Adaptive crosstalk compensation of optical signal detection

Abstract

Systems, methods, computers, and instruments programmed with software to compensate for fluorescence crosstalk from one channel of a fluorometer into a different channel of the fluorometer during nucleic acid amplification. An adaptive crosstalk compensation factor specific for a paired set of Emitter channel and Receiver channel amplification run curves can be calculated and used to reduce false-positive errors.

Description

Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 641,886, filed May 2, 2024. The entire disclosure of this related application is hereby incorporated by reference. Government Rights

[0002] This invention was made with government support under contract No. 75A50122C00074 awarded by the U.S. Department of Health & Human Services Biomedical Advanced Research & Development Authority (BARDA). The government has certain rights in the invention. Technical Field

[0003] The present disclosure relates generally to the field of biotechnology. More specifically, the disclosure relates to the field of multiplex nucleic acid detection. Still more specifically, the disclosure relates to detection of a target nucleic acid using a fluorescent detection channel with reduced interference from a crosstalk signal. Background

[0004] Modern laboratory instruments that detect biological analytes in a multiplex format commonly employ fluorescent signals to indicate the presence or amount of one or more analytes. Highly complex nucleic acid amplification reactions may involve amplification and detection of multiple targets, each being associated with a different fluorescent signal generated in an in vitro nucleic acid amplification reaction. For example, some multiplex assays amplify and detect four different target nucleic acids using four different fluorescently labeled probes. Detection of nucleic acid analytes typically is based on sequence-specific hybridization of a fluorescently labeled probe to a nucleic acid amplification product (i.e., an “amplicon”).

[0005] Fluorescence-based multiplex assays have unique issues that must be controlled to avoid erroneous results. Multiplex assays commonly employ a plurality of probes labeled with different fluorophores for detecting a like number of target analytes. Difficulty arises when the emission spectra of the different fluorescent labels overlap with each other, a problem obviously exacerbated when the number of fluorophores used in the assay increases. When this is the case, the signal measured in one detection channel undesirably may also be measured in a different channel, possibly leading to a false-positive result. Physically distinct hardware typically is used for each different channel that monitors fluorescence emission from a different fluorophore, although all hardware for the different channels may be contained within a single housing or fixed on a single chassis. This hardware may include, for example, light sources, excitation filters, dichroic filters, emission filters, and detectors.

[0006] Data processing adjustments to measured fluorescent signals can be used to control for undesirable crosstalk between different optical detection channels. Crosstalk becomes significant when emission spectra of fluorophores used in an assay exhibit sufficient overlap that signal emitted by one fluorophore is perceived as having been emitted by a different fluorophore. This obviously confuses interpretation of the experimental results and can lead to false conclusions. In the context of biological sample analysis, crosstalk may lead to the conclusion that a pathogen is present when it really is absent (i.e., a “false-positive” result).

[0007] Undesirable impacts of crosstalk conventionally have been minimized either by requiring strict tolerances on manufacturing the instrumentation used for stimulating and detecting fluorescent signals, by selective choice of fluorophores used as detectable labels to minimize spectral overlaps, or by adjustment by a fixed fraction of the detected signal (e.g., by subtraction or multiplication) to remove contributions from an irrelevant crosstalk signal. The use of multiple fluorophores in a single procedure constrains the range of available solutions because crosstalk issues become more likely.

[0008] Despite the existing approaches to address crosstalk correction, there remains a need for techniques and ancillary hardware and software that better isolate and quantify signals attributed to fluorescent labels in multiplex analyte detection systems. The present disclosure addresses this need. Summary of the Disclosure

[0009] Provided herein are the following embodiments.

[0010] Embodiment 1 is a system that quantifies a target nucleic acid. The system includes a real-time nucleic acid analyzer, wherein the real-time nucleic acid analyzer amplifies target nucleic acids in a multiplex amplification reaction and monitors synthesis of amplification products with a fluorometer, wherein the fluorometer is configured to detect multiple wavelengths of fluorescent dye emissions in different channels, and wherein the real-time nucleic acid analyzer produces each of a Receiver channel run curve that measures amplification of a first target nucleic acid, and an Emitter channel run curve that measures amplification of a second target nucleic acid. The system further includes a computer in communication with the real-time nucleic acid analyzer. The computer is programmed with software instructions which, when executed by the computer, cause the computer to: (a) direct the real-time nucleic acid analyzer to perform the multiplex amplification reaction and monitor synthesis of amplification products by measuring fluorescent signals using the fluorometer; (b) quantify each of a magnitude of the Emitter channel run curve, and a correlation of the Emitter channel run curve and the Receiver channel run curve, whereby it is determined that (i) the magnitude of the Emitter channel run curve indicates amplification of the second target nucleic acid took place in the multiplex amplification reaction, and (ii) the Emitter channel run curve and the Receiver channel run curve are highly correlated; (c) adjust fluorescent signal values of the Receiver channel run curve by subtracting therefrom a quantity calculated by multiplying an adaptive crosstalk compensation factor by fluorescent signal values of the Emitter channel run curve, thereby preparing an adjusted Receiver channel run curve, wherein the adaptive crosstalk compensation factor is specific for the Emitter channel run curve and the Receiver channel run curve; (d) quantify the first target nucleic acid using the adjusted Receiver channel run curve; and (e) record the result of (d) in a non-transient form.

[0011] Embodiment 2 is the system of embodiment 1, wherein the real-time nucleic acid analyzer includes a temperature-controlled incubator that undergoes thermal cycling, and wherein the multiplex nucleic acid amplification reaction includes a multiplex real-time PCR amplification reaction.

[0012] Embodiment 3 is the system of embodiment 2, wherein the temperature-controlled incubator includes a temperature-controlled block or a temperature-controlled plate.

[0013] Embodiment 4 is the system of any one of embodiments 1 to 3, wherein the multiplex nucleic acid amplification reaction includes a plurality of reaction cycles.

[0014] Embodiment 5 is the system of any one of embodiments 1 to 4, wherein the fluorometer monitors fluorescent signals indicating amplification of nucleic acids as a function of reaction cycle number.

[0015] Embodiment 6 is the system of any one of embodiments 1 to 5, wherein the realtime nucleic acid analyzer is an automated real-time nucleic acid analyzer that isolates nucleic acids used as templates in the multiplex nucleic acid amplification reaction.

[0016] Embodiment 7 is the system of any one of embodiments 1 to 6, wherein the fluorometer detects fluorescent signals for the different channels in serial fashion, so that readings for the different channels are spaced apart in time.

[0017] Embodiment 8 is the system of any one of embodiments 1 to 7, wherein the realtime nucleic acid analyzer includes one or more fluid transfer devices that transfer fluids to and from containers contained within the real-time nucleic acid analyzer.

[0018] Embodiment 9 is the system of any one of embodiments 1 to 8, wherein the computer is an integral component of the automated real-time nucleic acid analyzer.

[0019] Embodiment 10 is the system of any one of embodiments 1 to 9, wherein the magnitude of the Emitter channel run curve in (b) is calculated as the difference between a maximum fluorescence value of the Emitter channel run curve and a minimum fluorescence value of the Emitter channel run curve.

[0020] Embodiment 11 is the system of embodiment 10, wherein the minimum fluorescence value of the Emitter channel run curve is a minimum fluorescence value of the Emitter channel run curve after baseline adjustment that removes fluctuations in fluorescence measurements.

[0021] Embodiment 12 is the system of any one of embodiments 1 to 11, wherein (b) includes quantify the correlation of the Emitter channel run curve and the Receiver channel run curve by calculating a measure of linearity of the correlation of the Emitter channel run curve and the Receiver channel run curve.

[0022] Embodiment 13 is the system of embodiment 12, wherein calculating the measure of linearity includes calculating a Pearson correlation coefficient to confirm the Emitter channel run curve and the Receiver channel run curve are highly correlated.

[0023] Embodiment 14 is the system of embodiment 13, wherein the Pearson correlation coefficient is calculated to be at least 0.95, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

[0024] Embodiment 15 is the system of embodiment 13, wherein the Pearson correlation coefficient is calculated to be at least 0.98, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

[0025] Embodiment 16 is the system of embodiment 13, wherein the Pearson correlation coefficient is calculated to be at least 0.99, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

[0026] Embodiment 17 is the system of any one of embodiments 1 to 16, wherein the adaptive crosstalk compensation factor is calculated by dividing a Receiver channel signal value of the Receiver channel run curve by an Emitter channel signal value of the Emitter channel run curve.

[0027] Embodiment 18 is the system of any one of embodiments 1 to 16, wherein the adaptive crosstalk compensation factor is calculated by dividing a signal value from the Receiver channel run curve by a signal value from the Emitter channel run curve, and wherein the Emitter channel run curve is first synchronized with the Receiver channel run curve by optimizing correlation of the two run curves before calculating the adaptive crosstalk compensation factor.

[0028] Embodiment 19 is the system of any one of embodiments 1 to 17, wherein (d) includes comparing a magnitude of the adjusted Receiver channel ran curve to a static threshold fluorescence value to determine whether the first target nucleic acid amplified in the multiplex amplification reaction.

[0029] Embodiment 20 is the system of embodiment 19, wherein the software instructions, when executed by the computer, further cause the computer to determine a cycle number of the multiplex amplification reaction at which the adjusted Receiver channel run curve exceeds the static threshold fluorescence value, and then determine the quantity of the first target nucleic acid in the multiplex amplification reaction before the reaction began.

[0030] Embodiment 21 is the system of any one of embodiments 1 to 20, wherein the result of (d) indicates the first target nucleic acid was amplified in the multiplex amplification reaction.

[0031] Embodiment 22 is a computer programmed with software instructions to quantify a target nucleic acid. The software instructions, when executed by the computer, cause the computer to: (a) receive results from a real-time nucleic acid analyzer that performs a multiplex amplification reaction and monitors synthesis of amplification products by measuring fluorescent signals using a fluorometer, wherein the real-time nucleic acid analyzer produces a Receiver channel run curve that measures amplification of a first target nucleic acid, and an Emitter channel ran curve that measures amplification of a second target nucleic acid; (b) quantify each of a magnitude of the Emitter channel run curve, and a correlation of the Emitter channel ran curve with the Receiver channel run curve, whereby it is determined that (i) the magnitude of the Emitter channel run curve indicates amplification of the second target nucleic acid took place in the multiplex amplification reaction, and (ii) the Emitter channel run curve and the Receiver channel run curve are highly correlated; (c) adjust fluorescent signal values of the Receiver channel run curve by subtracting therefrom a quantity calculated by multiplying an adaptive crosstalk compensation factor by fluorescent signal values of the Emitter channel ran curve, thereby preparing an adjusted Receiver channel run curve, wherein the adaptive crosstalk compensation factor is specific for the Emitter channel run curve and the Receiver channel run curve; (d) quantify the first target nucleic acid using the adjusted Receiver channel run curve; and (e) record the result of (d) in a non-transient form.

[0032] Embodiment 23 is the computer of embodiment 22, wherein the software instructions, when executed by the computer, further cause the computer to direct the realtime nucleic acid analyzer to perform the multiplex amplification reaction and monitor synthesis of amplification products by measuring fluorescent signals using the fluorometer.

[0033] Embodiment 24 is the computer of embodiment 23, wherein the software instructions, when executed by the computer, further cause the computer to direct the realtime nucleic acid analyzer to carry out a plurality of temperature cycling steps to perform the multiplex nucleic acid amplification reaction.

[0034] Embodiment 25 is the computer of any one of embodiments 22 to 24, wherein the magnitude of the Emitter channel run curve in (b) is calculated as the difference between a maximum fluorescence value of the Emitter channel run curve and a minimum fluorescence value of the Emitter channel run curve.

[0035] Embodiment 26 is the computer of embodiment 25, wherein the minimum fluorescence value of the Emitter channel run curve is a minimum fluorescence value of the Emitter channel run curve after baseline adjustment that removes fluctuations in fluorescence measurements.

[0036] Embodiment 27 is the computer of any one of embodiments 22 to 26, wherein (b) includes quantify the correlation of the Emitter channel run curve and the Receiver channel run curve by calculating a measure of linearity of the correlation of the Emitter channel run curve and the Receiver channel run curve.

[0037] Embodiment 28 is the computer of embodiment 27, wherein calculating the measure of linearity includes calculating a Pearson correlation coefficient to confirm the Emitter channel run curve and the Receiver channel run curve are highly correlated.

[0038] Embodiment 29 is the computer of embodiment 28, wherein the Pearson correlation coefficient is calculated to be at least 0.95, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

[0039] Embodiment 30 is the computer of embodiment 28, wherein the Pearson correlation coefficient is calculated to be at least 0.98, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

[0040] Embodiment 31 is the computer of embodiment 28, wherein the Pearson correlation coefficient is calculated to be at least 0.99, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

[0041] Embodiment 32 is the computer of any one of embodiments 22 to 31, wherein the adaptive crosstalk compensation factor is calculated by dividing a Receiver channel signal value of the Receiver channel run curve by an Emitter channel signal value of the Emitter channel run curve.

[0042] Embodiment 33 is the computer of any one of embodiments 22 to 31, wherein the adaptive crosstalk compensation factor is calculated by dividing a signal value from the Receiver channel run curve by a signal value from the Emitter channel run curve, and wherein the Emitter channel run curve is first synchronized with the Receiver channel run curve by optimizing correlation of the two run curves before calculating the adaptive crosstalk compensation factor.

[0043] Embodiment 34 is the computer of any one of embodiments 22 to 33, wherein (d) includes comparing a magnitude of the adjusted Receiver channel run curve to a static threshold fluorescence value to determine whether the first target nucleic acid amplified in the multiplex amplification reaction.

[0044] Embodiment 35 is the computer of embodiment 34, wherein the software instructions, when executed by the computer, further cause the computer to determine a cycle number of the multiplex amplification reaction at which the adjusted Receiver channel run curve exceeds the static threshold fluorescence value, and then determine the quantity of the first target nucleic acid in the multiplex amplification reaction before the reaction began.

[0045] Embodiment 36 is the computer of embodiment 34, wherein the result of (d) qualitatively indicates the first target nucleic acid was amplified in the multiplex amplification reaction.

[0046] Embodiment 37 is a method of quantifying a target nucleic acid after compensating for crosstalk of signals from an Emitter channel of a fluorometer into a Receiver channel of the fluorometer, the fluorometer being a component of a real-time nucleic acid analyzer. The method includes the steps of: (a) performing or having performed a multiplex amplification reaction using the real-time nucleic acid analyzer to obtain an Emitter channel run curve and a Receiver channel run curve, wherein the Receiver channel run curve measures amplification of a first target nucleic acid, and wherein the Emitter channel run curve measures amplification of a second target nucleic acid; (b) quantifying or having quantified each of a magnitude of the Emitter channel run curve, and a correlation of the Emitter channel run curve with the Receiver channel run curve, whereby it is determined that (i) the magnitude of the Emitter channel run curve indicates amplification of the second target nucleic acid took place in the multiplex amplification reaction, and (ii) the Emitter channel run cun e and the Receiver channel run curve are highly correlated; (c) adjusting or having adjusted fluorescent signal values of the Receiver channel run curve by subtracting therefrom a quantity calculated by multiplying an adaptive crosstalk compensation factor by fluorescent signal values of the Emitter channel run curve, thereby preparing an adjusted Receiver channel run curve, wherein the adaptive crosstalk compensation factor is specific for the Emitter channel run curve and the Receiver channel run curve; and (d) quantifying or having quantified the first target nucleic acid using the adjusted Receiver channel run curve.

[0047] Embodiment 38 is the method of embodiment 37, wherein the real-time nucleic acid analyzer is an automated real-time nucleic acid analyzer controlled by a computer programmed with software, and wherein the automated real-time nucleic acid analyzer performs the multiplex amplification reaction and monitors synthesis of amplification products as the reaction is occurring.

[0048] Embodiment 39 is the method of embodiment 38, wherein the computer is an integral component of the automated real-time nucleic acid analyzer.

[0049] Embodiment 40 is the method of any one of embodiments 37 to embodiment 39 wherein the multiplex amplification reaction amplifies nucleic acids by a process including thermal cycling.

[0050] Embodiment 41 is the method of any one of embodiments 37 to 40, wherein the multiplex amplification reaction includes a multiplex PCR reaction.

[0051] Embodiment 42 is the method of any one of embodiments 37 to 41, wherein at least one of the first target nucleic acid and the second target nucleic acid is either a bacterial nucleic acid or a viral nucleic acid.

[0052] Embodiment 43 is the method of any one of embodiments 37 to 42, wherein the magnitude of the Emitter channel run curve in step (b) is calculated as the difference between a maximum fluorescence value of the Emitter channel run curve and a minimum fluorescence value of the Emitter channel run curve.

[0053] Embodiment 44 is the method of embodiment 43, wherein the minimum fluorescence value of the Emitter channel run curve is a minimum fluorescence value of the Emitter channel run curve after baseline adjustment that removes fluctuations in fluorescence measurements.

[0054] Embodiment 45 is the method of any one of embodiments 37 to 44, wherein step (b) includes quantifying the correlation of the Emitter channel run curve and the Receiver channel run curve by calculating a measure of linearity of the correlation of the Emitter channel run curve and the Receiver channel run curve.

[0055] Embodiment 46 is the method of embodiment 45, wherein calculating the measure of linearity includes calculating a Pearson correlation coefficient to confirm the Emitter channel run curve and the Receiver channel run curve are highly correlated.

[0056] Embodiment 47 is the method of embodiment 46, wherein the Pearson correlation coefficient is calculated to be at least 0.95, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

[0057] Embodiment 48 is the method of embodiment 46, wherein the Pearson correlation coefficient is calculated to be at least 0.98, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

[0058] Embodiment 49 is the method of embodiment 46, wherein the Pearson correlation coefficient is calculated to be at least 0.99, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

[0059] Embodiment 50 is the method of embodiment 37, wherein the adaptive crosstalk compensation factor is calculated by dividing a Receiver channel signal value of the Receiver channel run curve by an Emitter channel signal value of the Emitter channel run curve.

[0060] Embodiment 51 is the method of embodiment 37, wherein the adaptive crosstalk compensation factor is calculated by dividing a signal value from the Receiver channel run curve by a signal value from the Emitter channel run curve, and wherein the Emitter channel run curve is first synchronized with the Receiver channel run curve by optimizing correlation of the two run curves before calculating the adaptive crosstalk compensation factor.

[0061] Embodiment 52 is the method of any one of embodiments 37 to 51, wherein step (d) includes comparing a magnitude of the adjusted Receiver channel run curve to a static threshold fluorescence value to determine whether the first target nucleic acid amplified in the multiplex amplification reaction.

[0062] Embodiment 53 is the method of embodiment 52, further including the step of determining a cycle number of the multiplex amplification reaction at which the adjusted Receiver channel run curve exceeds the static threshold fluorescence value, and then determining the quantity of the first target nucleic acid in the multiplex amplification reaction before the reaction began.

[0063] Embodiment 54 is the method of embodiment 52, wherein the result of step (d) qualitatively indicates the first target nucleic acid was amplified in the multiplex amplification reaction. Brief Description of the Drawings

[0064] Fig. 1 shows absorption spectra (upper panel) and emission spectra (lower panel) for the CAL FLUOR RED 610 dye (stippled) and QUASAR 670 dye (hatched) used to illustrate the adaptive crosstalk compensation technique.

[0065] Figs. 2A-2D show graphical plots used to assess correlations between Emitter and Receiver run curves for real-time multiplex amplification reactions. Fig. 2A shows results from an amplification reaction that did not amplify either of the SARS or Flu B target nucleic acids. The upper panel presents fluorescence readings as a function of cycle number for the Emitter channel (SARS target; filled circles) and Receiver channel (Flu B target; open circles). The lower panel presents results for the Emitter and Receiver channels on different axes to assess linearity of the correlation. Fig. 2B shows results from an amplification reaction that amplified the Flu B target nucleic acid but not the SARS target nucleic acid. The upper panel presents fluorescence readings as a function of cycle number for the Emitter channel (SARS target; filled circles) and Receiver channel (Flu B target; open circles). The lower panel presents results for the Emitter and Receiver channels on different axes to assess linearity of the correlation. Fig. 2C shows results from an amplification reaction that amplified the SARS target nucleic acid but did not amplify the Flu B target nucleic acid. The upper panel presents fluorescence readings as a function of cycle number for the Emitter channel (SARS target; filled circles) and Receiver channel (Flu B target; open circles). The lower panel presents results for the Emitter and Receiver channels on different axes to assess linearity of the correlation. Fig. 2D shows results from an amplification reaction that amplified both of the SARS and Flu B target nucleic acids. The upper panel presents fluorescence readings as a function of cycle number for the Emitter channel (SARS target; filled circles) and Receiver channel (Flu B target; open circles). The lower panel presents results for the Emitter and Receiver channels on different axes to assess linearity of the correlation.

[0066] Fig. 3 shows real-time run curves for each of the Emitter channel (SARS target; filled circles) and Receiver channel (Flu B target; open circles) in a multiplex reaction that amplified the SARS target nucleic acid but not the Flu B target nucleic acid. Fluorescence intensity before crosstalk compensation is plotted as a function of cycle number. The Emitter channel signal is substantially greater than the Receiver channel signal.

[0067] Fig. 4 is a graphical plot of ratio data as a function of reaction cycle number. The average of selected data points was used as an adaptive crosstalk compensation factor. Data points represented by open circles were included in the ratio calculation, while other data points (filled circles) were excluded. The average of the included data points was 0.03975.

[0068] Fig. 5 is a graphical plot of Receiver channel (Flu B target) run curve data processed two ways, where the amplification reaction did not amplify Flu B nucleic acids. The upper curve (filled circles) that crosses the horizontal threshold represents data processed for crosstalk compensation using a preset default compensation factor, and indicates a falsepositive result. Notably, the preset default crosstalk compensation factor underestimates the crosstalk compensation required for a proper result. The lower curve (open circles) represents data processed for crosstalk compensation using the adaptive crosstalk compensation technique, and indicates a true-negative result.

[0069] Figs. 6A and 6B are perspective views of an automated nucleic acid analyzer. Detailed Description of Certain Embodiments Definitions

[0070] The following terms have the indicated meanings in the specification unless expressly indicated to have a different meaning.

[0071] The terms "a," "an," and "the" include plural referents, unless the context clearly indicates otherwise. For example, "a nucleic acid" as used herein is understood to represent one or more nucleic acids. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0072] A “nucleic acid” is a polymeric form of nucleotides, including ribonucleotides and / or deoxy ribonucleotides, of any length. This term refers only to the primary structure of the molecule. Thus, this term embraces double- and single-stranded DNA and RNA (e.g., nucleic acids). It also includes known types of modifications including labels known in the art, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, and inter-nucleotide modifications such as uncharged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), as well as unmodified forms of the nucleic acid.

[0073] As used herein, a “nucleic acid analyzer” is an apparatus or instrument that amplifies, detects, and optionally quantifies nucleic acid analytes. Certain preferred nucleic acid analyzers include a temperature-controlled incubator (e.g., a block, plate, or chamber), a fluorometer in optical communication with contents of the temperature-controlled incubator, and one or more computers or processors that process data gathered by the fluorometer to quantify a nucleic acid analyte of interest. In some embodiments, the temperature-controlled incubator undergoes temperature cycling. In some embodiments, preferred nucleic acid analyzers perform enzyme-based reactions that amplify or increase the number of copies of a target nucleic acid that is to be quantified. In other embodiments, “signal amplification” is used to detect and / or quantify the target nucleic acid that is to be quantified. An example signal amplification system is provided by the “serial invasive signal amplification reaction” disclosed by Hall et al., in Proc. Natl. Acad. Sci. USA 97:8272-8277 (2000). Preferred nucleic acid analyzers are automated instruments that may include robotic fluid transfer devices, and may be subject to process control by a computer or processor. The computer or processor can be either a stand-alone device or an integral component of the nucleic acid analyzer.

[0074] As used herein, a “fluorometer” is an instrument that measures the intensity of fluorescent optical signals. Fluorometer components typically include each of a light source (sometimes an “LED” or light-emitting diode), an excitation filter, a dichroic filter, an emission filter, and a detector. In some embodiments, a fluorometer is configured to detect or measure a select band or range of wavelengths. Multiple fluorometers can be combined or bundled together on a single chassis or in a single instrument (e.g., a nucleic acid analyzer) to provide multiple detection “channels.” A “channel” refers to the band of wavelengths detected by a single fluorometer. Fluorometer channels are commonly referred to by a fluorophore that is detected (e.g., a “ROX channel”).

[0075] As used herein, a “cognate” channel of a fluorometer is the channel that most efficiently detects and / or monitors fluorescent emission of a pre-specified band of wavelengths associated with a particular fluorophore. As an example, the “ROX-channel” of a fluorometer is the cognate channel for detecting a fluorescent emission produced by a ROX-labeled or CAL FLUOR RED 610-labeled nucleic acid probe to indicate hybridization. A “non-cognate” channel of a fluorometer is a channel other than the cognate channel, and may be used for detecting fluorescent emission from a different fluorescent label or fluorophore. For example, the RED 647-channel would be a non-cognate channel for detecting emission from a CAL FLUOR RED 610 fluorophore, but could be the cognate channel for detecting a QUASAR 670 (Biosearch Technologies, Inc; California) label. Thus, a fluorometer channel can be either “cognate” or “non-cognate” for detection of a fluorescent emission from a particular fluorescent label.

[0076] As used herein, “crosstalk” is a phenomenon by which an optical signal produced by a fluorophore or fluorescent dye and measured in a cognate detection channel of a fluorometer is also measured to some extent in a non-cognate fluorescence detection channel of the fluorometer. In the context of the disclosure, crosstalk can occur when two fluorophores in the same reaction mixture have emission spectra that are partially shared or overlapping, and wherein the emission signal predominantly detected in one channel (the Emitter channel) of the fluorometer is detected to a lesser extent by “bleed-through” into a different channel (the Receiver channel) of the fluorometer.

[0077] As used herein, a “run curve” (sometimes “growth curve”) refers to the characteristic pattern of appearance of a synthetic product, such as an amplicon, in a reaction as a function of time or cycle number (i.e., reaction progress parameters). A run curve is conveniently represented as a two-dimensional plot of time or cycle number (x-axis) against some indicator of product amount, such as a fluorescence measurement (y-axis). Run curves in accordance with the disclosure represent a plot of fluorescence as a function of reaction time or cycle number. Some, but not all, run curves have a sigmoid-shape.

[0078] The terms “Emitter channel” and “Receiver channel” are used herein to describe a functional relationship between different channels of a fluorometer. This relationship is dependent on the fluorescent dyes selected for detection in each of the channels. The “Emitter channel” is the channel of a fluorometer that detects emission of a fluorescent dye in a procedure where the emission also is detected to a lesser extent in a different channel of the same fluorometer. The “Receiver channel” is the channel of the fluorometer that, in addition to detection of its intended fluorescent dye, also detects emission of the fluorescent dye that is primarily detected in a different channel (i.e., the Emitter channel) of the fluorometer.

[0079] An “Emitter channel run curve” refers to a run curve representing fluorescence measured in the Emitter channel of the fluorometer. A “Receiver channel run curve” refers to a run curve representing fluorescence measured in the Receiver channel of the fluorometer. The Receiver channel run curve detects or measures crosstalk signal (bleed-through) from the Emitter channel as well as signal from a fluorophore for which the Receiver channel is the cognate or primary channel for detection. An “Emitter channel signal value” is a fluorescent signal value measured in an Emitter channel of a fluorometer, or determined from an Emitter channel run curve. A “Receiver channel signal value” is a fluorescent signal value measured in a Receiver channel of a fluorometer, or determined from a Receiver channel run curve.

[0080] As used herein, a “correlation” or “correlation factor” is a quantitative measure or gauge of similarity shared between two run curves. Typically, a correlation factor is a numerical value that is a measure of the linear relationship between two graphs, run curves, plots, etc.

[0081] As used herein, “Pearson correlation” refers to a particular mathematical approach for quantifying linearity of a data set, where the approach is amenable to implementation using software. A “Pearson correlation factor” is a numerical value that quantifies the correlation. In this equation, r is the correlation factor, xi and yi are the fluorescence datapoints, x and y are the mean of the fluorescence curves for the Emitter (x) and the Receiver (y), respectively.

[0082] As used herein, a “compensation factor” is a numerical value that can be multiplied by the value of an Emitter channel signal (or adjusted or shifted version thereof) to determine a quantity to be subtracted from a Receiver channel signal or adjusted version thereof to compensate for crosstalk from the Emitter channel.

[0083] As used herein, “adaptive crosstalk compensation technique” refers to a procedure wherein the same multi-channel fluorometer was used to establish a compensation factor and the Receiver channel results that are to be adjusted using the compensation factor. Typically, the multi-channel fluorometer will be a component of an instrument operated by an end-user.

[0084] As used herein, an “adaptive crosstalk compensation factor” is a compensation factor determined using Emitter channel and Receiver channel run curve results determined using results obtained using one fluorometer, where the compensation factor is used to adjust the Receiver channel results that were used to determine the adaptive crosstalk compensation factor.

[0085] As used herein, a “preset” or “preset static” or “preset default” or simply “default” compensation factor refers to a compensation factor that was determined using one or more multi-channel fluorometers other than from the fluorometer that generated Receiver channel signal data that is to be adjusted to compensate for crosstalk from an Emitter channel. Typically, the multi-channel fluorometer used to establish the preset compensation factor will be operated by an instrument manufacturer, and not the end-user.

[0086] As used herein, the “baseline phase” of a growth curve refers to the initial phase of the curve wherein the amount of product (such as an amplicon) increases at a substantially constant rate, this rate being less than the rate of increase characteristic of the growth phase (which may have a log-linear profile) of the growth curve. The baseline phase of a growth curve typically has a very shallow slope, frequently approximating zero, and may exhibit “noise” or random fluctuations in fluorescence readings.

[0087] As used herein, the “growth phase” of a growth curve refers to the portion of the curve wherein the measurable product substantially increases with time. Transition from the baseline phase into the growth phase in a typical nucleic acid amplification reaction is characterized by the appearance of amplicon at a rate that increases with time. Transition from the growth phase to the plateau phase of the growth curve begins at an inflection point where the rate of amplicon appearance begins to decrease.

[0088] As used herein, the “plateau phase” of a triphasic growth curve refers to the final phase of the curve. In the plateau phase, the rate of measurable product formation generally is substantially lower than the rate of amplicon production in the log-linear phase, and may even approach zero.

[0089] As used herein, the phrase “indicia of amplification” refers to features of real-time run curves which indicate a predetermined level of progress in nucleic acid amplification reactions. In some embodiments, the time or cycle number at which a threshold level of fluorescence is exceeded serves as the indicia of amplification. Such indicia are commonly determined by mathematical analysis of run curves, sometimes referred to as “growth curves,” which display a measurable signal (such as a fluorescence reading) whose intensity is related to the quantity of an amplicon present in a reaction mixture as a function of time, cycle number, etc.

[0090] As used herein, “time-dependent” monitoring of nucleic acid amplification, or monitoring of nucleic acid amplification in “real-time” refers to a process wherein the amount of amplicon present in an amplification reaction is measured as a function of reaction time or cycle number, and can then be used to determine the presence or absence, or even the starting amount of template that was present in the reaction mixture at the time the amplification reaction was initiated. For example, the amount of amplicon can be measured prior to commencing each complete cycle of an amplification reaction that comprises thermal cycling, such as PCR. Alternatively, isothermal amplification reactions that do not require physical intervention to initiate the transitions between amplification cycles can be monitored continuously, or at regular time intervals to obtain information regarding the amount of amplicon present as a function of time.

[0091] As used herein, the phrase “time-dependent indicia of amplification” refers generally to indicia of amplification (e.g., a reaction progress parameter, such as a Fluorescence RFU reading) that are measured in time units (e.g., minutes). Time-dependent indicia of amplification are commonly used for monitoring progress in isothermal nucleic acid amplification reactions that are not characterized by distinct “cycles.” All of TTime, TArc and OTArc are examples of time-dependent indicia of amplification.

[0092] As used herein, a “test sample” is any sample to be investigated for the presence of a particular nucleic acid sequence. Test samples include any nucleic acid-containing material obtained from a human, animal, environmental, or laboratory-derived or synthetic sample. Preferred test samples include bodily fluid samples. Preferred test samples include swab samples (e.g., oral, nasal, throat, or vaginal swab samples) saliva, urine, whole blood, plasma, and serum.

[0093] As used herein, an “analyte” is a chemical or biochemical species that is to be detected and / or quantified. For example, a “nucleic acid analyte” refers to a nucleic acid (e.g., a segment of a viral nucleic acid, or of a bacterial nucleic acid) that is to be detected or quantified in a test procedure.

[0094] The term "amplify" is used in the broad sense to mean creating an amplification product that can be synthesized enzymatically with a DNA or RNA polymerase (including a reverse transcriptase). By “amplification” or “nucleic acid amplification” or “nucleic acid amplification” and the like is meant any known procedure for obtaining multiple copies, allowing for RNA and DNA equivalents, of a target nucleic acid sequence or its complement or fragments thereof. "Multiple copies" mean at least two copies. A "copy" does not necessarily mean perfect sequence complementarity or identity to the template sequence. Methods for amplifying mRNA are generally known in the art, and include reverse transcription PCR (RT-PCR). Another method which may be used is quantitative PCR (or Q-PCR).

[0095] An "amplification product" (sometimes “amplicon”) is a nucleic acid product of an amplification reaction, wherein a target nucleic acid sequence of a nucleic acid analyte served as the template for synthesis of nucleic acid copies or amplification products. Preferred amplification products include or comprise DNA.

[0096] As used herein, the terms “coamplify” and “coamplifying” and variants thereof refer to a process wherein different target nucleic acid sequences are amplified in a single (i.e., the same) amplification reaction. For example, a nucleic acid analyte and an unrelated internal calibrator nucleic acid are “coamplified” when both nucleic acids are amplified in reactions taking place in a single tube or reaction vessel, and when both amplification reactions share at least one reagent (e.g., deoxyribonucleotide triphosphates, enzyme, primer(s), etc.) in common.

[0097] As used herein, a “multiplex” reaction is one that processes (e.g., amplifies and / or detects) more than one analyte. For example, a multiplex nucleic acid amplification reaction may amplify and detect two or more nucleic acid analytes.

[0098] As used herein, "thermal cycling" refers to repeated changes of temperature, (i.e., increases or decreases of temperature) in a reaction mixture. Samples undergoing thermal cycling may shift from one temperature to another, stabilize at that temperature, transition to a second temperature or return to the starting temperature. The temperature cycle may be repeated as many times as required to study or complete the particular chemical reaction of interest.

[0099] By ‘‘target” or “target nucleic acid” or “target polynucleotide” is meant a nucleic acid containing a sequence that is to be amplified, detected, and / or quantified. A target nucleic acid sequence that is to be amplified preferably will be positioned between two oppositely disposed amplification oligonucleotides (e.g., primers), and will include the portion of the target nucleic acid that is complementary to each of the oligonucleotides.

[00100] By “target nucleic acid sequence” or “target sequence” or “target region” is meant a specific deoxyribonucleotide or ribonucleotide sequence comprising all or part of the nucleotide sequence of a single-stranded target nucleic acid molecule, and the deoxyribonucleotide or ribonucleotide sequence complementary thereto.

[00101] As used herein, an "oligonucleotide" or “oligomer” or “oligo” is a polymeric chain of at least two, generally between about five and about 100, chemical subunits, each subunit comprising a nucleotide base moiety, a sugar moiety, and a linking moiety that joins the subunits in a linear spatial configuration. Common nucleotide base moieties are guanine (G), adenine (A), cytosine (C), thymine (T) and uracil (U), although other rare or modified nucleotide bases able to hydrogen bond are well known to those skilled in the art. Oligonucleotides may optionally include analogs of any of the sugar moieties, the base moieties, and the backbone constituents. Preferred oligonucleotides of the present disclosure fall in a size range of about 10 to about 100 residues. Oligonucleotides may be purified from naturally occurring sources, but preferably are synthesized using any of a variety of well-known enzymatic or chemical methods.

[00102] By “amplification oligonucleotide” or “amplification oligomer” is meant an oligomer that hybridizes to a target polynucleotide, or its complement and participates in a nucleic acid amplification reaction. Examples of amplification oligomers include primers that contain a 3’-end that is extended as part of the amplification process, but also include oligomers that are not extended by a polymerase (e.g., a 3’-blocked oligomer), but may participate in, or facilitate efficient amplification from a primer. An amplification oligomer may optionally include modified nucleotides or analogs, or additional nucleotides that participate in an amplification reaction but are not complementary to or contained in the target polynucleotide. An amplification oligomer that is 3’-blocked but capable of hybridizing to a target polynucleotide and providing an upstream promoter sequence that serves to initiate transcription is referred to as a “promoter provider” oligomer.

[00103] A “primer” is an amplification oligomer that hybridizes to a target nucleic acid template and has a 3’-OH end that can be extended by a DNA polymerase. The 5' region of the primer may be non-complementary to the target nucleic acid (e.g., a non-complementary promoter sequence), resulting in an oligomer referred to as a “promoter-primer.” Those skilled in the art will appreciate that any oligomer that can function as a primer can be modified to include a 5' promoter sequence, and thus could function as a promoter-primer. Similarly, any promoter-primer can be modified by removal of, or synthesis without, a promoter sequence and still function as a primer.

[00104] As used herein, a “probe” is an oligonucleotide that hybridizes specifically to a target sequence in a nucleic acid, preferably in an amplified nucleic acid, under conditions that promote or allow hybridization, to form a detectable hybrid. Preferred probes include a detectable label (e.g., a fluorescent label). Hydrolysis probes conventionally include a fluorescent label and a quencher moiety.

[00105] The term "label" refers to a composition capable of producing a detectable signal indicative of the presence of the labeled molecule. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Fluorescent molecules or “fluorophores” are particularly preferred labels in accordance with the disclosure.

[00106] "Detection" includes any means of detecting, including direct and indirect detection of gene expression and changes therein. For example, "detectably less" products may be observed directly or indirectly, and the term indicates any reduction (including the absence of detectable signal). Similarly, "detectably more" product means any increase, whether observed directly or indirectly.

[00107] As used herein, the phrase “as a function of” describes the relationship between a dependent variable (i.e., a variable that depends on one or more other variables) and an independent variable (i.e., a variable that may have its value freely chosen without considering the values of any other variables), wherein each input value for the independent variable relates to exactly one output value for the dependent variable. Conventional notation for an equation that relates a y-value (i.e., the dependent variable) “as a function of’ an x-value (i.e., the independent variable) is y = f(x).

[00108] As used herein, a “computer” or “processor” is an electronic device capable of receiving and processing input information using software instructions to generate an output. The computer may be a standalone device (e.g., a personal computer), or may be an integral component of an instrument (e.g., a nucleic acid analyzer that amplifies a target nucleic acid and monitors synthesis of amplification products as a function of reaction cycle number or time). Particularly embraced by the term is an embedded processor resident within an analyzer instrument, and harboring embedded software instructions (sometimes referred to a “firmware”).

[00109] As used herein, “optimizing” or “fitting” an equation refers to a process, as commonly practiced in mathematical modeling or curve fitting procedures, for obtaining numerical values for coefficients in an equation to yield an expression that “fits” or approximates experimental measurements. Typically, an optimized equation will define a best-fit curve.

[00110] As used herein, the terms “optimized equation,” and “fitted equation” are alternative references to an equation containing fixed numerical values for coefficients as the result of an optimizing procedure. “Fitted” curves (e.g., the products of curve fitting procedures) result from optimizing an equation.

[00111] As used herein, a “system” is an arrangement of parts or components organized to cooperate with one another. For example, a system may include an instrument that detects nucleic acids in a sequence-specific manner, and a computer programmed with software to analyze results, where the computer and the instrument are in communication with each other.

[00112] As used herein, an “instrument” is a tool, device, or implement for performing a task. The term embraces the collection of equipment needed for a particular purpose or function. In some embodiments, an instrument is a device contained within a single housing or situated on common support structure (e.g., a single chassis).

[00113] By “kit” is meant a packaged combination of materials, typically intended for use in conjunction with each other. Kits in accordance with the present disclosure may include instructions or other information in a “tangible” form (e.g., printed information, electronically recorded on a computer-readable medium, or otherwise recorded on a machine-readable medium such as a bar code for storing numerical values).

[00114] Conditions that "allow" an event to occur or conditions that are "suitable" for an event to occur, such as hybridization, strand extension, and the like, or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and / or are conducive to the event. Such conditions, known in the art and described herein, depend upon, for example, the nature of the nucleotide sequence, temperature, and buffer conditions. These conditions also depend on what event is desired, such as hybridization, cleavage, strand extension or transcription.

[00115] As used herein, the term "comprising" and its cognates are used in their inclusive sense; that is, equivalent to the term "including" and its corresponding cognates.

[00116] By “consisting essentially of’ is meant that additional component(s), composition(s) or method step(s) that do not materially change the basic and novel characteristics of the present invention may be included in the present invention. Any component(s), composition(s), or method step(s) that have a material effect on the basic and novel characteristics of the present invention would fall outside of this term.

[00117] Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Introduction and Overview

[00118] Disclosed herein are methods, instrument systems, tangible embodiments of software, and programmed computers or embedded processors in communication with automated nucleic acid analyzers, where each of these can be used to compensate or correct for the effects of fluorescent “crosstalk” detected or measured in different channels of a fluorometer. The technique is illustrated in the framework of a correction applied to results obtained with multiplex real-time in vitro nucleic acid amplification reactions. One benefit of the disclosed technique is enhanced accuracy in target identification without the need for hardware modifications. A particular benefit is reduction of false-positive results that arise because of crosstalk from optical signals intended for detection in one channel of a fluorometer that are also received by a different channel of the fluorometer. Crosstalk corrected by the disclosed technique may result from any number of different factors, including changes in fluorometers that may result from extended use or aging.

[00119] In accordance with a previous crosstalk compensation approach (e.g., a “default” technique herein), a fixed or constant percentage of the Emitter channel (e.g., ROX-channel) signal used for detecting the “Analyte-2” amplicon was subtracted from the Receiver channel signal (e.g., RED 647-channel) used to indicate the presence of the “Analyte-1” amplicon. Throughout this disclosure, a SARS nucleic acid served as a model for Analyte-2, and a Flu B nucleic acid served as a model for Analyte-1. The default technique ultimately was found to contribute to false-positive detection of Analyte-1 (Flu B). The adaptive crosstalk compensation technique disclosed herein can determine whether adjustment of the Receiver channel signal should be effected using a dynamically calculated adjustment value (i.e., an adaptive crosstalk compensation factor) rather than a preset value used by default on multiple instruments.

[00120] The adaptive crosstalk compensation technique disclosed herein followed from the discovery that false-positive results obtained for multiplex nucleic acid amplification reactions could be attributed to variations in the performance characteristics of fluorometer components of instruments that amplify nucleic acids and monitor synthesis of amplification products. The technique can be used to correct results obtained with multiplex reactions employing as few as two different fluorophores, where each fluorophore is predominantly detected by a different channel of a fluorometer.

[00121] The present technique was developed to control the undesired effects of fluorescent crosstalk between detection channels within a fluorometer, where crosstalk can lead to erroneous conclusions about the presence of pathogenic organisms or agents. More specifically, the technique was developed using a real-time multiplex nucleic acid amplification assay as a model system, where the assay employed different fluorescent dyes (fluorophores) for each of four different targets that could be detected.

[00122] During development of the disclosed technique, it was observed that certain automated nucleic acid analyzers running a fourplex real-time PCR assay (e.g., amplifies and detects Analytes 1-4) occasionally yielded false-positive calls for detection of Analyte-1 (Flu B). All instances of Analyte-1 (Flu B) false-positive results arose from reactions that amplified Analyte-2 (SARS) nucleic acids. The false-positive results were obtained using a small number of instruments, but were not observed with a larger number of similar instruments. The false-positive results were obtained using multiple different reagent lots, where cartridges used on the automated nucleic acid analyzer were preloaded with reagents needed to perform PCR reactions following sample addition. Thus, reagents were not likely responsible for the false-positive errors. Ultimately, replacement of fluorometers in the problematic instruments corrected the false identification issue, thereby implicating these hardware components as root causes of the problem.

[00123] Further assessment of result profiles for the affected instruments revealed that fluorescent signal primarily detected in the ROX-channel used for monitoring synthesis of Analyte-2 (SARS) amplification products was also being detected in the RED 647-channel that was used for monitoring synthesis of Analyte-1 (Flu B) amplification products. To clarify this arrangement, the two channels were referred to as the “Emitter channel” (sometimes “ROX Emitter” channel) and the “Receiver channel” (sometimes “RED 647 Receiver” channel), to indicate that signal from the ROX-labeled probe was received in the non-cognate RED 647-channel. Since only a small fraction of all monitored instruments yielded false-positive determinations or calls for Analyte-1 (Flu B), and since there was evidence that fluorescent signal from the ROX-labeled probe in a reaction that amplified the Analyte-2 (SARS) nucleic acid was also detected in the non-cognate RED 647 Receiver channel, it was reasoned that the observed false-positive calls might be due to reception of fluorescent signal in the non-cognate channel of the fluorometers in the affected instruments. Reception and measurement of fluorescent signal in a non-cognate channel of a fluorometer is sometimes referred to as “crosstalk.”

[00124] As demonstrated below, a crosstalk signal from a fluorescent dye primarily or predominantly detected in a ROX Emitter channel also was detected in a RED 647 Receiver channel. This crosstalk could be compensated to some extent by software using a fixed or constant compensation factor to adjust the Receiver channel signal. Analyte-1 (Flu B) detection using a QUASAR 670 fluorophore detected in the RED 647 channel was very sensitive to crosstalk from a CAL FLUOR RED 610 signal measured in a ROX channel of the instrument fluorometer. This was due to the overlap in emission spectra of the two dyes, together with the relative fluorescence intensity of the two dyes in the reaction mixture (e.g., very strong CAL FLUOR RED 610 signal in the ROX channel, and weak QUASAR 670 signal in the RED 647 detection channel).

[00125] Instrument-to-instrument variability for crosstalk into the RED 647-channel was sufficient in some instances to produce false-positive Analyte-1 (Flu B) calls in reactions that amplified Analyte-2 (SARS) but not Analyte-1 (Flu B), and that employed the ROX channel output for detecting amplicons using a CAL FLUOR RED 610 fluorescent label on an Analyte-2 (SARS) sequence-specific nucleic acid probe. Variability in the magnitude of required crosstalk compensation across different instruments was attributed to slight differences in the tolerances of the LEDs, filters, and detectors used to manufacture the fluorescence detector channels of certain fluorometers. Notably, all of the fluorometers, including those associated with false-positive calls, had been released within manufacturing specifications. Thus, differences in hardware between instruments that did and did not produce false-positive results must have been minimal. Simply increasing the crosstalk compensation factor used to adjust signals in the Receiver channel by an arbitrary amount caused most results to be overcompensated, thereby leading to Analyte-1 (Flu B) falsenegative results in cases of true dual-analyte amplification. A more comprehensive solution was needed. The Adaptive Crosstalk Compensation Technique

[00126] The adaptive crosstalk compensation technique provides an improvement over previous crosstalk compensation procedures by determining - for different Emitter and Receiver channel paired data sets - the compensation factor that should be used to adjust the Receiver channel fluorescence signal. In some embodiments, the technique further provides an option for determining whether an adaptive crosstalk compensation factor or a preset default compensation factor should be used to adjust Receiver channel signal data. According to prior techniques conventionally used for crosstalk compensation, a fixed or constant percentage of the Emitter channel signal (e.g., the ROX-channel monitoring amplification of Analyte-2 (SARS)) in the case illustrated herein was subtracted from the signal measured in the Receiver channel (e.g., the RED 647-channel monitoring amplification of the Analyte-1 (Flu B) analyte in the case illustrated herein). By this previous approach, the same crosstalk compensation factor was applied to adjust all Receiver channel signals on all similar nucleic acid analyzers by subtracting the same fraction or percentage of the Emitter channel signals. This general approach undesirably led to false-positive Analyte-1 (Flu B) assignments when fluorescent signal measured in the Emitter channel indicating the presence of Analyte-2 (SARS) (ROX-channel) was high, and when the reaction did not include Analyte-1 (Flu B) target nucleic acid templates. The improved adaptive crosstalk compensation procedure optionally identifies this situation and then uses a different compensation factor specific for the Emitter and Receiver channel run curves to calculate the magnitude of the Emitter channel signal that should be subtracted from the Receiver channel signal to compensate for crosstalk. As described below, this approach advantageously reduced false-positive assignments resulting from fluorescence crosstalk.

[00127] The adaptive crosstalk compensation technique described herein particularly addressed cases where the fluorescence run curve monitored in the Receiver channel was substantially due to crosstalk from the Emitter channel. In this instance the Receiver channel fluorescence run curve demonstrated a high linear correlation with the Emitter channel fluorescence run curve. It is to be understood that two fluorescence run curves are considered highly correlated when their signal profiles for cycle-dependent amplification are similar. In these highly correlated cases, the crosstalk compensation factor could be calculated directly as the ratio of the two amplification curves at different points along the curves. In cases where the curves were not highly correlated, the confidence in calculating the crosstalk compensation factor from the curve ratio was lower, and so a preset default crosstalk compensation factor value was used instead. The disclosed technique is illustrated herein for use with an automated instrument that amplified and detected nucleic acids, but this application is merely exemplary. The technique disclosed herein can be used wherever two or more fluorescence emission spectra are used for detecting analytes exhibit wavelength overlap. Exemplary analytes include nucleic acids, peptides or proteins, analytes bound directly or indirectly by protein receptors (e.g., antibodies, etc.) and the like.

[00128] Since automated nucleic acid analyzers employed in the procedure collected data across all fluorometer channels in a time-dependent serial fashion using a mechanical rotary motion, fluorescence measurements for each channel exhibited a time offset. Shifting the Emitter channel run curve relative to the Receiver channel run curve compensated for this time offset by synchronizing both curves, thereby maximizing their correlation to increase accuracy in calculating the compensation factor. This synchronization step is considered optional, and is not needed or required for instrument platforms that do not exhibit a time or cycle number offset.

[00129] Processing of real-time run curve data optionally involves a series of adjustments between raw fluorescent signal acquisition and calculation of a Ct value for individual amplifying species. These adjustments can involve one or more of data smoothing (e.g., by calculation of a moving average or other mathematical technique), baseline subtraction, crosstalk correction (e.g., adaptive crosstalk compensation), etc. Adaptive crosstalk compensation is applied to correct run curve data before calculating a Ct (i.e., cycle threshold) value using the corrected data. The Adaptive crosstalk compensation technique is generally applied after data smoothing, after baseline subtraction, and before calculating a Ct value.

[00130] The adaptive crosstalk compensation technique includes calculation of a difference between Relative Fluorescent Unit (RFU) values measured for the Emitter channel (sometimes “Delta RFU”), together with calculation of a correlation (e.g., a “correlation factor”) between run curves representing outputs of the Emitter channel and Receiver channel. The magnitude of the Delta RFU value is an indicator of possible crosstalk into the Receiver channel. If the Delta RFU value is substantially zero, then crosstalk would not be possible. Conversely, if the Delta RFU value is high (e.g., exceeds a pre-established threshold value needed to establish amplification), then crosstalk into the Receiver channel would be possible. The correlation factor provides a quantitative or mathematical gauge of similarity shared between two run curves. In some embodiments, the Delta RFU calculated for the Emitter channel is simply the difference between the highest and lowest measured Emitter channel signals, preferably after smoothing the Emitter channel fluorescence readings (e.g., to remove random fluctuations in fluorescence readings). It will be apparent from this description that the magnitude of any crosstalk signal detected in the Receiver channel of a fluorometer cannot exceed the magnitude of the Emitter channel signal.

[00131] If the Delta RFU value and the calculated correlation factor each exceed respective established threshold values, then a “crosstalk ratio” can be calculated. This preferably is accomplished by first synchronizing (e.g., “aligning”) the Emitter channel and Receiver channel run curves to within +1- 1 Ct unit, and then calculating an average ratio of the amplifying curves. In one embodiment, determining the average ratio of the two amplifying run curves involves averaging a plurality of calculated ratio values on a point-by-point basis at different cycle numbers. The plurality of calculated ratios can be selected to allow for removal of data points at cycle numbers where the calculated ratio values exhibit significant fluctuation. Typically, the removed data points correspond to cycle numbers associated with baseline regions of the Emitter and Receiver channel run curves. Fig. 4 illustrates how calculated ratio values from selected data points can be used to find an average ratio.

[00132] The magnitude of a calculated average ratio of the Receiver and shifted Emitter channel results can be used to determine an “adaptive crosstalk compensation factor.” The adaptive crosstalk compensation factor can be multiplied by the magnitude of the Emitter channel signal to determine a quantity that should be subtracted from the Receiver channel signal to correct for the effects of crosstalk. In some preferred embodiments, the average ratio is used as the adaptive crosstalk compensation factor. In other preferred embodiments, the average ratio is used to select an adaptive crosstalk compensation factor from two or more predetermined values. For example, the magnitude of the average ratio can be used to select an adaptive crosstalk compensation factor falling into one of a plurality of ranges or zones. The selected adaptive crosstalk compensation factor may fall into a range such as: (A) below 1%; (B) between 1% and 4%; (C) between 4% and 10%; and (D) greater than 10%. The identity of the zone can be used to indicate the value of the adaptive crosstalk compensation factor to be used as the adaptive crosstalk compensation factor. Evidence for Crosstalk Between two Fluorescence Detection Channels of a Fluorometer

[00133] The present technique optionally includes a step whereby evidence of crosstalk may be obtained before a crosstalk compensation factor is calculated or determined. In a preferred embodiment, this is accomplished by comparing real-time run curves from different channels of a fluorometer to determine whether the curves are linearly correlated. In a preferred embodiment this involves assessing correlation of signal increases and decreases of the two curves with each other. A convenient approach for accomplishing this in a manner amenable to processing by computer software involves first preparing data for two run curves and then quantifying the extent of the relationship between shapes of the two run curves by calculating a Pearson correlation factor. Optionally, the two curves being compared are “synchronized” before the comparison is performed, as described above. Synchronized curves will have maximum shape similarity. One approach for comparing the synchronized curves involves calculating a ratio of one curve to the other, point-by-point (e.g., at periodic Ct increments using fitted splines or other fitted curves).

[00134] Reasons for synchronizing run curves measured in different channels of a fluorometer include correction or accommodation of time offsets for recording fluorescence readings. This may occur when fluorescence readings are taken by mechanically moving the sample and detector or other fluorometer component relative to each other in order to take readings at different wavelengths. Some fluorometers employ a rotary motion, where only one channel is engaged at a time. Synchronizing two run curves can give improved results when assessing run curve correlations. Determination of the Correlation Factor

[00135] Basis for the problem solved by the present technique can be understood with reference to Fig. 1, which shows overlapping absorption spectra and overlapping emission spectra for two fluorescent dyes. The particular dyes shown in the figure are the CAL FLUOR RED 610 dye (stippled) and the QUASAR 670 dye (hatched) used in the Examples. Inspection of the upper panel of the figure reveals that wavelengths that excite the CAL FLUOR RED 610 dye will likely also excite the QUASAR 670 dye, at least somewhat, when the two dyes are present in the same reaction mixture. The substantial overlap of emission spectra for the two dyes shown in the lower panel of the figure means that a fluorometer channel configured to detect fluorescence from the QUASAR 670 dye might also detect fluorescent emission from the CAL FLUOR RED 610 dye. This is because detection is typically not limited to a single wavelength, but instead extends over a band or range of wavelengths. If this band encompasses a substantial portion of the shared or overlapping emission spectrum of the adjacent CAL FLUOR RED 610 dye, then the CAL FLUOR RED 610 dye emission will be received in the RED 647 channel that monitors emission from the QUASAR 670 dye. If the two dyes were used to label oligonucleotide probes specific for different target nucleic acids, and if the fluorometer used for detecting a signal produced by the QUASAR 670 dye received crosstalk signals from the CAL FLUOR RED 610 dye emission, then fluorometer readings might lead to false conclusions regarding the presence of the target nucleic acid detected using the QUASAR 670 dye.

[00136] To be clear, the dyes and fluorometer channels chosen to illustrate the disclosed technique are merely exemplary, and the technique is not limited to these. Indeed, the technique is generally useful whenever two fluorophores are combined in a single reaction, and when the two fluorophores exhibit a shared or overlapping range of emission wavelengths.

[00137] The origin of the correlation factor that gauges similarity of run curves can be understood with reference to Figs. 2A-2D. Illustrated in the upper plots of the figures are pairs of real-time amplification run curves, where each curve represents fluorescence readings from differentially labeled probes as a function of reaction cycle number. The ROX-channel (used for detecting CAL FLUOR RED 610 dye emission; filled circles) represented a model Emitter channel, and the RED 647-channel (used for detecting QUASAR 670 dye emission; open circles) represented a model Receiver channel. This arrangement results from the fact that emission wavelengths of the CAL FLUOR RED 610 dye detected in the ROX-channel overlap with emission wavelengths of the QUASAR 670 dye detected in the RED 647-channel, so that at least some of the signal measured or detected in the ROX-channel could also be received in the RED 647-channel and interpreted as a QUASAR 670 emission signal. Illustrated in the lower parts of the figures are plots correlating the real-time data from two run curves in the upper plots.

[00138] In each of Figs. 2A-2D, a Pearson correlation factor greater than 0.95, more preferably greater than 0.98, and still more preferably greater than 0.99 was regarded as indicating a high correlation between Emitter and Receiver run curves. These numerical values are examples of useful threshold cutoffs for gauging correlation between run curves.

[00139] Fig. 2A illustrates results obtained for reactions that did not amplify either the Analyte-2 (SARS) or the Analyte-1 (Flu B) target nucleic acid. The upper plot shows increases in background fluorescence readings for both targets as the reaction cycle number increased. It will be apparent that the Delta RFU value for the Emitter channel plot measuring fluorescence signal produced by the CAL FLUOR RED 610 dye is less than about 250 RFU (i.e., very low), and so did not exceed a 500 RFU threshold or cutoff needed to indicate target nucleic acid amplification, or a substantial opportunity for crosstalk. The lower correlation plot shows the magnitude of the RED 647 Receiver channel (QUASAR 670 dye) signal (Analyte-1 (Flu B) target) on the y-axis plotted against the magnitude of the ROX Emitter channel (CAL FLUOR RED 610 dye) signal (Analyte-2 (SARS) target) on the x-axis. Also shown in the lower plot is a best-fit line and associated R2 value. The correlation factor (R=0.929) was calculated using the Pearson correlation equation, with R2 as its squared value. This correlation factor did not exceed the minimum required threshold (i.e., 0.95) needed to indicate a high correlation.

[00140] Fig. 2B illustrates results obtained for reactions that amplified the Analyte-1 (Flu B) target nucleic acid, but not the Analyte-2 (SARS) target nucleic acid. The upper plot shows a sigmoid increase in the fluorescence reading for the Analyte-1 (Flu B) target (QUASAR 670 label measured in the RED 647-channel), and a substantially linear increase in the background fluorescence reading for the Analyte-2 (SARS) target (CAL FLUOR RED 610 label measured in the ROX-channel) as the reaction cycle number increased. It will be apparent that the Delta RFU value for the Emitter channel plot is about 250 RFU, and so did not exceed the 500 RFU threshold or cutoff needed to indicate target nucleic acid amplification, or a substantial opportunity for crosstalk. The lower correlation plot shows the magnitude of the RED 647 Receiver channel signal (Analyte-1 (Flu B) target) on the y-axis plotted against the magnitude of the ROX Emitter channel signal (Analyte-2 (SARS) target) on the x-axis. Also shown in the lower plot is a best-fit line and associated R2 value. The correlation factor (R=0.936) was calculated using the Pearson correlation equation, with R2 as its squared value. This correlation factor did not exceed the minimum required threshold (i.e., 0.95) needed to indicate a high correlation.

[00141] Fig. 2C illustrates results obtained for reactions that amplified the Analyte-2 (SARS) target nucleic acid, but not the Analyte-1 (Flu B) target nucleic acid. The upper plot clearly shows a sigmoid increase in fluorescence readings for the Analyte-2 (SARS) target (CAL FLUOR RED 610 label measured in the ROX-channel) as the reaction cycle number increased. An increase in the fluorescence readings for the Analyte-1 (Flu B) target (QUASAR 670 label measured in the RED 647-channel) also is apparent, but appears to be an increase in background signal. It will be apparent that the Delta RFU value for the Emitter channel plot is greater than about 25,000 RFU, and so exceeded the 500 RFU threshold or cutoff needed to indicate target nucleic acid amplification, or a substantial opportunity for crosstalk. The lower correlation plot shows the magnitude of the RED 647 Receiver channel QUASAR 670 signal (Analyte-1 (Flu B) target) on the y-axis plotted against the magnitude of the ROX Emitter channel CAL FLUOR RED 610 signal (Analyte-2 (SARS) target) on the x-axis. Also shown in the lower plot is a best-fit line and associated R2 value. The correlation factor (R=0.9998) was calculated using the Pearson correlation equation, with R2 as its squared value. The correlation factor clearly exceeded the minimum required threshold (i.e., 0.95) needed to indicate high correlation. Indeed, the ROX-channel and RED 647-channel curves were substantially identical even though the reaction amplified the Analyte-2 (SARS) target nucleic acid, but the reaction did not include or amplify Analyte-1 (Flu B) target nucleic acid. Thus, the RED 647 Receiver channel curve reflected only crosstalk from the CAL FLUOR RED 610 fluorophore.

[00142] Fig. 2D illustrates results obtained for reactions that amplified both Analyte-2 (SARS) and Analyte-1 (Flu B) target nucleic acids. The upper plot shows sigmoid increases in fluorescence readings for both targets as the reaction cycle number increased. It will be apparent that the Delta RFU value for the ROX Emitter channel plot is about 6,000 RFU, and so exceeded the 500 RFU threshold or cutoff needed to indicate target nucleic acid amplification, or a substantial opportunity for crosstalk. The lower correlation plot shows the magnitude of the RED 647 Receiver channel (QUASAR 670 dye) signal (Analyte-1 (Flu B) target) on the y-axis plotted against the magnitude of the ROX Emitter channel (CAL FLUOR RED 610 dye) signal (Analyte-2 (SARS) target) on the x-axis. Also shown in the lower plot is a best-fit line and associated R2 value. The correlation factor (0.921) was calculated using the Pearson correlation equation, with R2 as its squared value.

[00143] Here the correlation factor did not exceed the minimum required threshold or cutoff (i.e., 0.95) needed to indicate high correlation. However, in view of the high Delta RFU value, calculation and use of the adaptive crosstalk compensation factor to adjust the Receiver channel signal was considered optional. In certain preferred cases where the Delta RFU value exceeded the 500 RFU threshold, and where the correlation factor was low (i.e., below 0.95), the preset default crosstalk compensation factor, and not the adaptive crosstalk compensation factor is used to adjust the Receiver channel signal.

[00144] As an alternative to assessing linearity of the correlation of the Emitter channel run curve and the Receiver channel run curve directly, linearity of the correlation of mathematical transformations of the Emitter and Receiver channel run curves can be assessed with good results. Example mathematical transformations include first, second, and nth order derivatives.

[00145] The preceding discussion illustrates how correlations between real-time Emitter channel and Receiver channel run curves were assessed. Highly correlated run curves indicated crosstalk of an Emitter channel signal into a Receiver channel. This, together with evidence for a high Delta RFU value in the Emitter channel supports adjustment of the Receiver channel signal using an adaptive crosstalk compensation factor as opposed to a different preset default factor.

[00146] As described above, the disclosed technique makes it possible to avoid replacing fluorometer hardware or requiring more stringent manufacturing tolerances on fluorometer components that would be difficult and costly to achieve. More particularly, because fluorometers are constructed from several critical components, and because each component can have slightly different performance characteristics as the result of differences in materials or manufacturing, these differences contribute to variation in the magnitude of crosstalk between channels of the fluorometer. As a result, a fluorometer in a first instrument may permit slightly different crosstalk of optical signals from a fluorescent label into noncognate channels compared to a different fluorometer in another instrument. The impact may be so insignificant that no difference will be detected in some multiplex systems that rely on a plurality of fluorescent channels. One advantage of the present technique is that a problematic instrument having a fluorometer prone to crosstalk can be retrofitted or upgraded using data processing software rather than replacing and discarding fluorometer hardware. Thus, in one embodiment, the disclosed technique can provide a confidence level that signal detected in a Receiver channel is actually (e.g., entirely) due to fluorescent signal detected in the Emitter channel.

[00147] In certain embodiments, the technique is applied between two channel pairs of a fluorometer with no prior knowledge about evidence for crosstalk. Synchronizing Run Curves - Maximizing Correlation Using Curve Fitting and Signal Value Interpolation

[00148] Although different approaches can be used to smooth run curve data (e.g., moving averages, four parameter logistic regression, etc.), spline interpolation was used to identify signal values between measured data points along the Emitter channel run curve in an optional procedure to synchronize Emitter and Receiver run curves. Essentially, RFU readings for measured cycle numbers were used for spline fitting, and the calculated spline was broken into subunits (e.g., each incremental subunit representing 0.1 cycles along the cycle number progress parameter dimension). Next, the Emitter channel run curve was repeatedly or iteratively shifted by single subunit intervals. The effect of this was to shift the original Emitter run curve relative to the Receiver run curve. The correlation between the two run curves was calculated at each new increment. The point at which maximum correlation was achieved was then identified. Typically, this procedure was carried out using 0.1 cycle increments between 0.9 cycle faster and 0.9 cycle slower than the original Emitter run curve. Again, recalculated correlation values for each shifted curve were determined, and the curve with the highest correlation to the Receiver run curve was established. By this procedure the Emitter and Receiver run curves were maximally correlated or “synchronized.” Calculating Ratios of the Receiver / Emitter Curves

[00149] Using the optionally synchronized run curves prepared by monitoring the Emitter and Receiver channels during a nucleic acid amplification reaction, ratios of the two curves at a plurality of points across the range of cycle numbers were calculated. As described below, ratios were calculated point-by-point along the reaction progress parameter (i.e., cycle numbers) of the Receiver / Emitter curves. Data points where the RFU was below a threshold value of the smoothed background-subtracted curve were excluded. In one embodiment, this threshold value was determined by calculating MinRFUPercent*MaxRFU. In this equation, MinRFUPercent is a ratio threshold on the fluorescence and MaxRFU is the maximum observed fluorescence from the Emitter channel. A “shifted ratio” was calculated as the mean of the non-excluded data for the synchronized run curves (e.g., see Fig. 4). This shifted ratio was useful as the adaptive crosstalk compensation factor. In some preferred embodiments, an average of a plurality of shifted ratio values was used as the adaptive crosstalk compensation factor. Preferred Systems and Apparatus

[00150] The methods disclosed herein are conveniently implemented using a computer or similar processing device (“computer” hereafter). In different preferred embodiments, software or machine-executable instructions can be loaded or otherwise held in a memory component of a freestanding computer, or in a memory component of a computer linked to a device used for monitoring, preferably as a function of a reaction progress parameter (e.g., either reaction time or reaction cycle number), the amount of a product undergoing analysis. In a highly preferred embodiment, software for executing the disclosed procedure is held in a memory component of a computer that is linked to, or that is an integral part of a device or apparatus capable of monitoring the amount of an amplicon present in a reaction mixture as a function of reaction cycle number. This includes a processing device component on an electronic circuit board (e.g., embedded software) of an automated nucleic acid analyzer. Generally speaking, the computer is said to be “in communication with” the apparatus that detects and / or quantifies a target nucleic acid when information from the nucleic acid analyzer is transferred from the apparatus to the computer, by any means. In some embodiments, results generated by the computer can be delivered to an output device that displays or records a result of a calculation or comparison. Exemplary output devices include a video monitor and a printer. In some embodiments, the output device is a recording device that produces a “non-transient” record (e.g., a “tangible” record). The non-transient record may be printed on paper, or stored electronically (such as on a computer hard drive or flash drive, magnetic tape or other computer-readable media, etc.).

[00151] In some embodiments, the computer can be in communication with, either by wired or wireless means, a fluorometer that detects fluorescent signals, where the fluorometer is arranged or configured to monitor fluorescent signals generated in one or more reaction vessels contained within a temperature-controlled incubator. The incubator can be a temperature-controlled block (e.g., a metal block configured for receiving and containing one or more tubes, or even a multi-well plate), or a chamber that exposes one or more reaction vessels to controlled temperature conditions.

[00152] In some embodiments, either or both of a controller system for controlling a real-time amplification device and / or the detection system of the real-time amplification device can be coupled to an appropriately programmed computer that functions to instruct the 32 operation of these instruments in accordance with preprogrammed or user input instructions. The computer preferably also can receive data and information from these instruments, and interpret, manipulate, and report this information to the user.

[00153] In some embodiments, the computer also can include appropriate software for receiving user instructions, either in the form of user input into a set of parameter fields, or in the form of preprogrammed instructions (e.g., preprogrammed for a variety of different specific operations). The software then converts these instructions to appropriate language for instructing the operation of the real-time amplification controller to carry out the desired operation. Preferably, the computer also is capable of receiving data from one or more sensors or detectors included within the system, and interpreting the data in accordance with the programming. The system preferably includes software that correlates a feature of a growth curve representing the quantity of amplified copies of the nucleic acid of interest as a function of time or cycle number, as detected by the detector, to the number of copies of the nucleic acid of interest present in a test sample.

[00154] Preferably, when the computer used for executing the disclosed technique is an integral component of an apparatus for performing and analyzing real-time nucleic acid amplification reactions, the apparatus preferably comprises a temperature-controlled incubator, a detection device for collecting signals (e.g., a fluorometer), and an analyzing device (e.g., a computer or processor) for analyzing signals. The apparatus optionally can further include an output device for displaying data obtained or generated. The analyzing device may be connected to the temperature-controlled incubator through an input device known in the art, and / or connected to an output device known in the art for data display. In one embodiment, the temperature-controlled incubator is capable of temperature cycling, and may be configured as a block for receiving one or more tubes, or reaction receptacles (e.g., cartridges or multi-tube units).

[00155] Generally speaking, the various components of an apparatus for performing the real-time nucleic acid amplification useful in connection with the disclosed methods will be conventional components that will be familiar to those having an ordinary level of skill in the art. The temperature-controlled incubator used to perform and analyze real-time nucleic acid amplification may be of a conventional design which can hold a plurality of reaction tubes, or reaction samples in a temperature-controlled block in standard amplification reaction tubes or in wells of a multiwell plate. In one aspect, the detection system is suitable for detecting optical signals from a plurality of fluorescent labels. The output of the detection system (e.g., signals corresponding to those generated during the amplification reaction) can 33 be fed to the computer for data storage and manipulation. In one embodiment, the system detects multiple different types of optical signals, such as multiple different types of fluorescent labels and has the capabilities of a microplate fluorescence reader. The detection system is preferably a multiplexed fluorimeter containing an excitation light source, which may be a visible light laser (e.g., an LED) or an ultraviolet lamp or a halogen lamp, a multiplexer device for distributing the excitation light to the individual reaction tubes and for receiving fluorescent light from the reaction tubes, a filtering means for separating the fluorescence light from the excitation light by their wavelengths, and a detection means for measuring the fluorescence light intensity. Preferably, the detection system of the temperature-controlled incubator provides a broad detection range that allows flexibility of fluorophore choice, high sensitivity and excellent signal-to-noise ratio. Optical signals received by the detection system are generally converted into signals which can be operated on by the computer or processor to provide data which can be viewed by a user on a display of a user device in communication with the computer or processor. The user device may comprise a user interface or may be a conventional commercially available computer system with a keyboard and video monitor. Examples of data which can be displayed by the user device include amplification plots, scatter plots, sample value screens for all the tubes or reaction vessels in the assembly and for all labels used, an optical signal intensity screen (e.g., fluorescent signal intensity screen), final call results, text reports, and the like. Illustrative Nucleic Acid Analyzers and Systems

[00156] Figs. 6A and 6B illustrate an exemplary automated analytical system 1000 that may be used to simultaneously analyze a plurality of samples. Fig. 6A is a perspective view of system 1000, while Fig. 6B is view of system 1000 with its canopy removed to show features within. In the discussion below, reference will be made to both Figs. 6A and 6B. System 1000 is configured to isolate and purify nucleic acid obtained from a plurality of samples introduced into the system, and to amplify and detect targeted nucleic acid contained in any of the samples using differently configured assay reagents. In some embodiments, system 1000 may be a random-access system that allows in vitro diagnostic (IVD) assays and laboratory developed tests (LDTs) to be performed in an interleaved manner. System 1000 may be configured to perform any type of molecular assay. In some embodiments, system 1000 may be configured to perform a plurality of different (e.g., differently configured) molecular assays on a plurality of samples. For example, a plurality of samples may be loaded in system 1000, processed to specifically or non-specifically isolate and purify targeted nucleic acids, subject a first subset of the samples to a first set of conditions for performing a first nucleic acid amplification, and, simultaneously, subject a second subset of the samples to a second set of conditions for performing a second nucleic acid amplification, where the reagents for performing the first and second nucleic acid amplifications are differently configured. In some such embodiments, system 1000 may prompt the user for information using, for example, a graphical user interface (GUI) displayed on a display device 50 (e.g., a computer monitor or a video monitor) of system 1000 (see Fig. 6A) or another display associated with system 1000 (e.g., a remote computer), defining one or more parameters of an assay protocol that can be saved and used later.

[00157] In some embodiments, system 1000 may have a modular structure and may be comprised of multiple modules operatively coupled together. However, it should be noted that the modular structure of system 1000 is only exemplary, and in some embodiments, system 1000 may be an integrated system having multiple regions or zones, with each region or zone, for example, performing specific steps of an assay which may be unique to that region. System 1000 includes a first module 100 and a second module 400 operatively coupled together. First module 100 and second module 400 may each be configured to perform one or more steps of an assay. In some embodiments, first and second modules 100, 400 may be separate modules selectively coupled together. That is, first module 100 can be selectively and operatively coupled to second module 400, and first module 100 can be selectively decoupled from second module 400 and coupled to a different second module 400. First and second modules 100, 400 may be coupled together by any method. For example, fasteners (e.g., bolts or screws), clamps, belts, straps, or any combination of fastening / attachment devices may be used to couple these modules together. As explained above, the modular structure of system 1000 is only exemplary, and in some embodiments, system 1000 may be an integral, self-contained structure (with, for example, the first module 100 forming a first region and the second module forming a second region within the integrated structure). It should be noted that in this disclosure, the term “module” is used to refer to a region (zone, location, etc.) of the analytical system. In some embodiments, each such region may be configured to perform specific steps of an assay which may be unique to that region of the system.

[00158] In some embodiments, power, data, and / or utility lines or conduits (air, water, vacuum, etc.) may extend between first and second modules 100, 400. In some embodiments, first module 100 may be a system that was previously purchased by a customer, and second module 400 may be a later acquired module that expands the analytical capabilities of the combined system. For example, in one embodiment the first module 100 may be a Panther® system (Hologic Inc.; Marlborough, MA) configured to perform sample processing and isothermal, transcription-based amplification assays (e.g., TMA or NASBA) on samples provided to the system, and module 400 may be a bolt-on that is configured to extend the functionality of the Panther® system by, for example, adding thermal cycling capabilities to enable, for example, real-time PCR reactions. An exemplary system 1000 with exemplary first and second modules 100, 400 is the Panther Fusion® system (Hologic Inc., Marlborough, MA), which is described in U.S. Patent Nos. 9,732,374, 9,465,161, and 9,604,185, and U.S. Patent Publication No. 2016 / 0032358. Exemplary systems, functions, devices or components, and capabilities of first and second modules 100, 400 are described in the above-referenced publications (and in the publications identified below), and are therefore not described in detail herein for the sake of brevity.

[00159] In some embodiments, first module 100 may include multiple vertically stacked decks. As illustrated, first module 100 may be configured to perform one or more steps of a multi-step molecular assay designed to detect at least one analyte (e.g., target nucleic acid). First module 100 may include receptacle-receiving components configured to receive and hold the reaction receptacles and, in some instances, to perform process steps on the contents of the receptacles. Exemplary process steps may include: dispensing sample and / or reagents into reaction receptacles, including, for example, target capture reagents, buffers, oils, primers and / or other amplification oligomers, probes, polymerases, etc.; aspirating material from the reaction receptacles, including, for example, non-immobilized components of a sample or wash solutions; mixing the contents of the reaction receptacles; maintaining and / or altering the temperature of the contents of reaction receptacles; heating or chilling the contents of the reaction receptacles or reagent containers; altering the concentration of one or more components of the contents of the reaction receptacles; separating or isolating constituent components of the contents of the reaction receptacles; detecting a signal, such as electromagnetic radiation (e.g., visible light) from the contents of the reaction receptacles; and / or deactivating nucleic acid or halting on-going reactions.

[00160] In some embodiments, first module 100 may include a receptacle drawer or compartment 102 adapted to receive and support a plurality of empty reaction receptacles. Compartment 102 may include a cover or door for accessing and loading the compartment with the reaction receptacles. Compartment 102 may further include a receptacle feeding device for moving the reaction receptacles into a receptacle pick-up position (e.g., a registered or known position) to facilitate removal of the reaction receptacles by a receptacle distributor. First module 100 may further include one or more compartments configured to store containers that hold bulk reagents (i.e., reagent volumes sufficient to perform multiple assays) or are configured to receive and hold waste material. The bulk reagents may include fluids such as, for example, water, buffer solutions, target capture reagents, and nucleic acid amplification and detection reagents. In some embodiments, the bulk reagent container compartments may be configured to maintain the containers at a desired temperature (e.g., at a prescribed storage temperature), and include holding structures that hold and / or agitate the containers to maintain their contents in solution or suspension. An exemplary holding structure for supporting and agitating fluid containers is described in U.S. Patent No. 9,604,185.

[00161] First module 100 may further include a sample bay supporting one or more sample holding racks with sample-containing receptacles. First module 100 may also include one or more fluid transfer devices for transferring fluids, for example, sample fluids, reagents, bulk fluids, waste fluids, etc., to and from reaction receptacles and / or other containers. In some embodiments, the fluid transfer devices may comprise one or more robotic pipettors configured for controlled, automated movement and access to the reaction receptacles, bulk containers holding reagents, and containers holding samples. In some embodiments, the fluid transfer devices may also include fluid dispensers, for example, nozzles, disposed within other devices and connected by suitable fluid conduits to containers, for example, bulk containers holding reagents, and to pumps or other devices for causing fluid movement from the containers to the dispensers. First module 100 may further include a plurality of load stations (e.g., heated load stations) configured to receive sample receptacles and other forms of holders for supporting sample receptacles and reagent containers. An exemplary load station and receptacle holder is described in U.S. Patent No. 8,309,036.

[00162] In some embodiments, first module 100 may include one or more magnetic parking stations and heated incubators 112, 114, 116 configured to heat (and / or maintain) the contents of reaction receptacles at a temperature higher than ambient temperature, and one or more chilling modules configured to cool (and / or maintain) the contents of reaction receptacles at a temperature lower than ambient temperature. Chilling modules may be used to aid in oligo hybridization and / or to cool a receptacle before performing luminescence measurements. In some embodiments, incubator 112 (which may be referred to as a transition incubator) may be set at a temperature of about 43.7°C and may be used for process 37 steps such as, for example, lysis, target capture, and hybridization. Incubator 114 may be a high temperature incubator which, in some embodiments, may be set at a temperature of about 64°C and used for process steps such as, for example, lysis, target capture, and hybridization. Incubator 116 (referred to as an amplification incubator) may be set at a temperature of about 42°C, and may be an incubator used for amplification during an assay. Incubator 116 may include real time fluorometers for the detection of fluorescence during amplification. Exemplary temperature ramping stations are described in U.S. Patent No. 8,192,992, and exemplary incubators are described in U.S. Patent Nos. 7,964,413 and 8,718,948. First module 100 may include sample-processing devices, such as magnetic wash stations adapted to separate or isolate a target nucleic acid or other analyte (e.g., immobilized on a magnetically-responsive solid support) from the remaining contents of the receptacle.

[00163] In some assays, samples are treated to release materials capable of interfering with the detection of an analyte (e.g., a targeted nucleic acid) in a magnetic wash station. To remove these interfering materials, samples may be treated with a target capture reagent that includes a magnetically-responsive solid support for immobilizing the analyte. Suitable solid supports may include paramagnetic particles (0.7-1.05 micron particles, Sera-Mag™ MG-CM (available from Seradyn, Inc., Indianapolis, Indiana). When the solid supports are brought into close proximity to a magnetic force, the solid supports are drawn out of suspension and aggregate adjacent a surface of a sample holding container, thereby isolating any immobilized analyte within the container. Non-immobilized components of the sample may then be aspirated or otherwise separated from immobilized analyte. Exemplary magnetic wash stations are described in U.S. Patent Nos. 6,605,213 and 9,011,771.

[00164] First module 100 may include a detector configured to receive a reaction receptacle and detect a signal (e.g., an optical signal) emitted by the contents of the reaction receptacle. In one implementation, the detector may comprise a luminometer for detecting luminescent signals emitted by the contents of a reaction receptacle and / or a fluorometer for detecting fluorescent emissions from the contents of the reaction receptacle. First module 100 may also include one or more signal detecting devices, such as, for example, fluorometers (e.g., coupled to one or more of incubators 112, 114, 116) configured to detect (e.g., at periodic intervals) signals emitted by the contents of receptacles contained In the incubators while a process, such as nucleic acid amplification, is occurring within the reaction receptacles. Exemplary luminometers and fluorometers are described in U.S. Patent Nos. 7,396,509 and 8,008,066.

[00165] First module 100 may further include a receptacle transfer device, which includes a receptacle distributor configured to move receptacles between various devices of first module 100 (e.g., incubators 112, 114, 116, load stations, magnetic parking stations, wash stations, and chilling modules). These devices may include a receptacle transfer portal (e.g., a port covered by an openable door) through which receptacles may be inserted into or removed from the devices. The receptacle distributor may include a receptacle distribution head configured to move in an X direction along a transport track assembly, rotate in a theta (0) direction, and move in an R direction, to move receptacles into and out of the devices of first module 100. An exemplary receptacle distributor, exemplary receptacle transfer portal doors, and mechanisms for opening the doors are described in U.S. Patent No. 8,731,712.

[00166] In an exemplary embodiment, second module 400 is configured to perform nucleic acid amplification reactions (such as, for example, PCR), and to measure fluorescence in real-time. System 1000 may include a controller that directs system 1000 to perform the different steps of a desired assay. The controller may accommodate LIS (“laboratory information system”) connectivity and remote user access. In some embodiments, second module 400 houses component modules that enable additional functionalities, such as melt analyses. An example of a melt station that could be adapted for use in the second module is described In U.S. Patent No. 9,588,069. Other devices may include a computer or controller, a computer hard drive or other memory device, a printer, and an optional uninterruptible power supply.

[00167] With reference to Fig. 6B, in some embodiments, second module 400 includes multiple vertically stacked levels (or decks) including devices configured for different functions. These levels include an amplification processing deck 430 and a receptacle processing deck 600. In the illustrated embodiment, receptacle processing deck 600 is positioned below amplification processing deck 430. However, this is not a requirement, and the vertical order of the decks (and their devices) may vary according to the intended use of analytical system 1000. Second module 400 may include devices positioned at different levels. These devices include, among others, a fluid transfer device in the form of one or more robotic pipettor(s) 410 (see Fig. 6B), a thermal cycler 432 with a signal detector, tip compartments 580 configured to store trays of disposable tips for pipettor(s) 410, cap / vial compartments 440 configured to store trays 460 of disposable processing vials and associated caps, a bulk reagent container compartment 500, a bulk reagent container transport, a receptacle distribution system including a receptacle handoff device and a receptacle distribution system including a receptacle distributor (which, in the exemplary embodiment shown, comprises a rotary distributor), receptacle storage units configured to store receptacles and / or multi-receptacle units (MRUs) (that, for example, includes multiple receptacles joined together as a single piece, integral unit), magnetic slots, a waste bin coupled to one or more trash chutes, a centrifuge 588, a reagent pack changer, reagent pack loading stations, and one or more compartments 450 (see Fig. 6B) configured to store accessories, such as, for example, consumables and / or storage trays for post-cap / vial assemblies. Robotic pipettor 410 attaches a disposable fluid transfer tip from a disposable tip tray 582 to a mounting end of its aspirator probe.

[00168] Exemplary embodiments of trays 460 for disposable processing vials and caps are disclosed in U.S. Patent Publication No. US 2017 / 0297027 Al. Several devices and features of system 1000 are described in U.S. Patent No. 9,732,374 and other references that are identified herein. Therefore, for the sake of brevity, these devices and features are not described in detail herein.

[00169] In the illustrated embodiment, robotic pipettor 410 is disposed near the top of second module 400. Below robotic pipettor 410, amplification processing deck 430 includes bulk reagent container compartment 500, centrifuge 588, the top of thermal cycler 432, tip compartments 580, and cap / vial compartments 440. Below amplification processing deck 430, receptacle processing deck 600 includes receptacle handoff device, receptacle distributor, receptacle storage units, magnetic slots, reagent pack changer, and reagent pack loading stations. Magnetic slots and reagent pack loading stations on receptacle processing deck 600 are accessible by robotic pipettor 410 through a gap between the devices of amplification processing deck 430. With reference to Fig. 6B, second module 400 may include a compartment 590 for storing accessories or to accommodate expansion of second module 400 (for example, to add additional reagent compartments for storage of reagents, add analytical capabilities to system 1000, etc.). Trash bin 650 collects and holds used materials, such as used disposable fluid transfer tips. The front surface of second module 400 preferably includes at least one drawer, where each drawer can include a drawer front 720.

[00170] The receptacles in the receptacle storage units may include individual receptacles (e.g., a container configured to store a fluid) having an open end and an opposite closed end, or multiple receptacles (e.g., five) coupled together as a unit (MRU). These MRUs may include a manipulating structure that is configured to be engaged by an engagement member (e.g., a hook) of a robotically controlled receptacle distribution system for moving the receptacle between different devices of system 1000. Exemplary receptacles 40 are described in U.S. Patent Nos. 6,086,827 and 9,732,374. In some embodiments, the receptacle distribution system, including receptacle handoff device and receptacle distributor, is configured to receive a receptacle or an MRU from the receptacle distributor of first module 100 and transfer the receptacle to second module 400, and then move the receptacle into different positions in second module 400. Computer Program Products

[00171] Included within the scope of the disclosure are software-based products (e.g., tangible embodiments of software for instructing a computer to execute various procedural steps) that can be used for performing the data processing method. These include software instructions stored on a computer or computer-readable media, such as magnetic media, optical media, “flash” memory devices, and computer networks or cloud storage.

[00172] The disclosure further embraces a system or an apparatus that amplifies nucleic acids, detects nucleic acid amplification products, and processes results to indicate a quantitative result for target in a test sample. Although the various components of the apparatus preferably function in a cooperative fashion, there is no requirement for the components to be part of an integrated assembly (e.g., on a single chassis). However, in a preferred embodiment, components of the apparatus are connected together. Included within the meaning of “connected” are connections via wired and wireless connections.

[00173] Particularly falling within the scope of the disclosure is an apparatus or system that includes a computer linked to a device that amplifies nucleic acids and monitors amplicon synthesis as a function of cycle number or time, where the computer is programmed to execute the algorithmic steps disclosed herein. An exemplary system in accordance with the disclosure will include a temperature-controlled incubator, and a fluorometer capable of monitoring and distinguishing at least two wavelengths of fluorescent emissions. These emissions may be used to indicate target amplicon synthesis, and internal control or internal calibrator amplicon synthesis.

[00174] In connection with computer-implemented or software-implemented embodiments of the disclosure, a result can be recorded or stored in a “non-transient” format where it can be accessed for reference at a later time than when the data analysis to be recorded was carried out or performed. For example, a computed result can be recorded in a non-transient format by printing on paper, or by storing on a computer-readable memory device (e.g., a hard drive, flash memory device, file in cloud storage, etc.).

[00175] Software instructions in accordance with the disclosure can direct a computer to carry out different steps. Algorithmic steps carried out by software instructions include: (1) causing the nucleic acid analyzer to carry out a multiplex amplification reaction such that a real-time fluorescent Receiver channel run curve (representing amplification of a first target) and a real-time fluorescent Emitter channel run curve (representing amplification of a second target) are obtained; (2) causing a computer or processor to quantify a magnitude of the Emitter channel run curve, and quantify a correlation between the Emitter channel run curve and the Receiver channel run curve, whereby it is determined that (i) the magnitude of the Emitter channel run curve indicates amplification of the second target nucleic acid took place in the multiplex amplification reaction, and (ii) the Emitter channel run curve and the Receiver channel run curve are highly correlated; (3) causing a computer or processor to adjust fluorescent signal values of the Receiver channel run curve by subtracting therefrom a quantity calculated by multiplying an adaptive crosstalk compensation factor by fluorescent signal values of the Emitter channel run curve, to prepare an adjusted Receiver channel run curve, the adaptive crosstalk compensation factor being specific for the Emitter channel run curve and the Receiver channel run curve; and (4) causing a computer or processor to quantify the first target nucleic acid that amplified in the amplification reaction using the adjusted Receiver channel run curve. Example Procedures

[00176] The following Examples employ a Flu B nucleic acid to represent the Analyte-1 nucleic acid target, and a SARS nucleic acid to represent the Analyte-2 nucleic acid target. The Emitter channel was represented by the ROX-channel of a fluorometer that detected fluorescence emission from a CAL FLUOR RED 610 dye-labeled probe, and the Receiver channel was represented by the RED 647-channel that detected fluorescence emission from a QUASAR 670 dye-labeled probe.

[00177] Example 1 illustrates a procedure that established evidence for crosstalk of an Emitter channel signal into a Receiver channel. More specifically, it was demonstrated that the Emitter channel signal exceeded a cutoff, thereby indicating target nucleic acid amplification and potential crosstalk into the Receiver channel. Generally, the cutoff was set equal to the positivity threshold of the Emitter channel signal. If the Emitter channel signal is sufficient to indicate analyte amplification, then it is determined to be high enough for crosstalk impact. Still further, it was demonstrated that the Emitter and Receiver channel run curves were closely correlated. Example 1 Obtaining Evidence for Crosstalk Between Channels of a Fluorometer

[00178] A real-time multiplex PCR assay was performed using a PANTHER FUSION® automated nucleic acid analyzer (Gen-Probe Incorporated; San Diego, CA), with results being collected in different channels of a fluorometer to indicate synthesis of amplification products (i.e., “amplicons”). Each multiplex amplification reaction was capable of amplifying SARS and Flu B nucleic acid targets, with each amplicon being detected by a different dual-labeled fluorescent hydrolysis probe in the amplification reaction. A SARS-specific amplicon was detected using a CAL FLUOR RED 610 dye-labeled probe with monitoring in the ROX-channel of a fluorometer of the automated instrument, and a Flu B-specific amplicon was detected using a QUASAR 670-labeled probe with monitoring in a RED 647-channel of the fluorometer onboard the automated nucleic acid analyzer. In this illustrative procedure, the amplification reaction included the SARS template, but did not include any Flu B template. Only the SARS amplicon, and not the Flu B amplicon was synthesized in the multiplex amplification reaction. Fluorescence readings were recorded during each round of temperature cycling for the multiplex PCR reaction. Results of the procedure are presented among Tables 1-3.

[00179] Table 1 presents results of cycle-dependent fluorescence monitoring for each of the RED 647-channel (column 2) and the ROX-channel (column 3). The calculated results appearing in column 4 have been adjusted for crosstalk compensation by a default method that included subtracting a preset 2.3% of the value of the signal measured in the ROX Emitter channel from the signal measured in the RED 647 Receiver channel. This approach had been successfully applied across different instruments running the same multiplex assay. This crosstalk compensation approach occasionally yielded false-positive detection of the Flu B nucleic acid in reactions that amplified the SARS target nucleic acid. The results appearing in columns 2 and 3 of Table 1 are graphically presented in Fig. 3. The results appearing in column 4 of Table 1 are included in the graphical presentation of Fig. 5. Table 1 Crosstalk Correction Using a Static Compensation Factor Cycle # RED 647-Channel Fluorescence Before Crosstalk Compensation ROX-Channel Fluorescence Before Crosstalk Compensation RED 647-Channel Fluorescence with Default Crosstalk Compensation from ROX (2.3%) 10 0.70 1.83 0.66 11 -0.50 0.00 -0.50 12 0.00 -2.53 0.06 13 0.23 2.94 0.17 14 2.63 38.94 1.74 15 4.33 113.34 1.73 16 9.73 247.64 4.04 17 20.63 505.44 9.01 18 38.80 982.44 16.20 19 70.20 1858.34 27.46 20 127.10 3291.24 51.40 21 215.57 5440.04 90.45 22 333.87 8266.14 143.75 23 472.17 11583.47 205.75 24 616.17 15018.97 270.73 25 748.17 18264.57 328.08 26 853.90 20979.47 371.37 27 928.90 23085.57 397.93 28 981.90 24574.14 416.69 29 1015.40 25626.84 425.98 30 1040.40 26322.54 434.98 31 1061.40 26828.74 444.34 32 1076.20 27153.14 451.68 33 1088.50 27414.74 457.96 34 1099.40 27595.84 464.70 35 1107.80 27771.44 469.06 36 1113.50 27895.64 471.90 37 1119.20 28037.24 474.34 38 1121.83 28131.54 474.81 39 1125.23 28236.74 475.79 40 1125.03 28275.04 474.71 41 1125.23 28318.94 473.90 42 1125.73 28317.64 474.43 43 1129.00 28324.94 477.53 44 1131.20 28306.04 480.16 45 1133.40 28305.84 482.37

[00180] Fig. 3 graphically illustrates a very strong ROX-channel signal (filled circles) in a run curve having a sigmoid shape characteristic of a PCR amplification profile for the SARS nucleic acid target, as expected. Also illustrated in the figure is a substantially weaker fluorescence run curve representing the RED 647-channel signal (open circles) before any crosstalk compensation. This latter curve seemingly indicated amplification of the Flu B target nucleic acid, even though none was included in the amplification reaction mixture. Both run curves illustrated in the figure appear to rise above a background fluorescence level, and both run curves exhibit sigmoid shapes, although the RED 647-channel run curve (Flu B target nucleic acid) is somewhat less pronounced as a result of the lower signal strength. After obtaining cycle-dependent fluorescence readings for each of the two fluorometer channels, there remained the question whether a default static crosstalk compensation (shown in col. 4 of Table 1) would be adequate, or whether the adaptive crosstalk compensation technique would be appropriate to make the necessary correction. The graph in Fig. 5 representing results from the default static crosstalk compensation approach (filled circles) reasonably would lead to the conclusion that the Flu B target nucleic acid had been amplified in the reaction mixture, even though that target nucleic acid truly was absent. This falsepositive result was the consequence of improper crosstalk compensation of the Receiver channel signal.

[00181] To investigate evidence for crosstalk into the Receiver channel, quantitative results obtained in the foregoing procedures were used to determine: (1) a Delta RFU value for the Emitter channel signal, and (2) a correlation factor for the Emitter channel and Receiver channel run curves. The Delta RFU for the Emitter channel signal was determined as the difference between the maximum and minimum RFU readings of the ROX-channel signal (filled circles) shown in Fig. 3. This indicated a substantial opportunity for crosstalk of signal detected in the Emitter channel into the Receiver channel. This value was about 28,300 RFU, a quantity that exceeded the cutoff value of 500 RFU (i.e., the cutoff for positivity of the SARS analyte) confirmed that the target nucleic acid amplified and was detected in the ROX Emitter channel of the fluorometer. Although not from the same amplification trial, results very similar to those presented in the lower panel of Fig. 2C were obtained by plotting results of the run curves on different axes. The calculated Pearson correlation factor for the resulting plot was greater than 0.95, thereby indicating a very high correlation between the two run curves. The fact that Delta RFU for the Emitter channel signal exceeded a cutoff value, and the fact that the correlation factor was sufficiently high as to indicate the Emitter and Receiver channel run curves were highly correlated indicated that 45 calculation and use of an adaptive crosstalk compensation factor for adjusting the Receiver channel signal was appropriate. Stated differently, these results were consistent with a falsepositive Flu B call being attributed to improper use of the preset default crosstalk compensation factor.

[00182] The preceding Example demonstrated how real-time run curve data was processed to obtain evidence that an Emitter channel signal had the potential to be detected in a Receiver channel (e.g., Delta RFU exceeded a fluorescent threshold cutoff indicating target amplification) to an extent that could cause false-positive detection of the target nucleic acid indicated by the Receiver channel signal. Further evidence supporting crosstalk of a fluorescent signal into the Receiver channel was provided by the high correlation between the Emitter and Receiver channel run curves. In view of these findings, it was necessary to establish and use an adaptive crosstalk compensation factor to adjust the Receiver channel signal to avoid false-positive results. More specifically, the adaptive crosstalk compensation factor was determined and used to adjust the Receiver channel signal to avoid false-positive determination that the reaction mixture included nucleic acid detected by the Receiver channel signal.

[00183] Example 2 demonstrates how use of a preset static compensation factor led to false-positive detection of an Analyte-1 (Flu B) target nucleic acid in a multiplex amplification reaction that amplified the Analyte-2 (SARS) target nucleic acid. The Example further demonstrates how use of the adaptive crosstalk compensation technique yielded a correct result after processing the same starting data. Example 2 Adaptive Crosstalk Compensation Corrects False-Positive Results

[00184] Real-time fluorescence data from Example 1 was further processed to assess the benefit of Receiver channel data adjustment using an adaptive crosstalk compensation factor in comparison to adjustment using a preset static crosstalk compensation factor.

[00185] Table 2 presents cycle-dependent fluorescence monitoring results used for determining the adaptive crosstalk compensation factor specific for a nucleic acid amplification reaction performed on a particular PANTHER FUSION® automated nucleic acid analyzer. Numerical results appearing in columns 2 and 3 of Table 2 are identical to results before crosstalk compensation appearing in Table 1 because the same data was used in the analysis. Column 4 of Table 2 presents ROX-channel shifted fluorescence values resulting from synchronizing the ROX-channel and RED 647-channel run curves. Column 5 presents calculated ratios determined as the RED 647-channel signal (column 2) divided by the ROX-channel shifted fluorescence signal (column 4). Column 6 indicates whether the calculated ratio of column 5 was used for calculating an “average ratio.” In an alternative embodiment, only a single ratio value is used to establish a compensation factor. Preferably however, the average ratio is established using a plurality of ratio values. In yet another alternative embodiment, linear regression is used to establish a compensation factor.

[00186] Various approaches can be used for determining whether a calculated ratio should be selected for inclusion in the averaging calculation. Included values preferably correspond to points along the reaction progress parameter where robust amplification has already begun. In one embodiment, selected points may occur after the endpoint of the baseline phase. In another embodiment, selected points include and / or follow the maximum of the first derivative of either the ROX Emitter run curve or the RED 647 Receiver run curve. In yet another embodiment, selected points may correspond to the highest of a specified percentage range of the Emitter channel signal or the Receiver channel signal. For example, selected points may represent signal values greater than any of 3%, 5%, 10%, 15%, 20%, 25%, 30% or more of the maximum signal measured in the Emitter channel. For illustrative purposes, the last column of Table 2 identifies calculated ratio values that were included in the averaging calculation, where the ratio values were based on ROX Emitter channel signals that were greater than about 20% of the maximum value of the ROX fluorescence value before crosstalk compensation (column 3 of Table 2). In this instance, the optional curve synchronization step was first performed because fluorometer readings were recorded at shifted time points in a serial fashion (e.g., one after the other). The average of these values, which corresponded to cycle numbers 22-45, was 3.975%. Fig. 4 provides a graphical illustration of data points that were used to calculate the average ratio of Receiver channel signals to Emitter channel signals.

[00187] This calculated average ratio value served as the adaptive crosstalk compensation factor for adjusting Receiver channel signals to compensate for the effects of crosstalk from the ROX-labeled probe. More specifically, this compensation factor (3.975%) was multiplied by the ROX Emitter channel signal value to determine the quantity that should be subtracted from RED 647 Receiver channel signal value to compensate for crosstalk from the CAL FLUOR RED 610 dye-labeled probe detected in the ROX-channel. Table 2 Determining an Adaptive Crosstalk Compensation Factor Cycle # RED 647-Channel Fluorescence Before Crosstalk Compensation ROX-Channel Fluorescence Before Crosstalk Compensation ROX-Channel “Shifted” Fluorescence Calculated Ratio Ratio Included in Average? 10 0.70 1.83 -29.10 -0.02406 FALSE 11 -0.50 0.00 -15.84 0.03157 FALSE 12 0.00 -2.53 1.49 0.00000 FALSE 13 0.23 2.94 25.18 0.00927 FALSE 14 2.63 38.94 64.85 0.04061 FALSE 15 4.33 113.34 140.29 0.03089 FALSE 16 9.73 247.64 282.14 0.03450 FALSE 17 20.63 505.44 554.06 0.03724 FALSE 18 38.80 982.44 1070.13 0.03626 FALSE 19 70.20 1858.34 1998.82 0.03512 FALSE 20 127.10 3291.24 3514.50 0.03616 FALSE 21 215.57 5440.04 5735.12 0.03759 FALSE 22 333.87 8266.14 8618.32 0.03874 TRUE 23 472.17 11583.47 11945.62 0.03953 TRUE 24 616.17 15018.97 15374.35 0.04008 TRUE 25 748.17 18264.57 18559.84 0.04031 TRUE 26 853.90 20979.47 21225.19 0.04023 TRUE 27 928.90 23085.57 23267.07 0.03992 TRUE 28 981.90 24574.14 24723.35 0.03972 TRUE 29 1015.40 25626.84 25729.31 0.03946 TRUE 30 1040.40 26322.54 26411.68 0.03939 TRUE 31 1061.40 26828.74 26884.42 0.03948 TRUE 32 1076.20 27153.14 27210.42 0.03955 TRUE 33 1088.50 27414.74 27453.25 0.03965 TRUE 34 1099.40 27595.84 27640.98 0.03977 TRUE 35 1107.80 27771.44 27801.32 0.03985 TRUE 36 1113.50 27895.64 27937.36 0.03986 TRUE 37 1119.20 28037.24 28063.92 0.03988 TRUE 38 1121.83 28131.54 28169.55 0.03982 TRUE 39 1125.23 28236.74 28255.86 0.03982 TRUE 40 1125.03 28275.04 28306.44 0.03974 TRUE 41 1125.23 28318.94 28336.36 0.03971 TRUE 42 1125.73 28317.64 28343.29 0.03972 TRUE 43 1129.00 28324.94 28340.69 0.03984 TRUE 44 1131.20 28306.04 28330.07 0.03993 TRUE 45 1133.40 28305.84 28326.24 0.04001 TRUE Average Ratio = 0.03975

[00188] Table 3 presents numerical results from implementing the adaptive crosstalk compensation technique. The above-described adaptive crosstalk compensation factor (0.03975) was multiplied by the value of the ROX-channel signal (column 3), and the resulting quantity was subtracted from the RED 647-channel signal (column 2) to give the RED 647-channel signal adjusted for crosstalk from the CAL FLUOR RED 610 dye-labeled probe (column 4). Fig. 5 includes a graphical representation of the results from column 4 of Table 3, together with results from column 4 of Table 1 for comparison. Table 3 Crosstalk Correction Using an Adaptive Crosstalk Compensation Factor Cycle # RED 647-Channel Fluorescence Before Crosstalk Compensation ROX-Channel Fluorescence Before Crosstalk Compensation RED 647-Channel Fluorescence with Adaptive Crosstalk Compensation from ROX-Channel signal (3.975%) 10 0.70 1.83 0.63 11 -0.50 0.00 -0.50 12 0.00 -2.53 0.10 13 0.23 2.94 0.12 14 2.63 38.94 1.09 15 4.33 113.34 -0.17 16 9.73 247.64 -0.11 17 20.63 505.44 0.54 18 38.80 982.44 -0.25 19 70.20 1858.34 -3.67 20 127.10 3291.24 -3.73 21 215.57 5440.04 -0.67 22 333.87 8266.14 5.29 23 472.17 11583.47 11.72 24 616.17 15018.97 19.16 25 748.17 18264.57 22.15 26 853.90 20979.47 19.97 27 928.90 23085.57 11.25 28 981.90 24574.14 5.08 29 1015.40 25626.84 -3.27 30 1040.40 26322.54 -5.92 31 1061.40 26828.74 -5.04 32 1076.20 27153.14 -3.14 33 1088.50 27414.74 -1.24 34 1099.40 27595.84 2.47 35 1107.80 27771.44 3.89 36 1113.50 27895.64 4.65 37 1119.20 28037.24 4.72 38 1121.83 28131.54 3.60 39 1125.23 28236.74 2.82 40 1125.03 28275.04 1.10 41 1125.23 28318.94 -0.44 42 1125.73 28317.64 0.11 43 1129.00 28324.94 3.08 44 1131.20 28306.04 6.03 45 1133.40 28305.84 8.24

[00189] Fig. 5 graphically illustrates how the adaptive crosstalk compensation technique represented an improvement over the prior approach that employed a static compensation factor, where the improvement eliminated false-positive results. Data is plotted for both of the preset static crosstalk compensation (column 4 of Table 1; filled circles) and for the adaptive crosstalk compensation (column 4 of Table 3; open circles) techniques. All data plotted in the figure are from the same experiment, meaning that the same real-time fluorescence results were processed two ways. Both run curves drawn in the figure indicate fluorescence values measured in the RED 647 Receiver channel, where the measured values reflect production of Flu B amplicons. A horizontal threshold drawn at 175 RFU marks the minimum RFU needed to indicate detection of the Flu B target nucleic acid. This positivity threshold for the Flu B target nucleic acid was established using linearity data and receiver operating characteristic (ROC) curve analysis. The upper curve that crosses the threshold represents fluorescence measurements that were adjusted using the preset default compensation factor (2.3%). By this approach, the run curve exceeded the horizontal threshold and so indicated false-positive detection of the Flu B target nucleic acid. Conversely, application of the adaptive crosstalk compensation factor (3.975%) gave the correct result. This was indicated because the RED 647-channel signal adjusted by this latter technique never exceeded the horizontal threshold line. The fluorometer of the particular instrument used in this demonstration made the instrument system susceptible to falsepositive detection of the Flu B target nucleic acid when the SARS target nucleic acid amplified in the multiplex amplification reaction. Implementation of the adaptive crosstalk compensation technique yielded the correct result (i.e., no false-positivity) and avoided replacement of fluorometers in the system used for amplifying and processing results of the multiplex reaction.

[00190] All of the compositions, kits, and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the disclosure describes preferred embodiments, it will be apparent to those of skill in the art that variations may be applied without departing from the spirit and scope of the disclosure. All such variations and equivalents apparent to those skilled in the art, whether now existing or later developed, are deemed to be within the spirit and scope of the disclosure.

Claims

1. A system that quantifies a target nucleic acid, comprising:a real-time nucleic acid analyzer,wherein the real-time nucleic acid analyzer amplifies target nucleic acids in a multiplex amplification reaction and monitors synthesis of amplification products with a fluorometer,wherein the fluorometer is configured to detect multiple wavelengths of fluorescent dye emissions in different channels, andwherein the real-time nucleic acid analyzer produces each of a Receiver channel run curve that measures amplification of a first target nucleic acid, and an Emitter channel run curve that measures amplification of a second target nucleic acid; anda computer in communication with the real-time nucleic acid analyzer, the computer being programmed with software instructions which, when executed by the computer, cause the computer to(a) direct the real-time nucleic acid analyzer to perform the multiplex amplification reaction and monitor synthesis of amplification products by measuring fluorescent signals using the fluorometer;(b) quantify each of a magnitude of the Emitter channel run curve, and a correlation of the Emitter channel run curve and the Receiver channel run curve, whereby it is determined that(i) the magnitude of the Emitter channel run curve indicates amplification of the second target nucleic acid took place in the multiplex amplification reaction, and(ii) the Emitter channel run curve and the Receiver channel run curve are highly correlated;(c) adjust fluorescent signal values of the Receiver channel run curve by subtracting therefrom a quantity calculated by multiplying an adaptive crosstalk compensation factor by fluorescent signal values of the Emitter channel run curve, thereby preparing an adjusted Receiver channel run curve,wherein the adaptive crosstalk compensation factor is specific for the Emitter channel run curve and the Receiver channel run curve;(d) quantify the first target nucleic acid using the adjusted Receiver channel run curve; and(e) record the result of (d) in a non-transient form.

2. The system of claim 1, wherein the real-time nucleic acid analyzer comprises a temperature-controlled incubator that undergoes thermal cycling, and wherein the multiplex nucleic acid amplification reaction comprises a multiplex real-time PCR amplification reaction.

3. The system of claim 2, wherein the temperature-controlled incubator comprises a temperature-controlled block or a temperature-controlled plate.

4. The system of any one of claims 1 to 3, wherein the multiplex nucleic acid amplification reaction comprises a plurality of reaction cycles.

5. The system of any one of claims 1 to 4, wherein the fluorometer monitors fluorescent signals indicating amplification of nucleic acids as a function of reaction cycle number.

6. The system of any one of claims 1 to 5, wherein the real-time nucleic acid analyzer is an automated real-time nucleic acid analyzer that isolates nucleic acids used as templates in the multiplex nucleic acid amplification reaction.

7. The system of any one of claims 1 to 6, wherein the fluorometer detectsfluorescent signals for the different channels in serial fashion, so that readings for the different channels are spaced apart in time.

8. The system of any one of claims 1 to 7, wherein the real-time nucleic acid analyzer comprises one or more fluid transfer devices that transfer fluids to and from containers contained within the real-time nucleic acid analyzer.

9. The system of any one of claims 1 to 8, wherein the computer is an integral component of the automated real-time nucleic acid analyzer.

10. The system of any one of claims 1 to 9, wherein the magnitude of the Emitter channel run curve in (b) is calculated as the difference between a maximum fluorescencevalue of the Emitter channel run curve and a minimum fluorescence value of the Emitter channel run curve.

11. The system of claim 10, wherein the minimum fluorescence value of theEmitter channel run curve is a minimum fluorescence value of the Emitter channel run curve after baseline adjustment that removes fluctuations in fluorescence measurements.

12. The system of any one of claims 1 to 11, wherein (b) comprises quantify the correlation of the Emitter channel run curve and the Receiver channel run curve by calculating a measure of linearity of the correlation of the Emitter channel run curve and the Receiver channel run curve.

13. The system of claim 12, wherein calculating the measure of linearity comprises calculating a Pearson correlation coefficient to confirm the Emitter channel run curve and the Receiver channel run curve are highly correlated.

14. The system of claim 13, wherein the Pearson correlation coefficient is calculated to be at least 0.95, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

15. The system of claim 13, wherein the Pearson correlation coefficient is calculated to be at least 0.98, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

16. The system of claim 13, wherein the Pearson correlation coefficient is calculated to be at least 0.99, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

17. The system of any one of claims 1 to 16, wherein the adaptive crosstalk compensation factor is calculated by dividing a Receiver channel signal value of the Receiver channel run curve by an Emitter channel signal value of the Emitter channel run curve.

18. The system of any one of claims 1 to 16, wherein the adaptive crosstalk compensation factor is calculated by dividing a signal value from the Receiver channel runcurve by a signal value from the Emitter channel run curve, and wherein the Emitter channel run curve is first synchronized with the Receiver channel run curve by optimizing correlation of the two run curves before calculating the adaptive crosstalk compensation factor.

19. The system of any one of claims 1 to 17, wherein (d) comprises comparing a magnitude of the adjusted Receiver channel run curve to a static threshold fluorescence value to determine whether the first target nucleic acid amplified in the multiplex amplification reaction.

20. The system of claim 19, wherein the software instructions, when executed by the computer, further cause the computer to determine a cycle number of the multiplex amplification reaction at which the adjusted Receiver channel run curve exceeds the static threshold fluorescence value, and then determine the quantity of the first target nucleic acid in the multiplex amplification reaction before the reaction began.

21. The system of any one of claims 1 to 20, wherein the result of (d) indicates the first target nucleic acid was amplified in the multiplex amplification reaction.

22. A computer programmed with software instructions to quantify a target nucleic acid, the software instructions, when executed by the computer, cause the computer to:(a) receive results from a real-time nucleic acid analyzer that performs a multiplex amplification reaction and monitors synthesis of amplification products by measuring fluorescent signals using a fluorometer,wherein the real-time nucleic acid analyzer produces a Receiver channel run curve that measures amplification of a first target nucleic acid, and an Emitter channel run curve that measures amplification of a second target nucleic acid;(b) quantify each of a magnitude of the Emitter channel run curve, and a correlation of the Emitter channel run curve with the Receiver channel run curve, whereby it is determined that(i) the magnitude of the Emitter channel run curve indicates amplification of the second target nucleic acid took place in the multiplex amplification reaction, and(ii) the Emitter channel run curve and the Receiver channel run curve are highly correlated;(c) adjust fluorescent signal values of the Receiver channel run curve by subtracting therefrom a quantity calculated by multiplying an adaptive crosstalk compensation factor by fluorescent signal values of the Emitter channel run curve, thereby preparing an adjusted Receiver channel run curve,wherein the adaptive crosstalk compensation factor is specific for the Emitter channel run curve and the Receiver channel run curve;(d) quantify the first target nucleic acid using the adjusted Receiver channel run curve; and(e) record the result of (d) in a non-transient form.

23. The computer of claim 22, wherein the software instructions, when executed by the computer, further cause the computer to direct the real-time nucleic acid analyzer to perform the multiplex amplification reaction and monitor synthesis of amplification products by measuring fluorescent signals using the fluorometer.

24. The computer of claim 23, wherein the software instructions, when executed by the computer, further cause the computer to direct the real-time nucleic acid analyzer to carry out a plurality of temperature cycling steps to perform the multiplex nucleic acid amplification reaction.

25. The computer of any one of claims 22 to 24, wherein the magnitude of the Emitter channel run curve in (b) is calculated as the difference between a maximum fluorescence value of the Emitter channel run curve and a minimum fluorescence value of the Emitter channel run curve.

26. The computer of claim 25, wherein the minimum fluorescence value of the Emitter channel run curve is a minimum fluorescence value of the Emitter channel run curve after baseline adjustment that removes fluctuations in fluorescence measurements.

27. The computer of any one of claims 22 to 26, wherein (b) comprises quantify the correlation of the Emitter channel run curve and the Receiver channel run curve bycalculating a measure of linearity of the correlation of the Emitter channel run curve and the Receiver channel run curve.

28. The computer of claim 27, wherein calculating the measure of linearity comprises calculating a Pearson correlation coefficient to confirm the Emitter channel run curve and the Receiver channel run curve are highly correlated.

29. The computer of claim 28, wherein the Pearson correlation coefficient is calculated to be at least 0.95, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

30. The computer of claim 28, wherein the Pearson correlation coefficient is calculated to be at least 0.98, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

31. The computer of claim 28, wherein the Pearson correlation coefficient iscalculated to be at least 0.99, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

32. The computer of any one of claims 22 to 31, wherein the adaptive crosstalk compensation factor is calculated by dividing a Receiver channel signal value of the Receiver channel run curve by an Emitter channel signal value of the Emitter channel run curve.

33. The computer of any one of claims 22 to 31, wherein the adaptive crosstalk compensation factor is calculated by dividing a signal value from the Receiver channel run curve by a signal value from the Emitter channel run curve, and wherein the Emitter channel run curve is first synchronized with the Receiver channel run curve by optimizing correlation of the two run curves before calculating the adaptive crosstalk compensation factor.

34. The computer of any one of claims 22 to 33, wherein (d) comprises comparing a magnitude of the adjusted Receiver channel run curve to a static threshold fluorescence value to determine whether the first target nucleic acid amplified in the multiplex amplification reaction.

35. The computer of claim 34, wherein the software instructions, when executed by the computer, further cause the computer to determine a cycle number of the multiplex amplification reaction at which the adjusted Receiver channel run curve exceeds the static threshold fluorescence value, and then determine the quantity of the first target nucleic acid in the multiplex amplification reaction before the reaction began.

36. The computer of claim 34, wherein the result of (d) qualitatively indicates the first target nucleic acid was amplified in the multiplex amplification reaction.

37. A method of quantifying a target nucleic acid after compensating for crosstalk of signals from an Emitter channel of a fluorometer into a Receiver channel of the fluorometer, the fluorometer being a component of a real-time nucleic acid analyzer, the method comprising:(a) performing or having performed a multiplex amplification reaction using the real-time nucleic acid analyzer to obtain an Emitter channel run curve and a Receiver channel run curve,wherein the Receiver channel run curve measures amplification of a first target nucleic acid, andwherein the Emitter channel run curve measures amplification of a second target nucleic acid;(b) quantifying or having quantified each of a magnitude of the Emitter channel run curve, and a correlation of the Emitter channel run curve with the Receiver channel run curve,whereby it is determined that(i) the magnitude of the Emitter channel run curve indicates amplification of the second target nucleic acid took place in the multiplex amplification reaction, and(ii) the Emitter channel run curve and the Receiver channel run curve are highly correlated;(c) adjusting or having adjusted fluorescent signal values of the Receiver channel run curve by subtracting therefrom a quantity calculated by multiplying an adaptive crosstalk compensation factor by fluorescent signal values of the Emitter channel run curve, thereby preparing an adjusted Receiver channel run curve,wherein the adaptive crosstalk compensation factor is specific for the Emitter channel run curve and the Receiver channel run curve; and(d) quantifying or having quantified the first target nucleic acid using the adjusted Receiver channel run curve.

38. The method of claim 37, wherein the real-time nucleic acid analyzer is an automated real-time nucleic acid analyzer controlled by a computer programmed with software, and wherein the automated real-time nucleic acid analyzer performs the multiplex amplification reaction and monitors synthesis of amplification products as the reaction is occurring.

39. The method of claim 38, wherein the computer is an integral component of the automated real-time nucleic acid analyzer.

40. The method of any one of claims 37 to claim 39 wherein the multiplex amplification reaction amplifies nucleic acids by a process comprising thermal cycling.

41. The method of any one of claims 37 to 40, wherein the multiplex amplification reaction comprises a multiplex PCR reaction.

42. The method of any one of claims 37 to 41, wherein at least one of the first target nucleic acid and the second target nucleic acid is either a bacterial nucleic acid or a viral nucleic acid.

43. The method of any one of claims 37 to 42, wherein the magnitude of the Emitter channel run curve in step (b) is calculated as the difference between a maximum fluorescence value of the Emitter channel run curve and a minimum fluorescence value of the Emitter channel run curve.

44. The method of claim 43, wherein the minimum fluorescence value of the Emitter channel run curve is a minimum fluorescence value of the Emitter channel run curve after baseline adjustment that removes fluctuations in fluorescence measurements.

45. The method of any one of claims 37 to 44, wherein step (b) comprises quantifying the correlation of the Emitter channel run curve and the Receiver channel run curve by calculating a measure of linearity of the correlation of the Emitter channel run curve and the Receiver channel run curve.

46. The method of claim 45, wherein calculating the measure of linearity comprises calculating a Pearson correlation coefficient to confirm the Emitter channel run curve and the Receiver channel run curve are highly correlated.

47. The method of claim 46, wherein the Pearson correlation coefficient is calculated to be at least 0.95, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

48. The method of claim 46, wherein the Pearson correlation coefficient is calculated to be at least 0.98, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

49. The method of claim 46, wherein the Pearson correlation coefficient is calculated to be at least 0.99, thereby confirming that the Emitter channel run curve and the Receiver channel run curve are highly correlated.

50. The method of claim 37, wherein the adaptive crosstalk compensation factor is calculated by dividing a Receiver channel signal value of the Receiver channel run curve by an Emitter channel signal value of the Emitter channel run curve.

51. The method of claim 37, wherein the adaptive crosstalk compensation factor is calculated by dividing a signal value from the Receiver channel run curve by a signal value from the Emitter channel run curve, and wherein the Emitter channel run curve is first synchronized with the Receiver channel run curve by optimizing correlation of the two run curves before calculating the adaptive crosstalk compensation factor.

52. The method of any one of claims 37 to 51, wherein step (d) comprises comparing a magnitude of the adjusted Receiver channel run curve to a static thresholdfluorescence value to determine whether the first target nucleic acid amplified in the multiplex amplification reaction.

53. The method of claim 52, further comprising the step of determining a cycle number of the multiplex amplification reaction at which the adjusted Receiver channel run curve exceeds the static threshold fluorescence value, and then determining the quantity of the first target nucleic acid in the multiplex amplification reaction before the reaction began.

54. The method of claim 52, wherein the result of step (d) qualitatively indicates the first target nucleic acid was amplified in the multiplex amplification reaction.

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