Method and system for improving the specificity of analyte detection using real-time nucleic acid amplification

Abstract

A method and system comprising a programmed computer that can be used to improve nucleic acid analyte detection using real-time amplification and monitoring, the method and system capable of discriminating closely related nucleic acid sequences from one another. In one embodiment, the detection of target sequences in which single nucleotides are different from each other was discriminated by applying a mathematical transformation. In another embodiment, two target sequences in which single nucleotides are different from each other were detected and discriminated from one another using a labeled probe specific to only one of the two target sequences.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 500,475, filed May 5, 2023. The entire disclosure of this related application is incorporated herein by reference. Field

[0002] The present disclosure generally relates to the field of nucleic acid detection. More specifically, the present disclosure relates to methods, systems, and software useful for detecting target nucleic acids with reduced false positive errors.

Background Art

[0003] Background Molecular diagnostic tests rely heavily on the detection of nucleic acid sequences with high specificity. Many different laboratory techniques, including allele-specific nucleic acid amplification, sequence-specific hybridization probe binding, signal amplification, and nucleic acid sequencing, are used for this purpose. Target sequences that can be targeted include single nucleotide sequence variants, insertions of one or more nucleotides, or deletions of one or more nucleotides.

[0004] One category of target sequences of particular interest for specific clinical applications involves very subtle base changes from conventional or wild-type starting sequences. For example, KRAS is a well-known oncogene that frequently mutates at characteristic nucleotide positions in various cancers (Forrester et al., Nature 327:298-303 (1987)). These cancers include pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and lung adenocarcinoma. Most oncogenic KRAS mutations occur at codons 12, 13, and 61. Using KRAS codon 12 as an example, at least four distinct point mutations within this single codon have been identified in humans (e.g., Gao et al., Theranostics 10:5137-5153 (2020)). Another example of point mutations with biological impact relates to drug resistance phenotypes. This category includes drug-resistant Mycobacterium tuberculosis (see Honore et al., Antimicrobial Agents and Chemotherapy 38:238-242 (1994)) and drug-resistant Mycoplasma genitalium (see International Publication No. 2022 / 016153).

[0005] To overcome the difficulty of obtaining sufficient sample material for analysis, processes for detecting and / or quantifying target nucleic acids often involve preliminary steps for synthesizing nucleic acid amplification products. While amplification reactions often synthesize more than one amplification product, detection of only one nucleic acid amplification product is sometimes desirable. For example, wild-type sequences and single nucleotide polymorphisms (SNPs) can be amplified in the same reaction mixture, but only the amplification product containing the SNP is targeted. Difficulties often arise when the detection of a non-target (e.g., wild-type) nucleic acid leads to the erroneous conclusion that the target nucleic acid (e.g., SNP) is present in the test sample. Such cases are called "false positive" results.

[0006] This disclosure details one approach that can be used to reduce the incidence of false positive results resulting from detection signals induced by hybridization of sequence-specific probes to non-target nucleic acid amplification products. Advantageously, this approach avoids the need to redesign assay chemistry by substituting modified amplification and detection oligonucleotides. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2022 / 016153 [Overview of the project] [Means for solving the problem]

[0008] overview This specification provides the following embodiments.

[0009] Embodiment 1 is a method for determining whether a test sample suspected to contain a nucleic acid template for a nucleic acid amplification reaction contains a target nucleic acid, the method comprising: (a) obtaining or obtaining a real-time run curve dataset containing signal data representing the production of an amplification product in a nucleic acid amplification reaction as a function of reaction progress parameters, wherein the nucleic acid amplification reaction uses either a target nucleic acid or a non-target nucleic acid that may be present in the test sample as a template for producing an amplification product; (b) calculating or having calculated the first derivative of the real-time run curve dataset, including the magnitude value of the first derivative; (c) comparing or having compared the calculated magnitude value of the first derivative to a first threshold; and (d) determining or having determined that (i) the test sample contains a target nucleic acid if any of the calculated magnitude values ​​of the first derivatives meet or exceed the first threshold, or (ii) the test sample does not contain a target nucleic acid if none of the calculated magnitude values ​​of the first derivatives meet or exceed the first threshold.

[0010] Embodiment 2 is the method of Embodiment 1, wherein the real-time run curve dataset includes fluorescence magnitude readings, and the method further comprises comparing the real-time run curve dataset with a fluorescence threshold to determine that at least one data point in the real-time run curve dataset has a fluorescence magnitude exceeding the fluorescence threshold, and determining that the test sample contains a non-target nucleic acid if it is determined in step (d) that the test sample does not contain a target nucleic acid.

[0011] Embodiment 3 is the method of Embodiment 1 or Embodiment 2, wherein the target nucleic acid and non-target nucleic acid differ from each other only at a single nucleotide position.

[0012] Embodiment 4 is one of the methods from Embodiments 1 to 3, wherein the test sample comprises a target nucleic acid, and the method further comprises the step of quantifying or having quantified the target nucleic acid present in the test sample.

[0013] Embodiment 5 is the method of Embodiment 4, wherein the quantification or quantified step includes first determining the maximum value of the first derivative from step (b), and then using the maximum value of the first derivative, along with the reaction progress parameters, as an indicator of the amount of target nucleic acid present in the test sample.

[0014] Embodiment 6 is one of the methods from Embodiments 1 to 5 and further includes the step of creating a non-temporary record of the results from step (d).

[0015] Embodiment 7 is the method of Embodiment 6, and non-temporary recording includes printing on paper or recording on a computer-readable storage medium.

[0016] Embodiment 8 is one of the methods from Embodiments 4 to 7, further comprising the step of creating a non-temporary record of the results from a quantified step or from a quantified step.

[0017] Embodiment 9 is the method of Embodiment 8, and the non - temporary recording includes printing on paper or recording on a computer - readable storage medium.

[0018] Embodiment 10 is the method of any one of Embodiments 1 to 9, wherein the reaction progress parameter in step (a) is measured by the number of cycles, the nucleic acid amplification reaction includes a PCR reaction, and the first threshold value is a predetermined threshold value.

[0019] Embodiment 11 is the method of any one of Embodiments 1 to 9, wherein the reaction progress parameter in step (a) is either a measure of reaction time or a measure of the number of reaction cycles.

[0020] Embodiment 12 is the method of any one of Embodiments 1 to 11, and step (a) includes performing a nucleic acid amplification reaction and monitoring the synthesis of an amplification product while the nucleic acid amplification reaction is occurring.

[0021] Embodiment 13 is the method of any one of Embodiments 1 to 11, and step (a) includes receiving a real - time execution curve data set as a computer - readable data file.

[0022] Embodiment 14 is the method of any one of Embodiments 1 to 13, and before step (b), the real - time execution curve data set obtained in step (a) is processed using at least one of (i) baseline subtraction, (ii) curve normalization using curve parameters, and (iii) curve fitting.

[0023] Embodiment 15 is the method of Embodiment 14, and the real - time execution curve data set obtained in step (a) is processed using curve fitting, and the curve fitting includes optimizing the coefficients of an equation so as to yield an optimized equation.

[0024] Embodiment 16 is one of the methods from Embodiments 1 to 15, wherein the real-time run curve dataset includes fluorescence readings measured as a function of reaction progress parameters, the reaction progress parameters being measured over the reaction cycle.

[0025] Embodiment 17 is one of the methods from Embodiments 1 to 16, and includes step (b) calculating using a computer and step (c) comparing with the computer.

[0026] Embodiment 18 is one of the methods of Embodiments 1 to 17, wherein the nucleic acid amplification reaction is performed using an automated nucleic acid analyzer, which is configured to isolate nucleic acids from a test sample and then perform a nucleic acid amplification reaction using the isolated nucleic acids, and step (b) includes calculation using a computer communicating with the automated nucleic acid analyzer, and step (c) includes comparison using a computer.

[0027] Embodiment 19 is one of the methods from Embodiments 1 to 18, wherein the first threshold is a numerical constant.

[0028] Embodiment 20 is one of the methods from Embodiments 1 to 19, wherein the target nucleic acid is a target nucleic acid isolated from a human pathogen.

[0029] Embodiment 21 is the method of Embodiment 20, wherein the human pathogen is either a bacterial pathogen or a viral pathogen.

[0030] Embodiment 22 is a computer programmed with software instructions for determining whether a target nucleic acid is present in a test sample, the software instructions, when executed by the computer, cause the computer to (a) receive a real-time run curve dataset containing signal data showing the amplification of target and non-target nucleic acids in a nucleic acid amplification reaction as a function of reaction progress parameters; (b) calculate the first derivative of the real-time run curve dataset or a processed version thereof, including the magnitude value of the first derivative; (c) compare the calculated magnitude value of the first derivative with a first threshold; and (d) determine that (i) the test sample contains the target nucleic acid if any of the calculated magnitude values ​​of the first derivatives meet or exceed the first threshold, or (ii) the test sample does not contain the target nucleic acid if none of the calculated magnitude values ​​of the first derivatives meet or exceed the first threshold.

[0031] Embodiment 23 is the computer of Embodiment 22, and the software instructions, when executed by the computer, further cause the computer to compare a real-time execution curve dataset with a fluorescence threshold to determine whether any signal data in the real-time execution curve dataset has a magnitude that satisfies or exceeds the fluorescence threshold, and if the computer determines that the magnitude satisfies or exceeds the fluorescence threshold, and if the computer determines in (d) that the test sample did not contain the target nucleic acid, the computer causes the computer to determine that the test sample contains a non-target nucleic acid different from the target nucleic acid.

[0032] Embodiment 24 is a computer of Embodiment 22 or Embodiment 23, and the signal data in (a) showing amplification of target nucleic acids and non-target nucleic acids includes fluorescence signal data.

[0033] Embodiment 25 is a computer from any one of Embodiments 22 to 24, wherein the software instruction, when executed by the computer, further causes the computer to generate a non-temporary record of the results from (e) and (d).

[0034] Embodiment 26 is a computer from any one of Embodiments 22 to 25, wherein, when executed by the computer, the software instruction causes the computer to create a processed version of the real-time running curve dataset, and then (b) calculate the first derivative of the processed version of the real-time running curve dataset.

[0035] Embodiment 27 is a computer of any one of Embodiments 22 to 25, wherein a software instruction, when executed by the computer, causes the computer to create a processed version of a real-time running curve dataset by performing at least one of (i) baseline subtraction, (ii) curve normalization using curve parameters, and (iii) curve fitting, and (b) calculating the first derivative of the processed version of the real-time running curve dataset.

[0036] Embodiment 28 is a computer from any one of Embodiments 22 to 27, where the first threshold in (c) is a numerical constant.

[0037] Embodiment 29 is a computer from any one of Embodiments 22 to 28, wherein the non-temporary records in (e) are electronically stored on the computer hard drive.

[0038] Embodiment 30 is a computer from any one of Embodiments 22 to 29, the computer communicates with a thermal cycling device equipped with a fluorometer.

[0039] Embodiment 31 is a system for determining whether a test sample contains a target nucleic acid, wherein the system comprises a nucleic acid analyzer configured to amplify a target nucleic acid and a non-target nucleic acid in a nucleic acid amplification reaction, the nucleic acid amplification reaction producing an amplified product using either the target nucleic acid or the non-target nucleic acid that may be present in the test sample as a template, the nucleic acid analyzer monitoring the synthesis of the amplified product in the nucleic acid amplification reaction as a function of reaction progress parameters, thereby generating a real-time execution curve dataset containing signal data as a function of reaction progress parameters, and a computer communicating with the nucleic acid analyzer and programmed with a set of software instructions. The system comprises a set of software instructions which causes the computer to (a) calculate the first derivative of a real-time execution curve dataset or a processed version thereof, including the magnitude of the first derivative; (b) compare the calculated magnitude of the first derivative to a first threshold; (c) determine that (i) the test sample contains the target nucleic acid if any of the calculated magnitudes of the first derivatives meets or exceeds the first threshold, or (ii) the test sample does not contain the target nucleic acid if none of the calculated magnitudes of the first derivatives meet or exceed the first threshold; and (d) generate a non-temporary record of the results from (c).

[0040] Embodiment 32 is the system of Embodiment 31, wherein the set of software instructions further causes the computer to compare a real-time execution curve dataset with a fluorescence threshold to determine whether any signal data in the real-time execution curve dataset has a magnitude that satisfies or exceeds the fluorescence threshold, and if the computer determines that the magnitude satisfies or exceeds the fluorescence threshold, and if the computer determines in (d) that the test sample did not contain the target nucleic acid, the computer causes the computer to determine that the test sample contains a non-target nucleic acid different from the target nucleic acid.

[0041] Embodiment 33 is the system of Embodiment 31 or Embodiment 32, and the set of software instructions further causes the computer to calculate the amount of target nucleic acid contained in the test sample.

[0042] Embodiment 34 is any one of Embodiments 31 to 33, wherein a set of software instructions causes a computer to create a processed version of a real-time running curve dataset by performing at least one of (i) baseline subtraction, (ii) curve normalization using curve parameters, and (iii) curve fitting, (a) including calculating the first derivative of the processed version of the real-time running curve dataset.

[0043] Embodiment 35 is one of the systems from Embodiments 31 to 34, wherein the computer is a standalone computer that is not physically connected to the nucleic acid analyzer.

[0044] Embodiment 36 is one of the systems from Embodiments 31 to 35, wherein a computer communicates with an electronic storage device, and the electronic storage device stores an electronic form of non-temporary records generated by the computer.

[0045] Embodiment 37 is one of the systems described in Embodiments 31 to 36, in which a computer communicates with a printer that produces non-temporary records.

[0046] Embodiment 38 is one of the systems from Embodiments 31 to 37, wherein the nucleic acid analyzer includes a fluorometer that detects a fluorescent signal produced in a nucleic acid amplification reaction, and the fluorometer is used to monitor the synthesis of the amplified product in the nucleic acid amplification reaction. [Brief explanation of the drawing]

[0047] [Figure 1]Figure 1 schematically illustrates nucleic acid target strands (thick horizontal rectangles) amplified using a pair of primers positioned in opposite directions (forward and reverse). A single amplification reaction involved only one nucleic acid target, which could be either a wild-type target or one of five SNP-containing nucleic acid targets, with the SNPs located in the packing region below the set of hydrolysis probes (thin horizontal rectangles; "x" indicates a single-nucleotide difference between probes). The reaction also involved all five SNP-specific probes, with only one probe in the illustrated set being perfectly complementary to the SNP-containing amplification product, and none of the illustrated SNP probes being perfectly complementary to the amplified wild-type sequence. Fluorophores and quenchers that were the same for all illustrated SNP probes are omitted from the schematic diagram. Wild-type probes with distinguishable fluorophore / quencher combinations are also omitted.

[0048] [Figure 2A] Figures 2A and 2B present graphical results obtained from nucleic acid amplification reactions using either a SNP-containing nucleic acid target (thin line), a wild-type nucleic acid target (thick line), or a negative control reaction without a template nucleic acid (dashed line). Figure 2A is a real-time run curve plot of fluorescence (y-axis) as a function of reaction cycle number (x-axis). A horizontal threshold line drawn at 1,000 RFU (relative fluorescence units) was used to determine the Ct value. Results from the negative control appear uniformly below the threshold. The amplified nucleic acid was detected using a SNP-specific hydrolysis probe containing FAM labeling. Figure 2B is a first-derived plot of the results appearing in Figure 2A, showing RFU / cycle (y-axis) plotted as a function of reaction cycle number (x-axis). Only the curve obtained from the first derivative of the run curve obtained using the SNP-containing variant nucleic acid target exceeded the horizontal threshold line drawn at 500 RFU. [Figure 2B]Figures 2A and 2B present graphical results obtained from nucleic acid amplification reactions using either a SNP-containing nucleic acid target (thin line), a wild-type nucleic acid target (thick line), or a negative control reaction without a template nucleic acid (dashed line). Figure 2A is a real-time run curve plot of fluorescence (y-axis) as a function of reaction cycle number (x-axis). A horizontal threshold line drawn at 1,000 RFU (relative fluorescence units) was used to determine the Ct value. Results from the negative control appear uniformly below the threshold. The amplified nucleic acid was detected using a SNP-specific hydrolysis probe containing FAM labeling. Figure 2B is a first-derived plot of the results appearing in Figure 2A, showing RFU / cycle (y-axis) plotted as a function of reaction cycle number (x-axis). Only the curve obtained from the first derivative of the run curve obtained using the SNP-containing variant nucleic acid target exceeded the horizontal threshold line drawn at 500 RFU.

[0049] [Figure 3A] Figures 3A and 3B are perspective views of an automated nucleic acid analyzer. [Figure 3B] Figures 3A and 3B are perspective views of an automated nucleic acid analyzer. [Modes for carrying out the invention]

[0050] definition The following terms have the meanings given herein unless explicitly indicated otherwise.

[0051] The terms “a,” “an,” and “the” refer to multiple objects unless the context clearly indicates otherwise. For example, as used herein, “nucleic acid” is understood to refer to one or more nucleic acids. Thus, the terms “a” (or “an”), “one or more,” and “at least one” are interchangeable herein.

[0052] A "polynucleotide" is a polymeric form of nucleotides containing ribonucleotides and / or deoxyribonucleotides of any length. This term refers only to the primary structure of the molecule. Therefore, this term encompasses double-stranded and single-stranded DNA and RNA (e.g., nucleic acids). This term also includes known types of modifications, including labels, methylated "caps," substitutions with one or more naturally occurring analogs of nucleotides, and internucleotide modifications, such as uncharged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), as well as the unmodified form of polynucleotides.

[0053] As used herein, “test sample” is any sample to be investigated for the presence of a particular polynucleotide sequence. A test sample includes any nucleic acid-containing material obtained from human, animal, environmental, or laboratory-derived or synthetic samples. Preferred test samples include bodily fluid samples. Whole blood, plasma, and serum are particularly preferred examples of test samples. Other test samples include swab samples (e.g., oral, nasal, throat, or vaginal swab samples), saliva, urine, and the like.

[0054] As used herein, “analyte” refers to a chemical or biochemical species to be detected and / or quantified. For example, “polynucleotide analyte” refers to a polynucleotide (e.g., a segment of viral nucleic acid or bacterial ribosomal nucleic acid) to be detected or quantified in the test procedure.

[0055] As used herein, “nucleic acid analyzer” is a device for amplifying, detecting, and, if necessary, quantifying a nucleic acid analyte. Specific preferred nucleic acid analyzers include a temperature-controlled incubator (e.g., a block, plate, or chamber), a fluorometer for optical communication with the contents of the temperature-controlled incubator, and one or more computers or processors for processing the data collected by the fluorometer to quantify the nucleic acid analyte of interest.

[0056] The "amplification product" (sometimes "amplicon") is the polynucleotide product of the amplification reaction, in which case the target polynucleotide sequence of the polynucleotide analyte served as a template in the synthesis of the polynucleotide copy or amplification product. Preferred amplification products contain or comprise DNA.

[0057] The term “amplify” is used in a broad sense to mean producing an amplification product that can be enzymatically synthesized using DNA or RNA polymerase (including reverse transcriptase). “Amplification,” “nucleic acid amplification,” or “polynucleotide amplification,” etc., means any known procedure for obtaining multiple copies of a target polynucleotide sequence or its complement or fragment thereof, enabling RNA and DNA equivalents. “Multiple copies” means at least two copies. “Copy” does not necessarily mean complete sequence complementarity or identity with respect to the template sequence. Methods for amplifying mRNA are generally known in the art and include reverse transcription PCR (RT-PCR). Another method that may be used is quantitative PCR (or Q-PCR).

[0058] As used herein, the terms “co-amplify” and “to co-amplify” and their variants refer to the process by which different target polynucleotide 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 “co-amplified” if both nucleic acids are amplified in a reaction carried out in a single tube, and both amplification reactions share at least one reagent in common (e.g., deoxyribonucleotide triphosphate, enzyme, primer, etc.).

[0059] As used herein, “thermal cycle” refers to repeated temperature changes (i.e., increases or decreases) in a reaction mixture. A sample undergoing a thermal cycle can 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 necessary to study or complete the particular chemical reaction of interest.

[0060] "Target" or "target nucleic acid" means a nucleic acid containing a sequence to be amplified, detected, and / or quantified. The target nucleic acid sequence to be amplified is preferably located between two opposing oligonucleotides and contains a portion of the target nucleic acid complementary to each oligonucleotide.

[0061] "Target nucleic acid sequence" or "target sequence" or "target region" means a specific deoxyribonucleotide or ribonucleotide sequence that includes all or part of the nucleotide sequence of a single-stranded target nucleic acid molecule and a complementary deoxyribonucleotide or ribonucleotide sequence.

[0062] "Transcription-associated amplification" refers to any type of polynucleotide amplification that uses RNA polymerase to produce multiple RNA transcripts from a polynucleotide template. Traditionally, these amplification reactions use at least one primer having a 3' end that can be extended by the activity of DNA polymerase. An example of transcription-associated amplification methods called "transcription-mediated amplification" (TMA) generally uses RNA polymerase, DNA polymerase, deoxyribonucleoside triphosphate, ribonucleoside triphosphate, and promoter-containing oligonucleotides complementary to the target polynucleotide. Variations of the TMA are well known in the art, as disclosed in detail in Burg et al., U.S. Patent No. 5,437,990; Kacian et al., U.S. Patents No. 5,399,491 and No. 5,554,516; Kacian et al., International Publication No. 93 / 22461; Gingeras et al., International Publication No. 88 / 01302; Gingeras et al., International Publication No. 88 / 10315; Malek et al., U.S. Patent No. 5,130,238; Urdea et al., U.S. Patents No. 4,868,105 and No. 5,124,246; McDonough et al., International Publication No. 94 / 03472; and Ryder et al., International Publication No. 95 / 03430. Other transcription-related amplification methods that use only a single primer that can be extended by a DNA polymerase, as disclosed in U.S. Patent No. 7,374,885, are specifically included in the definition and are very preferred for use in connection with the methods disclosed herein.

[0063] As used herein, “oligonucleotide” or “oligomer” or “oligo” is a polymer chain of at least two, generally about 5 to about 100, chemical subunits, each subunit comprising a nucleotide base moiety, a sugar moiety, and a link moiety that connects the subunits in a linear spatial arrangement. Common nucleotide base moieties are guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U), but other rare or modified nucleotide bases that can hydrogen bond are well known to those skilled in the art. Oligonucleotides may optionally contain analogues of any of the sugar moiety, base moiety, and skeletal components. Preferred oligonucleotides of this disclosure are in the size range of about 10 to about 100 residues. Oligonucleotides may be purified from naturally occurring sources, but are preferably synthesized using any of a variety of well known enzymatic or chemical methods.

[0064] An "amplifying oligonucleotide" or "amplifying oligomer" refers to an oligomer that hybridizes to a target polynucleotide or its complement and participates in a nucleic acid amplification reaction. Examples of amplifying oligomers include primers containing a 3' end that is extended as part of the amplification process, but also oligomers that are not extended by polymerase (e.g., 3'-block oligomers) but participate in or can facilitate efficient amplification from the primer. Preferred size ranges for amplifying oligomers are about 10 to about 80 nucleotides in length, or 10 to about 60 nucleotides in length, and include at least about 10 consecutive bases, more preferably at least 12 consecutive bases, that are complementary to the region of the target polynucleotide sequence (or its complementary strand). The consecutive bases are preferably at least about 80%, more preferably at least about 90%, and most preferably about 100% complementary to the target sequence to which the amplifying oligomer binds. The amplifying oligomer may optionally include modified nucleotides or analogs, or additional nucleotides, that participate in the amplification reaction but are not complementary to or not contained in the target polynucleotide. Amplified oligomers that are 3' blocked but can hybridize to a target polynucleotide and provide an upstream promoter sequence that helps initiate transcription are called “promoter provider” oligomers.

[0065] A "primer" is an amplified oligomer having a 3'-OH end that hybridizes to a target polynucleotide template and can be extended by DNA polymerase. The 5' region of the primer may be complementary to the target polynucleotide (e.g., a non-complementary promoter sequence) resulting in an oligomer called a "promoter-primer." Those skilled in the art will understand that any oligomer that can function as a primer can be modified to include a 5' promoter sequence and thus can function as a promoter-primer. Similarly, any promoter-primer can be modified by removing the promoter sequence or by synthesis without the promoter sequence and may still function as a primer.

[0066] As used herein, “probe” is an oligonucleotide that, under conditions promoting hybridization, specifically hybridizes to a target sequence in a polynucleotide, preferably an amplified polynucleotide, to form a detectable hybrid. Certain preferred probes include a detectable label (e.g., fluorescent or chemiluminescent label). Hydrolysis probes conventionally include a fluorescent label and a quencher moiety.

[0067] The term "label" refers to a composition that can produce a detectable signal indicating the presence of a labeled molecule. Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, and bioluminescent moieties. Therefore, a label is any composition that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.

[0068] "Detection" includes any means of detection, including the direct and indirect detection of gene expression and changes thereof. For example, a "detectably low" product may be observed directly or indirectly, and this term indicates any decrease (including the absence of a detectable signal). Similarly, a "detectably high" product means any increase, whether observed directly or indirectly.

[0069] As used herein, “nucleic acid analyzer” is a device or instrument for amplifying, detecting, and, if necessary, quantifying a nucleic acid analyte. Specific preferred nucleic acid analyzers include a temperature-controlled incubator (e.g., block, plate, or chamber), a fluorometer that optically communicates with the contents of the temperature-controlled incubator, and one or more computers or processors that process the data collected by the fluorometer to quantify the nucleic acid analyte of interest. In some embodiments, the preferred nucleic acid analyzer performs an enzyme-based reaction that amplifies or increases the copy number of the target nucleic acid to be quantified. In other embodiments, “signal amplification” is used to detect and / or quantify the target nucleic acid to be quantified. An example of a signal amplification system is the “sequential invasive signal amplification reaction” disclosed by Hall et al. in Proc.Natl.Acad.Sci.USA97:8272-8277 (2000).

[0070] As used herein, “time-dependent” monitoring of polynucleotide amplification, or “real-time” monitoring of polynucleotide amplification, refers to a process in which the amount of amplicon present during the amplification reaction is measured as a function of reaction time or number of cycles, and this is then used to determine the starting amount of template present in the reaction mixture at the time the amplification reaction was initiated. For example, the amount of amplicon can be measured before initiating each complete cycle of an amplification reaction, such as PCR, which involves thermal cycling. Alternatively, isothermal amplification reactions that do not require physical intervention to initiate transitions between amplification cycles can be monitored continuously or at regular time intervals to obtain information about the amount of amplicon present as a function of time.

[0071] As used herein, the “running curve” (sometimes “growth curve” as used herein) refers to a characteristic pattern of the appearance of synthetic products, such as amplicons, in a reaction as a function of time or number of cycles (i.e., reaction progress parameters). Running curves are conveniently represented as a two-dimensional plot of time or number of cycles (x-axis) against some indicator of product quantity, such as fluorescence measurement (y-axis). Some, but not all, running curves have a sigmoid shape.

[0072] As used herein, the “baseline stage” of a growth curve refers to the initial stage of the curve where the amount of product (such as an amplicon) increases at a substantially constant rate, slower than the rate of increase characteristic of the growth stage of the growth curve (which may have a log-linear profile). The baseline stage of a growth curve typically has a very shallow slope and often approaches zero.

[0073] As used herein, the “growth phase” of a growth curve refers to a portion of the curve in which the measurable product substantially increases over time. The transition from the baseline phase to the growth phase in a typical polynucleotide amplification reaction is characterized by the appearance of amplicons at a rate that increases over time. The transition from the growth phase to the plateau phase of the growth curve begins at an inflection point in which the rate of amplicon appearance begins to decrease.

[0074] As used herein, the “plateau phase” of a triphasic growth curve refers to the final phase of the curve. In the plateau phase, the measurable rate of product formation is generally substantially lower than the rate of amplicon production in the log-linear phase and may approach zero.

[0075] As used herein, the term “indicia of amplification” refers to a feature of the real-time run curve that indicates a given progress level of a polynucleotide amplification reaction. In some embodiments, the time or number of cycles at which a threshold level of fluorescence is achieved serves as the indicia of amplification. Such indicia is typically determined by a mathematical analysis of a run curve, sometimes called a “growth curve,” which displays a measurable signal (such as a fluorescence reading) whose intensity is related to the amount of amplicon present in the reaction mixture as a function of time, number of cycles, etc.

[0076] As used herein, the term “threshold-based amplification indicia” refers to amplification indicia that measures the time or number of cycles at which a growth curve signal crosses an arbitrary value or threshold (e.g., threshold fluorescence value). Cycle threshold (Ct) and TTime values ​​are examples of threshold-based amplification indicia, while TArc and OTArc determinations are examples of non-threshold amplification indicia.

[0077] As used herein, the term “time-dependent indicia of amplification” generally refers to the indicia of amplification (e.g., reaction progress parameters) measured over time units (e.g., minutes). Time-dependent indicia of amplification is typically used to monitor the progress of isothermal polynucleotide amplification reactions that do not feature distinct “cycles.” TTime, TArc, and OTArc are all examples of time-dependent indicia of amplification.

[0078] As used herein, the phrase “as a function of” describes a 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 can have a value of any choice without considering the values ​​of any other variable), where each input value of the independent variable relates to exactly one output value of the dependent variable. The conventional notation for an equation relating x values ​​(i.e., the independent variable) “as a function of” y values ​​(i.e., the dependent variable) is y = f(x).

[0079] As used herein, “computer” is an electronic device capable of receiving and processing input information using software instructions to produce an output. A computer may be a standalone device (e.g., a personal computer) or an integrated component of an instrument (e.g., a nucleic acid analyzer that amplifies a target nucleic acid and monitors the synthesis of the amplified product as a function of the number of reaction cycles or time). The term particularly includes embedded processors that reside within an analytical instrument and have built-in software instructions (sometimes called “firmware”).

[0080] As used herein, “optimizing” or “fitting” an equation refers to the process of obtaining numerical values ​​for the coefficients of an equation to “fit” or approximate an equation to experimental measurements, as is typically done in mathematical modeling or curve fitting procedures. Typically, the optimized equation defines the best-fitting curve.

[0081] As used herein, the terms “optimized equation” and “fitted equation” are alternative references to equations that contain fixed values ​​for coefficients as a result of an optimization procedure. A “fitted” curve (e.g., the product of a curve fitting procedure) is obtained by optimizing an equation.

[0082] As used herein, “system” is an arrangement of components or elements organized to work together. For example, a system may include a device that detects nucleic acids in a sequence-specific manner and a computer programmed with software to analyze the results, with the computer and the device communicating with each other.

[0083] As used herein, “apparatus” generally refers to a set of instruments (e.g., tools, equipment, etc.) necessary for a particular purpose or function.

[0084] As used herein, “apparatus” refers to a tool, device, or instrument for performing a task. In some embodiments, the apparatus is a device housed in a single housing or located on a common support structure (e.g., a single chassis).

[0085] "Kit" typically means a packaged combination of materials intended to be used in combination with each other. Kits according to this disclosure may include instructions or other information in "tangible" form (e.g., printed information, information electronically recorded on a computer-readable medium, or information recorded on a machine-readable medium such as a barcode for storing numerical values).

[0086] As used herein, the term “equipment” and its cogenus are used in their comprehensive sense, i.e., equivalent to the term “contains” and its corresponding cogenus.

[0087] "Essentially derived from" means that additional components, compositions, or method steps that do not substantially alter the fundamental and novel features of the present invention may be included in the present invention. Any component, composition, or method step that substantially affects the fundamental and novel features of the present invention is outside the scope of this term.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains.

[0089] Detailed description of a specific embodiment The specificity of analyte detection using real-time nucleic acid amplification techniques generally depends on matching primers and probes to complementary target sequences to be detected. Specificity can decrease when attempting to distinguish between nucleotide sequences that differ only slightly from each other. Indeed, the detection of single-nucleotide differences (e.g., "single nucleotide polymorphisms" or "SNPs") typically relies on the use of probes that precisely complement the sequence being detected. Non-specific detection of closely matching sequences (e.g., wild-type sequences and SNPs differing by a single nucleotide) can be problematic for real-time detection algorithms that rely on run curves exceeding a fluorescence threshold (sometimes referred to herein as the "cutoff") to demonstrate the presence of the target nucleic acid. In other words, undesirable signals from cross-hybridization between labeled probes and non-target amplification products may undesirably exceed the threshold used to measure the presence of the target nucleic acid. This technique overcomes this problem by using mathematical transformations of run curve data to enhance specific target detection.

[0090] Preferred polynucleotide amplification method Useful in vitro polynucleotide amplification methods related to this technology include, but are not limited to, polymerase chain reaction (PCR), transcription-mediated amplification (TMA), single-primer nucleic acid amplification, nucleic acid sequence-based amplification (NASBA), strand substitution amplification (SDA), self-persistent sequence replication (3SR), DNA ligase chain reaction (LCR), and amplification methods using self-replicating polynucleotide molecules and replication enzymes such as MDV-1 RNA and Q-beta enzyme. Methods for carrying out these various amplification techniques can be found in U.S. Patent No. 4,965,188, European Patent No. 0460828, U.S. Patent No. 5,399,491, U.S. Patent No. 7,374,885, Published European Patent Application No. 0525882, U.S. Patent No. 5,455,166, Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878 (1990), International Publication No. 89 / 09835, U.S. Patent No. 5,472,840, and Lizardi et al., Trends Biotechnol. 9:53-58 (1991). Disclosures of these documents describing methods for carrying out polynucleotide amplification reactions are incorporated herein by reference.

[0091] Preferred systems and apparatus The methods disclosed herein are conveniently carried out using a computer or similar processing device (hereinafter, "computer"). In different preferred embodiments, software or machine-executable instructions may be loaded or held in a memory component of an independent computer, or in a memory component of a computer linked to a device used to monitor the amount of product under analysis as a function of reaction progress parameters (e.g., reaction time or number of reaction cycles). In a very preferred embodiment, software for performing the disclosed procedure is held in a memory component of a computer linked to, or an integral part of, a device or apparatus capable of monitoring the amount of amplicon present in the reaction mixture as a function of the number of reaction cycles. This includes processing device components (e.g., embedded software) on the electronic circuit board of an automated nucleic acid analyzer. Generally speaking, the computer is said to "communicate" 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. Steps commanded by the software may include obtaining a real-time run curve dataset representing the production of amplification products in an amplification reaction; calculating the derivative of the real-time run curve dataset, or a processed version thereof, including the magnitude of the derivative; comparing the calculated magnitude of the derivative to a cutoff value or threshold (e.g., a predetermined threshold); determining that the test sample contained the target nucleic acid if any of the calculated magnitude values ​​exceeded the threshold, or determining that the test sample did not contain the target nucleic acid if none of the calculated magnitude values ​​exceeded the threshold. In some embodiments, the results generated by the computer may be delivered to an output device that displays or records the calculation or comparison results. Typical output devices include video monitors and printers. In some embodiments, the output device is a recording device that produces “non-temporary” records (e.g., “tangible” records).Non-temporary records may be printed on paper or stored electronically (for example, on a computer hard drive or flash drive, magnetic tape, or other computer-readable media).

[0092] In some embodiments, a computer can communicate by either wired or wireless means with a fluorometer that detects fluorescence signals, which are arranged or configured to monitor fluorescence signals generated in one or more reaction vessels housed in a temperature-controlled incubator. The incubator may be a temperature-controlled block (e.g., a metal block configured to receive and house one or more tubes, or even a multiwell plate), or a chamber that exposes one or more reaction vessels to controlled temperature conditions.

[0093] In some embodiments, either or both of the controller system for controlling the real-time amplification device and / or the detection system for the real-time amplification device may be connected to a appropriately programmed computer that functions to command the operation of these devices according to pre-programmed instructions or user input instructions. The computer may also preferably receive data and information from these devices, interpret this information, manipulate it, and report it to the user.

[0094] In some embodiments, the computer may also include appropriate software for receiving user instructions, either in the form of user input to a set of parameter fields or in the form of pre-programmed instructions (e.g., pre-programmed for a variety of different specific operations). The software then translates these instructions into an appropriate language to instruct the real-time amplification controller to perform the desired operation. Preferably, the computer may also receive data from one or more sensors or detectors included in the system and interpret the data according to the programming. The system preferably includes software that correlates the features of an amplification curve, which represents the amount of amplified copies of the nucleic acid of interest as a function of time, as detected by the detector, with the number of copies of the nucleic acid of interest present in the test sample.

[0095] Preferably, if the computer used to perform the disclosed technology is an integrated component of the apparatus for performing and analyzing real-time nucleic acid amplification reactions, the apparatus preferably comprises a temperature-controlled incubator, a detection device (e.g., a fluorometer) for collecting signals, and an analysis device (e.g., a computer or processor) for analyzing signals. The apparatus may optionally further include an output device for displaying obtained or generated data. The analysis device may be connected to the temperature-controlled incubator via an input device known in the art and / or to an output device known in the art for data display. In one embodiment, the temperature-controlled incubator may be temperature-cyclable and configured as a block for receiving one or more tubes or reaction receptacles (e.g., a multi-tube unit).

[0096] Generally speaking, various components of apparatus for performing real-time nucleic acid amplification useful in relation to the disclosed method are conventional components well known to those skilled in the art. A temperature-controlled incubator used for performing real-time nucleic acid amplification and analysis may be of a conventional design capable of holding reaction samples in temperature-controlled blocks within multiple reaction tubes or within the wells of a standard amplification reaction tube or multi-well plate. In one embodiment, a detection system is suitable for detecting optical signals from one or more fluorescent labels. The output of the detection system (e.g., a signal corresponding to a signal generated during the amplification reaction) can be supplied to a computer for data storage and manipulation. In one embodiment, the system has the capability of a microplate fluorescence reader, detecting multiple different types of optical signals, such as multiple different types of fluorescent labels. The detection system is preferably a multiplexed fluorometer comprising an excitation light source, which may be a visible light laser or an ultraviolet lamp or a halogen lamp; a multiplexer device for distributing the excitation light to individual reaction tubes and receiving fluorescence from the reaction tubes; filtering means for separating the fluorescence from the excitation light by their wavelengths; and detection means for measuring the fluorescence intensity. Preferably, a temperature-controlled incubator detection system provides a wide detection range that enables flexibility in fluorophore selection, high sensitivity, and a good signal-to-noise ratio. The optical signals received by the detection system are generally converted into signals that can be manipulated by a computer or processor to provide data that can be viewed by the user on the display of a user device communicating with the computer or processor. The user device may have a user interface or may be a conventional commercially available computer system with a keyboard and video monitor. Examples of data that can be displayed by the user device include amplification plots, scatter plots, sample value screens, optical signal intensity screens (e.g., fluorescence signal intensity screens), final call results, and text reports for all tubes or reaction vessels in the assembly and all labels used.

[0097] Exemplary nucleic acid analyzers and systems Figures 3A and 3B illustrate a typical automated analysis system 1000 that may be used to analyze multiple samples simultaneously. Figure 3A is a perspective view of system 1000, while Figure 3B is a diagram of system 1000 with the canopy removed to show its features. Both Figures 3A and 3B will be referenced in the following description. System 1000 is configured to isolate and purify nucleic acids obtained from multiple samples introduced into the system, and to amplify and detect target nucleic acids contained in any of the samples using assay reagents of different configurations. In some embodiments, system 1000 may be a random access system that allows in vitro diagnostic (IVD) assays and laboratory development 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 multiple different (e.g., differently configured) molecular assays on multiple samples. For example, multiple samples may be loaded into system 1000, processed to specifically or nonspecifically isolate and purify target nucleic acids, a first subset of the samples may be subjected to a first set of conditions for first nucleic acid amplification, and a second subset of the samples may be subjected to a second set of conditions for second nucleic acid amplification, with different reagents for the first and second nucleic acid amplifications. In some such embodiments, system 1000 may prompt the user with information using, for example, a graphical user interface (GUI) displayed on system 1000's display device 50 (e.g., a computer monitor or video monitor) (see Figure 3A) or another display associated with system 1000 (e.g., a remote computer) to define one or more parameters of an assay protocol that can be saved and used later.

[0098] In some embodiments, system 1000 may have a modular structure and may consist of a plurality of modules operably linked to one another. However, it should be noted that the modular structure of system 1000 is merely a typical example, and in some embodiments, system 1000 may be an integrated system having a plurality of regions or zones, each region or zone performing, for example, a specific step of an assay which may be specific to that region. System 1000 includes a first module 100 and a second module 400 operably linked to one another. The first module 100 and the second module 400 may each be configured to perform one or more steps of an assay. In some embodiments, the first and second modules 100 and 400 may be separate modules selectively linked to one another. That is, the first module 100 can be selectively and operably linked to the second module 400, and the first module 100 can be selectively detached from the second module 400 and linked to a different second module 400. The first and second modules 100,400 may be connected in any way. For example, these modules can be connected to each other using fasteners (e.g., bolts or screws), clamps, belts, straps, or any combination of fastening / attaching devices. As described above, the modular structure of system 1000 is merely a typical example, and in some embodiments, system 1000 may be an integrated, self-contained structure (e.g., the first module 100 forms a first region, and the second module forms a second region within the integrated structure). Note that in this disclosure, the term “module” is used to refer to a region (zone, location, etc.) of an analytical system. In some embodiments, each such region may be configured to perform a specific step of an assay that may be specific to that region of the system.

[0099] In some embodiments, power, data, and / or utility lines or conduits (such as air, water, or vacuum) may extend between the first and second modules 100 and 400. In some embodiments, the first module 100 may be a system previously purchased by the customer, and the second module 400 may be a module acquired later to extend the analytical capabilities of the combined system. For example, in one embodiment, the first module 100 may be a Panther® system (Hologic Inc.; Marlborough, Massachusetts) configured to perform sample processing and isothermal transfer-based amplification assays (e.g., TMA or NASBA) on samples provided to the system, and module 400 may be a bolt-on configured to extend the functionality of the Panther® system, in particular by adding thermal cycling capability to enable real-time PCR reactions, for example. A typical system 1000 with typical first and second modules 100,400 is the Panther Fusion® system (Hologic Inc., Marlborough, Massachusetts) described in U.S. Patent Nos. 9,732,374, 9,465,161, and 9,604,185, and U.S. Patent Application Publication No. 2016 / 0032358. Typical systems, functions, devices or components, and capabilities of the first and second modules 100,400 are described in the above publications (and the publications specified below) and are therefore not described in detail herein for the sake of brevity.

[0100] In some embodiments, the first module 100 may include a plurality of vertically stacked decks. As illustrated, the 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., a target nucleic acid). The first module 100 may include a receptacle receiving component configured to receive and hold a reaction receptacle and optionally perform processing steps on the contents of the receptacle. Typical process steps may include, for example, dispensing a sample and / or reagent into a reaction receptacle containing a target capture reagent, buffer, oil, primer and / or other amplification oligomers, probe, polymerase, etc.; aspirating material from the reaction receptacle, for example, including unimmobilized components of the sample or washing solution; mixing the contents of the reaction receptacle; maintaining and / or changing the temperature of the contents of the reaction receptacle; heating or cooling the contents of the reaction receptacle or reagent container; changing the concentration of one or more components of the contents of the reaction receptacle; separating or isolating components of the contents of the reaction receptacle; detecting signals from the contents of the reaction receptacle, such as electromagnetic radiation (e.g., visible light); and / or inactivating nucleic acids or stopping a reaction in progress.

[0101] In some embodiments, the first module 100 may include a receptacle drawer or compartment 102 adapted to receive and support a plurality of empty reaction receptacles. The compartment 102 may include a cover or door for accessing the compartment and loading reaction receptacles. The compartment 102 may further include a receptacle supply device for moving reaction receptacles to a receptacle pickup position (e.g., a registered position or a known position) to facilitate removal of reaction receptacles by a receptacle dispenser. The first module 100 may further include one or more compartments configured to house a container configured to hold bulk reagents (i.e., a sufficient volume of reagents to perform multiple assays) or to receive and hold waste. The bulk reagents may include fluids such as water, buffers, target capture reagents, and nucleic acid amplification and detection reagents. In some embodiments, the bulk reagent container compartment may be configured to maintain the container at a desired temperature (e.g., a predetermined storage temperature) and may include a holding structure that holds and / or agitates the container to maintain its contents in a solution or suspension. A typical holding structure for supporting and agitating a fluid container is described in U.S. Patent No. 9,604,185.

[0102] The first module 100 may further include a sample bay supporting one or more sample holding racks with sample-containing receptacles. The first module 100 may also include one or more fluid transfer devices for transferring fluids, such as sample fluids, reagents, bulk fluids, waste liquids, etc., to and from the reaction receptacles and / or other containers. In some embodiments, the fluid transfer device may comprise one or more robotic pipettes configured for controlled and automated movement and access to the reaction receptacles, bulk containers holding reagents, and containers holding samples. In some embodiments, the fluid transfer device may also include a fluid dispenser, such as a nozzle, located within another device and connected by appropriate fluid conduits to a container, such as a bulk container holding reagents, and a pump or other device for causing fluid transfer from the containers to the dispenser. The first module 100 may further include a plurality of loading stations (e.g., heated loading stations) configured to receive sample receptacles and other forms of holders for supporting sample receptacles and reagent containers. A typical loading station and receptacle holder is described in U.S. Patent No. 8,309,036.

[0103] In some embodiments, the first module 100 may include one or more magnetic parking stations and heating incubators 112, 114, 116 configured to heat (and / or maintain) the contents of the reaction receptacle at a temperature higher than the ambient temperature, and one or more cooling modules configured to cool (and / or maintain) the contents of the reaction receptacle at a temperature lower than the ambient temperature. The cooling modules may be used to assist oligohybridization and / or to cool the receptacle before performing luminescence measurements. In some embodiments, incubator 112 (sometimes called a transition incubator) may be set to a temperature of about 43.7°C and may be used for process steps such as dissolution, target capture, and hybridization. In some embodiments, incubator 114 may be a high-temperature incubator set to a temperature of about 64°C and may be used for process steps such as dissolution, target capture, and hybridization. Incubator 116 (referred to as the amplification incubator) may be set to a temperature of approximately 42°C and may be an incubator used for amplification during the assay. Incubator 116 may include a real-time fluorometer for detecting fluorescence during amplification. A typical temperature rise station is described in U.S. Patent No. 8,192,992, and a typical incubator is described in U.S. Patents No. 7,964,413 and No. 8,718,948. The first module 100 may include a sample handling device such as a magnetic washing station adapted to separate or isolate the target nucleic acid or other analyte (e.g., immobilized on a magnetically responsive solid support) from the remaining contents of the receptacle.

[0104] In some assays, the sample is processed to release substances that can interfere with the detection of the analyte (e.g., target nucleic acid) at the magnetic washing station. To remove these interfering substances, the sample may be treated with a target capture reagent containing a magnetically responsive solid support for immobilizing the analyte. Suitable solid supports may contain paramagnetic particles (0.7–1.05 micron particles, Sera-Mag® MG-CM (Seradyn, Inc., available from Indianapolis, Indiana)). When the solid support is brought into contact with a magnetic field, it is drawn out of the suspension and aggregates adjacent to the surface of the sample holding container, thereby isolating the immobilized analyte within the container. The unimmobilized components of the sample can then be aspirated or otherwise separated from the immobilized analyte. Typical magnetic washing stations are described in U.S. Patents 6,605,213 and 9,011,771.

[0105] The 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 embodiment, the detector may comprise a luminometer for detecting a light emission signal emitted by the contents of the reaction receptacle and / or a fluorometer for detecting fluorescence emission from the contents of the reaction receptacle. The first module 100 may also include one or more signal detection devices, such as a fluorometer (e.g., coupled to one or more of incubators 112, 114, 116), configured to detect a signal (e.g., at periodic intervals) emitted by the contents of the receptacle housed in an incubator while a process, such as nucleic acid amplification, is taking place within the reaction receptacle. Typical luminometers and fluorometers are described in U.S. Patents 7,396,509 and 8,008,066.

[0106] The first module 100 may further include a receptacle transfer device, which includes a receptacle distributor configured to move receptacles between various devices of the first module 100 (e.g., incubators 112, 114, 116, loading stations, magnetic parking stations, washing stations, and cooling modules). These devices may include a receptacle transfer portal (e.g., a port covered by an openable / closable door) through which receptacles can be inserted into or removed from the devices. The receptacle distributor may include a receptacle distribution head configured to move along a transport track assembly in the X direction, rotate in the theta (θ) direction, and move in the R direction for inserting and removing receptacles into and removing them from the devices of the first module 100. A typical receptacle distributor, a typical receptacle transfer portal door, and a mechanism for opening the door are described in U.S. Patent No. 8,731,712.

[0107] In a typical embodiment, the second module 400 is configured to perform nucleic acid amplification reactions (e.g., PCR) and measure fluorescence in real time. System 1000 may include a controller that instructs System 1000 to perform different steps of a desired assay. The controller may support LIS ("Laboratory Information System") connectivity and remote user access. In some embodiments, the second module 400 houses component modules that enable additional functions such as melt analysis. An example of a melt station that can be adapted for use with 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.

[0108] Referring to Figure 3B, in some embodiments, the second module 400 includes multiple vertically stacked levels (or decks) containing devices configured for different functions. These levels include an amplification processing deck 430 and a receptacle processing deck 600. In the illustrated embodiment, the receptacle processing deck 600 is located below the 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 the analysis system 1000. The second module 400 may include devices located at different levels. These devices include, among other things, a fluid transfer device in the form of one or more robotic pipettes 410 (see Figure 3B), a thermal cycler 432 with a signal detector, a tip compartment 580 configured to house a tray of disposable tips for the pipettes 410, a cap / vial compartment 440 configured to house a tray 460 of disposable processing vials and associated caps, a bulk reagent container compartment 500, a bulk reagent container transporter, a receptacle distribution system and a receptacle distributor (in a typical embodiment shown, a rotary distributor) including a receptacle transfer device. The system includes a receptacle distribution system (including a receptacle storage unit configured to house receptacles and / or multi-receptacle units (MRUs) (e.g., multiple receptacles linked together as a single integrated unit), a magnetic slot, a waste bin connected to one or more chutes, a centrifuge 588, a reagent pack changer, a reagent pack loading station, and one or more compartments 450 (see Figure 3B) configured to house accessories such as storage trays for consumables for post-cap / vial assemblies. The robotic pipette 410 loads disposable fluid transfer tips from a disposable tip tray 582 onto the mounting end of its aspiration probe.

[0109] A typical embodiment of tray 460 for disposable processing vials and caps is disclosed in U.S. Patent Application Publication No. 2017 / 0297027. Several devices and features of System 1000 are described in U.S. Patent No. 9,732,374 and other references specified herein. Therefore, for the sake of brevity, these devices and features are not described in detail herein.

[0110] In the illustrated embodiment, the robotic pipette 410 is located near the top of the second module 400. Below the robotic pipette 410, the amplification processing deck 430 includes a bulk reagent container compartment 500, a centrifuge 588, the top of a thermal cycler 432, a tip compartment 580, and a cap / vial compartment 440. Below the amplification processing deck 430, the receptacle processing deck 600 includes a receptacle handoff device, a receptacle distributor, a receptacle storage unit, magnetic slots, a reagent pack changer, and a reagent pack loading station. The magnetic slots and reagent pack loading station on the receptacle processing deck 600 are accessible by the robotic pipette 410 through gaps between devices in the amplification processing deck 430. Referring to Figure 3B, the second module 400 may include a compartment 590 for storing accessories or to accommodate expansions of the second module 400 (for example, adding an additional reagent compartment for storing reagents to add analytical capabilities to the system 1000). A waste bin 650 collects and holds used materials such as used disposable fluid transfer tips. The front of the second module 400 preferably includes at least one drawer, each drawer may include a drawer front 720.

[0111] The receptacles within the receptacle storage unit may include individual receptacles having an open end and a closed end on the opposite side (e.g., containers configured to store fluids), or a plurality of receptacles linked together as a unit (MRU) (e.g., 5). These MRUs may include operating structures configured to be engaged by engaging members (e.g., hooks) of a robot-controlled receptacle distribution system in order to move receptacles between different devices of system 1000. Typical receptacles are described in U.S. Patents 6,086,827 and 9,732,374. In some embodiments, a receptacle distribution system including a receptacle handoff device and a receptacle distributor is configured to receive receptacles or MRUs from a receptacle distributor in a first module 100, transfer the receptacles to a second module 400, and then move the receptacles to different locations within the second module 400.

[0112] Computer program products Within the scope of this disclosure are software-based products that can be used to perform data processing methods, including, for example, tangible embodiments of software for instructing a computer to perform various procedural steps. These include software instructions stored on computer or computer-readable media such as magnetic media, optical media, "flash" memory devices, and computer networks or cloud storage.

[0113] This disclosure further encompasses systems or apparatus for amplifying nucleic acids, detecting nucleic acid amplification products, and processing the results to indicate quantitative results of a target in a test sample. While the various components of the apparatus preferably function in cooperation, the components do not necessarily have to be parts of an integrated assembly (e.g., on a single chassis). However, in preferred embodiments, the components of the apparatus are connected to one another. The meaning of “connected” includes connections via wired and wireless connections.

[0114] In particular, within the scope of this disclosure is an apparatus or system including a computer linked to a device that amplifies nucleic acids and monitors amplicon synthesis as a function of cycle number or time, the computer being programmed to perform algorithmic steps disclosed herein. A typical system according to this disclosure includes a temperature-controlled incubator and a fluorometer capable of monitoring and distinguishing at least two wavelengths of fluorescence emission. These emissions may be used to demonstrate target amplicon synthesis and internal control or internal calibrator amplicon synthesis.

[0115] In relation to embodiments of the computer or software implementations of this disclosure, the results may be recorded or stored in a “non-temporary” format that can be accessed for reference at a later date than when the recorded data analysis was performed or carried out. For example, calculation results may be recorded in a non-temporary format by printing them on paper or by storing them in a computer-readable memory device (e.g., a hard drive, a flash memory device, or a file in cloud storage).

[0116] The software instructions provided in this disclosure can instruct a computer to perform different steps. For example, these steps may relate to receiving an input signal representing a real-time run curve dataset; calculating a derivative of the real-time run curve dataset or a processed version thereof (e.g., processed by baseline subtraction and / or curve fitting to smooth the data); comparing the magnitude of the derivative to a threshold (e.g., a given threshold); determining that the test sample contained the target nucleic acid if any of the calculated magnitude values ​​exceeded the threshold; or determining that the test sample did not contain the target nucleic acid if none of the calculated magnitude values ​​exceeded the threshold.

[0117] Curve fitting procedure According to the disclosed method for generating and evaluating a real-time running curve, plotted or fitted equation for determining the presence or absence of a target nucleic acid in a test sample, the procedure preferably involves obtaining one or more equations optimized to fit a real-time running curve dataset. The dataset may include signal values ​​(e.g., fluorescence signal values) processed as necessary using baseline subtraction and / or curve fitting, as a function of reaction progress parameters (e.g., number of reaction cycles) produced by a nucleic acid analyzer calibrated to determine the amount of target nucleic acid in a known volume of liquid sample. If necessary, the dataset may be normalized using curve parameters or processed using baseline subtraction. In some embodiments, normalization may involve dividing the fluorescence values ​​of the dataset by the curve or the maximum observed fluorescence value of the dataset or some other parameter. Processing can be achieved by applying standard mathematical curve fitting techniques to the dataset to obtain fitted equations that define the curve associated therewith. In some embodiments, the equations used in the curve fitting procedure are preferably nonlinear equations containing two or more, more preferably three or more, more preferably four or more coefficients that can be optimized or determined during the curve fitting procedure. Some highly favorable equations have exactly four coefficients, while others have exactly five. Optimizing the equations to fit the measured amplification indicia can be easily achieved using commercially available software packages such as the SOLVER program, available as an Excel add-in tool for finding optimal values ​​of equations, and solutions to the equations from Microsoft Corporation (Redmond, Washington).

[0118] While other equations can be used in the curve fitting procedure, a particular preferred method uses the four-parameter logistic (4-PL) equation, which has the following form: [ka] In this equation, the dependent variable (y) can represent the fluorescence signal observed or processed as a function of the number of reaction cycles (x). The four coefficients of the equation, which can be optimized by standard procedures, are identified as "a" to "d". It should be understood, of course, that success in using the techniques of this disclosure does not require the use of any particular equation.

[0119] In some embodiments, calculating the first derivative of a real-time running curve dataset involved first fitting the data to an optimized equation (e.g., an optimized 4-PL equation), and then taking the first derivative of the optimized equation. The derivative of the optimized equation is an equation that can be solved for different input cycle values ​​(e.g., x values) to obtain an output representing the calculated derivative. An exemplary equation representing the derivative of the fitted 4-PL equation may have the following form: [ka]

[0120] The derivative of a real-time run curve dataset can be determined according to the disclosed techniques using at least three different approaches. As described elsewhere in this specification, data points (x, y) in the real-time run curve dataset represent reaction progress parameter values ​​(e.g., time or number of cycles) and the magnitude of amplified product production (e.g., fluorescence magnitude). Firstly, the gradient values ​​between adjacent “fitted” data points can be calculated using raw or processed run curve data. Secondly, the equations optimized to fit the real-time run curve dataset in the curve fitting procedure can be solved for selected x values ​​to calculate the corresponding fluorescence values ​​(e.g., y values). This processed run curve data can then be used to calculate the gradient values ​​between adjacent data points. An exemplary equation that can be used to fit the run curve data is given by Equation 1. Thirdly, the equation for the derivative of the equation optimized to fit the real-time run curve dataset can be solved for different x values ​​(e.g., number of cycles) to give the corresponding gradient values. An exemplary equation for the derivative is given by Equation 2. In each of the three cases, the result of the calculation can be expressed in units such as RFU / cycle.

[0121] Alternative equations for curve fitting In particular, the 4-PL equation is preferred for fitting or modeling real-time execution curves, but other mathematical functions can also be used in the procedure with equally good results.

[0122] As those skilled in the art will see, numerous types of equations can be used in the procedures disclosed herein. Examples of symmetric transition functions include, but are not limited to, the sigmoid, Gaussian cumulative, Lorentz cumulative, and cumulative symmetric double sigmoid. Examples of asymmetric transition functions include, but are not limited to, the logistic dose-response (LDR), log-normal cumulative, extreme value cumulative, pulse cumulative, pulse cumulative with power term, Weibull cumulative, asymmetric sigmoid, asymmetric sigmoid inverse asymmetric, cascade formation, and cumulative exponential modified Gaussian. Furthermore, as detailed herein, simple linear and nonlinear equations such as multi-degree polynomials, powers, exponential and logarithmic functions can be used to model real-time data with subsequent adjustments to baseline coefficients. Kinetic functions with baseline coefficients can be used similarly. Typical fundamental kinetic equations including baseline coefficients include, but are not limited to, half-order decay and formation, first-order decay and formation, second-order decay and formation, second-order decay and formation (hyperbolic), and cubic decay and formation, variable-order decay and formation. Typical complex kinetic equations including baseline coefficients include, but are not limited to, simultaneous first- and second-order damping and formation, first-order sequential formation, two-component first-order damping, two first-order independent damping and formation, two second-order independent damping and formation, and first- and second-order independent damping and formation. Typical kinetic equilibrium equations including baseline coefficients include, but are not limited to, simple equilibrium (forward and inverse velocity), simple equilibrium (net velocity and equilibrium concentration), compound equilibrium A=B+C, and compound equilibrium A+B=C+D. Typical intermediate kinetic equations including baseline coefficients include, but are not limited to, first-order intermediates and first-order intermediates with equilibrium.

[0123] The disclosed method using fitted curves can be implemented using any of the types of equations listed above. This is because the success of the procedure depends not on the specific equation used, but on its ability to best fit the data. For example, running curve data can be fitted to an equation (e.g., the 4-PL equation), and derivative analysis can be performed using the fitted equation or curve instead of raw fluorescence data. (Examples)

[0124] In the following examples, SNP-containing variant nucleic acids represent "target" nucleic acids (which are desirable to be detected), and wild-type nucleic acids represent "non-target" nucleic acids.

[0125] Example 1 illustrates how undesirable cross-hybridization of a SNP-specific probe to amplified wild-type nucleic acid resulted in a false-positive result indicating the presence of a SNP-containing target nucleic acid in the test sample. Transformation based on the derivative of the real-time nucleic acid amplification run curve favorably distinguishes between true positive and false-positive results when compared to a fluorescence threshold.

[0126] Example 1 The data processing algorithm determines the cross-hybridization result. Samples of six different model nucleic acid targets (in vitro RNA transcripts and linearized DNA plasmids) containing ribosomal nucleic acid sequences with different single nucleotide positions were measured in 8.33 × 10⁶ units. 3 ~8.33×10 5Samples were prepared in pH-buffered sample transport medium (STM) at concentrations in the range of copies / mL. One model target represented a wild-type sequence, and the remaining five model targets represented SNPs (e.g., sequence variants; model mutant sequences). Aliquots of each sample were incubated separately in aqueous solutions containing sequence-specific oligonucleotide reagents to facilitate the capture of nucleic acid targets to magnetic microparticles exhibiting surface oligo-(dT). Magnetic microparticles and bound nucleic acids were separated from the bulk solution by applying a magnetic field. This allowed for the removal of the supernatant from the [captured target]:[magnetic microparticle] complex. The magnetic microparticles were washed twice using a cycle of resuspension in washing buffer followed by magnetic separation. The washed microparticles were then incubated in 50 μL of low ionic strength elution buffer. Magnetic particles were separated again by applying a magnetic field, and the nucleic acid target-containing eluate was collected.

[0127] Measured aliquots of each captured and purified model nucleic acid target were distributed into individual reaction vessels and subsequently combined with aliquots of the amplification reagent mixture. The amplification reagent mixture contained reverse transcriptase, DNA polymerase enzyme, 4 dNTPs, dUTP, inorganic salts, trehalose, and EDTA. The reagent mixture further contained a pair of opposingly positioned primers for amplifying six model nucleic acid targets using a common primer binding site for all target nucleic acid templates. In addition, the reagent mixture contained six hydrolysis probes of the same length. It should be noted that the use of the probe set was merely a design choice in the procedure. Probes of different lengths, each with a sequence complementary to a different SNP, could have been used instead. Each probe had an energy-transfer relationship between a fluorophore and a quencher, and each probe had a nucleotide sequence precisely complementary to only one of the model nucleic acid amplification products. The probe complementary to the amplification product representing the wild-type sequence contained the CalRed610 fluorophore and the BHQ-2 quencher moiety (Biosearch Technologies, Inc.; Petaluma, California). Each of the five probes complementary to the SNP variant contained the FAM fluorophore (i.e., the same fluorophore) and the BHQ-1 quencher moiety (Biosearch Technologies, Inc.; Petaluma, California). A typical arrangement of primers and probes used for detecting the amplification product is schematically illustrated in Figure 1. Real-time PCR amplification and detection reactions were performed using an automated Panther Fusion® System (Hologic, Inc.; San Diego, California), with thermal cycling and fluorescence monitoring. The Panther Fusion® System served as a typical nucleic acid analyzer in this procedure. Only one model nucleic acid target was used as a template to prime each amplification reaction, but all six hydrolysis probes were included.The selection of labels used for the probes meant that fluorescence resulting from the hydrolysis of the SNP-specific probe (used to detect the model variant sequence) could be detected in one fluorescence channel of the instrument, but was substantially undetectable in a different fluorescence channel used to detect the hydrolysis of the wild-type probe. Similarly, fluorescence resulting from the hydrolysis of the wild-type probe could be detected in one channel of the instrument, but was substantially undetectable in a different channel used to detect the fluorescence resulting from the hydrolysis of the SNP-specific probe. This allowed all probes to be used in the same reaction mixture while still enabling distinction between fluorescence resulting from the hydrolysis of the SNP-specific probe and fluorescence resulting from the hydrolysis of the wild-type probe. A negative control without the model nucleic acid target was amplified in two replicates. All other trials were amplified in four replicates. Fluorescence readings were collected at each cycle of the PCR amplification procedure to obtain a run curve showing fluorescence as a function of the number of reaction cycles. The graph results presented in Figures 2A and 2B illustrate how false-positive detection of SNP-containing target nucleic acids was substantially eliminated using the disclosed techniques with a real-time nucleic acid amplification platform and derivative analysis.

[0128] Figure 2A shows the run curve representing fluorescence as a function of reaction cycles (starting at cycle number 10), where fluorescence was induced by a FAM-labeled SNP-specific probe in an amplified reaction mixture containing one of the following: (1) 8.33 × 10 5 or 8.33 × 10 3 Nucleic acid targets containing SNPs at a rate of copies / mL (e.g., model variant sequences); (2) 8.33 × 10 5 or 8.33 × 10 3(3) A wild-type nucleic acid target at a concentration of copies / mL; or a negative control with both SNP-containing nucleic acid and wild-type nucleic acid completely omitted. The horizontal fluorescence threshold line shown in the figure was established to achieve a balance between sensitivity and specificity for the detection of the SNP-containing nucleic acid target. As those skilled in the art will know, such thresholds are conventionally selected to maximize correct results (e.g., true positive and true negative) while minimizing false results (e.g., false positive and false negative). This approach can be used to establish a threshold that can be used for subsequent analyses, and the threshold is referred to as the “predetermined threshold” in subsequent assays. As expected, the negative control reaction produced only very low levels of fluorescence, remaining below the horizontal threshold line used for scoring the detection of the amplified product, and therefore no Ct value was produced. Ideally, only run curves with a fluorescence signal magnitude above the horizontal line in Figure 2A (i.e., this intersection is the Ct value) can indicate the presence of the SNP-containing nucleic acid target. As evidenced by the run curve with an increased magnitude above the horizontal threshold line, the SNP-containing nucleic acid target was clearly amplified and detected in the reaction mixture. Importantly, the run curve of the reaction mixture containing the wild-type template nucleic acid was also observed to rise above the horizontal threshold between approximately cycle numbers 34 and 42. The examination in Figure 2A reveals that SNP-containing nucleic acid targets at lower test concentrations also rose above the horizontal threshold in almost the same cycle number range. This means that it was not possible to distinguish between the SNP-containing nucleic acid variant and the wild-type nucleic acid target based on the presence or absence of a Ct value (i.e., the point where the run curve intersects the threshold) in this cycle number range. False-positive errors resulting from cross-hybridization of SNP-specific probes against amplified wild-type nucleic acid targets that are non-complementary at a single nucleotide position can have devastating consequences in medical diagnostic applications.

[0129] Figure 2B illustrates a solution to the problem identified in Figure 2A. The plot in Figure 2B shows the mathematical derivative of the run curve results presented in Figure 2A. Generally speaking, the purpose of derivative analysis was to impose a standard on the run curve shape, in addition to the fluorescence magnitude, to establish the presence of SNP-containing nucleic acid targets in the test sample undergoing amplification. In this specification, the first derivative of the run curve is used to illustrate the disclosed technique, but second, third, or even higher-order derivatives may be used instead of the first derivative for evaluation. In the illustrations, any calculated derivative with a magnitude greater than the threshold (shown as a horizontal line) indicated the presence of an SNP-containing amplification product in the reaction mixture. Any trial resulting in a calculated derivative with a magnitude less than the threshold indicated that no SNP-containing amplification product was present in the reaction mixture.

[0130] Example 2 further illustrates how signals resulting from unwanted cross-hybridization between labeled SNP-specific probes and mismatched wild-type targets were distinguished from signals resulting from hybridization of SNP-specific probes to amplified variants containing SNPs. In this procedure, a collection of five detectably labeled probes, each having a sequence specific to five individual SNPs, was used in a real-time amplification reaction to amplify either the corresponding variant target ("SNP target") or a wild-type target not precisely complementary to any of the five probe sequences, one at a time. Fluorescent signals resulting from hybridization of labeled SNP-specific probes to mismatched wild-type target amplicons were identified using analysis based on the derivative of the run curve. All SNP-specific probes had the same fluorescent label (e.g., first fluorescent label). Wild-type target amplicons were detected using a control procedure with a wild-type probe having a second fluorescent label (data not shown). In some embodiments, the signal resulting from the second fluorescent label is not reported to the end-user of the assay. The techniques disclosed below illustrate how specific target nucleic acids (e.g., SNP-containing targets or variant sequences) can be detected and identified from closely matched non-target (e.g., wild-type) sequences that may be sensitive to cross-hybridization of labeled probes. This approach advantageously avoids the need to redesign oligonucleotide primers and probes to enable identification. Furthermore, the procedures disclosed below enable the detection of both variant and wild-type sequences using only labeled probes specific to the variant target nucleic acid. In certain preferred embodiments, the procedures are used to detect the presence or absence of a specific type of target nucleic acid (e.g., variant or mutant target nucleic acid).

[0131] Example 2 Derivative-based analysis identifies closely related target sequences detected in real-time nucleic acid amplification procedures. Real-time nucleic acid amplification procedure Each IVT corresponds to the same ribosomal nucleic acid sequence, but differs in the position of a single nucleotide. Six different in vitro transcript (IVT) samples were prepared using 8.33 × 10⁻⁶ samples. 3 ~8.33×10 6 Samples were prepared in pH-buffered sample transport medium (STM) at concentrations in the range of copies / mL. One of the IVTs represented a wild-type sequence, and the remaining five IVTs represented SNPs or variant sequences. Aliquots of each sample were incubated separately in an aqueous solution containing a sequence-specific oligonucleotide reagent to promote the capture of IVTs to magnetic microparticles. Magnetic microparticles and bound nucleic acids were separated from the bulk solution by applying a magnetic field. This allowed for the removal of the supernatant from the [captured target]:[magnetic microparticle] complex. The magnetic microparticles were washed twice using a cycle of resuspension in washing buffer followed by magnetic separation. The washed microparticles were then incubated in 50 μL of low ionic strength elution buffer. Magnetic particles were separated again by applying a magnetic field, and the IVT-containing eluate was collected.

[0132] Measured aliquots of each captured and purified IVT were distributed into individual reaction vessels and subsequently combined with aliquots of the amplification reagent mixture. The amplification reagent mixture contained reverse transcriptase, DNA polymerase enzyme, 4 dNTPs, dUTP, inorganic salts, trehalose, and EDTA. The reagent mixture further contained a pair of opposingly positioned primers for amplifying six IVTs using a common primer binding site for all of the target nucleic acid templates. In addition, the reagent mixture contained six hydrolysis probes of the same length. The nucleotide sequences of the probes were the same as those of the probes in Example 1, although the probes in this example contained several arbitrary nucleotide modifications. Each probe had an energy-transfer relationship between a fluorophore and a quencher, and each probe had a nucleotide sequence that was exactly complementary to only one of the IVT amplification products. In this example, the probe complementary to the amplification product representing the wild-type sequence contained a FAM fluorophore and a BHQ-1 quencher moiety. Five probes complementary to the SNP variant contained a CalRed610 fluorophore and a BHQ-2 quencher moiety. Real-time amplification and detection reactions were performed using an automated Panther Fusion® System (Hologic, Inc., San Diego, California), with thermal cycling and fluorescence monitoring. Emission from the CalRed610 fluorophore was monitored using the ROX detection channel of the Panther Fusion® System, which served as a typical nucleic acid analyzer in this procedure. Each amplification reaction was primed using only one of the IVT-targeted nucleic acid templates, but contained all six detectably labeled hydrolysis probes. The selection of labels used for the probes meant that fluorescence resulting from the hydrolysis of the SNP-specific probe (i.e., used to detect the model variant sequence) could be detected in one channel of the instrument ("Channel 1"), but was substantially undetectable in a different channel ("Channel 2") used to detect the hydrolysis of the wild-type probe. Similarly, fluorescence resulting from the hydrolysis of the wild-type probe could be detected in Channel 2, but was substantially undetectable in Channel 1.Negative controls, excluding IVT, were amplified in two replicates. All other trials were amplified in four replicates.

[0133] Quantitative results collected using a fluorometer optically connected to the reaction mixture undergoing amplification were processed to construct a run curve (fluorescence measured as a function of the number of reaction cycles), the derivative of the run curve was calculated, the magnitude of the calculated derivative was calculated, and the maximum value of the calculated magnitude was determined. These procedures were performed using a programmed computer that is a component of the instrument used for nucleic acid amplification with real-time fluorescence monitoring of amplification product formation, or that communicates with it. The first derivative was calculated, and the maximum value of the magnitude of the first derivative was determined for the run curve of channel 1. As described above, the Ct value was determined as the point at which the fluorescence run curve exceeds a threshold (i.e., fluorescence threshold or cutoff). If the calculated derivative value exceeded a second threshold (i.e., derivative threshold), it was determined that SNP-containing nucleic acid was present in the amplification reaction. If the derivative did not exceed the second threshold at any point, the SNP-containing target nucleic acid was not present in the amplification reaction. To simplify the presentation of results in tabular form, the presence or absence of variant nucleic acid targets in the reaction mixture was determined by comparing the maximum value of the calculated run curve derivative with the threshold.

[0134] As demonstrated by the results presented in Table 1, derivative analysis improved the specificity of variant sequence detection by distinguishing between cycle-dependent signals arising from the amplification of variant and non-variant (i.e., wild-type) target nucleic acid templates. Examination of the results in the table (see column 4) reveals that the mean first derivative maximums of variants 1–5 were clearly separated from the mean first derivative maximums of tests containing wild-type nucleic acid targets. For example, a threshold of 100 RFU / cycle distinguished the two groups of these results. Any first derivative maximum above the 100 RFU / cycle threshold was judged to indicate the detection of variant target nucleic acid. Indeed, tests containing any of variant IVT templates 1–5 met this criterion and were therefore determined to be positive for the presence of variant target nucleic acid templates containing SNPs. In tests containing only negative controls and wild-type templates (i.e., no SNPs), uniform first derivative maximums below the threshold were obtained, indicating the absence of variant template nucleic acids. This favorably excluded false positive results attributable to cross-hybridization between SNP-specific probes and wild-type amplification products. Clearly, if the maximum value of the first derivative did not exceed the threshold required for scoring positive detection, then other first derivative values ​​did not exceed that threshold. A similar protocol, based on determining whether the first derivative of the wild-type run curve obtained by monitoring the FAM-labeled probe (detected in channel 2 of the nucleic acid analyzer) exceeded a threshold, could be used to assess the presence or absence of wild-type template nucleic acid. In this case, different predetermined thresholds (i.e., 250 RFU / cycle) were used to detect the presence of amplified wild-type template nucleic acid.

[0135] The results presented in Table 1 also demonstrate a method for confirming the presence of either variant or wild-type target nucleic acids by using Ct values ​​determined using signals induced solely by hydrolysis of SNP-specific probes, in combination with derivative results. For example, Ct values ​​were determined by evaluating channel 1 fluorescence measured during amplification against a set threshold of 250 RFU. Detecting a Ct value meant that either variant or wild-type nucleic acids were present in the reaction mixture, without distinguishing between the two possibilities. Similarly, the maximum value of the first derivative of the fluorescence run curve data was compared to a threshold of 100 RFU / cycle. The calculated presence of Ct was used, along with the calculated maximum value of the run curve first derivative, to distinguish between variant target sequences and wild-type target sequences. More specifically, detection of a Ct value combined with a first derivative maximum value that did not exceed the threshold meant that the reaction contained a wild-type nucleic acid target. Detection of a Ct value combined with a first derivative maximum value that exceeded the threshold meant that the reaction contained a variant nucleic acid target. [Table 1]

[0136] The results from Example 2 demonstrated how data analysis involving the calculation and comparison of the magnitude of the derivative of the real-time run curve increases the specificity of variant sequence detection by distinguishing signals arising from the amplification of variant and wild-type target nucleic acid templates. In tests containing the wild-type template, Ct values ​​were obtained based on channel 1 fluorescence, but the signals contributing to the establishment of those Ct values ​​were due to cross-hybridization between the wild-type amplified product and the variant template-specific SNP probe. If the Ct value had been used as the sole criterion for detecting the SNP-containing variant nucleic acid target, tests primed with the wild-type template would have been incorrectly concluded to be positive for the variant template. Imposing an additional criterion requiring comparison with a threshold of the first derivative of the run curve preferably prevented false-positive assignments. More specifically, the fact that the maximum value of the first derivative of the run curve primed with the wild-type template was below a predetermined threshold of 100 RFU / cycle confirmed that the template was a wild-type target nucleic acid template and not a variant target nucleic acid template. In some embodiments, this latter observation was used to indicate the presence of the wild-type target nucleic acid template in the reaction mixture.

[0137] In summary, the results from Example 2 demonstrated how data analysis involving the calculation and comparison of the magnitude of the derivative of the real-time run curve, combined with threshold-based Ct determination of the run curve as needed, increases the specificity of variant-targeted nucleic acid detection. Amplification reactions producing a channel 1 (CalRed610) run curve with a first derivative maximum value greater than a predetermined threshold (e.g., 100 RFU / cycle) were sufficient to indicate the presence of the variant-targeted nucleic acid template in the reaction mixture. As briefly mentioned above, in some embodiments, the presence of amplified wild-type target nucleic acid was indicated by a channel 2 (FAM) first derivative maximum value greater than a predetermined threshold. In this case, a predetermined threshold of 250 RFU / cycle was used to accommodate the use of different fluorescent labels. In other embodiments (see Table 1 in columns 3 and 4), the detection of a Ct value in the channel 1 (CalRed610) run curve, along with a channel 1 (CalRed610) run curve first derivative maximum value that was below a predetermined threshold, indicated the absence of the variant-targeted nucleic acid in the reaction mixture and the presence of the wild-type target nucleic acid template in the reaction mixture. These approaches allowed for the detection of both variant and wild-type target nucleic acid templates using labeled probes specific to variant sequences only.

[0138] All patents, patent applications, and publications referenced herein represent the level of skill of those skilled in the art to whom this disclosure relates. All patents, patent applications, and publications are incorporated herein by reference in their entirety for any purpose, to the same extent as each individual publication is specifically and individually indicated to be incorporated herein by reference in its entirety for any purpose.

[0139] All compositions, kits, and methods disclosed herein and described in the claims can be prepared and performed without excessive experimentation in light of this disclosure. While this disclosure describes preferred embodiments, it will be apparent to those skilled in the art that variations can be applied without departing from the spirit and scope of this disclosure. All such variations and equivalents, whether currently existing or to be developed in the future, will be considered to be within the spirit and scope of this disclosure.

Claims

1. A method for determining whether a test sample suspected to contain a nucleic acid template for a nucleic acid amplification reaction contains a target nucleic acid, wherein the method is: (a) Obtaining or obtaining a real-time execution curve dataset that includes signal data representing the production of the amplification product in the nucleic acid amplification reaction as a function of the reaction progress parameters, The nucleic acid amplification reaction comprises the steps of using either the target nucleic acid or non-target nucleic acid that may be present in the test sample as a template for producing the amplification product, (b) A step of calculating or having calculated the first derivative of the real-time execution curve dataset, including the magnitude of the first derivative, (c) The step of comparing or comparing the calculated magnitude value of the first derivative with a first threshold, (d) If the calculated magnitude of any of the first derivatives satisfies or exceeds the first threshold, the test sample contains the target nucleic acid, or If none of the calculated magnitudes of the first derivatives satisfy or exceed the first threshold, the test sample does not contain the target nucleic acid. The step of determining or deciding that it is one of the following Methods that include...

2. The real-time execution curve dataset includes fluorescence magnitude readings, and the method is The real-time execution curve dataset is compared with a fluorescence threshold, and it is determined that at least one data point in the real-time execution curve dataset has a fluorescence magnitude exceeding the fluorescence threshold. If it is determined in step (d) that the test sample does not contain the target nucleic acid, then it is determined that the test sample contains the non-target nucleic acid. The method according to claim 1, further comprising:

3. The method according to claim 1 or claim 2, wherein the target nucleic acid and the non-target nucleic acid differ from each other only at a single nucleotide position.

4. The method according to any one of claims 1 to 3, wherein the test sample comprises the target nucleic acid, and the method further comprises the step of quantifying or having quantified the target nucleic acid present in the test sample.

5. The method according to claim 4, wherein the quantification or quantified step includes first determining the maximum value of the first derivative from step (b), and then using the maximum value of the first derivative together with the reaction progress parameter as an indicator of the amount of the target nucleic acid present in the test sample.

6. The method according to any one of claims 1 to 5, further comprising the step of creating a non-temporary record of the results from step (d).

7. The method according to claim 6, wherein the non-temporary record is printed on paper or recorded on a computer-readable storage medium.

8. The method according to any one of claims 4 to 7, further comprising the step of creating a non-temporary record of the results from the quantification step or the quantified step.

9. The method according to claim 8, wherein the non-temporary record is printed on paper or recorded on a computer-readable storage medium.

10. The method according to any one of claims 1 to 9, wherein the reaction progress parameter in step (a) is measured in terms of the number of cycles, the nucleic acid amplification reaction comprises a PCR reaction, and the first threshold is a predetermined threshold.

11. The method according to any one of claims 1 to 9, wherein the reaction progress parameter in step (a) is either a measure of reaction time or a measure of the number of reaction cycles.

12. The method according to any one of claims 1 to 11, wherein step (a) comprises carrying out the nucleic acid amplification reaction and monitoring the synthesis of the amplification product while the nucleic acid amplification reaction is occurring.

13. The method according to any one of claims 1 to 11, wherein step (a) includes receiving the real-time execution curve dataset as a computer-readable data file.

14. The method according to any one of claims 1 to 13, wherein, prior to step (b), the real-time run curve dataset obtained in step (a) is processed using at least one of (i) baseline subtraction, (ii) curve normalization using curve parameters, and (iii) curve fitting.

15. The method of claim 14, wherein the real-time running curve dataset obtained in step (a) is processed using curve fitting, the curve fitting comprising optimizing the coefficients of the equations to yield an optimized equation.

16. The method according to any one of claims 1 to 15, wherein the real-time execution curve dataset includes fluorescence readings measured as a function of the reaction progress parameter, the reaction progress parameter being measured during the reaction cycle.

17. The method according to any one of claims 1 to 16, wherein step (b) comprises performing calculations using a computer, and step (c) comprises performing a comparison using the computer.

18. The method according to any one of claims 1 to 17, wherein the nucleic acid amplification reaction is performed using an automated nucleic acid analyzer, the automated nucleic acid analyzer is configured to isolate nucleic acids from the test sample and then perform the nucleic acid amplification reaction using the isolated nucleic acids, step (b) comprises performing calculations using a computer that communicates with the automated nucleic acid analyzer, and step (c) comprises performing comparisons using the computer.

19. The method according to any one of claims 1 to 18, wherein the first threshold is a numerical constant.

20. The method according to any one of claims 1 to 19, wherein the target nucleic acid is a target nucleic acid isolated from a human pathogen.

21. The method according to claim 20, wherein the human pathogen is either a bacterial pathogen or a viral pathogen.

22. A computer programmed with software instructions for determining whether a target nucleic acid is present in a test sample, wherein, when the software instructions are executed by the computer, the computer will: (a) Receiving a real-time execution curve dataset that includes signal data showing the amplification of the target nucleic acid and non-target nucleic acid in the nucleic acid amplification reaction as a function of the reaction progress parameters, (b) Calculate the first derivative of the real-time execution curve dataset or its processed version, including the magnitude of the first derivative, (c) Comparing the calculated magnitude of the first derivative with a first threshold, (d) If the calculated magnitude of any of the first derivatives satisfies or exceeds the first threshold, the test sample contains the target nucleic acid, or If none of the calculated magnitudes of the first derivatives satisfy or exceed the first threshold, the test sample does not contain the target nucleic acid. To determine that it is one of the following A computer that performs a task.

23. When the aforementioned software instruction is executed by the computer, it further causes the computer to: The real-time execution curve dataset is compared with a fluorescence threshold to determine whether any signal data in the real-time execution curve dataset has a magnitude that satisfies or exceeds the fluorescence threshold. If the computer determines that the size satisfies or exceeds the fluorescence threshold, and if the computer determines in (d) that the test sample did not contain the target nucleic acid, then it is determined that the test sample contains a non-target nucleic acid different from the target nucleic acid. The computer according to claim 22, which causes the computer to perform the following.

24. The computer according to claim 22 or claim 23, wherein the signal data in (a) showing amplification of the target nucleic acid and the non-target nucleic acid includes fluorescence signal data.

25. The computer according to any one of claims 22 to 24, wherein when the software instruction is executed by the computer, it further causes the computer to generate a non-temporary record of the results from (e) and (d).

26. The computer according to any one of claims 22 to 25, wherein when the software instruction is executed by the computer, the computer causes the computer to create the processed version of the real-time execution curve dataset, and then (b) calculate the first derivative of the processed version of the real-time execution curve dataset.

27. The computer according to any one of claims 22 to 25, wherein when the software instruction is executed by the computer, the computer further causes the computer to produce the processed version of the real-time running curve dataset by performing at least one of (i) baseline subtraction, (ii) curve normalization using curve parameters, and (iii) curve fitting, and (b) calculates the first derivative of the processed version of the real-time running curve dataset.

28. The computer according to any one of claims 22 to 27, wherein the first threshold in (c) is a numerical constant.

29. The computer according to any one of claims 22 to 28, wherein the non-temporary record in (e) is stored electronically on a computer hard drive.

30. The computer according to any one of claims 22 to 29, wherein the computer communicates with a thermal cycle device equipped with a fluorometer.

31. A system for determining whether a target nucleic acid is present in a test sample, wherein the system is A nucleic acid analyzer configured to amplify the target nucleic acid and the non-target nucleic acid in a nucleic acid amplification reaction, The nucleic acid amplification reaction produces an amplified product using either the target nucleic acid or the non-target nucleic acid that may be present in the test sample as a template. The nucleic acid analyzer monitors the synthesis of the amplification product in the nucleic acid amplification reaction as a function of the reaction progress parameters, thereby generating a real-time execution curve dataset including signal data as a function of the reaction progress parameters. A computer communicating with the nucleic acid analyzer, wherein the computer is programmed with a set of software instructions, and the set of software instructions is provided to the computer. (a) Calculate the first derivative of the real-time execution curve dataset or its processed version, including the magnitude of the first derivative, (b) Comparing the calculated magnitude of the first derivative with a first threshold, (c) If the calculated magnitude of any of the first derivatives satisfies or exceeds the first threshold, the test sample contains the target nucleic acid, or If none of the calculated magnitudes of the first derivatives satisfy or exceed the first threshold, the test sample does not contain the target nucleic acid. To determine that it is one of the following, (d) To generate a non-temporary record of the results from (c) and To have a computer perform this task A system equipped with these features.

32. The set of software instructions is further provided to the computer, The real-time execution curve dataset is compared with a fluorescence threshold to determine whether any signal data in the real-time execution curve dataset has a magnitude that satisfies or exceeds the fluorescence threshold. If the computer determines that the size satisfies or exceeds the fluorescence threshold, and if the computer determines in (d) that the test sample did not contain the target nucleic acid, then it is determined that the test sample contains a non-target nucleic acid different from the target nucleic acid. The system according to claim 31, which causes the following to be performed.

33. The system according to claim 31 or 32, wherein the set of software instructions further causes the computer to calculate the amount of the target nucleic acid contained in the test sample.

34. The set of software instructions further causes the computer to create the processed version of the real-time running curve dataset by performing at least one of (i) baseline subtraction, (ii) curve normalization using curve parameters, and (iii) curve fitting. (a) comprises calculating the first derivative of the processed version of the real-time execution curve dataset, The system according to any one of claims 31 to 33.

35. The system according to any one of claims 31 to 34, wherein the computer is a standalone computer that is not physically connected to the nucleic acid analyzer.

36. The system according to any one of claims 31 to 35, wherein the computer communicates with an electronic storage device, and the electronic storage device stores the electronic format of the non-temporary records generated by the computer.

37. The system according to any one of claims 31 to 36, wherein the computer communicates with a printer that generates the non-temporary records.

38. The system according to any one of claims 31 to 37, wherein the nucleic acid analyzer comprises a fluorometer for detecting a fluorescent signal produced in the nucleic acid amplification reaction, and the fluorometer is used to monitor the synthesis of the amplification product in the nucleic acid amplification reaction.

Images