published in 2015); “CCR-GSVM: A boundary data generation algorithm for support vector machine in imbalanced majority noise problem” by Kai Huang & Xiaoguo Wang (Published in 2023); “SVM clustering” by Stephen Winters-Hilt and Sam Merat. (Published in 2007).

[0084] In either case, the decision boundary can be approximated to a straight line of the form: Ct < maxdRn * Ct + n (for mutant target nucleic acid detection); and Ct > maxdRn * Ct + n (for WT target nucleic acid detection).

[0085] However, it is contemplated as within the scope of the present disclosure that other mathematical functions for expressing the boundaries can have the form of an exponential function or a second or third degree polynomial.PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304

[0086] In various embodiments, the following heuristic can be utilized to determine the mathematical function to be used to determine boundary region and thereby output the presence or absence of either a target nucleic acid or an allele of the target nucleic acid.

[0087] To establish the boundary line, at least three or more samples can be used and undergo PCR amplification, one at a low concentration, near the limit of detection (LoD), and two others at higher concentrations, for example, at least two orders of magnitude higher each one from the next one for both samples containing the wild type target nucleic acid and three or more for sample containing the mutant target nucleic acid (e.g., a WT target nucleic acid and a mutant target nucleic acid). Accordingly, if the LoD is five copies per amplification reaction (cp / rxn), each of the two types of samples (i.e., containing the target and the allele) are at 5 cp / rxn, 5 x 102cp / rxn and 5 x 104cp / rxn. These concentrations assume that the Ct value of some of the samples for each of the wild type target nucleic acid and mutant target nucleic acid will be above 30, which is generally the case for concentrations near LoD and for most if not all the mutant target nucleic acid Ct values for typical concentrations. If that is not the case, the concentration may be adjusted accordingly so that that occurs, or instead of 30, a lower number can be used.

[0088] Next, the various samples can be subjected to an amplification reaction (e.g., qPCR) to establish the pairs [Ct, max(dRn)] for each sample, as discussed above with reference to FIG.4.

[0089] Then, using only the data corresponding to sample containing mutant target nucleic acid and in which where the Ct associated with a sample containing the mutant target nucleic acid is greater than 30 (Ct_A >30), perform a linear regression on that data to obtain a slope-intercept line function: Ct_A = m * max(dRn) + n, which can then be expressed as max(dRn) = (Ct_A – n) / m.

[0090] Values for max(dRn) can next be computed using the function above to establish putative mutant max(dRn) values for the Ct values of points (corresponding to the target nucleic acid (e.g., WT or other non-allelic form) that have Ct> 30. The values Ct>30 are used in various embodiments because for lower Ct values there may be a nonlinearPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 relation between Ct and max(dRn) due to early saturation. But those of ordinary skill in the art would understand that a different threshold could be used for comparison, and corresponding data used to compute the putative max(dRn) values depending on the saturation level and other contributing factors, such as, for example total number of cycles, different concentration ranges, and general dynamics of the amplification curves.

[0091] Finally, points in the decision boundary can be computed as points between the putative max(dRn) for the allele of the target nucleic acid and the max(dRn) of the target nucleic acid for the points when Ct_T <30. In one embodiment, the points between may be the middle point between a putative max(dRn) for the allele and the target. Linear regression on these computed boundary points can then be used as decision boundary mathematical function, F(Ct, maxdRn), for any future data point. Example 2

[0092] FIG.8 depicts an example of performing the above heuristic for determining the boundary mathematical function utilizing data obtained with samples containing Micoplasma Genitalium (MG) ParC either in its WT form or with the mutation A-260G. More specifically, FIG.8 shows the (max(dRn),Ct) pairs of values obtained for a sample containing WT target nucleic acid (the diagonally extending cluster of data points at the far right) and a sample containing mutant target nucleic acid (the cluster of diagonally extending cluster of data points at the far left) corresponding to each of the sample types at different concentrations (107, 106, 105, 104, 103, 100, 10, 5 copies per reaction) with 4 repeats for most concentrations and 8 repeats at the two lowest concentrations for WT and mutant samples (40 samples each). Only samples that reached threshold before cycle 40 were considered.

[0093] The left most dashed line in the results of FIG.8, is the linear regression of the left diagonally extending cluster of data points represented by closed circles in FIG.8, which are associated with the samples containing the mutant (A260G) target nucleic acid which resulted in the function: Ct_mutant = -0.29 max(dRn) + 48PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304

[0094] To determine a boundary to separate WT and mutants the previous function was used to estimate the max(dRn) for mutant samples if their Ct values would be those that correspond to the values obtained for WT samples according to MaxdRn_mut = (Ct WT– 48) / -0.29. This in turn allowed for each WT sample to estimate the midpoint between WT and mutant for the max(dRn) value, for each Ct value above 30. Midpoints were then estimated between the calculated mutant max(dRn) and the measured max(dRn) of the WT (represented by diamonds in FIG.8) datapoints as: max(dRn)_mut + max(dRn_WT) / 2, for the Ct values above 30 obtained for the WT samples, which are referred as boundary points in FIG.8.

[0095] The linear regression fit of these midpoints (shown by the middle diagonally extending cluster of points represented by open circles in FIG.8), determined the discrimination region, which was calculated as: Ct_boundary = -0.11 max(dRn) + 45.6. Thus, using the above equation, if the measured Ct is larger than Ct_boundary for a given measured max(dRn), then the sample is determined to contain the WT MG ParC nucleic acid and if it is smaller, then the sample is determined to contain the A260G mutant target nucleic acid.

[0096] In addition to what is described above, it is contemplated that other machine learning models can be used, including, but not limited to, logistic regression, support vector machines, multilayer neural networks, K-means clustering, Gaussian-mixture models, etc., to identify the discrimination region that separates WT from mutants. It is also contemplated that in addition to max(dRn) and Ct, other parameters of the curve could be used, like the amplitude, slope, and intercept of the sigmoid after fitting as well as higher order parameters, such as but not limited to for example, maximums, minimums, and the respective locations of the max, min and zero crossings of the first and second derivative of the amplification curves. Alternatively, instead of max(dRn), and to avoid issues related to saturation, a value at a specific late cycle (e.g. cycle 38), or a number of cycles after the Ct value can be utilized. For example, the number can be a predetermined number of cycles or can be based on the cycle corresponding to the negative peak that follows the positive peak of the amplification curve, whichPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 corresponds to a minimum value after the positive peak that can be found using the second derivative of a smoothed amplification curve of the calibrator probe. In this case, only minimum values of the second derivative that occur after the positive peak are considered. In order to make the decision boundary applicable across instruments with different brightness, the Rn signal can be normalized by its baseline prior to computation of dRn, which has shown to greatly reduce variability. Computer System Implementation

[0097] In some embodiments, at least a portion of the methods described herein may be implemented using one or more computer systems. In some instances, the techniques discussed herein are represented in computer-executable instructions that may be stored on one or more hardware storage devices. The computer-executable instructions may be executable by one or more processors to carry out (or to configure a system to carry out) the disclosed techniques. In some embodiments, a system may be configured to send the computer-executable instructions to a remote device to configure the remote device for carrying out the disclosed techniques.

[0098] In an example embodiment, a computer system comprises one or more processors, and a memory storing one or more instructions which, when executed by the one or more processors, cause the one or more processors to perform a process such as one or more actions of the workflow 200 and / or workflow 800.

[0099] Some embodiments include one or more computer-readable media storing one or more instructions which, when executed by one or more processors of at least one computing device, cause the one or more processors to perform the foregoing process or other computer-implemented process as described herein.

[0100] Systems for implementing the disclosed embodiments may include various components, such as, by way of non-limiting example, processor(s), storage, sensor(s), I / O system(s), communication system(s), and the like. The processor(s) may comprise one or more sets of electronic circuitries that include any number of logic units, registers, and / or control units to facilitate the execution of computer-readable instructions (e.g., instructions that form a computer program). Such computer-readablePCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 instructions may be stored within storage. The storage may comprise physical system memory and may be volatile, non-volatile, or some combination thereof. Furthermore, storage may comprise local storage, remote storage (e.g., accessible via communication system(s) or otherwise), or some combination thereof.

[0101] Furthermore, a system may comprise or be in communication with I / O system(s). I / O system(s) may include any type of input or output device such as, by way of non- limiting example, a touch screen, a mouse, a keyboard, a controller, a speaker and / or others, without limitation. For example, the I / O system(s) may include a display system that may comprise any number of display panels, optics, laser scanning display assemblies, and / or other components.

[0102] Disclosed embodiments may also include physical and other computer-readable media for carrying or storing computer-executable instructions and / or data structures. Such computer-readable media can be any available media that can be accessed by a general-purpose or special-purpose computer system. Computer storage media (aka “hardware storage device”) are computer-readable hardware storage devices, such as RAM, ROM, EEPROM, CD-ROM, solid state drives (“SSD”) that are based on RAM, Flash memory, phase-change memory (“PCM”), or other types of memory, or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store desired program code means in hardware in the form of computer-executable instructions, data, or data structures and that can be accessed by a general-purpose or special-purpose computer.

[0103] Those skilled in the art will appreciate that various aspects of the workflows and / or techniques described herein may be practiced in network computing environments with many types of computer system configurations, including, personal computers, desktop computers, laptop computers, message processors, hand-held devices, multi-processor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, mobile telephones, PDAs, pagers, routers, switches, wearable devices, and the like. The invention may also be practiced in distributed system environments where multiple computer systemsPCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 (e.g., local and remote systems), which are linked through a network (either by hardwired data links, wireless data links, or by a combination of hardwired and wireless data links), perform tasks. In a distributed system environment, program modules may be in local and / or remote memory storage devices.

[0104] Alternatively, or in addition, the functionality described herein can be performed, at least in part, by one or more hardware logic components. For example, and without limitation, illustrative types of hardware logic components that can be used include Field-programmable Gate Arrays (FPGAs), Program-specific Integrated Circuits (ASICs), Application-specific Standard Products (ASSPs), System-on-a-chip systems (SOCs), Complex Programmable Logic Devices (CPLDs), central processing units (CPUs), graphics processing units (GPUs), and / or others.

[0105] As used herein, the terms “executable module,” “executable component,” “component,” “module,” or “engine” can refer to hardware processing units or to software objects, routines, or methods that may be executed on one or more computer systems. The different components, modules, engines, and services described herein may be implemented as objects or processors that execute on one or more computer systems (e.g., as separate threads).

[0106] Those of ordinary skill in the art will also appreciate that any feature or operation disclosed herein may be combined with any one or combination of the other features and operations disclosed herein. Additionally, the content or feature in any one of the figures may be combined or used in connection with any content or feature used in any of the other figures. In this regard, the content disclosed in any one figure is not mutually exclusive and instead may be combinable with the content from any of the other figures. Moreover, the various actions of the workflows disclosed can be used in any combination and a workflow need not include all actions as depicted in the figures.

[0107] The described embodiments are to be considered in all respects only as illustrative and not restrictive. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 CLAIMS WHAT IS CLAIMED IS:

1. A method of biological sample analysis, the method comprising: subjecting a biological sample comprising nucleic acid to an amplification reaction conducted in the presence of a detector probe and a calibrator probe, wherein the detector probe is configured to specifically interact with a first region of a wild type target nucleic acid sequence and emit a first emission signal in response to the interaction, and wherein the calibrator probe is configured to specifically interact with a second region of the wild type target nucleic acid sequence and emit a second emission signal in response to the interaction, the first region and the second region differing from each other; measuring the first emission signal during the amplification reaction; measuring the second emission signal during the amplification reaction; calculating a ratio of the change in the first emission signal to the change of the second emission signal; comparing the ratio to a threshold ratio; and based on the comparing, determining one of a presence or absence of an mutation of the target nucleic acid sequence in the amplified product.

2. The method of claim 1, wherein the amplification reaction comprises a polymerase chain reaction (PCR).

3. The method of any one of claims 1 and 2, wherein the calculating the ratio corresponds to a predetermined point in time of the amplification reaction.

4. The method of claim 3, wherein the amplification reaction is a PCR amplification reaction and the predetermined point in time is at predetermined number of cycles of the PCR amplification reaction.PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 5. The method of claim 4, wherein the predetermined number of cycles is a last cycle of the PCR amplification reaction or a predetermined number of cycles after a cycle threshold value for the calibrator probe is reached, whichever occurs first.

6. The method of claim 4, wherein the predetermined point in time corresponds to a cycle at which the minimum value of the second derivative of a smoothed amplification curve for the calibrator probe is reached, wherein the minimum value is a value occurring after the maximum value.

7. The method of any one of claims 1-6, wherein each of the detector probe and the calibrator probe emit differing emission signals in response to interaction with the mutation of the target nucleic acid.

8. The method of claim 7, wherein the second region of the wild type target nucleic acid comprises a region of the wild type target nucleic acid that remains relatively unchanged in the presence of a mutation of the target nucleic acid.

9. The method of any of claims 1 to 8, wherein the first and second emission signals are fluorescence.

10. A method of biological sample analysis, the method comprising: receiving first data corresponding to a change of an amount of a first nucleic acid subsequence of a wild type target nucleic acid measured over a time period during an amplification reaction on the biological sample, the amplification reaction configured to produce an amplification product of the wild type target nucleic acid; receiving second data corresponding to a change of an amount of a second nucleic acid subsequence of the wild type target nucleic acid sequence measured over the number of cycles during the amplification reaction;PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 at a predetermined number of cycles, calculating a ratio of the change of the amount of the first nucleic acid subsequence to the change of the amount of the second nucleic acid subsequence; comparing the ratio to a predetermined threshold; and based on the comparing, determining one of a presence or absence of a mutation of the target nucleic acid sequence in the biological sample.

11. The method of claim 10, wherein the first data comprises first emission signal data from a detector probe and the second data comprises second emission signal data from a calibrator probe, wherein the detector probe is configured to specifically interact with the first nucleic acid subsequence and the calibrator probe is configured to specifically interact with the second nucleic acid subsequence.

12. The method of any of claim 10, wherein the first emission signal differs from the second emission signal in the presence of a mutation of the target nucleic acid in the sample.

13. The method of any one of claim 11 to 12, wherein the emission signals are fluorescence signals.

14. Computer-readable media storing one or more instructions which, when executed by one or more processors of at least one computing device, cause the one or more processors to perform a process of any of claims 1-13.

15. A system comprising: one or more processors of at least one computing device; and a memory storing one or more instructions which, when executed by the one or more processors, cause the one or more processors to perform a process of any one of claims 1-14.PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 16. A method of biological sample analysis, said method comprising: subjecting a biological sample to an amplification reaction comprising multiple cycles conducted in the presence of a detector probe, wherein the detector probe is configured to specifically interact with a wild type target nucleic acid and emit an emission signal in response to interaction with amplified product of the wild type target nucleic acid; measuring the emission signal during the amplification reaction; based on the measuring, identifying a value corresponding to a cycle of the amplification reaction at which the change in measured emission signal reaches a threshold and identifying a maximum amplitude value associated with the change of emission signal generated during the amplification reaction; comparing the value to a calculated cycle value, wherein the calculated cycle value is determined by a mathematical function of the maximum; and based on the comparing, determining one of a presence or absence of a mutation of the target nucleic acid sequence in the amplified product.

17. The method of claim 16, wherein the mathematical function is a linear slope intercept equation.

18. The method of claim any of claims 16 or 17, wherein the mathematical function is based on determining Ct values and maximum amounts of signal emitted for differing concentrations of sample containing wild target nucleic acid and sample containing a mutation of the target nucleic acid.

19. The method of any of claims 16 to 18, wherein the mathematical function is a boundary condition function, wherein calculated Ct from the mathematical function greater than a predefined value indicates presence of the wild type target nucleic acid and calculated Ct less than the predefined value indicates presence of the mutation of the target nucleic acid.PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 20. A method of biological sample analysis, the method comprising: determining, for a plurality of concentrations of a sample containing a wild type target nucleic acid, a respective plurality of wild type data points comprising pairs of a cycle threshold and maximum change in emission signal from a detector probe generated by an amplification reaction configured to produce amplified product of the wild type target nucleic acid; determining, for a plurality of concentrations of a sample containing a mutation of the target nucleic acid, a respective plurality of mutant data points comprising pairs of a cycle threshold and maximum change in emission signal from the detector probe generated by the amplification reaction; using at least some of the mutant data points, generating a mathematical function expressing maximum limit on change in amount of emission signal as a function of cycle threshold; using the mathematical function and the cycle thresholds from the wild type data points, calculating putative maximum limits of change in amount of generated amplified product of the mutation of target nucleic acid; calculating boundary points based on values between the calculated putative maximum limits and the measured target maximum limits corresponding to at least some of the wild type data points; based on the boundary points, determining a boundary mathematical function of cycle threshold and maximum limit of change of nucleic acid sequence in an amplification product.

21. The method of claim 20, further comprising storing instructions to execute the boundary function on computer readable media on amplification data of an amplification curve obtained from an amplification reaction of a biological sample, the amplification reaction configured to produce amplified product of the wild type target nucleic acid and of the mutation of the target nucleic acid.PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 22. The method of any of claims 21 or 20, further comprising outputting information regarding presence of wild type target nucleic acid in the amplified product or presence of the mutation of the target nucleic acid in the amplified product.

23. The method of any one of claims 20-22, wherein the amplification reaction is an amplification reaction of a qPCR assay using a detector probe configured to specifically interact with the wild type target nucleic acid.

24. The method of any one of claims 20-23, wherein the amplification data is based on emission signal data from the detector probe.

25. The method of any one of claims 20-24wherein the mathematical functions are determined using a linear regression analysis.

26. The method of any one of claims 20-24, wherein the mathematical functions are determined using machine learning models.

27. The method of any one of claims 20-24, wherein the mathematical functions are one of linear, exponential, or polynomial.

28. A method of biological sample analysis, the method comprising: obtaining emission signal data from an amplification reaction conducted on a biological sample, the amplification reaction configured to produce amplified product of a wild type target nucleic acid; identifying from the emission signal data: a cycle of the amplification reaction at which a change in the emission signal data measured over the course of the amplification reaction reaches a threshold, and a maximum value of the change in emission signal data measured over the course of the amplification reaction;PCT Application Attorney Docket No. TP385326WO1 Alternative Docket No.1168.0114.00304 utilizing a mathematical function expressing cycle as a function of maximum change in emission signal, calculating a cycle value from the identified maximum value; comparing the identified cycle to the calculated cycle value; and based on the comparing, outputting one of an indication that the biological sample contains the wild type target nucleic acid or an indication that the biological sample contains the mutation of the target nucleic acid.

29. The method of claim 28, wherein the emission signal data is fluorescence.

30. The method of claim 28 or 29, wherein the emission signal data is from a detector probe used in the amplification reaction and configured to specifically interact with the wild type target nucleic acid.

31. Computer-readable media storing one or more instructions which, when executed by one or more processors of at least one computing device, cause the one or more processors to perform a method of any of claims 16-30.

32. A system, comprising: one or more processors of at least one computing device; and a memory storing one or more instructions which, when executed by the one or more processors, cause the one or more processors to perform a process of any one of claims 16-30.

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