Nucleic acid quantification method for improving dynamic range of digital PCR

Abstract

The present invention relates to a nucleic acid quantification method comprising the steps of: (i) providing a sample comprising a plurality of nucleic acid molecules; (ii) amplifying at least two loci in the plurality of nucleic acid molecules wherein the at least two loci comprise at least one multi-copy locus and at least one single-copy locus, and wherein the amplification method is a digital polymerase chain reaction method; (iii) detecting at least two amplification products; and (iv) quantifying the amount of the plurality of nucleic acid molecules present in the sample; wherein the quantifiable nucleic acid concentration ranges of the at least one multi-copy locus and the at least one single-copy locus overlap. The invention also relates to a kit for use in the method of the invention.

Description

Field of the Invention

[0001] The present invention belongs to the field of molecular biology, particularly in the fields of analysis and forensic science. In addition, the present invention also relates to the fields of nucleic acid amplification and quantification, and more specifically, to the field of nucleic acid quantification of a wide range of DNA concentrations in digital PCR. Background Art

[0002] DNA-based diagnostic and analytical methods are becoming increasingly important in many fields, such as in the fields of diagnosis, genetics, forensic science, and food testing, or for the detection of genetically modified organisms (GMOs). The more accurate the determination of the amount of (amplifiable) DNA, the more successful the subsequent analytical and diagnostic methods will be.

[0003] Currently, quantitative real-time PCR (qPCR) is a widely used method for DNA or RNA quantification. As reviewed by Quan et al. (Quan et al., 2018, MDPI, "dPCR: A Technology Review"), quantitative real-time PCR is based on conventional PCR. In conventional PCR, the target DNA is amplified through multiple cycles, where at the end of each PCR cycle with 100% efficiency, the number of target DNA molecules doubles (exponential amplification). Thus, in theory, 2 n copies can be produced after n cycles. However, in practice, the PCR reagents will be depleted at a certain point, and the amplification products will self-anneal, resulting in a decrease in amplification efficiency until a plateau is reached and the amplification process reaches saturation. At the end of a conventional PCR reaction, the amplification products can be analyzed using agarose gel electrophoresis (endpoint determination). The specificity of conventional PCR depends on sequence hybridization. The sensitivity of conventional PCR depends on enzyme-based amplification (Quan et al., 2018, MDPI, "dPCR: A Technology Review").

[0004] As described above, quantitative real-time PCR is based on conventional PCR, but the amount of amplified PCR product is measured using fluorescence readings after each amplification cycle. A typical real-time PCR amplification plot shows an S-shaped curve (linear scale), including a baseline period, followed by an exponential period that reaches a plateau through a linear period. The exponential period represents the most efficient stage of amplification, where the amount of amplified PCR product doubles in each cycle (amplification efficiency of 100%). Real-time PCR can achieve relative quantification of the target relative to a calibrator. The "absolute" amount of the target sequence in a qPCR reaction is measured relative to a standard curve generated from samples with known amounts or copy numbers. This method implies that the amplification efficiencies of the standards and samples are equal. Differences in PCR efficiency can significantly affect the accuracy of quantification (Quan et al., 2018, MDPI, "dPCR: A Technology Review").

[0005] qPCR is a widely used method for DNA quantification because it has multiple advantages. These advantages include species-specific identification, a wide dynamic range, and ease of automation.

[0006] When using qPCR for DNA quantification, a standard curve is required to calculate the amount of DNA in a sample. Typically, the standard curve is prepared by the user through serial dilution. The preparation of the standard curve is crucial for the accurate quantification of sample DNA. Depending on the user's skills and the quality of the equipment used for standard curve preparation, the process of DNA dilution and DNA standard curve preparation itself can be very cumbersome, laborious, and error-prone, which may lead to incorrect quantification results.

[0007] Another method for quantifying DNA is to utilize digital polymerase chain reaction (dPCR; also abbreviated as digital PCR, DigitalPCR, ddPCR, or dePCR), which is an improvement over other polymerase chain reaction methods such as qPCR.

[0008] Digital polymerase chain reaction (dPCR) is capable of absolute quantification of the target nucleic acid present in a sample and remedies the deficiencies of qPCR. Different from qPCR, dPCR does not rely on a calibration curve for sample quantification. Therefore, it avoids the defects associated with variations in reaction efficiency. dPCR is an absolute nucleic acid quantification method, the key of which lies in the detection of endpoint fluorescence signals and the counting of binomial events (i.e., the presence or absence of fluorescence in partitions). In dPCR, the sample is first partitioned into multiple independent PCR sub-reactions, and each partition contains a small amount, one, or no target sequence. Such partitions or microreactors can be arranged as, for example (but not limited to), small water-in-oil droplets or microfluidic nanoplatelets.

[0009] After PCR, Poisson statistics is used to quantify the concentration of the target sequence with statistically defined accuracy, where the calculation requires each partition containing 0, 1, or more target sequences. Interestingly, sample partitioning can effectively concentrate the target sequence in isolated microreactors. This concentration effect reduces template competition, enabling the detection of rare mutations in the background of wild-type sequences (Quan et al., 2018, MDPI, "dPCR: A Technical Review").

[0010] dPCR can also improve the tolerance to inhibitors present in the sample because it does not require an amplification efficiency close to 100% for each cycle like qPCR. Instead, it is sufficient to detect a signal or no signal at the end of the amplification reaction.

[0011] PCR performs one reaction for each sample. dPCR also performs one reaction on the sample, but the sample is divided into multiple partitions, and the reaction is carried out separately in each partition. This separation allows for more reliable collection and sensitive measurement of the amount of nucleic acid.

[0012] Instead of performing a single reaction per well, digital polymerase chain reaction (dPCR) divides the PCR solution into at least several hundred, but in most cases several thousand, tens of thousands, or more nanoliter-sized partitions, and separate PCR reactions are carried out in each partition. The preparation method of the dPCR solution is similar to quantitative analysis, using fluorescence quenching probes or intercalating dyes, as well as PCR premixes (containing optimal concentrations of DNA polymerase, dNTPs, MgCl2, and reaction buffer).

[0013] There are various different methods for sample partitioning, including microplates, microfluidic nanoplatelets, capillaries, oil emulsions, and microcavity arrays with nucleic acid-binding surfaces.

[0014] After multiple PCR amplification cycles, the fluorescence of the sample is examined with binary readings of "0" (absent) or "1" (present). The proportion of fluorescent partitions is recorded. The partitioning of the sample allows for the estimation of the number of different molecules by assuming that the molecular population follows a Poisson distribution, thus taking into account the possibility of multiple target molecules being present in a single partition.

[0015] Different from qPCR reactions, dPCR reactions are end-point PCR reactions. dPCR uses the number of fluorescent positive partitions divided by the total number to back-calculate the target concentration. Different from qPCR, sample quantification in dPCR does not require a calibration curve. In summary, compared with qPCR, dPCR provides more robust quantification, is less susceptible to inhibitors, and is independent of quantification standards.

[0016] The benefits of dPCR include increased precision through large-scale sample partitioning, ensuring reliable measurement of target DNA sequences due to reproducibility. qPCR has a higher error rate when detecting small-fold change differences, while dPCR has a lower error rate because smaller-fold change differences in DNA sequences can be detected. In addition, dPCR is highly quantitative because it does not rely on the relative fluorescence of the solution to determine the amount of amplified target DNA.

[0017] Despite these advances, current dPCR systems are limited in their partitioning capacity per sample (typically ≤100,000) due to physical and manufacturing limitations, which in turn limits the dynamic range of the dPCR system (Shum et al., 2022, BioRxiv, "Next generation digital PCR: high dynamic range single molecule DNA counting via ultra-partitioning"), i.e., the dynamic range is determined by the total number of partitions (Jones et al., 2016, "Digital PCR dynamic range is approaching that of real-time quantitative PCR"). The detection dynamic range used in this article is defined as the range of sample concentrations that can be quantified in digital PCR, which, as described above, is directly related to the number of partitions available per sample. Thus, the dynamic range is proportional to the number of partitions in the reaction (dMIQE Group and Huggett, 2020, ClinChem., "The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020"), and increasing the number of partitions can expand the dynamic range (Basu, 2017, SLAS Technol., "Digital Assays Part I: Partitioning Statistics and Digital PCR"). Thus, ideally, a dPCR system should significantly increase the number of partitions to reduce or eliminate partition saturation, thereby improving quantification precision and dynamic range (Shum et al., 2022, BioRxiv, "Next generation digital PCR: high dynamic range single molecule DNA counting via ultra-partitioning"). Therefore, in some cases, prior knowledge of the template concentration may be required to avoid instrument saturation. When the concentration is high enough, common methods, such as those using fluorescence and spectrophotometry, can be used to quantify nucleic acids and guide dilution to a concentration for optimal measurement using digital PCR. It should be noted that such methods estimate the mass per unit volume of the constituent nucleic acid bases, rather than the mass of the macromolecule.Therefore, determining genomic copy number using methods that measure mass requires knowledge or assumptions about the composition, purity, and quality of the template in order to convert mass to moles. The user should also be aware of potential interfering factors that may affect the accuracy of such optical methods (dMIQE Group and Huggett, 2020, ClinChem., "Digital MIQE Guidelines Update: Essential Information for the Release of Quantitative Digital PCR Experiments in 2020").

[0018] Determining the amount of DNA recovered from forensic samples as well as other samples is a critical step in the overall DNA typing process or DNA testing in other scientific fields. To ensure that positive results are truly positive and / or negative results are negative due to DNA depletion, DNA quantification is crucial. For example, when using a multiplex DNA typing kit, a narrow range of DNA input of typically 0.5 to 2 ng is often required to obtain optimal results. Accurately measuring the amount of DNA in a sample is also essential for the success of DNA analysis (such as by short tandem repeat (STR) analysis). In fact, the more accurately the amount of amplifiable DNA can be determined, the more successful the DNA analysis will be.

[0019] Therefore, a sensitive DNA quantification method based on digital PCR with a high dynamic range is highly desirable, which can precisely detect and quantify DNA in a wide range of disciplines, overcome the limitation that the dynamic range of digital PCR is determined by the total number of partitions, and requires a method to overcome the drawbacks of currently available kits and methods. SUMMARY OF THE INVENTION

[0020] The present invention solves the above problems and provides the following solutions. Specifically, the present invention provides a method for expanding the dynamic range (the detection dynamic range used herein is defined as the sample concentration range in which quantification can be performed in digital PCR without increasing the number of available partitions per reaction per sample, thereby accommodating a larger range of samples without diluting the sample). Therefore, this method overcomes the above-mentioned drawbacks and limitations of digital PCR in terms of dynamic range. Surprisingly, compared with the above-mentioned prior art, the applicant has developed a method that can be used to expand the dynamic range by at least 10-fold.

[0021] In a first aspect, the present invention provides a method for nucleic acid quantification, the method comprising the following steps: a. Providing a sample containing a plurality of nucleic acid molecules; b. Amplifying at least one single-copy locus among the plurality of nucleic acid molecules, wherein the amplification method is digital polymerase chain reaction; c. Amplifying at least one multi-copy locus among the plurality of nucleic acid molecules, wherein the amplification method is digital polymerase chain reaction; d. Detecting the amplification products of steps b and c; and e. Quantify the amount of the plurality of nucleic acid molecules present in the sample.

[0022] In a second aspect, the present invention provides a method for nucleic acid quantification, the method comprising the steps of: a. Provide a sample comprising a plurality of nucleic acid molecules; b. Amplify at least two loci among the plurality of nucleic acid molecules, wherein the at least two loci comprise at least one multi-copy locus and at least one single-copy locus, and wherein the amplification method is a digital polymerase chain reaction method; c. Detect at least two amplification products; and d. Quantify the amount of the plurality of nucleic acid molecules present in the sample.

[0023] The quantifiable nucleic acid concentration ranges of the at least one multi-copy locus and the at least one single-copy locus overlap. This improves or extends the dynamic range of the method of the present invention without increasing the number of available partitions per reaction per sample.

[0024] Thus, in a third aspect, the present invention provides a method for nucleic acid quantification, the method comprising the steps of: a. Provide a sample comprising a plurality of nucleic acid molecules; b. Amplify at least two loci among the plurality of nucleic acid molecules, wherein the at least two loci comprise at least one multi-copy locus and at least one single-copy locus, and wherein the amplification method is a digital polymerase chain reaction method; c. Detect at least two amplification products; and d. Quantify the amount of the plurality of nucleic acid molecules present in the sample; wherein the quantifiable nucleic acid concentration ranges of the at least one multi-copy locus and the at least one single-copy locus overlap.

[0025] In a fourth aspect, the present invention provides a method for nucleic acid quantification, the method comprising the steps of: a. Provide a sample comprising a plurality of nucleic acid molecules; b. Amplify at least two loci among the plurality of nucleic acid molecules, wherein the at least two loci comprise at least one multi-copy locus and at least one single-copy locus, and wherein the amplification method is a digital polymerase chain reaction method; c. Detect at least two amplification products; and d. Quantify the amount of the plurality of nucleic acid molecules present in the sample; wherein the quantifiable nucleic acid concentration ranges of the at least one multi-copy locus and the at least one single-copy locus overlap; and wherein the detection accuracy is at least 70%.

[0026] In a fifth aspect, the present invention provides a method for nucleic acid quantification, the method comprising the following steps: a. Providing a sample comprising a plurality of nucleic acid molecules; b. Amplifying at least two loci among the plurality of nucleic acid molecules, wherein the at least two loci comprise at least one single-copy locus and at least one multi-copy locus, and wherein the amplification method is a digital polymerase chain reaction method; c. Detecting at least two amplification products; and d. Quantifying the amount of the plurality of nucleic acid molecules present in the sample; wherein the quantifiable nucleic acid concentration ranges of the at least one multi-copy locus and the at least one single-copy locus overlap; wherein, if the concentration of the plurality of nucleic acid molecules present in the sample is low and the amplification of the at least one single-copy locus does not produce a detectable amplification product, the amplification product of the at least one multi-copy locus is used for quantification; wherein, if the concentration of the plurality of nucleic acid molecules present in the sample is high and the amplification product of the at least one multi-copy locus reaches signal saturation, the amplification product of the at least one single-copy locus is used for quantification.

[0027] In a sixth aspect, the present invention provides a method for nucleic acid quantification, the method comprising the following steps: a. Providing a sample comprising a plurality of nucleic acid molecules; b. Amplifying at least two genomic loci among the plurality of nucleic acid molecules, wherein the at least two genomic loci comprise at least one single-copy locus and at least one multi-copy locus, and wherein the amplification method is a digital polymerase chain reaction method; c. Detecting at least two amplification products; and d. Quantifying the amount of the plurality of nucleic acid molecules in the sample; wherein the quantifiable nucleic acid concentration ranges of the at least one multi-copy locus and the at least one single-copy locus overlap; wherein, if the concentration of the plurality of nucleic acid molecules is less than 0.002 ng / reaction, quantification is based only on the steps performed on the at least one multi-copy locus; and / or wherein, if the concentration of the plurality of nucleic acid molecules is greater than 125 ng / reaction, quantification is based only on the steps performed on the at least one single-copy locus.

[0028] The sample can be partitioned into a plurality of partitions before the amplification step, and the method is performed in each of the plurality of partitions.

[0029] Optionally, the sample may be diluted prior to partitioning. This may be necessary, for example, if signal saturation is reached during the first run of the method of the invention due to a high nucleic acid concentration of the sample.

[0030] In certain embodiments of the above aspects, a multi-copy locus and a single-copy locus are amplified within one dPCR partition. In certain alternative embodiments of the above aspects, a multi-copy locus and a single-copy locus are amplified in different dPCR partitions. In certain other embodiments of the above aspects, more than one multi-copy locus and a single-copy locus are amplified within one dPCR partition or in different dPCR partitions. In certain other embodiments of the above aspects, more than one single-copy locus and a multi-copy locus are amplified within one dPCR partition or in different dPCR partitions. In certain other embodiments of the above aspects, more than one multi-copy locus and more than one single-copy locus are amplified within one dPCR partition or in different dPCR partitions.

[0031] In certain embodiments of the above aspects, at least one multi-copy locus and at least one single-copy locus are amplified within one dPCR partition.

[0032] In certain other embodiments of the above aspects, at least one multi-copy locus and at least one single-copy locus are amplified in different dPCR partitions.

[0033] In certain embodiments of the above aspects, the maximum quantifiable nucleic acid concentration of the sample is defined by the maximum quantifiable nucleic acid concentration of the at least one single-copy locus.

[0034] In certain embodiments of the above aspects, the minimum quantifiable nucleic acid concentration of the sample is defined by the minimum quantifiable nucleic acid concentration of the multi-copy locus having the largest number of copies among the at least one multi-copy locus.

[0035] In certain embodiments of the above aspects, the sample is selected from the group of samples including, but not limited to: eukaryotes, humans, animals, plants, bacteria, archaea, oomycetes, viruses, mitochondria, genomes, extrachromosomal, sex chromosomes, autosomes, human autosomes, or DNA or RNA or cells of fungi, environmental samples (e.g., containing microorganisms) or food samples (e.g., of animal or plant origin), or mixtures thereof.

[0036] In certain embodiments of the above aspects, the sample is a eukaryotic sample.

[0037] In certain embodiments of the above aspects, the detection step is performed using at least two probes, wherein the probes bind to the at least two amplification products.

[0038] In certain embodiments of the above aspects, the length of the amplification product is between 20 base pairs and 2000 base pairs.

[0039] In certain embodiments of the above aspects, the amplification is a triple dPCR amplification of a single-copy locus and two multi-copy loci, wherein the two multi-copy loci each occur at different copy numbers.

[0040] In certain other embodiments of the above aspects, the amplification is a multiplex dPCR amplification of a single-copy locus and multiple multi-copy loci, wherein the copy numbers of the multiple multi-copy loci are different from each other.

[0041] According to certain embodiments of the above aspects, the dPCR method is a nanoarray-based dPCR method, a water-in-oil droplet-based dPCR method, a microfluidic chip or nanofluidic chip-based dPCR method, or any other dPCR method.

[0042] In certain embodiments of the above aspects, the detection accuracy is at least 70%.

[0043] According to certain embodiments of the above aspects, the method is used for forensic analysis.

[0044] In certain embodiments of the above aspects, each genome and / or transcriptome of the at least one multi-copy locus has a copy number in the range of 2 to 260000, more preferably in the range of 5 to 200000, and even more preferably in the range of 10 to 130000.

[0045] When digital polymerase chain reaction contains at least 8500 partitions, the amount of the genome and / or transcriptome containing at least one multi-copy locus and at least one single-copy locus is between 0.000024 and 42500.

[0046] When digital polymerase chain reaction contains at least 22000 partitions, the amount of the genome and / or transcriptome containing the at least one multi-copy locus and the at least one single-copy locus is between 0.00001 and 100000.

[0047] When digital polymerase chain reaction contains at least 26000 partitions, the amount of the genome and / or transcriptome containing at least one multi-copy locus and at least one single-copy locus is between 0.000008 and 130000.

[0048] In certain embodiments of the above aspects, when there is one copy of MCL per genome and / or transcriptome at 130,000 copies in the digital amplification reaction, the lower limit of the dynamic range of the digital amplification reaction is reached.

[0049] The present invention also relates to a kit for the method of the present invention, the kit comprising: i. at least two primers and / or at least one probe for amplifying and / or quantifying at least one multi-copy locus; and ii. at least two primers and / or at least one probe for amplifying and / or quantifying at least one single-copy locus. Detailed embodiments

[0050] In a first aspect, the present invention provides a method for nucleic acid quantification, the method comprising the steps of: a. providing a sample comprising a plurality of nucleic acid molecules; b. amplifying at least one single-copy locus among the plurality of nucleic acid molecules, wherein the amplification method is digital polymerase chain reaction; c. amplifying at least one multi-copy locus among the plurality of nucleic acid molecules, wherein the amplification method is digital polymerase chain reaction; d. detecting the amplification products of steps b and c; and e. quantifying the amount of the plurality of nucleic acid molecules present in the sample.

[0051] In a second aspect, the present invention provides a method for nucleic acid quantification, the method comprising the steps of: a. providing a sample comprising a plurality of nucleic acid molecules; b. amplifying at least two loci among the plurality of nucleic acid molecules, wherein the at least two loci comprise at least one multi-copy locus and at least one single-copy locus, and wherein the amplification method is a digital polymerase chain reaction method; c. detecting at least two amplification products; and d. quantifying the amount of the plurality of nucleic acid molecules present in the sample.

[0052] The quantifiable nucleic acid concentration ranges of at least one multi-copy locus and at least one single-copy locus overlap. This improves or extends the dynamic range of the method of the present invention without increasing the number of available partitions per reaction per sample.

[0053] In a third aspect, the present invention provides a method for nucleic acid quantification, the method comprising the steps of: a. providing a sample comprising a plurality of nucleic acid molecules; b. Amplify at least two loci among the plurality of nucleic acid molecules, wherein the at least two loci comprise at least one multi-copy locus and at least one single-copy locus, and wherein the amplification method is a digital polymerase chain reaction method; c. Detect at least two amplification products; and d. Quantify the amount of the plurality of nucleic acid molecules present in the sample; wherein the quantifiable nucleic acid concentration ranges of the at least one multi-copy locus and the at least one single-copy locus overlap.

[0054] In a fourth aspect, the present invention provides a method for quantifying nucleic acids, the method comprising the steps of: a. Provide a sample comprising a plurality of nucleic acid molecules; b. Amplify at least two loci among the plurality of nucleic acid molecules, wherein the at least two loci comprise at least one multi-copy locus and at least one single-copy locus, and wherein the amplification method is a digital polymerase chain reaction method; c. Detect at least two amplification products; and d. Quantify the amount of the plurality of nucleic acid molecules present in the sample; wherein the quantifiable nucleic acid concentration ranges of the at least one multi-copy locus and the at least one single-copy locus overlap; and wherein the detection accuracy is at least 70%.

[0055] In a fifth aspect, the present invention provides a method for quantifying nucleic acids, the method comprising the steps of: a. Provide a sample comprising a plurality of nucleic acid molecules; b. Amplify at least two loci among the plurality of nucleic acid molecules, wherein the at least two loci comprise at least one multi-copy locus and at least one single-copy locus, and wherein the amplification method is a digital polymerase chain reaction method; c. Detect at least two amplification products; and d. Quantify the amount of the plurality of nucleic acid molecules present in the sample, wherein the quantifiable nucleic acid concentration ranges of the at least one multi-copy locus and the at least one single-copy locus overlap; wherein, if the concentration of the plurality of nucleic acid molecules present in the sample is low and the amplification of the at least one single-copy locus does not produce a detectable amplification product, the amplification product of the at least one multi-copy locus is used for quantification; wherein, if the concentration of the plurality of nucleic acid molecules present in the sample is high and the amplification product of the at least one multi-copy locus reaches signal saturation, the amplification product of the at least one single-copy locus is used for quantification.

[0056] In a sixth aspect, the present invention provides a method for nucleic acid quantification, the method comprising the following steps: a. providing a sample comprising a plurality of nucleic acid molecules; b. amplifying at least two loci among the plurality of nucleic acid molecules, wherein the at least two loci include at least one single-copy locus and at least one multi-copy locus, and wherein the amplification method is a digital polymerase chain reaction method; c. detecting at least two amplification products; and d. quantifying the amount of the plurality of nucleic acid molecules present in the sample, wherein the quantifiable nucleic acid concentration ranges of the at least one multi-copy locus and the at least one single-copy locus overlap; wherein, if the concentration of the plurality of nucleic acid molecules is lower than 0.002 ng / reaction, the quantification is based only on the steps performed on the at least one multi-copy locus; and / or if the concentration of the plurality of nucleic acid molecules is higher than 125 ng / reaction, the quantification is based only on the steps performed on the at least one single-copy locus.

[0057] As used herein, the "dynamic range" is defined as the sample concentration range that can be quantified in digital PCR.

[0058] The term "a plurality of nucleic acid molecules" includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) or mixtures thereof. Also covered are natural or artificially modified DNA and RNA.

[0059] The term "at least one" as used in the present invention refers to 1, 2, 3 or more. For example, at least one locus includes 1, 2, 3 or more loci respectively. Similarly, "at least two" as used herein refers to 2, 3, 4 or more. For example, at least two loci include 2, 3, 4 or more loci respectively.

[0060] The term "detectable amplification product" as used herein means that the amplification is successful, and the amplification product can be detected by the methods described herein (e.g., by a probe), and the amount of the plurality of nucleic acid molecules present in the sample can be quantified. Conversely, if the amplification product cannot be detected, there may be various reasons for this. For example, the amplification may fail due to too little nucleic acid per reaction. Therefore, the detection may fail if the signal intensity is not significantly higher than the background signal.

[0061] The term "quantify" as used herein means to determine or calculate the concentration. However, it may also include determining the absolute quantity value.

[0062] As used herein, "signal saturation" refers to a situation where the concentration of multiple nucleic acid molecules is so high that most or all partitions show positive signals. If this occurs, quantitative analysis can no longer be performed accurately.

[0063] The term "accuracy" as used herein refers to a measure of truthfulness. Accuracy refers to how closely a given set of measurements approximates its true value. It only describes systematic errors, i.e., a measure of the statistical deviation from a given measure of central tendency. Low accuracy results in a difference between the result and the true value.

[0064] "Accuracy" should not be confused with "precision", which defines the closeness between measured values. In other words, precision is a description of random errors, i.e., a measure of statistical variability.

[0065] If a locus is a multi-copy locus, it may occur multiple times in the genome or on a single chromosome. It may also be present on multiple DNA, RNA, or cDNA molecules being analyzed.

[0066] If a locus is a single-copy locus, it may occur only once on a single chromosome (e.g., a single-copy locus on the Y chromosome), or twice in the genome (e.g., on two alleles of a single-copy locus on an autosome or the X chromosome). It may also occur only once on the DNA, RNA, or cDNA molecules being analyzed.

[0067] Exemplary multi-copy loci include sex chromosome and / or autosomal multi-copy loci, non-limiting examples of which include MCL-Y, MCL-auto, repetitive DNA sequences (such as Alu sequences), retrotransposons, or any other multi-copy locus present on multiple nucleic acids in the sample being analyzed.

[0068] Exemplary single-copy loci include sex chromosome and / or autosomal single-copy loci, non-limiting examples of which include SRY, GRM7, ZFY, AMELY, TBL1Y, TSPY1, TSPY2, USP9Y, DDX3Y, UTY, TB4Y, EIF1AY, KDM5D, XKRY, HSFY1, HSFY2, PRY, PRY2, or any other single-copy locus present on multiple nucleic acids in the sample being analyzed.

[0069] All of the above gene examples are only exemplary genes and should not be considered as limiting the scope of the present invention. In fact, the methods of the present invention can be carried out using any multi-copy locus (or even more than one) and any single-copy locus (or even more than one), as long as the corresponding quantifiable nucleic acid concentration ranges overlap.

[0070] The present invention can also be achieved by using at least one low-copy multi-copy locus to replace at least one single-copy locus, and at least one MCL with a copy number higher than that of the low-copy multi-copy locus. In this case, the maximum copy number of the low-copy number locus is between 2 and 20, preferably between 2 and 10, and more preferably between 2 and 5.

[0071] Generally, the quantifiable range of nucleic acid molecules in a sample depends on the copy number of the locus being analyzed. The higher the copy number of the locus being analyzed, the lower the concentration of the input substance that can be quantified. The lower the copy number of the locus being analyzed, the higher the concentration of the input substance that can be quantified.

[0072] Therefore, the copy number information of a specific locus (SCL and MCL, or only SCL), combined with the number of positive and negative partitions obtained, can be used to calculate the nucleic acid concentration of the sample.

[0073] However, the quantifiable nucleic acid concentration ranges of at least one multi-copy locus and at least one single-copy locus need to overlap. For example, if one SCL and two MCLs are used, the quantifiable nucleic acid concentration range of the SCL needs to overlap with that of one of the MCLs (the one with the lower copy number), and the said MCL needs to overlap with the second MCL with a higher copy number, and so on for more MCLs.

[0074] In several embodiments of various aspects, each genomic and / or transcriptomic copy number of the at least one multi-copy locus is in the range of 2 to 260,000, more preferably in the range of 5 to 200,000, and even more preferably in the range of 10 to 130,000.

[0075] When the digital polymerase chain reaction contains at least 8,500 partitions, the amount of genomic and / or transcriptomic containing at least one multi-copy locus and at least one single-copy locus is between 0.000024 and 42,500.

[0076] When the digital polymerase chain reaction contains at least 22,000 partitions, the amount of genomic and / or transcriptomic containing the at least one multi-copy locus and the at least one single-copy locus is between 0.00001 and 100,000.

[0077] When the digital polymerase chain reaction contains at least 26,000 partitions, the amount of genomic and / or transcriptomic containing at least one multi-copy locus and at least one single-copy locus is between 0.000008 and 130,000.

[0078] The maximum quantifiable nucleic acid concentration of the sample is defined by the maximum quantifiable nucleic acid concentration of the at least one single-copy locus. Quantification may be limited by, for example (but not limited to), signal saturation.

[0079] The minimum quantifiable nucleic acid concentration of the sample is defined by the minimum quantifiable nucleic acid concentration of the multi-copy locus having the largest number of copies among the at least one multi-copy locus. Quantification may be limited, for example (but not limited to), by too few positive partitions.

[0080] In certain embodiments of the above aspects, when there is one copy of the MCL per genome and / or transcriptome at 130,000 copies in the digital amplification reaction, the lower limit of the dynamic range of the digital amplification reaction is reached.

[0081] According to the present invention, the maximum distance between the highest and lowest quantifiable amplification target concentrations is the distance between the maximum quantifiable amount of at least one SCL target and the minimum quantifiable amount of at least one MCL target before signal saturation. This is the case when at least 99.9% but less than 100% of the partitions are positive for the SCL target and at least one partition is positive for at least one MCL target, wherein the minimum quantifiable amount of at least one SCL target overlaps with the maximum quantifiable amount of at least one MCL target.

[0082] In certain embodiments of the aspects, the sample is dispensed into a plurality of partitions prior to the amplification step, and the method is performed in each of the plurality of partitions.

[0083] Optionally, the sample can be diluted prior to partitioning. This may be necessary, for example, when signal saturation is reached in the first run of the method of the present invention due to a high nucleic acid concentration of the sample.

[0084] Here, a variety of different types of digital PCR can be used.

[0085] Droplet digital PCR

[0086] Droplet digital PCR (ddPCR) is a dPCR method in which, for example, a 20 μl sample reaction (including assay primers and Taqman probe or intercalating dye) is divided into approximately 20,000 nanoliter-sized oil droplets by a water-oil emulsification technique, thermocycled to endpoint in a 96-well PCR plate, and then the fluorescence amplitude of all droplets in each sample well is read in a droplet flow cytometer.

[0087] Chip-based digital PCR

[0088] Chip-based digital PCR (dPCR) is also a dPCR method in which the reaction mixture (also used in qPCR) is partitioned on a chip into at least a few hundred, but in most cases several thousand, or about 10,000 to 45,000, or tens of thousands, or hundreds of thousands or more partitions. Amplification is then carried out using an end-point PCR thermocycler, and all partitions on each chip are read using a high-performance camera reader with fluorescence filters. A variety of fluorescent dyes can be used as long as the corresponding fluorescence channels can be separated from each other. Examples of available dyes include HEX, FAM, Cy5, Cy5.5, and Texas Red. This chip-based method relies on a nanofluidic chip.

[0089] Preferably, it is dPCR based on the QIAcuity plate (QIAGEN). For example, this method is based on the partitions in the plate rather than oil droplets. Here, ideally, each partition containing the target DNA molecule will produce a signal, while no signal will be produced in the partitions without the target DNA, hence the use of the term "digital", i.e., "yes / no".

[0090] According to several embodiments of various aspects, the dPCR method is a nanoplate-based dPCR method.

[0091] According to several embodiments of various aspects, the method is used for forensic analysis.

[0092] In several embodiments of various aspects, a multi-copy locus and a single-copy locus are amplified within one dPCR partition. In several alternative embodiments of various aspects, a multi-copy locus and a single-copy locus are amplified in different dPCR partitions. In several other embodiments of various aspects, more than one multi-copy locus and a single-copy locus are amplified within one dPCR partition or in different dPCR partitions. In several other embodiments of various aspects, more than one single-copy locus and a multi-copy locus are amplified within one dPCR partition or in different dPCR partitions. In several other embodiments of various aspects, more than one multi-copy locus and more than one single-copy locus are amplified within one dPCR partition or in different dPCR partitions.

[0093] In several embodiments of various aspects, at least one multi-copy locus and at least one single-copy locus are amplified within one dPCR partition.

[0094] In several other embodiments of various aspects, at least one multi-copy locus and at least one single-copy locus are amplified in different dPCR partitions.

[0095] In the method of the present invention, one or more multi-copy loci can be used, and the multi-copy loci occur 2 copies, or 10 copies, or 100 copies, or 1,000 copies, or 10,000 copies, or 100,000 copies, or more than 100,000 copies, or 2 to 11 copies, or 101 to 1000 copies, or 5,000 to 10,000 copies or 10,000 to 100,000 copies in each cell.

[0096] According to certain embodiments of various aspects, the sample is selected from the group of samples including but not limited to: eukaryotes, humans, animals, plants, bacteria, archaea, oomycetes, viruses, mitochondria, genomes, extrachromosomal, sex chromosomes, autosomes, human autosomes, or DNA or RNA or cells of fungi, environmental samples (such as those containing microorganisms) or food samples (such as those of animal or plant origin), or mixtures thereof.

[0097] The method of the present invention can also be used for human / animal pathogen tests (bacteria, fungi, oomycetes) and plant pathology (bacteria, fungi, oomycetes), and use the corresponding samples.

[0098] Preferably, the sample is a eukaryotic sample. Most preferably, the sample is a human sample. The sample can be derived from one of the following sample or tissue types, including whole blood, blood fractions, plasma, serum, body fluids, oral samples, oral fluid, saliva, sputum, swabs, urine, human biological tissues, clothing samples containing biological materials, vaginal swabs, sperm, skin or wound swabs, or other samples containing biological materials, or other parts of the human body samples that can be used to isolate nucleic acids. As used herein, the terms "oral fluid" and "body fluid" refer to the fluids excreted or secreted from the oral cavity and the body, respectively, from which nucleic acids can be isolated. As non-limiting examples, oral fluid and body fluid can include saliva, sputum, swabs, and urine.

[0099] For forensic samples, the sample can be a male or female sample. Alternatively, in the case of forensic samples, the sample may contain a mixture of male and female nucleic acids, where the amount of female nucleic acids (such as DNA or RNA) is several orders of magnitude higher than the amount of male nucleic acids (such as DNA or RNA), such as in sexual assault samples or pregnant women's blood samples containing male fetal nucleic acids. Thus, according to another embodiment, the sample contains one or more additional nucleic acids from different individuals.

[0100] According to certain embodiments of various aspects, the length of each dPCR amplification product of the at least two loci can be between 20 base pairs and 2000 base pairs.

[0101] Multiple nucleic acid molecules and / or probes (or other detection agents) and / or amplification products in a sample can be labeled or can contain modifications such as base modifications, sugar modifications, and / or backbone modifications, and can also contain fluorescent tags, barcodes, indices, peptides, proteins, and / or chemical moieties attached thereto.

[0102] In certain embodiments of various aspects, the amplification is a triple dPCR amplification of one single-copy locus and two multi-copy loci, wherein the two multi-copy loci each occur at a different copy number.

[0103] In certain other embodiments of various aspects, the amplification is a multiplex dPCR amplification of one single-copy locus and multiple multi-copy loci, wherein the copy numbers of the multiple multi-copy loci are different from each other.

[0104] Using multiple multi-copy loci (each with a different copy number) for multiplexing can improve the dynamic range more than using one single-copy locus and one multi-copy locus.

[0105] The target locus, i.e., the single-copy locus or multi-copy locus, can be located on an autosome or a sex chromosome.

[0106] The dPCR amplification method will include a buffer, dNTP or NTP in addition to the required enzyme.

[0107] The term "dNTP" as used herein refers to deoxyribonucleoside triphosphate. Non-limiting examples of such dNTPs include dATP, dGTP, dCTP, dTTP, dUTP, which can also exist in the form of labeled derivatives, such as those containing a fluorescent label, a radioactive label, a biotin label. dNTPs with modified nucleobases are also encompassed, wherein the nucleobase is, for example, hypoxanthine, xanthine, 7-methylguanine, inosine, xanthosine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, pseudouridine, dihydrouridine, 5-methylcytidine. In addition, the present invention also encompasses ddNTPs of the above molecules.

[0108] The term "NTP" as used herein refers to ribonucleoside triphosphate. Non-limiting examples of such NTPs include ATP, GTP, CTP, TTP, UTP, which can also exist in the form of labeled derivatives, such as those containing a fluorescent label, a radioactive label, a biotin label.

[0109] According to another embodiment of aspects of the present invention, the amplification reaction comprises: (a) Tris-HCl with a pH value between 8 and 8.8 (at 20°C) and / or; (b) a potassium salt (selected from potassium chloride and potassium sulfate) and / or; (c) an ammonium salt (preferably ammonium chloride or ammonium sulfate) and / or; (d) magnesium chloride and / or; (e) a hot-start polymerase.

[0110] Preferably, the concentration of Tris-HCl is in the range of 10 to 100 mM, most preferably in the range of 20 to 70 mM; the concentration of K + is in the range of 1 to 25 mM, most preferably in the range of 2.5 to 20 mM; the concentration of NH4 + is in the range of 1 to 40 mM, most preferably in the range of 2.5 to 30 mM; the concentration of Mg 2+ is 0.5 mM to 8 mM higher than the concentration of the four dNTPs, most preferably the concentration of Mg 2+ is 0.7 mM to 5 mM higher than the concentration of the four dNTPs; a hot-start polymerase is used, preferably a hot-start polymerase that allows a hot-start time of less than 5 minutes, most preferably less than 2 minutes.

[0111] At least two amplification products can be detected using a DNA probe, or an RNA probe, or a DNA intercalating dye or other known DNA labels or detection reagents. The detection of the amplification products can include direct or indirect fluorescence and / or chemical labeling and / or peptide- or protein-based labeling of the amplicons by a composition comprising one or more of the following: a probe and / or a guide molecule and / or a protein and / or a peptide and / or a nucleic acid and / or derivatives of the above-listed components. Preferably, at least two probes are used to detect at least two amplification products, wherein at least one of the at least two probes binds to one of the at least two amplification products, and at least one other of the at least two probes binds to another of the at least two amplification products. Thus, the detection step is carried out using at least two probes, wherein the probes bind to at least two amplification products.

[0112] Non-limiting examples of the probes include Taqman probes, Scorpion probes, molecular beacon probes, and / or fluorescence resonance energy transfer probes.

[0113] The quantitative analysis of multiple nucleic acid molecules in a sample is carried out using Poisson's law of small numbers. The background art section of this application describes in more detail how to quantify these amounts.

[0114] The present invention also relates to a kit for the method of the present invention, the kit comprising: i. at least two primers and / or at least one probe for amplifying and / or quantifying at least one multi-copy locus; and ii. At least two primers and / or at least one probe for amplifying and / or quantifying at least one single-copy locus.

[0115] The kit of the present invention may further comprise an operation manual, buffers and / or other reagents required for implementing the method of the present invention.

[0116] The method of the present invention has a short running time, high accuracy, high precision, low sensitivity to inhibitors and a wide dynamic range.

[0117] The detection accuracy of the method of the present invention should be at least 95%, or at least 90%, or at least 85%, or at least 80%, or at least 75%, or preferably at least 70%. Alternatively, the detection accuracy of the method of the present invention may be 70% to 100%, or 70% to 99%, or 71% to 99%, or 70% to 98%, or 70% to 97%, or 70% to 96%, or 70% to 95%, or 70% to 90%, or 70% to 85%, or 70% to 80%, or 75% to 99%, or 80% to 99%, or 85% to 99% or 90% to 99%.

[0118] The detection accuracy used herein also includes quantification accuracy, which is generally related to the overall accuracy of the method of the present invention.

[0119] Calculation of the dynamic range extension of dPCR by parallel use of a combination of single-copy and multi-copy targets

[0120] Currently commercially available digital PCR systems are based on sample partitioning in microfluidic / nanofluidic chips or nanoplate or water-in-oil droplets. For example, the QIAGEN QIAcuity dPCR system provides a nanoplate with 8500 or 26000 partitions per well, the ThermoFisher QuantStudio3D digital PCR system is based on a nanofluidic chip with 20000 partitions, and the Bio-Rad QX200 droplet digital PCR system has 20000 droplets (partitions) for every 20 µl of sample.

[0121] The theoretical upper and lower limits of the dynamic range can be calculated and expressed using Poisson statistics.

[0122] According to dMIQE Group and Huggett (dMIQE Group and Huggett, 2020, ClinChem., "Digital MIQE Guidelines Update: Essential Information for the Release of Quantitative Digital PCR Experiments in 2020"), the two assumptions for dPCR to fit the Poisson distribution are that the volumes of all partitions are equal and the target molecules are randomly distributed among the partitions. In practice, this means that each partition has an equal chance of containing the target molecule. The number of target molecules present in a positive partition may be one, two, or more molecules, and it is currently impossible to determine how many molecules a given positive partition may contain. However, the number of molecules in a negative partition is known. If the volumes of all partitions are equal, the average concentration of target molecules (λ) in each partition can be estimated based on the probability that a partition is negative, using the proportion of negative partitions and the Poisson distribution. This concentration is derived from the number of negative partitions (w) and the total number of partitions (n) in the reaction: 。

[0123] If all partitions contain the amplified target, i.e., all partitions have a positive signal and the dPCR experiment is saturated, quantification cannot be performed.

[0124] Therefore, for the above dPCR system, theoretically, when 8499 (=99.98%), 25999 (=99.99%), or 21999 (=99.99%) partitions contain at least one amplified target, the upper limit of the dynamic range is reached. Therefore, for the above dPCR system, λ should be (rounded to the nearest integer): 。

[0125] The above λ value marks the statistical / theoretical limit of the present invention. However, more practically, to reduce the sources of error, the value of λ = 5.

[0126] For DNA and / or RNA quantification systems based on single-copy loci, the minimum amount of genome and / or transcriptome per volume of each analytical sample required for one positive partition in dPCR is 1 (= the lower limit of the dynamic range or the lowest detectable DNA and / or RNA concentration in a dPCR experiment). Before reaching saturation, the maximum number (m) of genome and / or transcriptome per volume of each analytical sample is the copy number (c) of each amplified locus per genome and / or transcriptome (where, if it is a single-copy locus, c is 1; if a multi-copy locus is amplified, c is 1 divided by the copy number of the multi-copy locus) multiplied by λ, and then multiplied by 99.98% (for 8500 partitions) or 99.99% (for 26000 or 22000 partitions) of the number of partitions (n 饱和度 ): m = c * λ * n 饱和度 。

[0127] When at least one MCL is amplified in addition to the SCL in dPCR, the lower limit of the dynamic range of the DNA and / or RNA quantification experiment of dPCR can be extended. For example, if 100 copies of MCL are amplified per genome and / or transcriptome, theoretically, as long as the genome / transcriptome appears 0.01 times, a positive partition can be obtained in dPCR.

[0128] The terms "genome" and "transcriptome" refer to the total amount of DNA or RNA of each cell in the analyzed sample volume, respectively.

[0129] For more details of the good results obtained by the method of the present invention, please refer to the description of the drawings and the examples.

[0130] Examples

[0131] The following examples are used in combination with the figures to illustrate the present invention.

[0132] In this article, dPCR refers to digital PCR; qPCR refers to real-time quantitative polymerase chain reaction.

[0133] All experiments were carried out using QIAGEN digital PCR mix on QIAcuity (8.5k 96-well plate) with a 20 µl reaction system (total volume) according to the manufacturer's instructions. Any commercially available dPCR mixture capable of digitally amplifying SCL and MCL can be used in the method of the present invention. Non-limiting examples include QIAcuity Residual DNA Quantification Kit, QIAcuity UCP Probe PCR Kit, QIAcuity EG PCR Kit, and QIAcuity Probe PCR Kit.

[0134] The amplification of the present invention was carried out for 40 cycles. The cycling protocol was as follows: an initial heat activation step for 3 minutes at 95 °C, followed by a two-step amplification step for 5 seconds at 95 °C and 35 seconds at 60 °C.

[0135] All experiments shown in the figures used the Figure 5 primers and probes shown in. However, the loci amplified and detected by these sequences and the sequences themselves should not be regarded as limiting the scope of the present invention, but only provide exemplary MCL and SCL that can be used in the method of the present invention. However, any MCL (at least one) and any SCL (at least one) can be used in the context of the present invention as long as the corresponding quantifiable nucleic acid concentration ranges overlap.

[0136] The concentration ranges in the following paragraphs use human genomic DNA as the nucleic acid used by way of example. Although they well illustrate the dynamic range expansion achieved by the methods of the present invention, dynamic range expansion can also be achieved by using one or more single-copy loci and one or more multi-copy loci on other types of nucleic acids (such as RNA, cDNA, other DNA), and / or by using multiple and / or different MCLs and / or SCLs. The degree of dynamic range expansion depends on various factors, such as but not limited to the type of nucleic acid used and / or the copy number or frequency of occurrence of one or more multi-copy loci used. Thus, to provide examples of the methods of the present invention, but without wishing to be bound thereby, the following paragraphs relate to exemplary / specific embodiments of specific MCLs (MCL-auto or MCL-Y), specific SCLs (SCL-auto or SCL-Y), and human genomic DNA.

[0137] In specific embodiments of various aspects, the total measurable concentration range of multiple nucleic acid molecules can be from 0.00001 ng / reaction to 2000 ng / reaction. Preferably, it ranges from 0.00005 ng / reaction to 1800 ng / reaction. More preferably, it ranges from 0.0001 ng / reaction to 1600 ng / reaction. Even more preferably, it ranges from 0.00019 ng / reaction to 1300 ng / reaction. Most preferably, it ranges from 0.00025 ng / reaction to 1000 ng / reaction.

[0138] In specific embodiments of various aspects, when amplifying multi-copy loci, the concentration range of the measurable multiple nucleic acid molecules can be from 0.00001 ng / reaction to 100 ng / reaction. Preferably, it ranges from 0.00005 ng / reaction to 90 ng / reaction. More preferably, it ranges from 0.0001 ng / reaction to 80 ng / reaction. Even more preferably, it ranges from 0.00019 ng / reaction to 70 ng / reaction. Most preferably, it ranges from 0.00025 ng / reaction to 62.5 ng / reaction. When amplifying multi-copy loci, the concentration of the measurable multiple nucleic acid molecules can be less than 125 ng / reaction, or less than 66 ng / reaction, or less than 26 ng / reaction, or less than 2.6 ng / reaction, or less than 0.26 ng / reaction, or less than 0.026 ng / reaction, or less than 0.0026 ng / reaction, or less than 0.00026 ng / reaction.

[0139] In specific embodiments of various aspects, when amplifying multi-copy loci, when the concentration range of multiple nucleic acid molecules is from 0.0039 ng / reaction to 40 ng / reaction, at least 70% detection accuracy can be achieved. In specific embodiments of various aspects, when amplifying multi-copy loci, when the concentration range of multiple nucleic acid molecules is from 0.125 ng / reaction to 40 ng / reaction (autosomal multi-copy loci) or from 0.125 ng / reaction to 20 ng / reaction (sex chromosome multi-copy loci), at least 90% detection accuracy can be achieved.

[0140] In specific embodiments of various aspects, when amplifying single-copy loci, the concentration range of measurable multiple nucleic acid molecules is from 0.00001 ng / reaction to 2000 ng / reaction. Preferably, the range is from 0.0001 ng / reaction to 1800 ng / reaction. More preferably, the range is from 0.001 ng / reaction to 1600 ng / reaction. Even more preferably, the range is from 0.0015 ng / reaction to 1300 ng / reaction. Most preferably, the range is from 0.002 ng / reaction to 1000 ng / reaction. When amplifying single-copy loci, the concentration of measurable multiple nucleic acid molecules can be greater than 0.0005 ng / reaction, or greater than 0.001 ng / reaction, or greater than 0.0099 ng / reaction, or greater than 0.099 ng / reaction, or greater than 0.99 ng / reaction, or greater than 9.9 ng / reaction, or greater than 99 ng / reaction, or greater than 999 ng / reaction.

[0141] In specific embodiments of various aspects, when amplifying single-copy loci, when the concentration range of multiple nucleic acid molecules is from 0.125 ng / reaction to 1000 ng / reaction, or from 0.125 ng / reaction to 640 ng / reaction (autosomal single-copy loci), or from 0.25 ng / reaction to 1000 ng / reaction (sex chromosome single-copy loci), the detection accuracy can reach at least 70%. In specific embodiments of various aspects, when amplifying single-copy loci, when the concentration range of multiple nucleic acid molecules is from 1 ng / reaction to 160 ng / reaction (autosomal single-copy loci), or from 2 ng / reaction to 80 ng / reaction (sex chromosome single-copy loci), the detection accuracy can reach at least 90%.

[0142] Table 1 shows the quantitative data according to Figure 1 and Figure 2 of.

[0143]

[0144] In specific embodiments of various aspects of the present invention, for single-copy loci, in the absence of complete signal saturation, the DNA concentration that can still be detected and quantified is at least 1000 ng / reaction.

[0145] In a specific embodiment of various aspects of the present invention, for a multi-copy locus, in the absence of a quantification failure, the DNA concentration that can still be detected and quantified is 0.00025 ng / reaction, and may even be lower.

[0146] In a specific embodiment of various aspects of the present invention, there is also a concentration range of multiple nucleic acid molecules for which amplification of at least one single-copy locus and at least one multi-copy locus can both be carried out and quantified. This range may include multiple nucleic acid molecules at a concentration of 0.002 ng / reaction to 62.5 ng / reaction, preferably 0.002 ng / reaction to 31.25 ng / reaction, or 0.008 ng / reaction to 31.25 ng / reaction, or 0.008 ng / reaction to 62.5 ng / reaction.

[0147] However, as described above, the methods of the present invention also cover other concentration ranges for other types of nucleic acids, other SCLs, or other (optionally more than one) multi-copy loci (each having a greater or lesser number of copies). Generally, according to the methods of the present invention, the following applies: If the concentration of multiple nucleic acid molecules is too high, amplification may still be successfully carried out, but quantification may not be successful due to signal saturation. If the concentration is too low, amplification and / or detection and / or quantification may fail. However, there is a certain range of nucleic acid input for which quantification can be carried out using both MCL and SCL or either one of them, thereby increasing the dynamic range of nucleic acid quantification.

[0148] Example 1

[0149] Expanding the dynamic range of dPCR by using a combination of single-copy and multi-copy autosomal targets in parallel

[0150] A human DNA dilution series from 1000 ng / reaction to 0.00025 ng / reaction was generated. To detect copy number, the single-copy autosomal target SCL-auto and the multi-copy autosomal target MCL-auto were combined in a duplex dPCR reaction. The DNA dilution samples were run in quadruplicate, with DNA concentrations ranging from 0.00025 ng / RxN to 1.95 ng / RxN. Replicate experiments were performed for all higher DNA concentrations. For 0.002 ng / reaction, all 4 replicates showed signals. For the multi-copy target, the highest quantifiable amount was up to 31.25 ng / reaction (4 logs). Due to signal saturation, all higher DNA concentrations could not be detected. All partitions were positive. For the single-copy target SCL-auto, the highest detectable DNA concentration without complete signal saturation was 1000 ng / reaction. When the two targets were applied in parallel, the ranges of the two targets overlapped, with a total detection range of 0.002 ng / reaction to 1000 ng / reaction and a dynamic range of 5 logs. The quantification results are as Figure 1 shown.

[0151] Example 2

[0152] Expanding the dynamic range of dPCR by using a combination of single-copy and multi-copy sex chromosome targets in parallel

[0153] A human male DNA dilution series from 1000 ng / reaction to 0.00025 ng / reaction was generated. To detect copy number, the single-copy Y chromosome target SCL-Y and the multi-copy Y chromosome target MCL-Y were combined in a duplex dPCR reaction. The DNA dilution samples were run in quadruplicate, with DNA concentrations ranging from 0.00025 ng / RxN to 1.95 ng / RxN. Replicate experiments were performed for all higher DNA concentrations. For 0.002 ng / reaction, all 4 replicates showed signals. For the multi-copy target, the highest quantifiable amount was up to 62.5 ng / reaction (4 logs). Due to signal saturation, all higher DNA concentrations could not be detected. All partitions were positive. For the single-copy Y chromosome target SCL-Y, the highest detectable DNA concentration without complete signal saturation was 1000 ng / reaction. When the two targets were applied in parallel, the ranges of the two targets overlapped, with a total detection range of 0.002 ng / reaction to 1000 ng / reaction and a dynamic range of 5 logs. The quantification results are as Figure 2 shown.

[0154] Example 3

[0155] Amplifying the dynamic range of dPCR by using a combination of single-copy and multi-copy autosomal targets in parallel and its impact on detection accuracy

[0156] A human DNA dilution series from 1000 ng / reaction to 0.002 ng / reaction was generated. To detect copy number, the single-copy autosomal target SCL-auto and the multi-copy autosomal target MCL-auto were combined in a duplex dPCR reaction. The DNA dilution samples were run in quadruplicate for the multi-copy target and also in quadruplicate for the single-copy target. The detection accuracy (ratio of detected copy number to expected copy number) of the MCL-auto target exceeded 70% in the range from 0.0039 ng / rxn to 40 ng / rxn and exceeded 90% for the highest accuracy in the range from 0.125 ng / rxn to 40 ng / reaction. The accuracy of the SCL-auto target exceeded 70% in the range from 0.125 ng / rxn to 640 ng / rxn and exceeded 90% for the highest accuracy in the range from 1 ng / rxn to 160 ng / rxn. If more than 40 ng / rxn was used, the MCL-auto detection system could not report accurate copy numbers (accuracy exceeding 70%), while the second detection system was able to detect copy numbers with an accuracy exceeding 70% at DNA amounts up to 640 ng / rxn. The same was true for the lowest DNA amounts. SCL-auto could not report copy numbers below 0.125 ng / rxn with an accuracy exceeding 70%, while MCL-auto was able to detect copy numbers as low as 0.0039 ng / rxn with an accuracy exceeding 70%. These data clearly show that combining at least two detection systems with different optimal detection ranges can expand the dynamic range. The quantitative results are as Figure 3 shown.

[0157] Example 4

[0158] Amplifying the dynamic range of dPCR and its effect on detection accuracy by using a combination of single-copy and multi-copy sex chromosome targets in parallel

[0159] A human DNA dilution series from 1000 ng / reaction to 0.002 ng / reaction was generated. To detect copy numbers, the single-copy sex chromosome target SCL-Y and the multi-copy sex chromosome target MCL-Y were combined in a duplex dPCR reaction. The DNA dilution samples were run in quadruplicate for the multi-copy target and also in quadruplicate for the single-copy target. The detection accuracy (ratio of detected copy number to expected copy number) of the MCL-Y target exceeded 70% in the range from 0.0039 ng / rxn to 40 ng / rxn and exceeded 90% for the highest accuracy in the range from 0.125 ng / rxn to 20 ng / reaction. The SCL-Y target showed an accuracy exceeding 70% from 0.25 ng / rxn to 1000 ng / rxn and exceeded 90% for the highest accuracy from 2 ng / rxn to 80 ng / rxn. If more than 40 ng / rxn was used, the MCL-Y detection system could not report accurate copy numbers (accuracy exceeding 70%), while the second detection system was able to detect copy numbers with an accuracy exceeding 70% at DNA amounts up to 1000 ng / rxn. The same was true for the lowest DNA amounts. Here, SCL-Y could not report copy numbers with an accuracy exceeding 70% below 0.25 ng / rxn, while MCL-Y was able to detect copy numbers as low as 0.0039 ng / rxn with an accuracy exceeding 70%. These data clearly show that combining at least two detection systems with different optimal detection ranges can extend the dynamic range. Quantitative results are as Figure 4 shown.

[0160] Example 5

[0161] Table 2 shows the calculation of the dynamic range extension for DNA and / or RNA quantification in dPCR using a combination of amplification of a single-copy locus (SCL) and at least one multi-copy locus (MCL), where the copy number per genome and / or transcriptome and per analyzed sample volume is 2 (MCL-2) or up to 300,000 (MCL-300,000), and the dPCR system has 8500 partitions (Table 2A), 26,000 partitions (Table 2B), or 20,000 partitions (Table 2C).

[0162] Table 2A)

[0163] In a dPCR system with 8,500 partitions, DNA and / or RNA quantification is performed by dPCR based on single-copy loci (SCL). The upper limit of the dynamic range is reached when there are 76,484 (λ = 9; black background color) or 42,491 (λ = 5; dark gray background color) genomes and / or transcriptomes containing the single-copy locus in each analyzed sample in the dPCR experiment. The lower limit of the dynamic range is reached when there is a single genome and / or transcriptome containing the single-copy locus in each analyzed sample in the dPCR experiment (gray background color).

[0164] Due to the overlapping dynamic ranges of SCL and MCL, in the dPCR DNA and / or RNA quantification assay, the amplification of the SCL is combined with the amplification of at least one multi-copy locus (MCL), significantly extending the lower limit of the dynamic range. In this example, the lowest possible value of the dynamic range is reached when one copy of the MCL with 76,484 (λ = 9) or 42,491 (λ = 5) copies per genome and / or transcriptome is amplified in the dPCR (black background color).

[0165]

[0166] Table 2B)

[0167] In a dPCR system with 26,000 partitions, DNA and / or RNA quantification is performed by dPCR based on single-copy loci (SCL). The upper limit of the dynamic range is reached when there are 259,974 (λ = 10; black background color) or 129,987 (λ = 5; dark gray background color) genomes and / or transcriptomes containing the single-copy locus in each analyzed sample in the dPCR experiment. The lower limit of the dynamic range is reached when there is a single genome and / or transcriptome containing the single-copy locus in each analyzed sample in the dPCR experiment (gray background color).

[0168] Due to the overlapping dynamic ranges of SCL and MCL, in the dPCR DNA and / or RNA quantification assay, the amplification of the SCL is combined with the amplification of at least one multi-copy locus (MCL), significantly extending the lower limit of the dynamic range. In this example, the lowest possible value of the dynamic range is reached when one copy of the MCL with 259,974 (λ = 10) or 129,987 (λ = 5) copies per genome and / or transcriptome is amplified in the dPCR (black background color).

[0169]

[0170] Table 2C)

[0171] In a dPCR system with 22,000 partitions, DNA and / or RNA quantification is performed based on single-copy locus (SCL) dPCR. When there are 179,964 (λ = 10; black background color) or 99,980 (λ = 5; dark gray background color) genomes and / or transcriptomes containing the single-copy locus in each analyzed sample in the dPCR experiment, the upper limit of the dynamic range is reached; when there is a single genome and / or transcriptome containing the single-copy locus in each analyzed sample in the dPCR experiment (gray background color), the lower limit of the dynamic range is reached.

[0172] Since the dynamic ranges of SCL and MCL overlap, therefore, in dPCR DNA and / or RNA quantification assays, the amplification of the SCL is combined with the amplification of at least one multi-copy locus (MCL), significantly expanding the lower limit of the dynamic range. In this example, when one copy of an MCL with 179,964 (λ = 10) or 99,980 (λ = 5) copies of each genome and / or transcriptome is amplified in dPCR (black background color), the lowest possible value of the dynamic range is reached.

[0173]

[0174] Example 6

[0175] The dynamic range of dPCR is expanded by using a combination of single-copy and at least two multi-copy autosomal targets in parallel.

[0176] A human DNA dilution series from 500 ng / reaction to 0.00000008192 ng / reaction was generated. To detect the copy number, the single-copy autosomal target SCL-auto and two multi-copy autosomal targets (mid-MCL-auto and high-MCL-auto) were combined in a triple dPCR reaction. DNA dilution samples at all DNA concentrations were performed in triplicate. For the high multi-copy target (high-MCL-auto), reliable quantification could be performed from 0.00001024 ng / reaction to 0.8 ng / reaction (4 log). All higher DNA concentrations could not be detected due to signal saturation. All partitions were positive. For the mid multi-copy target (mid-MCL-auto), reliable quantification could be performed from 0.00128 ng / reaction to 20 ng / reaction (4 log) until saturation was reached. Using the single-copy target (SCL-auto), reliable quantification could be performed in the range of 0.032 ng / reaction to 500 ng / reaction (4 log). When all three targets were applied in parallel, the ranges of all targets overlapped, and the total detection range was from 0.00001024 ng / reaction to 500 ng / reaction, with a dynamic range of 7 log. The quantification results are as Figure 7 shown. Description of the Drawings

[0177] Figure 1 : Using target combinations with different copy numbers to expand the dynamic range of digital PCR.

[0178] Shown are DNA quantification data for a human DNA dilution series from 0.00025 ng / reaction to 1000 ng / reaction. To detect copy numbers, the single-copy autosomal target SCL-auto and the multi-copy autosomal target MCL-auto were combined in a dual reaction. The DNA dilution samples were run in quadruplicate, with DNA concentrations ranging from 0.00025 ng / RxN to 1.95 ng / RxN. Replicates were performed for all higher DNA concentrations. For 0.002 ng / reaction, all 4 replicates showed signals. For the multi-copy target, the highest quantifiable amount was up to 31.25 ng / reaction (4 log). Due to signal saturation, all higher DNA concentrations could not be detected. All partitions were positive. For the single-copy target SCL-auto, the highest detectable DNA concentration without complete signal saturation was 1000 ng / reaction. When two targets were applied in parallel, the ranges of the two targets overlapped, with a total detection range of 0.002 ng / reaction to 1000 ng / reaction and a dynamic range of 5 log.

[0179] Figure 2 : Using target combinations with different copy numbers to expand the dynamic range of digital PCR.

[0180] Shown are DNA quantification data for a human male DNA dilution series from 0.00025 ng / reaction to 1000 ng / reaction. To detect copy numbers, the single-copy Y-chromosome target SCL-Y and the multi-copy Y-chromosome target MCL-Y were combined in a dual reaction. The DNA dilution samples were run in quadruplicate, with DNA concentrations ranging from 0.00025 ng / RxN to 1.95 ng / RxN. Replicates were performed for all higher DNA concentrations. For 0.002 ng / reaction, all 4 replicates showed signals. For the multi-copy target, the highest quantifiable amount was up to 62.5 ng / reaction (4 log). Due to signal saturation, all higher DNA concentrations could not be detected. All partition results were positive. For the single-copy Y-chromosome target SCL-Y, the highest detectable DNA concentration without complete signal saturation was 1000 ng / reaction. When two targets were applied in parallel, the ranges of the two targets overlapped, with a total detection range of 0.002 ng / reaction to 1000 ng / reaction and a dynamic range of 5 log.

[0181] Figure 3: Digital PCR dynamic range extension by parallel use of single-copy and multi-copy target combinations and its impact on detection accuracy.

[0182] Shown are DNA quantification data for a human DNA dilution series from 0.002 ng / reaction to 1000 ng / reaction. To detect copy number, the single-copy autosomal target SCL-auto and the multi-copy autosomal target MCL-auto were combined in a dual reaction. The DNA dilution samples were run in quadruplicate for the multi-copy target and also in quadruplicate for the single-copy target. Shown in the figure is the detection accuracy exceeding 70% (ratio of detected copy number to expected copy number) for the DNA template amounts tested. The MCL-auto target showed accuracy exceeding 70% in the range from 0.0039 ng / rxn to 40 ng / rxn and a peak accuracy exceeding 90% in the range from 0.125 ng / rxn to 40 ng / reaction. The SCL-auto target showed accuracy exceeding 70% in the range from 0.125 ng / rxn to 640 ng / rxn and a peak accuracy exceeding 90% in the range from 1 ng / rxn to 160 ng / rxn. If more than 40 ng / rxn was used, the MCL-auto detection system could not report accurate copy numbers (accuracy exceeding 70%), while the second detection system was able to detect copy numbers with accuracy exceeding 70% at DNA amounts up to 640 ng / rxn. The same was true for the lowest DNA amounts. SCL-auto could not report copy numbers with accuracy exceeding 70% below 0.125 ng / rxn, while MCL-auto was able to detect copy numbers as low as 0.0039 ng / rxn with accuracy exceeding 70%. These data clearly demonstrate that combining at least two detection systems with different optimal detection ranges can extend the dynamic range.

[0183] Figure 4 : Digital PCR dynamic range extension by parallel use of single-copy and multi-copy target combinations and its impact on detection accuracy.

[0184] Shown are DNA quantification data of a human DNA dilution series from 0.002 ng / reaction to 1000 ng / reaction. To detect copy numbers, the single-copy sex chromosome target SCL-Y and the multi-copy sex chromosome target MCL-Y were combined in a dual reaction. The DNA dilution samples were run in quadruplicate for the multi-copy target and also in quadruplicate for the single-copy target. Shown in the figure is that the detection accuracy exceeds 70% (the ratio of detected copy number to expected copy number) for the tested amounts of DNA template. The MCL-Y target shows an accuracy exceeding 70% in the range from 0.0039 ng / rxn to 40 ng / rxn, with a maximum accuracy exceeding 90% in the range from 0.125 ng / rxn to 20 ng / reaction. The SCL-Y target shows an accuracy exceeding 70% in the range from 0.25 ng / rxn to 1000 ng / rxn, with a maximum accuracy exceeding 90% in the range from 2 ng / rxn to 80 ng / rxn. If more than 40 ng / rxn is used, the MCL-Y detection system cannot report accurate copy numbers (accuracy exceeding 70%), while the second detection system can detect copy numbers with an accuracy exceeding 70% for DNA amounts up to 1000 ng / rxn. The same is true for the lowest DNA amounts. SCL-Y cannot report copy numbers below 0.25 ng / rxn with an accuracy exceeding 70%, while MCL-Y can detect copy numbers as low as 0.0039 ng / rxn with an accuracy exceeding 70%. These data clearly show that combining at least two detection systems with different optimal detection ranges can expand the dynamic range.

[0185] Figure 5 : Sequence

[0186] Shown in the figure are exemplary sequences of primers and probes used in the method of the present invention. The "+" sign indicates that the base after the sign is a locked nucleic acid nucleotide (LNA). LNA can enhance the template binding strength.

[0187] Figure 6 : Calculated copy number per haploid genome of a multi-copy locus with unknown copy number

[0188] Shown is the calculated copy number of the target multi-copy locus (MCL-auto or MCL-Y) based on the copy number of the corresponding single-copy locus. Using Poisson's law of small numbers, the concentration of the amplified locus is calculated using the number of positive and negative partitions, and based on the knowledge that SCL appears once in each haploid genome, the copy number of the MCL used is calculated by backtracking. These results show that the method of the present invention can perform constant and accurate quantification over a wide range of input sample nucleic acid concentrations (at least from about 1.9 ng / reaction to about 31.3 ng / reaction).

[0189] Figure 7:Expanding the dynamic range of dPCR by using a combination of single-copy and at least two multi-copy autosomal targets in parallel.

[0190] A human DNA dilution series from 500 ng / reaction to 0.00000008192 ng / reaction was generated. To detect copy number, the single-copy autosomal target SCL-auto and two multi-copy autosomal targets (mid-MCL-auto and high-MCL-auto) were combined in a triple dPCR reaction. DNA dilution samples at all DNA concentrations were performed in triplicate. For the high multi-copy target (high-MCL-auto), reliable quantification was possible from 0.00001024 ng / reaction to 0.8 ng / reaction (4 logs). All higher DNA concentrations could not be detected due to signal saturation. All partitions were positive. For the mid multi-copy target (mid-MCL-auto), reliable quantification was possible from 0.00128 ng / reaction to 20 ng / reaction (4 logs) until saturation was reached. Using the single-copy target (SCL-auto), reliable quantification was possible in the range of 0.032 ng / reaction to 500 ng / reaction (4 logs). When all three targets were applied in parallel, the ranges of all targets overlapped, and the total detection range was from 0.00001024 ng / reaction to 500 ng / reaction, with a dynamic range of 7 logs. The quantification results are shown in the figure.

Claims

1. A nucleic acid quantification method, the method comprising the following steps: a. Providing a sample comprising a plurality of nucleic acid molecules; b. Amplifying at least two loci among the plurality of nucleic acid molecules, wherein the at least two loci comprise at least one multi-copy locus and at least one single-copy locus, and wherein the amplification method is a digital polymerase chain reaction method; c. Detecting at least two amplification products; And d. Quantifying the amount of the plurality of nucleic acid molecules present in the sample; wherein the quantifiable nucleic acid concentration ranges of the at least one multi-copy locus and the at least one single-copy locus overlap.

2. The method according to any one of the preceding claims, wherein the sample is partitioned into a plurality of partitions before the amplification step, and wherein the method is performed in each of the plurality of partitions.

3. The method according to any one of the preceding claims, wherein the maximum quantifiable nucleic acid concentration of the sample is defined by the maximum quantifiable nucleic acid concentration of the at least one single-copy locus.

4. The method according to any one of the preceding claims, wherein the minimum quantifiable nucleic acid concentration of the sample is defined by the minimum quantifiable nucleic acid concentration of the multi-copy locus having the largest number of copies among the at least one multi-copy locus.

5. The method according to any one of the preceding claims, wherein the sample is selected from the group of samples including but not limited to: eukaryotes, humans, animals, plants, bacteria, archaea, oomycetes, viruses, mitochondria, genomes, extrachromosomal, sex chromosomes, autosomes, human autosomes, or DNA or RNA or cells or environmental samples or food samples of fungi, or mixtures thereof.

6. The method according to any one of the preceding claims, wherein the detection step is performed using at least two probes, wherein the probes bind to the at least two amplification products.

7. The method according to any one of the preceding claims, wherein the length of the amplification product is between 20 base pairs and 2000 base pairs.

8. The method according to any one of the preceding claims, wherein the amplification is a triple dPCR amplification of one single-copy locus and two multi-copy loci, wherein the two multi-copy loci each occur in different copy numbers.

9. The method according to any one of the preceding claims, wherein the dPCR method is a nanoarray-based dPCR method.

10. The method according to any one of the preceding claims, wherein the method is used for forensic analysis.

11. The method according to any one of the preceding claims, wherein each genome and / or transcriptome of the at least one multi-copy locus contains a copy number in the range of 2 to 260000, more preferably in the range of 5 to 200000, even more preferably in the range of 10 to 130000.

12. The method according to any one of the preceding claims, wherein when the digital polymerase chain reaction comprises at least 8500 partitions, the amount of the genome and / or transcriptome comprising at least one multi-copy locus and at least one single-copy locus is between 0.000024 and 42500.

13. The method according to any one of claims 1-11, wherein when the digital polymerase chain reaction comprises at least 22,000 partitions, the amount of the genome and / or transcriptome comprising the at least one multi-copy locus and the at least one single-copy locus is between 0.00001 and 100,000.

14. The method according to any one of claims 1-11, wherein when the digital polymerase chain reaction comprises at least 26,000 partitions, the amount of the genome and / or transcriptome comprising at least one multi-copy locus and at least one single-copy locus is between 0.000008 and 130,000.

15. A kit for the method according to any one of the preceding claims, the kit comprising (i) at least two primers and / or at least one probe for amplifying and / or quantifying at least one multi-copy locus, and (ii) at least two primers and / or at least one probe for amplifying and / or quantifying at least one single-copy locus.

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