Quantification of polynucleotide analytes from dried samples

Abstract

Methods, systems, and software products are presented that are useful for determining the concentration of an analyte in a fluid sample used to prepare a dried sample, which serves as the source of the analyte in a detection and quantification procedure. Particularly demonstrated is the use of dried blood spots to quantify polynucleotide analytes.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 946,270, filed December 10, 2019, and U.S. Provisional Patent Application No. 62 / 945,685, filed December 9, 2019, the entire disclosures of which are incorporated herein by reference.

[0002] The present disclosure relates generally to the field of biotechnology. More specifically, the present disclosure relates to methods, systems, and software products for determining the concentration of an analyte in a liquid sample used to prepare a dried sample, which serves as the source of the analyte in a detection and quantification procedure. [Background technology]

[0003] The use of dried body fluid samples, such as dried blood samples, for analyte detection significantly increases the availability of advanced clinical testing by simplifying sample collection, transportation, and processing requirements. Dried blood spots (DBS) represent a specific type of dried sample. More specifically, DBS is a form of biosampling in which 50–70 μl of blood is blotted onto a circle of filter paper, allowed to dry, and then used to detect one or more bioanalytes in the blood sample. This approach allows blood spots to be conveniently prepared, dried, and then shipped to a remote testing location. The location where the blood sample is collected for spotting does not require the resources to perform traditional blood collection. The advantage here is that it makes bioanalyte testing available not only in resource-challenged settings but also in the anonymous donation and home testing categories.

[0004] The use of DBS sampling has many advantages. For example, samples are easy to collect, store, and transport without the need for refrigeration. Sample collection is less invasive than exsanguination because only a very small amount of blood is required. Dried samples can be stable for several months at ambient temperature. Therefore, this type of sample offers a convenient way to provide laboratory access to patients outside of traditional clinical settings. Dried samples can also be used as a source of template for priming in vitro nucleic acid amplification reactions, such as real-time nucleic acid amplification reactions.

[0005] Unfortunately, translating quantitative DBS results (e.g., measured in copies / ml using reconstituted DBS samples) into accurate "wet sample" results (e.g., measured in copies / ml) for different types of liquid or fluid samples (e.g., whole blood or plasma) can be very challenging. Quantitative outputs from nucleic acid analyzers processing liquid samples (e.g., plasma samples) typically differ significantly from quantitative outputs generated using reconstituted DBS samples. Indeed, the efficiency of sample preparation can differ significantly for direct sampling of fluid blood products and reconstituted DBS samples, thereby affecting the amount of native target that enters the amplification and detection pathway.

[0006] If DBS results are used to inform decisions about medical treatment or treatment changes, close correspondence between DBS and wet sample quantitative results can be important. For example, if DBS test results are used to determine antiretroviral treatment failure for HIV-1, according to World Health Organization guidelines (WHO Consolidated Guidelines for the Use of Antiretroviral Medications for the Treatment or Prevention of HIV Infection, 2016), they must be able to detect when the HIV viral load exceeds 1,000 copies / mL in plasma.

[0007] The present disclosure addresses the need to convert results from DBS testing to a wet sample standard, thereby correlating the two results in a manner that is both highly accurate and precise. Summary of the Invention [Means for solving the problem]

[0008] The following embodiments are provided herein:

[0009] Embodiment 1 is a method for quantifying polynucleotide analytes present in a fluid blood sample that is dried to produce a dried blood spot (DBS), the method comprising: (a) performing a nucleic acid amplification reaction using the DBS as a source of template to obtain a measurement result, the measurement result indicating the concentration or amount of the polynucleotide analyte; and (b) multiplying the measurement result by a correction factor to obtain a corrected result, the correction factor being the solution of an equation that specifies the correction factor as a function of the measurement result, thereby quantifying the polynucleotide analyte present in the fluid blood sample.

[0010] Embodiment 2 is a method for quantifying polynucleotide analytes present in a fluid blood sample from which dried blood spots (DBS) have been prepared, the method comprising the steps of: (a) performing a nucleic acid amplification reaction using DBS as a source of template to generate an amplification product and obtain a measurement result, the measurement result indicating the concentration or amount of the polynucleotide analyte; (b) solving an equation to determine a correction factor, the equation specifying the correction factor as a function of the measurement result; and (c) multiplying the measurement result by the correction factor to obtain a corrected result, thereby quantifying the polynucleotide analyte present in the fluid blood sample.

[0011] Embodiment 3 is the method of embodiment 1 or 2, wherein the equation in step (b) comprises a nonlinear equation.

[0012] Embodiment 4 is the method of embodiment 3, wherein the nonlinear equation comprises coefficients optimized in a mathematical curve fitting procedure to define the approximate curve.

[0013] Embodiment 5 is the method of embodiment 4, wherein the nonlinear equation includes four coefficients.

[0014] Embodiment 6 is the method of any one of embodiments 1 to 5, wherein step (a) comprises performing the step using an automated nucleic acid analyzer to amplify the polynucleotide analyte and detect the amplification product as the nucleic acid amplification reaction is occurring.

[0015] Embodiment 7 is the method of embodiment 6, wherein the equation in step (b) is a nonlinear equation created using results obtained from an automated nucleic acid analyzer different from the automated nucleic acid analyzer used to perform the nucleic acid amplification reaction in step (a).

[0016] Embodiment 8 is the method of any one of embodiments 1 to 5, wherein step (a) comprises isolating polynucleotide analytes and then performing the step using an automated nucleic acid analyzer that amplifies the isolated polynucleotide analytes.

[0017] Embodiment 9 is the method of embodiment 8, wherein the automated nucleic acid analyzer further detects synthesis of amplification products as the nucleic acid amplification reaction occurs.

[0018] Embodiment 10 is the method according to any one of embodiments 1 to 9, wherein the measurement result indicates the concentration of the polynucleotide analyte in the plasma sample.

[0019] Embodiment 11 is the method according to any one of embodiments 1 to 10, wherein the nucleic acid amplification reaction is an isothermal nucleic acid amplification reaction.

[0020] Embodiment 12 is the method of embodiment 11, wherein the isothermal nucleic acid amplification reaction is a transcription-associated nucleic acid amplification reaction.

[0021] Embodiment 13 is the method of embodiment 12, wherein the transcription-associated nucleic acid amplification reaction comprises a transcription-mediated amplification (TMA) reaction.

[0022] Embodiment 14 is the method of any one of embodiments 1 to 13, wherein the polynucleotide analyte comprises a segment of a viral genome.

[0023] Embodiment 15 is the method of embodiment 14, wherein the viral genome comprises RNA.

[0024] Embodiment 16 is the method of any one of embodiments 1 to 15, wherein the polynucleotide analyte comprises a segment of the HIV-1 genome.

[0025] Embodiment 17 is the method of any one of embodiments 1 to 16, wherein the fluid blood sample comprises whole blood.

[0026] Embodiment 18 is a computer programmed with software instructions for quantifying polynucleotide analytes present in a dried fluid blood sample to produce a dried blood spot (DBS), the software instructions, when executed by the computer, causing the computer to: (a) receive a measurement result; (b) solve an equation to determine a correction factor, the equation specifying the correction factor as a function of the measurement result; (c) multiply the measurement result by the correction factor to calculate the corrected result; and (d) record the corrected result in a non-transitory form, thereby quantifying the polynucleotide analyte.

[0027] Embodiment 19 is the computer of embodiment 18, wherein the measurement result is determined from the result of a real-time nucleic acid amplification reaction, the real-time nucleic acid amplification reaction being performed using DBS as a source of template for generating an amplification product, and the measurement result indicates the concentration or amount of a polynucleotide analyte.

[0028] Embodiment 20 is the computer of embodiment 18 or 19, wherein both the measurement result and the corrected result are expressed in concentration units.

[0029] Embodiment 21 is the computer according to any one of embodiments 18 to 20, wherein the equation is a nonlinear equation.

[0030] Embodiment 22 is the computer of embodiment 21, wherein the nonlinear equation includes coefficients optimized in a mathematical curve fitting procedure to define the approximate curve.

[0031] Embodiment 23 is the computer of embodiment 22, wherein the nonlinear equation includes four coefficients.

[0032] Embodiment 24 is a computer according to any one of embodiments 18 to 23, wherein the non-transitory form includes storage on a computer-readable memory device.

[0033] Embodiment 25 is a computer according to any one of embodiments 18 to 24, wherein the fluid blood sample comprises whole blood.

[0034] Embodiment 26 is a computer-readable medium containing instructions that, when executed by a computer, cause the computer to perform the method of embodiment 18.

[0035] Embodiment 27 is a system for quantifying polynucleotide analytes that may be present in a test sample, the system including a nucleic acid analyzer comprising: a temperature-controlled incubator; a fluorometer in optical communication with the temperature-controlled incubator, the fluorometer configured to measure production of nucleic acid amplification products contained within the temperature-controlled incubator as a function of time or cycle number; and a computer in communication with the fluorometer, the computer being programmed with software instructions to cause the computer to: (a) calculate a measurement result using measurements from the fluorometer; (b) solve an equation to determine a correction factor, the equation specifying the correction factor as a function of the measurement result; (c) multiply the measurement result by the correction factor to calculate the corrected result; and (d) record the corrected result in a non-transitory form, thereby quantifying target polynucleotide analytes present in the test sample.

[0036] Embodiment 28 is the system described in embodiment 27, wherein the temperature-controlled incubator is configured to maintain a constant temperature.

[0037] Embodiment 29 is the system of embodiment 27, wherein the temperature-controlled incubator is configured for temperature cycling.

[0038] Embodiment 30 is a system according to any one of embodiments 27 to 29, wherein the fluorometer is configured to detect light at a plurality of different wavelengths.

[0039] Embodiment 31 is a system according to any one of embodiments 27 to 30, wherein the temperature-controlled incubator, the fluorometer, and the computer are all essential components of the nucleic acid analyzer.

[0040] Embodiment 32 is the system of any one of embodiments 27 to 31, wherein the measurement result comprises a concentration value of the polynucleotide analyte.

[0041] Embodiment 33 is a method for quantifying an analyte present in a body fluid sample that is dried to produce a dried sample, comprising: (a) performing a reaction using the dried sample as a source of the analyte and obtaining a measurement result, the measurement result indicating the concentration or amount of the analyte; and (b) multiplying the measurement result by a correction factor, the correction factor being the solution of an equation that specifies the correction factor as a function of the measurement result, thereby quantifying the analyte present in the body fluid sample.

[0042] Embodiment 34 is the method of embodiment 33, wherein the equation in step (b) comprises a nonlinear equation.

[0043] Embodiment 35 is the method of embodiment 34, wherein the nonlinear equation comprises coefficients optimized in a mathematical curve fitting procedure to define an approximate curve.

[0044] Embodiment 36 is the method of embodiment 35, wherein the nonlinear equation includes four coefficients.

[0045] Embodiment 37 is the method of any one of embodiments 33 to 36, wherein the analyte is a polynucleotide analyte, and step (a) comprises performing with an automated nucleic acid analyzer that amplifies the polynucleotide analyte and detects the amplification product as the nucleic acid amplification reaction occurs.

[0046] Embodiment 38 is the method of embodiment 33 or 37, wherein the equation in step (b) comprises a nonlinear equation, the nonlinear equation comprising coefficients optimized in a mathematical curve fitting procedure to define an approximate curve, and the nonlinear equation is created using results obtained from an automated nucleic acid analyzer different from the automated nucleic acid analyzer used to perform the nucleic acid amplification reaction in step (a).

[0047] Embodiment 39 is the method of embodiment 37, wherein step (a) comprises isolating polynucleotide analytes and then performing the step using an automated nucleic acid analyzer that amplifies the isolated polynucleotide analytes.

[0048] Embodiment 40 is the method of embodiment 39, wherein the automated nucleic acid analyzer further detects synthesis of amplification products as the nucleic acid amplification reaction occurs.

[0049] Embodiment 41 is the method of any one of embodiments 37, 39, or 40, wherein the measurement indicates the concentration of the polynucleotide analyte in the plasma sample.

[0050] Embodiment 42 is the method according to any one of embodiments 37 to 41, wherein the nucleic acid amplification reaction is an isothermal nucleic acid amplification reaction.

[0051] Embodiment 43 is the method of embodiment 42, wherein the isothermal nucleic acid amplification reaction is a transcription-associated nucleic acid amplification reaction.

[0052] Embodiment 44 is the method of embodiment 43, wherein the transcription-associated nucleic acid amplification reaction comprises a transcription-mediated amplification (TMA) reaction.

[0053] Embodiment 45 is the method of any one of embodiments 37 to 44, wherein the polynucleotide analyte comprises a segment of a viral genome.

[0054] Embodiment 46 is the method of embodiment 45, wherein the viral genome comprises RNA.

[0055] Embodiment 47 is the method of any one of embodiments 37 to 46, wherein the polynucleotide analyte comprises a segment of the HIV-1 genome.

[0056] Embodiment 48 is the method of any one of embodiments 33 to 47, wherein the body fluid sample is selected from the group consisting of a whole blood sample, a plasma sample, a urine sample, and a saliva sample. (Mode for Carrying Out the Invention)

[0057] Introduction and Overview Disclosed herein is an approach for accurately converting quantitative results obtained using dried body fluid samples into corresponding results measured in concentration units for the liquid sample used to create the dried sample. In the exemplary procedure, HIV nucleic acid served as a model analyte. The chemistry used for nucleic acid amplification and detection does not need to be changed to achieve excellent results. This means that a single assay chemistry can be used to quantify polynucleotide analytes using either liquid samples or reconstituted dried samples over a wide dynamic range. Dried blood spot sampling was used to illustrate the technique.

[0058] Rather than modifying the assay chemistry, a numerical "correction factor" (hereafter, CF) multiplier is used to achieve the desired result. The CF can be multiplied by the output, or "measured," result of a quantitative assay using reconstituted DBS as the analyte source. This converts the measurement result to a corresponding concentration (e.g., copies / ml) for different sample types. For example, results obtained using a reconstituted DBS sample can be converted or adjusted to a corresponding concentration for a whole blood sample. Thus, a single assay chemistry (e.g., a single type of amplification and detection reaction mixture, or a single type of assay kit) can be used to amplify and quantify samples regardless of their type (e.g., whole blood, plasma, reconstituted DBS, etc.).

[0059] An important feature of this technology involves the manner in which the CF is determined. During the development of this technology, it was discovered that the required CF is not constant across the quantitative dynamic range of the assay. Instead, the CF varies as a function of the output of a nucleic acid analyzer calibrated using a liquid sample (e.g., when the output can be measured in concentration units). As the amount of analyte present in a dry sample (such as DBS) during reconstitution and testing decreases, the CF required for accurate quantification increases.

[0060] In some embodiments, the CF used as a numerical multiplier is calculated using an equation fitted to the collection of data. In some embodiments, the equation is a nonlinear equation. In other embodiments, the equation can include one or more linear equations. The data used to obtain the fitted equation represents a correction factor calculated as a function of quantitative output values ​​derived from a nucleic acid analyzer calibrated to process liquid samples (such as plasma). In some embodiments, the fitted equation used to determine the CF used on one instrument can be determined on the same instrument. However, it is more convenient, and therefore preferred, to determine the fitted equation using one or more instruments and then use the fitted equation on another instrument (i.e., an instrument not used to determine the fitted equation). For example, the fitted equation can be created by the manufacturer of the assay kit and transferred to and used by a customer or end user on another instrument (sometimes referred to as a "local" instrument).

[0061] In some embodiments, the CF is calculated from multiple fitted curves or lines, and the appropriate curve or line for the CF determination depends on the analyzer output before applying a CF multiplier. Again, the CF is selected as a function of the measured concentration or amount of analyte shown to be present when testing a dried sample (such as a DBS sample) after reconstitution. The selected CF is then multiplied by the measured concentration to generate an adjusted quantitative result that quantifies the analyte.

[0062] definition The following terms have the meanings specified herein unless expressly indicated to have a different meaning.

[0063] The terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, as used herein, "a polynucleotide" is understood to refer to one or more polynucleotides. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0064] As used herein, a "dried body fluid sample" is a sample of a body fluid, such as a sample of whole blood or other blood product, from which the water component of the fluid has been substantially removed. Typically, the body fluid is applied to a solid matrix (e.g., filter paper, glass fiber filter, cloth, flocked swab, sponge material, etc.) prior to removing the water component.

[0065] As used herein, "dried blood spot" (sometimes "DBS") refers to a sample of blood or blood products that is dried before being analyzed for the presence or amount of an analyte. Preferably, the blood sample is applied to a solid matrix and then dried to create or produce the DBS. In some embodiments, the solid matrix is ​​a filter, such as a paper filter or glass fiber filter, a cloth, a flocked swab, or a sponge material. Preferably, the DBS comprises a sample of dried whole blood. Preferred analytes for testing using DBS samples include polynucleotide analytes.

[0066] As used herein, a "reconstituted" sample is a liquid or fluid sample that results from combining a dried biological sample (e.g., DBS) with a liquid (e.g., extraction solution) that dissolves, liquefies, or resuspends the dried biological sample. In some embodiments, the reconstituted sample results from combining or contacting a dried blood spot on a solid support matrix (e.g., a filter paper "card") with an extraction buffer that may include a pH buffer and a surfactant. Thus, the dried blood spot can serve as a source of analyte (e.g., polynucleotide analyte) to be detected when the analyte of the reconstituted sample is used for detection. In some embodiments, the procedure for detecting the polynucleotide analyte of the reconstituted sample may include an in vitro nucleic acid amplification procedure.

[0067] As used herein, "polynucleotide" refers to a chimeric molecule containing either RNA, DNA, or both RNA and DNA. The term also includes molecules containing nucleotide analogs of RNA or DNA.

[0068] As used herein, a "test sample" is any sample investigated for the presence of a specific polynucleotide sequence. Test samples include polynucleotide-containing materials obtained from humans, animals, the environment, 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 saliva, urine, etc.

[0069] As used herein, an "analyte" is a chemical or biochemical species to be detected and / or quantified. For example, a "polynucleotide analyte" refers to a polynucleotide (e.g., a segment of an HIV-1 polynucleotide) that is detected or quantified in a testing procedure.

[0070] As used herein, a "nucleic acid analyzer" (or "polynucleotide analyzer") is an apparatus that amplifies, detects, and quantifies polynucleotide analytes. Certain preferred nucleic acid analyzers include a temperature-controlled incubator (e.g., a block, plate, or chamber), a fluorometer in optical communication with the contents of the temperature-controlled incubator, and one or more computers or processors that process data collected by the fluorometer to quantify the polynucleotide analytes of interest.

[0071] "Analyte polynucleotide standard" refers to a composition containing a known amount of a polynucleotide analyte or fragment thereof. For example, an HIV-1 analyte polynucleotide standard may contain a known number of copies of the HIV-1 genome, an HIV-1 transcript, or an in vitro synthesized transcript representing a portion of the viral genome. A "WHO" standard (e.g., an HIV-1 WHO standard) is an analyte polynucleotide standard of established concentration provided by the World Health Organization.

[0072] A "calibration standard" refers to a composition containing a known or predetermined amount of an analyte polynucleotide standard in combination with a known, fixed amount of an internal calibrator polynucleotide. Two different calibration standards may contain different amounts of a polynucleotide analyte or fragment thereof, but will contain the same amount of an internal calibrator polynucleotide. The polynucleotide analyte of an analyte polynucleotide standard and the internal calibrator polynucleotide are distinguishable from one another, for example, by having different nucleotide base sequences. A testing instrument (e.g., a nucleic acid analyzer) is said to be "calibrated" when a calibration standard is used to ensure that the instrument provides accurate results. For example, an instrument can be calibrated to provide accurate results when processing plasma samples.

[0073] An "amplicon" (sometimes "amplification product") is a polynucleotide product of an amplification reaction in which the target polynucleotide sequence of the polynucleotide analyte served as a template for the synthesis of the polynucleotide copy or amplification product.

[0074] "Amplification" or "nucleic acid amplification" or "polynucleotide amplification," etc., refers to any known procedure for obtaining multiple copies of a target polynucleotide sequence, or its complement, or a fragment thereof, allowing for RNA and DNA equivalents. Amplification of a "fragment thereof" refers to the production of an amplified nucleic acid (i.e., polynucleotide) containing a nucleic acid sequence or its complement that is less than the entire target region. Such fragments can be generated by amplifying a portion of the target nucleic acid, for example, by using amplification oligonucleotides that hybridize to and initiate polymerization at an internal position of the target polynucleotide.

[0075] As used herein, the terms "co-amplification" and "co-amplifying," and variations thereof, refer to a process in which different target polynucleotide sequences are amplified in a single (i.e., the same) amplification reaction. For example, a polynucleotide analyte and an unrelated internal calibrator polynucleotide are "co-amplified" when both polynucleotides are amplified in a reaction performed in a single tube and when both amplification reactions share at least one reagent (e.g., deoxyribonucleotide triphosphates, enzymes, primers, etc.).

[0076] As used herein, "thermal cycling" refers to repeated changes in temperature (i.e., increasing or decreasing temperature) in a reaction mixture. A sample undergoing thermal cycling may shift from one temperature to another, stabilize at that temperature, transition to a second temperature, or return to the starting temperature. Temperature cycling can be repeated as many times as necessary to study or complete a particular chemical reaction of interest.

[0077] "Target" or "target nucleic acid" or "target polynucleotide" refers to a polynucleotide containing a sequence to be amplified, detected, and quantified. The target polynucleotide sequence to be amplified will preferably be located between two oppositely positioned oligonucleotides and will include a portion of the target polynucleotide that is complementary to each of the oligonucleotides.

[0078] "Target nucleic acid sequence" or "target sequence" or "target region" means a specific deoxyribonucleotide or ribonucleotide sequence comprising all or part of the nucleotide sequence of a single-stranded polynucleotide molecule, and complementary deoxyribonucleotide or ribonucleotide sequences thereto.

[0079] "Transcription-associated amplification" refers to any type of polynucleotide amplification that uses an RNA polymerase to generate multiple RNA transcripts from a polynucleotide template. Traditionally, these amplification reactions use at least one primer with a 3' end that can be extended by the activity of a DNA polymerase. One example of a transcription-associated amplification method, called "transcription-mediated amplification" (TMA), generally uses an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a promoter-containing oligonucleotide complementary to the target polynucleotide. Variations of TMA are well known in the art, as disclosed in detail in Burg et al., U.S. Pat. No. 5,437,990; Kacian et al., U.S. Pat. Nos. 5,399,491 and 5,554,516; Kacian et al., PCT No. WO 93 / 22461; Gingeras et al., PCT No. WO 88 / 01302; Gingeras et al., PCT No. WO 88 / 10315; Malek et al., U.S. Pat. No. 5,130,238; Urdea et al., U.S. Pat. Nos. 4,868,105 and 5,124,246; McDonough et al., PCT No. WO 94 / 03472; and Ryder et al., PCT No. WO 95 / 03430. Other transcription-associated amplification methods that use only a single primer that can be extended by DNA polymerase, such as those disclosed in U.S. Pat. No. 7,374,885, are specifically included in the definition and are highly preferred for use in connection with the methods disclosed herein.

[0080] As used herein, an "oligonucleotide" or "oligomer" is a polymeric chain of at least two, and generally about 5 to about 100, chemical subunits, each of which contains a nucleotide base moiety, a sugar moiety, and a linking 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), although other rare or modified nucleotide bases capable of hydrogen bonding are well known to those of skill in the art. Oligonucleotides may optionally contain analogs of any of the sugar moieties, base moieties, and backbone components. Preferred oligonucleotides of the present invention fall within 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.

[0081] "Amplification oligonucleotide" or "amplification oligomer" refers to an oligomer that hybridizes to a target polynucleotide or its complement and participates in a polynucleotide amplification reaction. Examples of amplification oligomers include primers containing a 3' end that are extended as part of the amplification process, but also oligomers that are not extended by a polymerase (e.g., 3' block oligomers) but may participate in or facilitate efficient amplification from the primer. Preferred size ranges for amplification oligomers include those about 10 to about 80 nucleotides in length, or 10 to about 60 nucleotides in length, containing at least about 10 contiguous bases, more preferably at least 12 contiguous bases, complementary to a region of the target polynucleotide sequence (or its complement). The contiguous 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 amplification oligomer binds. Amplification oligomers may optionally contain modified nucleotides or analogs, or additional nucleotides that participate in the amplification reaction but are not complementary to or contained in the target polynucleotide. Amplification oligomers that are 3' blocked but can hybridize to the target polynucleotide and provide an upstream promoter sequence that serves to initiate transcription are referred to as "promoter provider" oligomers.

[0082] A "primer" is an amplification oligomer that hybridizes to a template polynucleotide and has a 3'-OH end that can be extended by a DNA polymerase. The 5' region of the primer can be non-complementary to the target polynucleotide (e.g., a promoter sequence), resulting in an oligomer referred to as 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 function as a promoter primer. Similarly, any promoter primer can be modified by removing the promoter sequence or by synthesizing it without the promoter sequence and still function as a primer.

[0083] As used herein, a "probe" is an oligonucleotide that specifically hybridizes to a target sequence of a polynucleotide, preferably an amplified polynucleotide, under conditions that promote hybridization to form a detectable hybrid. Certain preferred probes include a detectable label (e.g., a fluorescent or chemiluminescent label).

[0084] As used herein, " time-dependent " monitoring of polynucleotide amplification or " real-time " monitoring of polynucleotide amplification refers to the process in which the amount of amplicon present in polynucleotide amplification reaction is measured as a function of reaction time or cycle number, and then used to determine the starting amount of template present in the reaction mixture at the time the amplification reaction is initiated.For example, the amount of amplicon can be measured before starting each full cycle of amplification reaction, including thermal cycles, such as PCR.Alternatively, isothermal amplification reaction, which does not require physical intervention to initiate the transition 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.

[0085] As used herein, a "growth curve" refers to the characteristic pattern of appearance of a synthetic product, such as an amplicon, in a reaction as a function of time or cycle number. A run curve is conveniently represented as a two-dimensional plot of either time or cycle number (x-axis) against some measure of output, such as fluorescence readings (y-axis). Some, but not all, run curves have a sigmoidal shape.

[0086] As used herein, the "baseline phase" of a growth curve refers to the initial phase of the curve in which the amount of product (e.g., amplicon) increases at a substantially constant rate that is less than the rate of increase characteristic of the growth phase of the growth curve (which may have a log-linear profile). The baseline phase of a growth curve typically has a very shallow slope, often approaching zero.

[0087] As used herein, the "growth phase" of a growth curve refers to the portion of the curve where measurable product increases substantially with time.The transition from baseline phase to growth phase in a typical polynucleotide amplification reaction is characterized by the appearance of amplicons at a rate that increases with time.The transition from the growth phase to the plateau phase of a growth curve begins at the inflection point where the rate of appearance of amplicons begins to decrease.

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

[0089] As used herein, the phrase "amplification indicia" refers to a feature of a real-time run curve that indicates a predetermined level of progression in a polynucleotide amplification reaction. Such indicia are generally determined by mathematical analysis of the run curve, sometimes referred to as a "growth curve." The curve displays a signal (e.g., a fluorescent reading) of measurable intensity related to the amount of amplicon present in the reaction mixture as a function of time, number of cycles, etc.

[0090] As used herein, "threshold-based indicia of amplification" refers to an indicia of amplification that measures the time or cycle number when a growth curve signal crosses a given value or threshold. Determining TTime is an example of a threshold-based indicia of amplification, while determining TArc and OTArc are examples of non-threshold-based indicia of amplification.

[0091] As used herein, a "time-dependent" amplification indicia generally refers to an amplification indicia (e.g., a reaction progress parameter) measured in time units (e.g., minutes). Time-dependent amplification indicia are commonly used to monitor the progress of isothermal polynucleotide amplification reactions that are not characterized by distinct "cycles." TTime, TArc, and OTarc are all examples of time-dependent amplification indicia.

[0092] As used herein, an "internal calibrator" (sometimes referred to herein as "IC") is a polynucleotide that can be amplified in an in vitro polynucleotide amplification reaction and is distinguishable from a polynucleotide analyte co-amplified in the same reaction. "Internal" means that the calibrator polynucleotide is amplified, detected, and quantified in the same reaction mixture as the polynucleotide analyte or fragment thereof. Generally speaking, the amount or concentration of the internal calibrator will be constant across the various reactions used to generate the calibration curve and quantify the polynucleotide analyte. Preferably, the constant amount or concentration of the internal calibrator is a known amount or concentration of the internal calibrator. In certain preferred embodiments, the internal calibrator and the polynucleotide analyte are co-amplified in an in vitro polynucleotide amplification reaction using one or more different amplification oligomers or primers. For example, the analyte and internal calibrator polynucleotides used in the examples detailed below were amplified using amplification oligonucleotides that were not shared. In other preferred embodiments, the internal calibrator and the polynucleotide analyte are co-amplified in an in vitro polynucleotide amplification reaction using one or more identical amplification oligomers or primers.

[0093] 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 whose value may be freely chosen without consideration of the values ​​of the other variables) in which each input value of the independent variable is related to exactly one output value of the dependent variable. The conventional notation for an equation relating y values ​​(i.e., the dependent variable) "as a function" of x values ​​(i.e., the independent variable) is y=f(x).

[0094] As used herein, a "computer" is an electronic device that can receive and process input information and generate output. A computer can 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 polynucleotide target and monitors the synthesis of amplification products as a function of reaction cycle number or time). Specifically included within this term are embedded processors that reside within the analyzer instrument and contain embedded software instructions (sometimes referred to as "firmware").

[0095] As used herein, "optimizing" or "fitting" an equation refers to the process of obtaining numerical values ​​for coefficients in an equation to find an expression that "fits" or approximates experimental measurements, as is commonly done in mathematical modeling or curve-fitting procedures. Typically, the optimized equation will define a best-fit curve.

[0096] As used herein, the terms "optimized equation" and "fitted equation" are alternative references to an equation with fixed numerical values ​​for the coefficients as a result of an optimization procedure. A "fitted" curve results from optimizing the equation.

[0097] "Local" means relative to the end user. For example, a local instrument refers to the instrument of the end user. A local calibration plot refers to a calibration plot that uses results obtained by the end user, for example, by performing an amplification reaction on a local instrument.

[0098] A "kit" generally refers to a packaged combination of materials intended for use in combination with one another. Kits according to the invention may include instructions or other information in "tangible" form (e.g., printed information, electronically recorded on a computer-readable medium, or recorded on a machine-readable medium such as a bar code for storing numerical values).

[0099] "Consisting essentially of" means that the invention may include additional components, compositions, or method steps that do not substantially change the basic and novel characteristics of the invention. Any components, compositions, or method steps that substantially affect the basic and novel characteristics of the invention would be excluded from the scope of this term. [Brief explanation of the drawings]

[0100] [Figure 1] This figure shows a plot of calculated correction factor (CF) values ​​(vertical axis) as a function of the "observed" or "measured" concentration (measured in copies / ml) of HIV-1 polynucleotide analyte (horizontal axis). Open data points represent calculated CF values ​​at various target concentrations measured in a procedure that amplified polynucleotide analytes from reconstituted DBS samples. A solid curve was fitted to the collected data points by mathematically optimizing the 4-PL equation. DETAILED DESCRIPTION OF THE INVENTION

[0101] Description of Specific Embodiments The currently disclosed technology was demonstrated using the Aptima™ HIV-1 QuantDx assay from Hologic, Inc. (Marlborough, MA) as a model system. This viral load monitoring assay is both sensitive and specific and can be used to assess response to antiretroviral therapy by monitoring changes in HIV-1 RNA concentrations. The assay is an in vitro polynucleotide amplification test for the detection and quantification of human immunodeficiency virus type 1 (HIV-1) RNA groups M, N, and O that can be run on the fully automated Panther™ system (Hologic, Inc.). The system that performs the viral load assay is calibrated to output the viral concentration measured in copies / ml using a 500 μl test sample. For example, the model assay can be used to monitor the effectiveness of antiviral therapy by measuring changes in HIV-1 RNA concentrations in plasma. It is advantageous to accurately correlate quantitative results obtained using DBS and plasma samples using the same calibration and reagents, and target capture, amplification, and detection protocols.

[0102] The model assay involves three major steps performed in a single tube on an automated Panther system for polynucleotide analysis: target capture, target amplification by transcription-mediated amplification, and detection of the amplified product (amplicon) with a fluorescently labeled hybridization probe (torch). During target capture, the specimen is treated with detergent to solubilize the viral envelope, denature proteins, and release viral genomic RNA. Capture oligonucleotides, if present in the test specimen, hybridize to highly conserved regions of the HIV-1 genome. The hybridized target is then captured on magnetic microparticles, which separate it from the specimen in a magnetic field. Washing steps remove excess components from the reaction tube. Target amplification is then performed via TMA, a transcription-mediated polynucleotide amplification method that utilizes two enzymes: MMLV (Moloney murine leukemia virus) reverse transcriptase and T7 RNA polymerase. Reverse transcriptase is used to generate a DNA copy of the target sequence (containing a promoter sequence for T7 RNA polymerase). T7 RNA polymerase generates multiple copies of an RNA amplicon from a DNA copy template. The model assay utilizes the TMA method to amplify two regions of HIV-1 RNA (pol and LTR). Amplification of these specific regions is achieved using specific primers designed to amplify HIV-1 groups M, N, and O. The primer design and dual-target approach ensure accurate detection and quantification of HIV-1. Detection is achieved using single-stranded polynucleotide torches, which are present during target amplification and specifically hybridize to the amplicon in real time. Each torch contains a fluorophore and a quencher. When the torch is not hybridized to the amplicon, the quencher is in close proximity to the fluorophore, suppressing fluorescence. When the torch binds to the amplicon, the quencher moves further away from the fluorophore and emits a signal of a specific wavelength when excited by a light source. As more torches hybridize to the amplicon, a higher fluorescent signal is generated. The time it takes for the fluorescent signal to reach a specified threshold is proportional to the starting HIV-1 concentration.Each reaction includes an internal calibrator / internal control (IC) that co-amplifies with the HIV-1 analyte to control for variations in sample processing, amplification, and detection. Sample concentrations are determined by the Panther system software using the HIV-1 and IC signals from each reaction and comparing them to calibration information. Determined concentrations are calibrated against HIV-1 in plasma samples, not reconstituted DBS samples. The concentrations determined here are alternatively referred to as "observed" or "measured" results.

[0103] There are various types of DBS samples, each of which can be used to perform the quantitative techniques described herein. Blood samples can be obtained from infants using standard heelstick or fingerstick techniques. Here, the infant's skin surface is disinfected and then pricked with a sterile needle or lancet. Next, 3–5 drops of blood are added to each of multiple (e.g., 5) spots on the DBS "card," ensuring that the entire surface of the circle is completely covered. The fingerstick technique can also be used to obtain DBS samples in adults. Conveniently, whole blood can be stored at 2°C–30°C for up to 24 hours before application to the DBS card. In this case, for example, 70 μl of stored whole blood can be applied to the center of the filter circle on the DBS card using a calibrated 200 μl pipette. Regardless of how the spotted blood sample is obtained, it can be allowed to dry at ambient temperature for 4–24 hours. Individual cards containing the dried sample can then be placed in an envelope (e.g., glassine envelope) for storage or transportation. Multiple glassine envelopes can be packaged in a resealable plastic bag along with one or more desiccant packs. Even when packaged, DBS samples can be stored or shipped at ambient temperature for subsequent processing.

[0104] Preferred Polynucleotide Amplification Methods Examples of in vitro polynucleotide amplification methods useful in connection with the present technology include, but are not limited to, transcription-mediated amplification (TMA), single primer nucleic acid amplification, nucleic acid sequence-based amplification (NASBA), polymerase chain reaction (PCR), strand displacement amplification (SDA), self-sustained sequence replication (3SR), DNA ligase chain reaction (LCR), and amplification methods that use self-replicating polynucleotide molecules and replicative enzymes (such as MDV-1 RNA and Q-beta enzymes). Methods for carrying out these various amplification techniques can be found in U.S. Patent No. 5,399,491, U.S. Patent Application No. 11 / 213,519, European Patent Application No. EP0525882, U.S. Patent Nos. 4,965,188, 5,455,166, Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878 (1990), International Patent Application No. 89 / 09835, U.S. Patent No. 5,472,840, and Lizardi et al., Trends Biotechnol. 9:53-58 (1991), respectively. The disclosures of these documents describing methods for carrying out nucleic acid amplification reactions are incorporated herein by reference. Thus, although the model system used to demonstrate the correction factor adjustment technique used TMA as the amplification reaction mechanism, alternative amplification reaction mechanisms can be used with similarly good results.

[0105] Examples of preferred real-time quantitative methods Generally speaking, real-time polynucleotide amplification and detection procedures involve monitoring the production of amplification reaction products as the amplification reaction occurs. As shown above, any number of different amplification methods can be used to create amplification products. In some embodiments, the synthesis of amplification products as a function of time or cycle number is indicated by detecting a fluorescent signal generated in the amplification reaction mixture. Examples of methods useful for calibrating instruments performing real-time amplification reactions are described in U.S. Patent Nos. 9,932,628 and 9,976,175, the disclosures of which are incorporated herein by reference for all purposes. The success of these methods is independent of the manner in which the run curve of the real-time procedure is obtained. In other words, different signs of amplification can be used to determine when the amplification reaction has reached a desired threshold level of amplification progression.

[0106] Before applying CF adjustment to the data, various indicia of amplification can be used to quantify the analyte. Real-time amplification and detection for quantifying polynucleotide analytes are highly preferred for use in conjunction with the disclosed CF adjustment techniques, and alternative data processing procedures are amenable to successful results in each case. For example, mathematical and computing techniques familiar to those skilled in the art can be used to identify the time of occurrence of the maximum of the first derivative or the maximum of the second derivative of a real-time running curve. Approaches for determining these features of a growth curve are detailed in Wittwer et al., U.S. Pat. No. 6,503,720, the disclosure of which is incorporated herein by reference. Another useful approach involves calculating the derivative of the running curve, identifying features of the growth curve, and then determining a threshold time or cycle number corresponding to the derivative feature. Such techniques are disclosed in U.S. Pat. No. 6,783,934, the disclosure of which is incorporated herein by reference. Still other useful indicia of amplification include "TTime" and "TArc." Different approaches to determining the TArc value use a directionally similar vector (i.e., resulting in a value identified simply as "TArc") and a directionally opposite vector (i.e., resulting in a value identified as "OTArc"). Yet other techniques involve identifying the cycle threshold (e.g., "Ct") as the time or cycle number during the reaction at which the signal, preferably the fluorescent signal, equals a static threshold (e.g., a predetermined static threshold).

[0107] Preferred Systems and Apparatus The methods disclosed herein are conveniently implemented using a computer or similar processing device (hereinafter "computer"). In different preferred embodiments, the software or machine-executable instructions for executing the algorithm can be loaded or stored in the memory component of a stand-alone computer or in the memory component of a computer linked to a device used for monitoring, preferably as a function of time, the amount of product being analyzed. In a highly preferred embodiment, the software for performing the correction factor adjustment procedure is stored in the memory component of a computer that is linked to or is an integral part of a device that can monitor the amount of amplicon present in a reaction mixture as a function of time. This includes processing device components (e.g., embedded software) on the electronic circuit board of an automated nucleic acid analyzer.

[0108] In some embodiments, the computer can communicate, either by wired or wireless means, with a fluorometer that detects fluorescent signals, the fluorometer being positioned or configured to monitor fluorescent signals generated in one or more reaction vessels housed within a temperature-controlled incubator, which can be a temperature-controlled block (e.g., a metal block configured to receive and house one or more tubes or a multiwell plate) or a chamber that exposes one or more reaction vessels to controlled temperature conditions.

[0109] In some embodiments, either or both of the controller system for controlling the real-time amplification device and / or the detection system of the real-time amplification device can be coupled to a suitably programmed computer that functions to direct the operation of these devices according to pre-programmed or user-entered instructions. The computer is preferably capable of receiving data and information from these devices, interpreting, manipulating, and reporting this information to the user.

[0110] In some embodiments, the computer can also include appropriate software for receiving user instructions, either in the form of user input into a set of parameter fields or in the form of preprogrammed instructions (e.g., preprogrammed for a variety of different specific operations). The software then translates these instructions into an appropriate language to direct the operation of the real-time amplification controller to perform the desired operation. Preferably, the computer can also receive data from one or more sensors / detectors included within the system and interpret the data according to its programming. The system preferably includes software that correlates a growth curve characteristic representing the amount of amplified copies of the polynucleotide of interest as a function of time, as detected by the detector, to the number of copies of the polynucleotide of interest present in the test sample.

[0111] Preferably, when the computer used to perform the disclosed CF determination and adjustment procedures is an essential component of an apparatus for performing and analyzing real-time polynucleotide amplification reactions, the apparatus preferably includes 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 the signals. Optionally, the apparatus may further include an output device for displaying acquired or generated data. The analysis device may be connected to the temperature-controlled incubator via an input device known in the art and / or may be connected to an output device known in the art for data display. In one embodiment, the temperature-controlled incubator is capable of temperature cycling.

[0112] Generally speaking, the various components of an apparatus for performing real-time polynucleotide amplification useful in connection with the disclosed methods are conventional components with which one of skill in the art would be familiar. The temperature-controlled incubator used to perform and analyze real-time polynucleotide amplification can be of conventional design, capable of holding reaction samples in multiple reaction tubes, or in a temperature-controlled block of standard amplification reaction tubes or multi-well plates. In one aspect, the detection system is suitable for detecting optical signals from one or more fluorescent labels. The output of the detection system (e.g., signals corresponding to those generated during the amplification reaction) can be provided to a computer for data storage and manipulation. In one embodiment, the system detects multiple different types of optical signals, such as multiple different types of fluorescent labels, and has the functionality of a microplate fluorescence reader. The detection system is preferably a multiplexed fluorometer including an excitation light source, preferably a visible laser, ultraviolet lamp, or halogen lamp; a multiplexer device for distributing the excitation light to individual reaction tubes and receiving fluorescent light from the reaction tubes; filtering means for separating the fluorescent light from the excitation light by wavelength; and detection means for measuring the fluorescence intensity. Preferably, the temperature-controlled incubator detection system provides a wide detection range, allowing for flexibility in fluorophore selection, high sensitivity, and an excellent signal-to-noise ratio. The optical signals received by the detection system are generally converted into signals that can be manipulated by a processor to provide data that can be viewed by a user on the display of a user device in communication with the processor. The user device may include a user interface or a conventional commercially available computer system with a keyboard and video monitor. Examples of data that can be displayed on the user device include amplification plots, scatter plots, sample value screens for all tubes or reaction vessels in the assembly and all labels used, optical signal intensity screens (such as fluorescent signal intensity screens), final results, text reports, etc.

[0113] computer program products Included within the scope of the present invention are software-based products (e.g., tangible embodiments of software for instructing a computer to perform various procedural steps) that can be used to perform data processing methods. These include software instructions stored on computer-readable media, such as magnetic media, optical media, "flash" memory devices, computer networks, or cloud storage. Similarly, the present invention encompasses systems or devices that amplify polynucleotides, detect polynucleotide amplification products, and process the results to provide a quantitative result of the target in a test sample. The various components of the device preferably function cooperatively, although the components need not be part of an integrated assembly (e.g., on a single chassis). However, in preferred embodiments, the components of the device are connected together. The term "connected" includes connections via wired and wireless connections.

[0114] Particularly within the scope of the present invention is an apparatus or system comprising a computer linked to a device that amplifies polynucleotides and monitors amplicon synthesis as a function of cycle number or time, the computer being programmed to execute the quantitative algorithms disclosed herein. An exemplary system according to the present invention would include a temperature-controlled incubator and a fluorometer capable of monitoring and distinguishing at least two wavelengths of fluorescent emission. These emissions may be used to indicate target amplicon synthesis and IC amplicon synthesis.

[0115] In connection with computer- or software-implemented embodiments of the present disclosure, results may be recorded or stored in a "non-transitory" format and accessed for reference at a later time than when the recorded data analysis was performed or carried out. For example, calculation results may be recorded in non-transitory form by printing them on paper or saving them to a computer-readable memory device (such as a hard drive, a flash memory device, a file in cloud storage, etc.).

[0116] Curve fitting procedure According to the disclosed methods for creating a curve, plot, or fitted equation for determining a correction factor, the relevant procedure or step preferably includes obtaining one or more equations optimized to fit a dataset. The dataset includes CF values ​​calculated as a function of results generated by a nucleic acid analyzer calibrated to determine the amount of analyte in a known volume of liquid sample. This can be achieved by applying standard mathematical curve-fitting techniques to each dataset to create a fitted equation that defines its associated curve. In some embodiments, one or more linear equations can be used to determine the appropriate CF from the output of a nucleic acid analyzer calibrated to provide quantitative results for sample types other than reconstituted DBS samples (e.g., plasma samples). In other embodiments, the equation used in the curve-fitting procedure is a nonlinear equation, preferably containing two or more, more preferably three or more, and more preferably four or more coefficients, which can be optimized or determined during the curve-fitting procedure. Some highly preferred equations have exactly four coefficients, while other highly preferred equations have exactly five coefficients. Optimizing the equation to fit the measured amplification signature can be easily performed using commercially available software packages, such as the SOLVER program, available as an Excel add-in tool for finding the optimum value of the equation, and the Equation Solver from Microsoft Corporation (Redmond, WA). Specific curves generated by this procedure can be shaped so that increasing levels of polynucleotide analyte input to the reaction correlate with decreasing CF values.

[0117] Although other equations can be used in the curve fitting procedure, the method described below used a four parameter logistic (4-PL) equation of the form:

number

[0118] Alternative equations for performing curve fitting In particular, although the 4-PL equation was used to explain the disclosed technique, other mathematical functions can also be used in the procedure to simulate the trend of CF values ​​versus measured power.

[0119] Those skilled in the art will understand that many types of equations can be used in the procedures disclosed herein. Examples of symmetric transformation functions include, but are not limited to, sigmoid, Gaussian cumulative, Lorentzian cumulative, and cumulative symmetric double sigmoid. Examples of asymmetric transformation functions include, but are not limited to, logistic dose response (LDR), log normal cumulative, extreme value cumulative, pulse cumulative, pulse cumulative with power term, Weibull cumulative, asymmetric sigmoid, asymmetric sigmoid reverse asymmetry, cascade formation, and cumulative exponentially modified Gaussian. Additionally, as described in detail herein, simple linear and nonlinear equations, such as multi-order polynomials, power, exponential, and logarithmic functions, can be used to model real-time data with subsequent adjustment of baseline coefficients. Rate functions with baseline coefficients can also be used in the same manner. Exemplary basic rate equations that include 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 Forms), Third Order Decay and Formation, and Variable Order Decay and Formation.Exemplary complex rate equations that include baseline coefficients include, but are not limited to, simultaneous first- and second-order decay and formation, first-order sequential formation, two-component first-order decay, two first-order independent decay and formation, two second-order independent decay and formation, and first- and second-order independent decay and formation. Exemplary rate equilibrium equations that include baseline coefficients include, but are not limited to, simple equilibrium (forward and reverse rates), simple equilibrium (net rate and equilibrium concentration), complex equilibrium A = B + C, and complex equilibrium A + B = C + D. Exemplary intermediate rate equations that include baseline coefficients include, but are not limited to, first-order intermediate and first-order intermediate with equilibrium. Those skilled in the art will readily appreciate that the success of the disclosed CF tuning method does not depend on the use of any particular equation to perform the curve fitting step. Indeed, any equation having coefficients that can be optimized in a curve fitting procedure for the disclosed CF tuning procedure is contemplated.

[0120] All of the above equation types can be used to perform the disclosed method, as the success of the procedure depends on the ability to best fit the data, rather than the particular equation used.

[0121] Example As noted above, the disclosed techniques improved the quantitative capabilities of assays performed using dried body fluid samples by providing reliable results that correlate with the concentration of polynucleotide analytes in the starting sample used to create the dried sample. The examples provided below are intended to be illustrative and not limiting of the present disclosure.

[0122] Those skilled in the art will understand that the lower limit of quantitation ("LLOQ") in an assay is the lowest concentration of analyte that can be quantified with a particular level of precision and accuracy and has at least 95% reactivity. Similarly, those skilled in the art will understand that the limit of detection ("LOD") in an assay is the lowest concentration of analyte that can be consistently detected in at least 95% of the samples tested.

[0123] The LLOQ of an assay is the lowest concentration at which two requirements are met: (1) reactivity must be at least 95% and (2) the total error (TE) must meet the assay precision specification. For the model viral load assay used to illustrate current CF adjustment techniques, the TE specification is a precision of 1 log or less at the LLOQ. Two different "total error" evaluation approaches were used to measure the impact of different correction factor approaches on the lower limit of quantification (LLOQ). These approaches were the CLSI EP-17-A2 guideline recommended by Westgard, and the root mean square (RMS) model for calculating the LLOQ. The procedure involved determining the precision and accuracy of quantification at low HIV concentrations using reconstituted DBS samples as the source of analyte. DBS samples were prepared using a diluted WHO HIV standard stock with assigned concentration values, which served as the "gold standard" for quantification. More specifically, various amounts of the HIV WHO standard stock were spiked into various aliquots of whole blood, and the resulting dilutions were used to prepare DBS samples.

[0124] TE can be calculated according to the Westgard model above using the following formula: Bias + (2 × standard deviation) ≦ 1 log (Equation 2)

[0125] According to the RMS model cited above, TE can be calculated using the following formula:

number

[0126] In the context of these equations, "bias" is the difference between the expected (i.e., actual) assay result and the "recovered" assay result. As used herein, a "recovered" result has been adjusted using a CF multiplier and therefore differs from a measurement result that has not been adjusted using a CF multiplier. Simply put, a recovered result may be calculated by multiplying the measurement result by the CF. CF adjustment can improve assay quantification at low analyte concentrations by reducing bias (improving precision) and improving precision (by lowering standard deviation).

[0127] Initial approaches to improving DBS quantification included the use of static (i.e., constant) CF value multipliers. More specifically, preselected constants ranging from 15 to 33 were multiplied by the measurements of a calibrated real-time polynucleotide amplification assay to provide quantitative results for a 500 μl liquid sample (e.g., plasma). Achieving precision targets at concentrations below 1,000 copies / ml is critical for assays measuring HIV viral load. This is because 1,000 copies / ml is the WHO-recommended medical decision point for monitoring the effectiveness of antiretroviral therapy. Therefore, the clinical sensitivity and specificity of the assay were calculated at the 1,000 copies / ml medical decision point using the recovered assay results for DBS calculated using various static CFs. No significant differences in assay sensitivity or specificity were observed when using CFs ranging from 25 to 33. According to one approach, the LOD determined for the reconstituted DBS sample (i.e., 873 copies / ml) was divided by the LLOQ for a plasma sample of the same assay chemistry (i.e., 30 copies / ml) to establish a constant CF value of 29.1 to use as a multiplier. Thus, an assay performed using a 500 μl aliquot of reconstituted DBS sample (e.g., a filter spotted with 70 μl of whole blood, then dried, and then reconstituted with 1 ml of buffer) would yield a "measured" or observed output of 35 copies / ml, which, when multiplied by 29.1, would yield a corrected (i.e., "recovered") result of 1,019 copies / ml.

[0128] Example 1 describes a real-time polynucleotide amplification assay that quantified HIV-1 polynucleotides using reconstituted DBS samples. The automated nucleic acid analyzer used in the procedure was calibrated to provide results measured in copies / ml of plasma sample.

[0129] Example 1 Static correction factors quantify polynucleotide analytes with excess error DBS samples containing known amounts of HIV-1 polynucleotides were prepared using experimental procedures familiar to those skilled in the art. Whole blood was spiked with HIV-1 from a value assigned to a WHO standard virus stock to produce samples with concentrations ranging from 50 copies / ml to 1,200 copies / ml. Whole blood samples (70 μl each) with various HIV concentrations were individually applied to standard filter paper cards and allowed to dry. Dried blood spots were punched from the cards, and each DBS was combined with 1 ml of buffered detergent solution (i.e., DBS extraction buffer). Half of each sample (500 μl) was then used for testing in the Aptima HIV-1 Quant Dx Real-Time Viral Load Assay on a Panther automated nucleic acid analyzer (Hologic, Inc.; Marlborough, MA). At least 90 replicates of DBS samples tested using different HIV-1 reagent lots on the platform yielded essentially equivalent results. Table 1 shows exemplary results obtained using one of the reagent lots. Columns 1 and 2 list the actual stock concentrations of the analytes in whole blood used to prepare the DBS samples. Column 3 ("Reactivity") indicates the percentage of tests that resulted in a positive result (i.e., the HIV-1 analyte was detected). Column 4 ("Mean Recovery") indicates the averaged product of the static CF multiplied by the measured concentration of the analyte output by the automated analyzer. Column 5 ("Bias") indicates the magnitude of deviation of the resulting mean recovered concentration from the actual analyte concentration. Column 6 ("Standard Deviation Log Copies") indicates the standard deviation of the recovered results shown in Column 4. Column 7 ("Total Error (Westgard)") indicates the results calculated according to the standard Westgard analytical protocol. Column 8 ("Total Error (RMS)") indicates the results calculated according to the RMS analytical protocol described above. [Table 1]

[0130] The results shown in Table 1 indicate that regardless of the method used to determine it, the total error exceeded the acceptable 1.0 threshold target, which is undesirable. Although not shown, a different constant CF value substituted in place of 29.1 also produced unacceptable results.

[0131] Example 2 presents experimental results demonstrating that a single (i.e., constant) CF cannot be used to quantify analytes across the dynamic range of the assay, particularly at lower analyte concentrations. As is evident from the results shown below, the lower concentration values ​​output by a nucleic acid analyzer calibrated to process plasma samples had to be multiplied by a higher CF to recover the correct starting concentrations used in preparing the DBS samples. Similarly, higher output values ​​and lower CFs were required to recover the correct starting concentrations used in preparing the DBS samples.

[0132] Example 2 Correction factors are not constant across the dynamic range of quantitative real-time assays DBS samples were prepared using whole blood spiked with different levels of HIV-1 analytes, essentially according to the procedure described in Example 1. The DBS samples were processed as described above, and eluted polynucleotides were amplified and detected using the Aptima HIV-1 Quant Dx real-time viral load assay on an automated Panther nucleic acid analyzer (Hologic, Inc.). The target HIV-1 concentrations used to prepare the DBS were compared to the concentrations measured in the assay, and the appropriate CF for each input HIV-1 concentration was calculated according to Equation 4.

number

[0133] The results shown in Table 2 clearly demonstrate that a single, fixed, or static CF value cannot be used to properly quantify polynucleotide analytes across the dynamic range of the assay. The last column of the table shows that, in general, higher CF values ​​were required to properly quantify samples with lower concentrations of polynucleotide analytes.

[0134] Example 3 describes the development of a quantitative approach that uses a CF value that varies as a function of a measurement result calibrated to a liquid sample (e.g., plasma) different from the liquid sample under test (i.e., an extracted DBS sample). This type of variable CF is sometimes referred to as "non-static."

[0135] Example 3 Development of non-static correction factors A total of 747 DBS samples were prepared using whole blood stocks with various known analyte HIV-1 concentrations spanning the quantification range of the model real-time quantitative assay. For completeness, the known analyte HIV-1 concentrations used to create the DBS samples were the same as those listed in the first column of Table 2. DBS samples were reconstituted with 1 ml of buffered detergent solution (i.e., DBS extraction buffer), and 500 μl of the resulting solution was used for nucleic acid isolation and target amplification and detection by the model real-time quantitative assay. The target (i.e., actual) HIV-1 concentrations were compared to the concentrations measured in the assay, and the appropriate CF for each HIV-1 concentration was calculated using Equation 4. The CF multiplier required to adjust the measured HIV-1 analyte concentration values ​​to equal the known input analyte concentrations was then plotted as a function of the measured copy number. The resulting data was then used to optimize a nonlinear equation according to standard mathematical curve-fitting techniques with which those skilled in the art would be familiar. While many different nonlinear equations can be used for this purpose, this approach is illustrated in Figure 1 using the approximated 4-PL equation. It will be appreciated that curve fitting using the 4-PL equation is frequently used to process data that exhibit biphasic or sigmoidal curve characteristics.

[0136] The results shown in Figure 1 graphically confirmed that CF values ​​are not static or constant but rather vary as a nonlinear function of the measured concentration values ​​output by a nucleic acid analyzer calibrated for processing plasma samples. Clusters of data points, displayed as spaced crescents, indicate variability in CF results calculated for a single level of input. Stated differently, a collection of DBS samples containing substantially the same amount of polynucleotide analyte (i.e., dried blood spots prepared using a single stock of diluted analyte) naturally yielded a range of CF values. The coefficients of the optimized 4-PL equation (i.e., Equation 1), shown as a fitted curve in Figure 1, are as follows: b = 3.705178, c = 47.21279, d = 486.6657, and e = 0.506889. Notably, as shown in the following example, the fact that the data in this case did not particularly fit a sigmoidal shape did not preclude the usefulness of the 4-PL equation.

[0137] Example 4 demonstrates the use of CF values ​​determined by the fitted nonlinear curve. More specifically, the determined CF values ​​were multiplied by the measured quantitative results (measured in copies / ml) output from a real-time nucleic acid analyzer to indicate the analyte concentration in the liquid sample used to prepare the dried blood spot.

[0138] Example 4 Correction factors calculated from improved analyte quantification with nonlinear curve fits DBS samples were prepared using whole blood spiked with different levels of HIV-1 analytes, following essentially the procedure described in Example 1. The DBS samples were processed as described above, and the eluted polynucleotides were amplified and detected using the Aptima HIV-1 Quant Dx real-time viral load assay on a Panther automated nucleic acid analyzer. The output (i.e., measured) quantitative results were multiplied by the CF values ​​obtained from the fitted curve shown in Figure 1. More specifically, the equation for the fitted curve shown in the figure was solved, and the quantitative results output on the horizontal axis were used as the independent variable (x value) in the equation to determine the CF values. The determined CF values ​​were then multiplied by the same output quantitative results (i.e., x value) to calculate the "recovered" (i.e., adjusted) concentrations. The results are shown in Table 3. [Table 3]

[0139] The results shown in Table 3 confirm that using the CF calculated from the fitted nonlinear equation significantly improved the quantitative capability of the assay. As shown in the last two columns of the table, the TE values ​​were significantly reduced compared to the results shown in Table 1. In other words, using the CF calculated from the fitted nonlinear equation resulted in a significant improvement compared to a similar process using a fixed value (CF = 29.1). The LLOQ of the assay in this example was 813 copies / ml (i.e., 2.91 Log copies / ml). Among all results obtained using three different reagent lots, the highest LLOQ determined using the calculated CF values ​​obtained from the fitted curve shown in Figure 1 was 883 copies / ml (i.e., 2.95 Log copies / ml).

[0140] Example 5 Use of correction factor equations improves the assay Precision and Accuracy Using the procedures disclosed herein, DBS samples were prepared from whole blood stocks spiked with HIV-1 analytes at 900 copies / ml, 1,000 copies / ml, or 1,200 copies / ml. Polynucleotides eluted from the samples were amplified using the Aptima HIV-1 Quant DX real-time viral load assay on a Panther automated nucleic acid analyzer. The reported results were obtained using procedures implemented in our laboratory, intended to analyze performance near the medically relevant decision point (1,000 copies / ml). Average results obtained using three different reagent lots are shown in Table 4. [Table 4]

[0141] The results shown in Table 4 demonstrate that multiplying the CF calculated from the nonlinear equation by the results measured in copies / ml of the plasma sample advantageously yields greater precision with greater accuracy in quantitative assignment. Columns 1 and 2 of the table show the HIV-1 target concentrations of the stock samples used to prepare the DBS samples. Columns 3 and 4 show the prepared HIV concentrations determined by multiplying the CF (29.1 for column 3, or the value calculated using the equation from the fitted curve in Figure 1 for column 4) by the output results of a model viral load assay that had been calibrated to quantify plasma samples rather than DBS samples. The difference between the values ​​displayed under column 2 and those displayed under columns 3 and 4 reflects the precision of the assay using a different correction factor approach. In both cases, the magnitude of the difference was smaller when the CF equation was used instead of the static CF. These smaller differences indicate more accurate quantification. Columns 5 and 6 show a measure of precision (i.e., the standard deviation of the measured concentrations between replicates). Again, the standard deviation was lower in both cases when the CF equation was used instead of the static CF. This indicated that the use of the CF equation was associated with greater accuracy of the quantitative results.

[0142] Example 5 presents clinical data showing how improved assay quantification resulting in higher clinical sensitivity at the medical decision point of 1,000 copies / ml for HIV-1 was achieved by using a CF multiplier calculated using the equation for the fitted curve shown in Figure 1. Notably, the data used to obtain the fitted curve was not the same clinical data processed in the Examples. This further demonstrated how one data set can be used to create a fitted curve (or its equation), which can then be used to determine a CF value and process another data set (e.g., a data set obtained using a different instrument to run the assay).

[0143] Example 5 Improving clinical outcomes at the WHO-recommended medical decision point of 1,000 copies / ml for HIV-1 viral load monitoring Paired plasma and DBS specimens were collected from HIV-1-positive patients undergoing antiretroviral therapy. Two replicates of plasma specimens were tested, and the viral load results obtained using the procedure described herein were averaged and used as a reference. Approximately five reconstituted DBS samples from each patient were also tested, and the measured quantitative results were multiplied by a static CF (i.e., 29.1) or a CF calculated using the nonlinear equation for the fitted curve shown in Figure 1 to obtain recovered concentration values. Results were then compared to plasma reference standards at a medically relevant decision point of 1,000 copies / ml. Note that 90% of the results in this study had plasma viral loads below 10,000 copies / ml, making this dataset ideal for evaluating HIV clinical outcomes around 1,000 copies / ml.

[0144] As shown in the results presented in Tables 5 and 6, assay sensitivity at 1,000 copies / ml improved from 79.83% to 90.56% when the CF was determined from the nonlinear equation compared to the static value. Changing from one CF value to another did not have a significant adverse effect on specificity. This is evident from the 94.30% specificity obtained using a static CF of 29.1 and the 91.36% specificity obtained using the CF equation. The close correspondence between these latter values ​​indicated minimal impact on specificity. [Table 5] [Table 6]

[0145] While the present disclosure has been described and illustrated in considerable detail with reference to certain illustrative embodiments including various combinations and sub-combinations of features, those skilled in the art will readily recognize other embodiments, and variations and modifications thereof, that are encompassed within the scope of the present disclosure. Moreover, the description of such embodiments, combinations, and sub-combinations is not intended to convey that the present disclosure requires features or combinations of features other than those expressly recited in the claims. Accordingly, the present disclosure is deemed to include all modifications and variations that are encompassed within the spirit and scope of the following numbered embodiments.

[0146] While various embodiments of the present disclosure have been shown and described in detail, it will be readily apparent to those skilled in the art that various modifications can be made therein without departing from the scope of the present disclosure or the appended claims.

Claims

1. 1. A method for quantifying polynucleotide analytes present in a fluid blood sample that is dried to produce a dried blood spot (DBS), comprising: (a) performing a nucleic acid amplification reaction using the DBS as a source of template to generate an amplification product and obtain a measurement result, the measurement result indicating the concentration or amount of the polynucleotide analyte; (b) multiplying the measurement result by a correction factor to obtain a corrected result; the correction factor is a solution to an equation specifying the correction factor as a function of the measurement; the equations include nonlinear equations; thereby quantifying said polynucleotide analytes present in said fluid blood sample.

2. 1. A method for quantifying polynucleotide analytes present in a fluid blood sample from which a dried blood spot (DBS) has been prepared, comprising: (a) performing a nucleic acid amplification reaction using the DBS as a source of template to generate an amplification product and obtain a measurement result, the measurement result indicating the concentration or amount of the polynucleotide analyte; (b) solving an equation to determine a correction factor; the equation specifying the correction coefficient as a function of the measurement, the equation comprising a non-linear equation; (c) multiplying the measurement result by the correction coefficient to obtain a corrected result; thereby quantifying said polynucleotide analytes present in said fluid blood sample.

3. The method of claim 1 or 2, wherein the nonlinear equation comprises coefficients optimized in a mathematical curve fitting procedure to define an approximate curve.

4. The method of claim 3 , wherein the nonlinear equation includes four coefficients.

5. 5. The method of any one of claims 1 to 4, wherein step (a) comprises performing the method using an automated nucleic acid analyzer to amplify the polynucleotide analyte and detect the amplification product as the nucleic acid amplification reaction is occurring.

6. 6. The method of claim 5, wherein the nonlinear equation is created using results obtained from an automated nucleic acid analyzer that is different from the automated nucleic acid analyzer used to perform the nucleic acid amplification reaction in step (a).

7. 5. The method of any one of claims 1 to 4, wherein step (a) comprises performing the method using an automated nucleic acid analyzer to isolate the polynucleotide analytes and then amplify the isolated polynucleotide analytes.

8. 8. The method of claim 7, wherein the automated nucleic acid analyzer further detects synthesis of an amplification product as the nucleic acid amplification reaction occurs.

9. The method of any one of claims 1 to 8, wherein the measurement result indicates the concentration of the polynucleotide analyte in the plasma sample.

10. The method according to any one of claims 1 to 9, wherein the nucleic acid amplification reaction is an isothermal nucleic acid amplification reaction.

11. The method of claim 10, wherein the isothermal nucleic acid amplification reaction is a transcription-associated nucleic acid amplification reaction.

12. 12. The method of claim 11, wherein the transcription-associated nucleic acid amplification reaction comprises a transcription-mediated amplification (TMA) reaction.

13. The method of any one of claims 1 to 12, wherein the polynucleotide analyte comprises a segment of a viral genome.

14. 14. The method of claim 13, wherein the viral genome comprises RNA.

15. The method of any one of claims 1 to 14, wherein the polynucleotide analyte comprises a segment of the HIV-1 genome.

16. The method of any one of claims 1 to 15, wherein the fluid blood sample comprises whole blood.

17. 1. A computer programmed with software instructions for quantifying polynucleotide analytes present in a fluid blood sample that is dried to produce a dried blood spot (DBS), the software instructions, when executed by the computer, causing the computer to: (a) receiving a measurement result; (b) solving a nonlinear equation to determine a correction factor; determining the equation specifying the correction factor as a function of the measurement results; (c) multiplying the measurement result by the correction coefficient to calculate a corrected result; (d) recording the correction results in a non-transitory manner, thereby quantifying the polynucleotide analyte.

18. the measurement result is determined from the result of a real-time nucleic acid amplification reaction; the real-time nucleic acid amplification reaction is performed using the DBS as a source of template for generating amplification products; 18. The computer of claim 17, wherein the measurement result indicates a concentration or amount of the polynucleotide analyte.

19. 19. The computer of claim 17 or 18, wherein both the measurement result and the corrected result are expressed in concentration units.

20. The computer of any one of claims 17 to 19, wherein the non-linear equation comprises coefficients optimized in a mathematical curve fitting procedure to define an approximate curve.

21. 21. The computer of claim 20, wherein the nonlinear equation includes four coefficients.

22. The computer of any one of claims 17 to 21, wherein the non-transitory form comprises storage on a computer-readable memory device.

23. The computer of any one of claims 17 to 22, wherein the fluid blood sample comprises whole blood.

24. 20. A computer-readable storage medium comprising instructions that, when executed by a computer, cause the computer to perform the computer-implemented steps (a) to (d) of claim 17.

25. 1. A system for quantifying a polynucleotide analyte that may be present in a test sample, comprising: a temperature-controlled incubator; a fluorometer in optical communication with the temperature-controlled incubator, a fluorometer configured to measure production of nucleic acid amplification products contained in the temperature-controlled incubator as a function of time or cycle number; a computer in communication with the fluorometer, The computer (a) calculating a measurement result using the fluorometer measurements; (b) solving an equation to determine a correction factor; determining an equation specifying the correction factor as a function of the measurement, the equation including a non-linear equation; (c) multiplying the measurement result by the correction coefficient to calculate a corrected result; (d) recording the correction results in a non-transitory manner, thereby quantifying the polynucleotide analytes present in the test sample.

26. 26. The system of claim 25, wherein the temperature-controlled incubator is configured to maintain a constant temperature.

27. 26. The system of claim 25, wherein the temperature-controlled incubator is configured for temperature cycling.

28. 28. The system of any one of claims 25 to 27, wherein the fluorometer is configured to detect light at a plurality of different wavelengths.

29. 29. The system of any one of claims 25 to 28, wherein the temperature-controlled incubator, the fluorometer, and the computer are all integral components of the nucleic acid analyzer.

30. 30. The system of any one of claims 25 to 29, wherein the measurement result comprises a concentration value of the polynucleotide analyte.

31. 1. A method for quantifying an analyte present in a body fluid sample that is dried to produce a dried sample, comprising: (a) performing a reaction using the dried sample as a source of an analyte to obtain a measurement result, the measurement result indicating the concentration or amount of the analyte; (b) multiplying the measurement result by a correction factor to obtain a corrected result; the correction coefficients are solutions to equations that specify the correction coefficients as a function of the measurement results, the equations including nonlinear equations; thereby quantifying the analyte present in the bodily fluid sample.

32. 32. The method of claim 31, wherein the nonlinear equation comprises coefficients optimized in a mathematical curve fitting procedure to define an approximate curve.

33. 33. The method of claim 32, wherein the nonlinear equation includes four coefficients.

34. 34. The method of any one of claims 31 to 33, wherein the analyte is a polynucleotide analyte and the reaction is a nucleic acid amplification reaction, and step (a) comprises carrying out using an automated nucleic acid analyzer that amplifies the polynucleotide analyte and detects the amplification product as the nucleic acid amplification reaction is occurring.

35. 35. The method of claim 34, wherein the nonlinear equation comprises coefficients optimized by a mathematical curve fitting procedure to define an approximate curve, and the nonlinear equation is created using results obtained from an automated nucleic acid analyzer that is different from the automated nucleic acid analyzer used to perform the nucleic acid amplification reaction in step (a).

36. 35. The method of claim 34, wherein step (a) comprises isolating the polynucleotide analyte and then performing the step using an automated nucleic acid analyzer to amplify the isolated polynucleotide analyte.

37. 37. The method of claim 36, wherein the automated nucleic acid analyzer further detects synthesis of an amplification product as the nucleic acid amplification reaction occurs.

38. 38. The method of any one of claims 34, 36, or 37, wherein the measurement indicates the concentration of the polynucleotide analyte in the plasma sample.

39. The method according to any one of claims 34 to 38, wherein the nucleic acid amplification reaction is an isothermal nucleic acid amplification reaction.

40. 40. The method of claim 39, wherein the isothermal nucleic acid amplification reaction is a transcription-associated nucleic acid amplification reaction.

41. 41. The method of claim 40, wherein the transcription-associated nucleic acid amplification reaction comprises a transcription-mediated amplification (TMA) reaction.

42. 42. The method of any one of claims 34 to 41, wherein the polynucleotide analyte comprises a segment of a viral genome.

43. 43. The method of claim 42, wherein the viral genome comprises RNA.

44. The method of any one of claims 34 to 43, wherein the polynucleotide analyte comprises a segment of the HIV-1 genome.

45. The method of any one of claims 34 to 44, wherein the body fluid sample is selected from the group consisting of a whole blood sample, a plasma sample, a urine sample, and a saliva sample.

Images