Bioanalysis process making use of nucleic acid fragmentation

Abstract

A method for detecting target nucleic acid molecules in a sample comprises fragmenting target nucleic acid molecules in the sample into multiple target nucleic acid fragments The sample is then subjected to at least one pre-analysis operation comprising extraction and / or purification of nucleic acid molecules to get a processed sample or an aliquot thereof comprising target nucleic acid fragments. The target nucleic acid fragments are detected in the processed sample or in the aliquot thereof and the method concludes the target nucleic acid molecules were present in the processed sample are quantified based on the detected target nucleic acid fragments. The invention improves the sensitivity and reproducibility in bioanalysis processed and in particular for samples containing very few target nucleic acid molecules.

Description

[0001] BIOANALYSIS PROCESS

[0002] TECHNICAL FIELD

[0003] The present invention generally relates to bioanalysis and in particular to a method for detecting target nucleic acid molecules in a sample.

[0004] BACKGROUND

[0005] Bioanalysis relates to the quantitative measurement of xenobiotics, such as drugs and their metabolites, and biotics, such as proteins and nucleic acid molecules, in biological systems. Bioanalysis generally involves a pre-analysis phase prior to the actual quantitative measurement of the target molecules, see Fig. 1. Such a pre-analysis phase may prepare the sample for quantification and include, for instance, extraction, sampling, and purification but may also include transport, storage, freezing / thawing, analyte concentrating, aliquoting, etc. The pre-analysis phase incurs losses to the target molecules in the sample as the yield is never 100 %. As an illustrative example, extraction of nucleic acid molecules from biological samples, such as blood or tissue, typically has a yield of 10-30 %.

[0006] The theoretical analytical sensitivity, also referred to as limit of detection (LoD), for any protocol involving subsampling is independent of the analytical method and is 3 target molecules at 95 % confidence (Forootan et al., Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR), Biomolecular Detection and Quantification (2017) 12: 1-6). In practice, this means that 95 % of repeated analyses will be positive while 5 % will be negative when analyzing a sample with an average concentration of three target molecules per mL if the pre-analysis involves a subsampling so that only 1 mL of the sample is analyzed at a time. Correspondingly, if the pre-analysis phase involves an extraction with a yield of 20 % and half of the extracted material is analyzed, while keeping the remaining half as back-up in case of analytical failure, for confirmation or for more extensive analysis in the case of a positive result, etc., and working at 95 % confidence interval, then the original sample must contain a minimum of 30 target molecules to reach the LoD.

[0007] The loss of target molecules during pre-analysis thereby limits the type of samples that can be analyzed and quantified even if the analysis method used is very sensitive. There is therefore a need to increase the sensitivity and improve the reproducibility in bioanalysis.

[0008] SUMMARY

[0009] It is a general objective to increase the sensitivity and improve the reproducibility in bioanalysis. This and other objectives are met by embodiments of the present invention.

[0010] The present invention is defined in the independent claim. Further embodiments of the invention are defined in the dependent claims.

[0011] An aspect of the invention relates to a method for detecting target nucleic acid molecules in a sample. The method comprises fragmenting target nucleic acid molecules in the sample into multiple target nucleic acid fragments and subjecting the sample to at least one pre-analysis operation comprising extraction and / or purification of nucleic acid molecules to get a processed sample or an aliquot thereof comprising target nucleic acid fragments. The method also comprises detecting the target nucleic acid fragments in the processed sample or in the aliquot thereof and concluding the target nucleic acid molecules were present in the processed sample based on the detected target nucleic acid fragments.

[0012] Another aspect relates to a kit for detecting target nucleic acid molecules in a sample. The kit comprises beads configured to fragment target nucleic acid molecules in the sample into multiple target nucleic acid fragments. The kit also comprises multiple primer pairs each comprising a unique forward primer and a unique reverse primer, and multiple labelled probes. Each primer pair of the multiple primer pairs is designed to amplify a respective target nucleic acid fragment. Each labelled probe of the multiple labelled probes is complementary or identical to a sequence of a respective target nucleic acid fragment of the multiple target nucleic acid fragments. The multiple labelled probes have a same label.

[0013] The pre-processing of the sample prior to the pre-analysis operation(s) by fragmenting any target nucleic acid molecules in the sample into multiple target nucleic acid fragments improves the sensitivity and reproducibility of bioanalysis processes and in particular when analyzing samples expected to contain minute amounts of target nucleic acid molecules. The pre-processing of the sample described further improves the precision of the detection and quantification of the target nucleic acid molecules.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The embodiments, together with further objects and advantages thereof, may best be understood by making reference to the following description taken together with the accompanying drawings, in which:

[0016] Fig. 1 is a flow chart illustrating a bioanalysis process; Fig. 2 schematically illustrates a bioanalysis process according to prior art;

[0017] Fig. 3 is a flow chart illustrating a method for detecting target nucleic acid molecules according to an embodiment;

[0018] Fig. 4 schematically illustrates a bioanalysis process according to an embodiment;

[0019] Fig. 5 is a graph illustrating Poisson distributions for different expected values (Y=Y(0));

[0020] Fig. 6 compares the samples vortexed in the presence (circles) and absence (triangles) of silica glass beads;

[0021] Fig. 7 compares the samples pipetted 50 times in the presence (circles) and absence (tringles) of silica glass beads; and

[0022] Fig. 8 compares the samples vortexed (circles) with those pipetted 50 times (tringles) in the presence of silica glass beads;

[0023] Fig. 9 illustrates the results from digital PCR with one (A, C, E) or ten assays (B, D, F) when samples are exposed to vortexing with beads (A, B, E, F) or pipetting with beads (C, D); and

[0024] Fig. 10 illustrate images of digital PCR experiments for A, B, E and F in Fig. 9.

[0025] DETAILED DESCRIPTION

[0026] The present invention generally relates to bioanalysis and in particular to a method for detecting target nucleic acid molecules in a sample.

[0027] Fig. 1 is a flow chart illustrating a general bioanalysis process. Such a bioanalysis process typically involves two main phases, the pre-analysis phase and the analysis phase. The pre-analysis phase typically starts with sampling followed by sample preparation, extraction, purification and sample distribution, and may often include further steps, such as transport, storage, freezing / thawing, quality control, sample concentrating, aliquoting, etc. prior to the actual analysis. The pre-analysis phase is commonly adapted based on the particular type of analysis. There are various analysis methods that can be used depending on the type of target nucleic acid molecule, type of sample, desired sensitivity and specificity, etc. As an example, real-time polymerase chain reaction (qPCR), digital PCR (dPCR) and next generation sequencing (NGS) are common analysis methods for detecting and quantifying nucleic acid molecules. In case of RNA targets, conversion to complementary DNA (cDNA) by reverse transcription may be used prior to analysis. Other detection methods include hybridization to immobilized probes on microarray, beads or other surfaces, rolling circle amplification, padlock probe hybridization, antibody binding and aptamer binding.

[0028] The analysis phase typically involves detection, and optionally quantifying, the target nucleic acid molecules in the sample. Detection and optional quantification are important for various types of diagnostics, prognostics but also for monitoring treatments, detecting relapses, and for screening. Detection and optional quantification of target nucleic acid molecules are further important for the treatment of diseases and other conditions.

[0029] The pre-analysis phase, however, incurs losses of target nucleic acid molecules as the yield of such preanalysis is never 100 %. Further, the pre-analysis phase often involves multiple, i.e., at least two, preanalysis operations, such as sampling, extraction, purification and sub-sampling, each such operation contributing to target nucleic acid molecule losses. The combined yield of such multiple pre-analysis operations may be low, such as 25 % or even lower. For instance, three pre-analysis operations with a yield of 50 % each result in a combined yield of merely 12.5 % and thereby an average loss of 87.5 % of the original target nucleic acid molecules during the pre-analysis phase. Such losses of target nucleic acid molecules during the pre-analysis phase put high demands on the sensitivity and LoD of the analysis method(s) used in the analysis phase and further limits the type of samples that can be analyzed using a given analysis method.

[0030] The present invention solves these limitations and shortcomings of prior art bioanalysis processes by introducing a process step prior to or at least early in the pre-analysis phase. This process step involves fragmenting the target nucleic acid molecules in the sample into multiple target nucleic acid fragments that can be quantified in the subsequent analysis phase. As a consequence of this fragmentation, the number of detectable and quantifiable molecules is increased and thereby at least partly compensates for the losses and the sub-100 % yield of the subsequent pre-analysis phase.

[0031] Most target nucleic acid molecules analyzed in bioanalysis process are macromolecules and thereby comparatively large. This means that they can be fragmented or split into smaller nucleic acid pieces or fragments that can be detected individually. As an example, nucleic acid molecules could be in the form of large chromosomes or pieces of chromosomes. Such long nucleic acid molecules can then be cut into smaller nucleic acid fragments, which are detected and optionally quantified in separate PCR analyses. As a consequence, instead of detecting one type of target DNA molecule with one PCR assay in Fig. 2, indicated by the single amplicon of the target DNA molecule, the target DNA molecule is divided into multiple DNA fragments, see Fig. 4, which each can be detection and optionally quantified in a respective PCR assay. The presence of the original target DNA molecule can then be concluded based on the detection of the multiple DNA fragments. In the illustrative example of Fig. 4, the original target DNA molecules are, on average, split into four DNA fragments each in the fragmentation step, which thereby quadruples the number of quantifiable molecules as compared to the prior art bioanalysis process in Fig. 2. This fragmentation increases the number of detectable and quantifiable molecules and at least partly compensates for the losses taking place during the pre-analysis operation(s). As a consequence, on average four times more quantifiable molecules are present at the analysis phase in the example of Fig. 4 as compared to the prior art bioanalysis in Fig. 2.

[0032] In an illustrative example, the sample is expected to contain four target nucleic acid molecules and the average yield of the pre-analysis phase in Fig. 1 is 50 %. In such an example, the processed sample following the pre-analysis phase contains, on average, two target nucleic acid molecules at the analysis phase. Fig. 5 illustrates the Poisson distribution for different average numbers of target nucleic acid molecules per sample. As is shown in Fig. 5, the probability that a processed sample to be detected and optionaly quantified in the analysis phase does not contain any target nucleic acid molecules is about 14 % for the case with an original sample with four target nucleic acid molecules and 50 % yield during the pre-analysis phase. This means that the probability that a sample, originally expected to contain four target nucleic acid molecules and processed in a pre-analysis phase with a 50 % yield, contains at least one target nucleic acid molecule at the analysis phase is 86 %. This is below the LoD, which is the amount or concentration that, in repeated analyses, gives a positive result in 95 % of the cases (Forootan et al., Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR), Biomolecular Detection and Quantification (2017) 12: 1-6).

[0033] Assume that the sample is processed in accordance with the present invention as shown in Fig. 3, involving fragmenting the target nucleic acid molecules into multiple target nucleic acid fragments and then detecting and optionally quantifying the different target nucleic acid fragments. Further assume that the fragmentation splits, on average, each target nucleic acid molecule into two target nucleic acid fragments. In such a case, the processed sample following the pre-analysis phase with 50 % yield is expected to contain four such target nucleic acid fragments. Referring to Fig. 5, the probability that the processed sample to be analyzed in the analysis phase does not contain any target fragment is now merely 1 .8 %. This is far below 5 % and thereby above the LoD. The present invention thereby increases the sensitivity compared to the situation above.

[0034] In typical examples, the target nucleic acid molecules are fragmented into more than two target nucleic acid fragments each. For instance, a genomic DNA molecule, as an example of a target nucleic acid molecule, could be fragmented into, for instance, 100 DNA fragments. In such an example, the processed sample following the pre-analysis phase with 50 % yield is expected to contain 200 (4x100x0.5) DNA fragments. The probability that the processed sample does not contain any DNA fragment upon detection and optional quantification in the analysis phase is negligible (1071).

[0035] In another example, a crude sample is analyzed directly after splitting it into 10 aliquots. Assume also that 50 % of the target nucleic acid molecules in the crude sample are lost in the sampling tube due to effects like surface adsorption, etc. If the original sample contained 40 target nucleic acid molecules, each aliquot will then in average contain 2 nucleic acid molecules. This is below the LoD. If the target nucleic acid molecules are fragmented into, on average, 10 nucleic acid fragments each and such a fragmentation is done immediately after sampling before any significant surface adsorption, the original sample will instead contain 400 nucleic acid fragments. Loosing 50 % by surface adsorption and then transferring 10 % to each aliquot, an average of 20 target nucleic acid fragments will be present in each aliquot. This number can reliably be detected and also detected and quantified with precision.

[0036] The present invention not only improves the sensitivity but also the reproducibility of the analysis. The reproducibility can be expressed as the relative error, which is the standard deviation (SD) reflecting the imprecision, i.e., is inversely proportional to the reproducibility, scaled by the expectation value. For Poisson distributed data, the SD is equal to the square root of the expectation value. This means that the However, if the target nucleic acid molecules are fragmented into two target nucleic acid fragments each, the SD is V4 = 2 and the relative error is 50 %. Correspondingly, if the target nucleic acid molecules are fragmented into 100 target nucleic acid fragments each, the SD is 14.1 and the relative error is 7 %. Thus, the relative error is thereby reduced tenfold if the target nucleic acid molecules are fragmented into 100 nucleic acid fragments prior to the pre-analysis phase. In theory, the precision improves with the square root of the number of nucleic acid fragments that are independently targeted. This means that for the prior art situation as shown in Fig. 2, the probability that the processed sample following the pre-analysis phase contains 0 nucleic acid molecules is 6.25 %, 1 nucleic acid molecule is 25.00 %, 2 nucleic acid molecules is 37.50 %, 3 nucleic acid molecules is 25.00 % and 4 nucleic acid molecules is 6.25 %. The expectation value is 2, the variance is 1 , the SD is 1 and the coefficient of variation is 0.5 (50 %). Further, the probability of a positive result during the subsequent analysis, i.e., that the analyzed process sample contains at least one nucleic acid molecule, is 93.75 %.

[0037] This should be compared to the situation as shown in Fig. 4. In such a case, the probability that the processed sample following the pre-analysis phase contains 0 nucleic acid molecules is 0.00 %, 1 nucleic acid molecule is 0.02 %, 2 nucleic acid molecules is 0.18 %, 3 nucleic acid molecules is 0.85 %, 4 nucleic acid molecules is 2.78 %, 5 nucleic acid molecules is 6.67 %, 6 nucleic acid molecules is 12.22 %, 7 nucleic acid molecules is 17.46 %, 8 nucleic acid molecules is 19.64 %, 9 nucleic acid molecules is 17.46 %, 10 nucleic acid molecules is 12.22 %, 11 nucleic acid molecules is 6.67 %, 12 nucleic acid molecules is 2.78 %, 13 nucleic acid molecules is 0.85 %, 14 nucleic acid molecules is 0.18 %, 15 nucleic acid molecules is 0.02 %, and 16 nucleic acid molecules is 0.00 %. The expectation value is 8, the variance is 4, the SD is 2 and the coefficient of variation is 0.25 (25 %). Further, the probability of a positive result during the subsequent analysis phase, i.e., that the analyzed process sample contains at least one nucleic acid molecule, is essentially 100 %.

[0038] An aspect of the invention relates to a method for detecting a target nucleic acid molecule in a sample, see Fig. 3. The method comprises fragmenting target nucleic acid molecules in the sample into multiple target nucleic acid fragments in step S1 . The sample is then subject to at least one pre-analysis operation, step or process comprising extraction and / or purification of nucleic acid molecules in step S2 to get a processed sample or an aliquot thereof comprising target nucleic acid fragments. The target nucleic acid fragments are then detected in step S3 in the processed sample or in the aliquot thereof. The method also comprises concluding the target nucleic acid molecules were present in the processed sample in step S4 based on the detected target nucleic acid fragments.

[0039] The present invention thereby performs fragmentation prior to at least one step that incurs losses, i.e., the at least one pre-analysis operation, as the increase of the number of target nucleic acid fragments that can be detected reduces the risk of all detectable molecules in the sample being lost. Fragmentation after all steps incurring losses might increase a detectable signal, by increasing the number of detectable. However, such an approach does not improve the assay sensitivity if an analytical method is used that detects a single target nucleic acid molecule. The fragmentation performed in step S1 produces, on average, at least two target nucleic acid fragments per target nucleic acid molecule that is fragmented. The fragmentation thereby increases the number of nucleic acid molecules in the sample. For instance, assume that the sample contains M target nucleic acid molecules and that the fragmentation in step S1 results, on average, in N target nucleic acid fragments per target nucleic acid molecule. In such a case, the number of detectable and optionally quantifiable nucleic acid molecules in the sample increases from M target nucleic acid molecules into A / xM target nucleic acid fragments.

[0040] The sample with the multiple target nucleic acid fragments obtained in step S1 is then subject to one or more pre-analysis operations comprising extraction and / or purification of nucleic acid molecules in step S2.

[0041] Step S2 could involve performing one pre-analysis operation, such an extraction operation or a purification operation, or performing multiple pre-analysis operations, such an extraction operation and a purification operation or at least one of an extraction operation and a purification operation and at least one additional pre-analysis operation. Each such pre-analysis operation is marred by a loss of nucleic acid molecules and thereby has a yield less than 100 %. If multiple pre-analysis operations are performed in step S2 then the combined molecule yield is a combination of the individual yields for each such preanalysis operations. For instance, assume that step S2 involves n pre-analysis operations where n is a positive integer equal to or larger than two and that pre-analysis operation no. k has a molecule yield of Y , wherein 0< Yx<1 for each =1...n, then the combined molecule yield for step S2 will be Yix Y2X..., i-e, n^i Yk.

[0042] In an embodiment, the at least one pre-analysis operation performed in step S2 has a combined molecule yield equal to or less than 50 %. For instance, if a single pre-analysis operation is performed in step S2 then such a single pre-analysis operation preferably has a molecule yield of 50 % or less. In an embodiment, the combined molecule yield of the one or more pre-analysis operations is equal to or less than 50 %, i.e., <0.5. In other words, if step S2 comprises a single pre-analysis operation and that preanalysis operation preferably has a molecule yield of equal to or less than 50 % and if step S2 comprises multiple pre-analysis operations then the combined molecule yield of these multiple pre-analysis operation is preferably equal to or less than 50 % (112=1 YK O.5) even if the molecule yield of one or more of these pre-analysis operations may be larger than 50 % (0< Y <1). The fragmentation in step S1 is preferably performed as early as possible in the bioanalysis process. For instance, if the pre-analysis phase contains multiple pre-analysis operations then the fragmentation in step S1 is preferably performed prior to such pre-analysis operations or at least prior to the pre-analysis operation(s) marred by the higher losses of target nucleic acid molecules.

[0043] In an embodiment, the method is a method for quantifying target nucleic acid molecules in a sample. In such an embodiment, step S3 of Fig. 3 comprises quantifying the target nucleic acid fragments in the processed sample or in the aliquot thereof. In this embodiment, step S4 comprises quantifying the target nucleic acid molecules in the processed sample based on the quantified target nucleic acid fragments.

[0044] Step S3 of the method shown in Fig. 3 comprises, in an embodiment, quantifying the target nucleic acid fragments in the processed sample, i.e., in the sample as obtained following the one or more pre-analysis operations in step S2. The quantification could be done directly in the processed sample or in an aliquot or subsample of the processed sample. The target nucleic acid molecules in the processed sample are then quantified in step S4 based on the target nucleic acid fragments as quantified in step S3. This quantification in step S4 could be performed in accordance with various embodiments depending on the type of quantification as performed in step S3.

[0045] As an example, assume fragmenting, on average, a target nucleic acid molecule into M target fragments in step S1 and that each resulting target nucleic acid fragment is quantified using a respective qPCR or dPCR assay but producing the same output signal, such as by using the same reporter dye, such as fluorescein amidites (FAM), for fragment-specific reporters or an unspecific dye, such as SYBR® or EvaGreen®, binding to all PCR products (amplified target fragments). In such an example, a single “combined” signal is obtained for all M target nucleic acid fragments representing a combined quantity of the M target nucleic acid fragments in the processed sample. The target nucleic acid molecules in the processed sample could then be quantified in step S4 based on this combined quantity, such as based on the combined quantity divided by M.

[0046] Alternatively, a standard or reference material or a standard or reference sample could be used for calibration to map or convert the combined quantity of the M target nucleic acid fragments in the processed sample into a quantity of the target molecules in the processed sample. In an embodiment, step S3 in Fig. 3 comprises quantifying the multiple target nucleic acid fragments in the processed sample or in the aliquot thereof by a respective fragment-specific quantification assay using a same detectable label to obtain a combined quantity of the multiple target nucleic acid fragments in the processed sample or in the aliquot thereof. Step S4 comprises, in this embodiment, quantifying the target nucleic acid molecules in the processed sample based on the combined quantity of the multiple target nucleic acid fragments.

[0047] Thus, this embodiment involves running separate quantification assays, such as qPCR or dPCR assays, for the different target nucleic acid fragments. Each such quantification assay is thereby specific for the given target nucleic acid fragments, such as by using PCR primers selected based on the nucleotide sequences of the target nucleic acid fragments. However, each such quantification assay uses the same detectable label, such as dye, and thereby produces the same type of output signal, such as fluorescence or color signal. The output signal will thereby be reflective of the combined quantity of the multiple target nucleic acid fragments in the processed sample or in the aliquot thereof.

[0048] In another example, each target nucleic acid fragment is individually quantified in step S3 such as by using fragment-specific reporters with different dyes for different fragment-specific report or by running the different quantification assays, such as qPCR or dPCR assays, in separate reaction vessels. In such an embodiment, M signals or quantities are obtained in step S3, i.e., one such signal or quantity per target nucleic acid fragment. The target nucleic acid molecules in the processed sample could then be quantified in step S4 based on these quantities, such as based on an average of the M quantities.

[0049] In an embodiment, step S3 in Fig. 3 comprises individually quantifying a respective target nucleic acid fragment of the multiple target nucleic acid fragments in the processed sample or in the aliquot thereof. In such an embodiment, step S4 comprises quantifying the target nucleic acid molecules in the processed sample based on the individually quantified target nucleic acid fragments.

[0050] The one or more pre-analysis operations performed in step S2 are typically dependent on the particular detection or quantification method used in the analysis phase of the bioanalysis process, the type of sample and / or the type of target nucleic acid molecule. Illustrative, but non-limiting, examples of such pre-analysis operations include extraction of target nucleic acid molecules from the sample, precipitation of the sample, purification of the sample or the target nucleic acid, sampling a portion of the sample (subsampling), aliquoting the sample, storing the sample, freeze-thawing the sample, exposing the sample to a target molecule compromising condition and eluting the sample. Exposing the sample to a target molecule compromising condition involves exposing, such during transport and / or storage, the sample to environmental conditions (temperature, light, ambient air, surfaces, etc.) that may compromise and deteriorate the target nucleic acid molecules. For instance, exposing heat-sensitive target nucleic acid molecules, like RNA, to high temperatures may cause deterioration of the target nucleic acid molecules. Other examples of such target nucleic acid molecule compromising conditions include exposing the target nucleic acid molecules to chemicals, enzymes or reagents that may induce deterioration of the target nucleic acid molecules.

[0051] Illustrative, but non-limiting, examples of extraction methods that could be used when extracting target nucleic acid molecules from the sample include organic extraction, silica resin-based extraction methods, magnetic bead-based extraction, precipitation, and affinity or ion exchange chromatography.

[0052] Illustrative, but non-limiting, examples of purification methods that could be used when purifying the sample include precipitation, chromatography, electrophoresis, magnetic bead-based purification, silica resin-based purification, ultracentrifugation and gradient centrifugation.

[0053] Elution is the process of extracting one material (target nucleic acid molecule) from an absorbent using an eluent.

[0054] Nucleic acid molecule as used herein include deoxyribonucleic acid (DNA) molecules, such as doublestranded DNA (dsDNA) molecules, single-stranded DNA (ssDNA) molecules, complementary DNA (cDNA) molecules, mitochondrial DNA, chromosomal DNA, chloroplast DNA, bacterial and viral DNA genomes, bacterial DNA inserts, viral vectors, DNA plasmids, synthetic or in vitro produced polynucleotides and oligonucleotides or derivatives thereof, and ribonucleic acid (RNA) molecules, such as messenger RNA (mRNA) molecules, transfer RNA (tRNA) molecules, ribosomal RNA (rRNA) molecules, non-coding RNA (ncRNA) molecules, long non-coding RNA (IncRNA) molecules, viral RNA genomes, synthetic or in vitro produced oligoribonucleotides and polyribonucleotides or derivatives thereof.

[0055] In an embodiment, step S1 of Fig. 3 comprises subjecting the sample to a physical fragmentation process to fragment the target nucleic acid molecules into multiple target nucleic acid fragments. Such physical fragmentation processes include, but are not limited to, sonication, bead beating, radiation, shearing forces, heat, acidic pH and basic pH. Sonication is the process of applying energy through sound waves to fragment the target nucleic acid molecules into the target nucleic acid fragments. During sonication, the sample is subjected to hydrodynamic shearing by exposure to brief periods of sonication. Most sonicators can be used to generate nucleic acid fragments of a size down to about 300-500 nucleotides (Sambrook and Russell, Fragmentation of DNA by sonication, Cold Spring Harbor Protocols (2006) 4: pdb.prot4538).

[0056] Nucleic acid molecules can be fragmented by exposure to radiation, such as ultraviolet (UV) radiation, beta radiation, gamma radiation, or laser light. For instance, ionizing radiation induces DNA double stranded-breaks (DSBs) and can thereby be used to produce target nucleic acid fragments (Pang et al., Radiation-generated short DNA fragments may perturb non-homologous end-joining and induce genomic instability, Journal of Radiation Research (2011) 52(3): 309-319).

[0057] Shearing forces to break target nucleic acid molecules into smaller target nucleic acid fragments can be applied using mechanical fragmentation processes. Illustrative, but non-limiting, examples of such fragmentation processes include nebulization, point-sink shearing, needle shearing, and French pressure cells.

[0058] Nucleic acid molecules can be further fragmented into target nucleic acid molecules by heating. For instance, double-stranded DNA can be melted by heating producing single strands that can be brought through the preanalytical process independently, each serving as template for PCR.

[0059] Single stranded nucleic acid molecules and especially RNAs exposed to high or low pH and optionally elevated temperature may be decomposed into nucleic acid fragments. Such a change in pH to acidic or basic pH can be used in step S1 to fragment the target nucleic acid molecules into target nucleic acid fragments. Generally, nucleic acid molecules are stable within the pH range of 5 to 9. Thus, pH-induced fragmentation is typically done at a pH below 5, preferably equal to or below 4, and more preferably equal to or below 3, such as equal to or below 2, or a pH above 9, preferably equal to or above 10, and more preferably equal to or above 11 .

[0060] In an embodiment, step S1 of Fig. 3 comprises contacting the sample with beads, preferably glass beads and / or silica beads, and inducing movement of the beads within the sample. Such an embodiment thereby involves bead beating as method to fragment the target nucleic acid molecules. Illustrative, but non-limiting, examples of such beads include silica beads, such as silica glass beads, ceramic beads, zirconia beads, and metal beads. Beads with various diameters could be used, preferably an average dimeter equal to or smaller than 1 mm, preferably equal to or smaller than 0.75 mm, more preferably equal to or smaller than 0.5 mm, and most preferably equal to or smaller than 0.25 mm, or even smaller, such as equal to or smaller than 0.2 mm, or equal to or smaller than 0.1 mm.

[0061] In this embodiment, beads are added to the sample and movement of the beads within the sample is induced, such as by vortexing the sample with the beads, pipetting up and down a portion of the sample, or any other operation that induces movement of the beads within the sample. The so-induced movement of the beads within the sample creates shearing forces that fragments the target nucleic acid molecules. An advantage of this embodiment is that it can be applied to samples containing cells, which in turn comprises the target nucleic acid molecules. The shearing forces by the moving beads will then not only break or lyse the cells to thereby cause release of the target nucleic acid molecules into the sample but will also fragment the so-released target nucleic acid molecules. Experimental data as presented herein show that moving beads can successfully be used to achieve a combined cell lysis and target nucleic acid molecule release and target nucleic acid molecule fragmentation.

[0062] The movement of the beads is therefore preferably sufficient vigorous to break cell membranes and / or walls and additionally fragment the target nucleic acid molecules present within the cell and released therefrom following breakage or lysis of the cell membrane and / or wall. As an example, the vortexing could be done at a speed of at least 1500 rpm, preferably at least 2000 rpm, at least 2500 rpm, more preferably at least 3000 rpm, such as at least 3500 rpm, at least 4000 rpm, and most preferably at least 4500 rpm, such as at least 5000 rpm, at least 5500 rpm or at least 6000 rpm. In general, a vortexing speed selected within an interval of from 1500 up to 6000 rpm would be sufficient to both break cell membranes and walls and fragment target nucleic acid molecules present in the cells. The vortexing is preferably performed for a time duration from about 1 min to about 5 min.

[0063] In an embodiment, the target nucleic acid molecules are target DNA molecules. In this embodiment, step S1 of Fig. 3 comprises contacting the sample with at least one endonuclease to fragment the target DNA molecules in the sample into multiple target DNA fragments.

[0064] Endonucleases are enzymes that cleave the phosphodiester bond within a DNA chain. Endonucleases may cut the DNA relatively non-specifically, such as deoxyribonuclease I or Micrococcal nuclease (MNase), or cleave only at very specific nucleotide sequences, typically called restriction endonucleases or restriction enzymes. In a particular embodiment, step S1 of Fig. 3 comprises contacting the sample with at least one restriction endonuclease to fragment the target DNA molecules in the sample into multiple target DNA fragments.

[0065] These restriction endonucleases cleave DNA into fragments at or near specific recognition sites within DNA molecules known as restriction sites. There are five categories of restriction endonucleases, generally referred to as type I, II, III, IV and V. Type l enzymes (EC 3.1.21.3) cleave at sites remote from a recognition site. Type II enzymes (EC 3.1.21.4) cleave within or at short specific distances from a recognition site. Type III enzymes (EC 3.1 .21 .5) cleave at sites a short distance from a recognition site. Type IV enzymes target modified DNA, e.g. methylated, hydroxymethylated and glucosylhydroxymethylated DNA, while type V enzymes utilize guide RNAs (gRNAs).

[0066] Step S1 could involve contacting the sample with a single endonuclease, preferably a single restriction endonuclease, or multiple different endonucleases, preferably multiple different restriction endonucleases.

[0067] In one embodiment restriction endonucleases are used to cleave within a sequence present in multiple copies in the genome. This creates fragments, each with one copy of sequence present in higher copy number. Those fragments can then be targeted with an assay for that sequence using primers that do not span the restriction site. A special case of this embodiment is targeting bacterial 16S rRNA gene, which often is present in multiple copies. In humans, targeting Alu-sequences after extensive fragmentation would give exceedingly high sensitivity and reproducibility even if the original sample contained a single molecule only.

[0068] The particular restriction endonuclease(s) used in step S1 can be selected based on the particular target DNA molecule to quantify and the desired number and length of the target DNA fragments.

[0069] In another embodiment, the target molecules are target dsDNA molecules. In this embodiment, step S1 of Fig. 3 comprises denaturing the target dsDNA molecules into multiple target ssDNA molecules. In such an embodiment, steps S2 and S3 of Fig. 3 are performed under denaturing conditions.

[0070] This particular embodiment thereby involves separating a target dsDNA molecule into two ssDNA molecules, thereby, on average, doubling the number of quantifiable molecules in the sample. Denaturing dsDNA molecules into ssDNA molecules can be performed by exposing the dsDNA molecules to heat, typically about 95°C, chemical treatment, such as dimethyl sulfoxide (DMSO) or a chaotropic agent, in particular a chaotropic salt, such as guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, and sodium dodecyl sulfate, and / or laser illumination.

[0071] In this embodiment, the subsequent pre-analysis operation(s) and quantification in steps S2 and S3 are performed under denaturing conditions to maintain the DNA molecules denatured, i.e., as ssDNA molecules. This means conducting these steps S2 and S3 during heating, in the presence of the chemical agent, such as DMSO or chaotropic agent, and / or upon laser illumination, or before the ssDNA renatures.

[0072] The above-described embodiments of fragmenting target dsDNA molecules, i.e., treatment with endonuclease(s), preferably restriction endonuclease(s), and denaturing dsDNA molecules can be combined. In such a case, target dsDNA molecules are separated into ssDNA molecules that are then fragmented into target ssDNA fragments by (restriction) endonuclease treatment or target dsDNA molecules are fragmented by (restriction) endonuclease treatment into smaller dsDNA fragments, which are then denatured into target ssDNA fragments.

[0073] In a further embodiment, the target molecules are target dsDNA molecules. In this embodiment, step S1 of Fig. 3 comprises contacting the sample with at least one nicking endonuclease to fragment at least one strand of the target dsDNA molecules. Step S1 also comprises denaturing the target dsDNA molecules into multiple single-stranded target fragments.

[0074] This embodiment involves using a nicking endonuclease, also referred to as nicking enzyme in the art, to cut one strand of a double-stranded DNA molecule, such as DNase I. This could be at a specific recognition nucleotide sequence, i.e., a restriction size, or non-specific. This enzyme thereby hydrolyzes, i.e., cuts, one strand of the DNA duplex to produce DNA molecules that are “nicked” rather than cleaved. Once at least one of the strands of the target dsDNA molecules have been nicked the two strands can separate from each other to form the multiple target fragments. Strand separation can be induced by, for example, heating or other means of denaturation like chaotropic salts.

[0075] In an embodiment, the target nucleic acid molecules are target RNA molecules. In such an embodiment, step S1 comprises contacting the sample with at least one endoribonuclease to fragment the target RNA molecules in the sample into multiple target RNA fragments. Endoribonuclease is a class of enzymes that cleaves either ssRNA or dsRNA. Illustrative, but nonlimiting, examples of such endoribonucleases include RNase III, RNase A, RNase T1 , RNase T2, RNase H, RNase P and endoribonuclease Xendoll.

[0076] In a particular embodiment, step S1 of Fig. 3 comprises contacting the sample with at least one DNA oligonucleotide complementary to a respective portion of the target RNA molecules. In such a particular embodiment, the sample is then contacted with ribonuclease H (RNase H).

[0077] RNase H is a family of non-sequence-specific ribonuclease enzymes that catalyze cleavage of RNA in an RNA / DNA substrate. This particular embodiment thereby uses one or more DNA oligonucleotides that is or are complementary and thereby hybridize to a respective portion of the target RNA molecules to form one or more RNA / DNA duplexes on the target RNA molecules. The RNase H will then cleave the target RNA molecules in the RNA / DNA duplex regions. The DNA oligonucleotides can thereby be used to control the fragmentation of the target RNA molecules to merely occur at specific region(s), to which the DNA oligonucleotide(s) hybridize(s).

[0078] Other examples of fragmenting target nucleic acid molecules include using clustered regularly interspaced short palindromic repeats (CRISPR) to the cut the target nucleic acid molecules into smaller target nucleic acid fragments typically using Cas nucleases (Swartjes et al., DNA cleavage by CRISPR RNA-guided nucleases Cas9 and Cas12a. Biochemical Society Transactions (2020) 48(1 ): 207-219) and variants, such as SpRY (Kathleen et al., Precise DNA cleavage using CRISPR-SpRYgests, Nature Biotechnology (2023) 41 : 409-416).

[0079] Preferred embodiments of step S3, include quantifying the target nucleic acid fragments using a PCR process or using a sequencing process.

[0080] In a preferred embodiment, the target nucleic acid fragments are quantified in step S3 using qPCR or dPCR. qPCR monitors amplification of the target nucleic acid fragment during PCR, i.e., in real time. The PCR products, i.e., amplified target nucleic acid fragments or amplified portions (“amplicon”) of the target nucleic acid fragments, can then be detected using a non-specific fluorescent dye that bind with any dsDNA, such as SYBR® or EvaGreen®, or using a sequence-specific labelled probe in the form of an oligonucleotide that is labelled with a fluorescent reporter or label, such as FAM. dPCR separates the processed sample or the aliquot thereof into a large number of partitions and the PCR reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. Several different methods can be used to partition the processed sample or the aliquot thereof, including microwell plates, capillaries, oil emulsion, and arrays of miniaturized chambers with nucleic acid binding surfaces. The partitions are then subjected to thermocycling so that each partition may independently undergo PCR amplification. After multiple PCR amplification cycles, the partitions are checked for fluorescence with a binary readout of “0” or “1”. The partitioning of the processed sample or the aliquot thereof allows one to estimate the number of different target nucleic acid fragments by assuming that the fragment population follows the Poisson distribution. Using Poisson's law of small numbers, the distribution of target nucleic acid fragments within the processed sample or aliquot thereof can be accurately approximated allowing for a quantification of the target nucleic acid fragments.

[0081] Fragmenting any target nucleic acid molecules in the sample into multiple target nucleic acid fragments increases the sensitivity in terms of producing a stronger output signal corresponding to a lower Cq value for qPCR and more positive partitions for dPCR.

[0082] Instead of using an amplification-based quantification, such as qPCR or dPCR, in step S3, the target nucleic acid fragments can be quantified by sequencing, preferably by next-generation sequencing (NGS) or third-generation sequencing.

[0083] NGS, also referred to as second-generation sequencing or massive parallel sequencing, allows massive parallel sequencing via spatially separated target nucleic acid fragments in a flow cell. Illustrative, but non-limiting, examples of such NGS platforms that can be used in step S3 include Roche 454, GS FLX titanium, Illumina MiSeq, HiSeq, NextSeq, NovaSeq or Genome Analyzer I IX, Life Technologies SOLi D4 or Ion Proton, Complete Genomics, Helicos Biosciences Heliscope, Pacific Biosciences SMRT and Onso, nanopore technology (Oxford Nanopore, Roche), Aviti (Element Biosciences), UG100 (Ultima Genomics), DNBSeq (BGI), Qitan Technology (Qi Carbon Tech), tSMS (SeqLL), G4X (Singular Genomics).

[0084] Third-generation sequencing, also referred to as long-read sequencing, is a class of DNA sequencing methods which produce longer sequence reads. Illustrative, but non-limiting, examples of third- generation sequencing include Pacific Biosciences SMRT and Oxford Nanopore MinlON and SmidglON. Also, third-generation sequencing platforms in development could be used, including 454.bio, Armonica Technologies, Axbio, Caerus Molecular, Centrillion, Cygbus Biosciences, Depixus, DNAe, Gene Sense, GeneMind Bioscience, Genia, Genvidia, IMEC, INanoBio, Mingyl Intelligent Manufacturer, Nooma Bio, One-Chip, Personal Genomics, Quantapore, Reticula, RH Genetech, Salus, Single Technologies, Universal sequencing Technologies.

[0085] The quantification used could also, or alternatively, include loop-mediated isothermal amplification, nucleic acid sequence-based amplification, rolling circle amplification, strand displacement amplification, recombinase polymerase amplification, multiple displacement amplification, transcript mediated amplification, ligase chain reaction.

[0086] In an embodiment, step S3 in Fig. 3 comprises amplifying a respective target nucleic acid fragment of the multiple nucleic acid target fragments in the processed sample or in the aliquot thereof in a respective PCR assay. This embodiment also comprises quantifying the amplified target nucleic acid fragments.

[0087] This particular embodiment preferably involves using qPCR or dPCR to quantify the target nucleic acid fragments.

[0088] In an embodiment, step S3 in Fig. 3 comprises contacting the processed sample or the aliquot thereof with multiple primer pairs each comprising a unique forward primer and a unique reverse primer, and multiple labelled probes. In this embodiment, each primer pair of the multiple primer pairs is designed to amplify a respective target nucleic acid fragment. Each labelled probe of the multiple labelled probes is complementary or identical to a sequence of a respective target nucleic acid fragment of the multiple target nucleic acid fragments and the multiple labelled probes have a same label. Step S3 also comprises, in this embodiment, amplifying the multiple target nucleic acid fragments by performing PCR amplification on the processed sample or the aliquot thereof. The embodiment comprises detecting labels bound to the amplified target nucleic acid fragments and quantifying the target nucleic acid fragments based on the detected labels bound to the amplified target nucleic acid fragments.

[0089] This embodiment thereby involves amplifying each target nucleic acid fragment in a respective PCR assay, i.e., using a respective primer pair with a forward primer and a reverse primer having sequences selected to amplify the given target nucleic acid fragment or an amplicon thereof. For instance, if the fragmentation in step S1 results in, on average, M target nucleic acid fragments per target nucleic acid molecule, then M primer pairs are used in this embodiment with unique forward and reverse primers to enable amplification of each of the M target nucleic acid fragments.

[0090] The embodiment also involves usage of a respective labelled probe per target nucleic acid fragment and primer pair. Each such labelled probe has a nucleotide sequence complementary to or identical to a nucleotide sequence of a respective target nucleic acid fragment. This means that the labelled probes can bind to the amplification products formed by amplification of the target nucleic acid fragments using the primer pairs. Although the labelled probes have different nucleotide sequences to thereby be able to bind to the amplification products of the different target nucleic acid fragments, the labelled probes preferably all have the same label, such as a same dye, preferably a same fluorescent dye, such as a FAM. The detection of labels bound to the amplified target nucleic acid fragments thereby results in a total quantification of all amplified target nucleic acid fragments since the labelled probes contain the same label. The target nucleic acid fragments can thereby be quantified based on the detected labels bound to the amplified target nucleic acid fragments.

[0091] In another embodiment, step S3 in Fig. 3 comprises contacting the processed sample or the aliquot thereof with multiple primer pairs each comprising a unique forward primer and a unique reverse primer, and a sequence-unspecific label. In this embodiment, each primer pair of the multiple primer pairs is designed to amplify a respective target nucleic acid fragment. Step S3 also comprises amplifying the multiple target nucleic acid fragments by performing PCR amplification on the processed sample or the aliquot thereof. The embodiment also comprises detecting sequence-unspecific labels bound to the amplified target nucleic acid fragments and quantifying the target nucleic acid fragments based on the detected sequence-unspecific labels bound to the amplified target nucleic acid fragments.

[0092] This embodiment involves the usage of a sequence-unspecific label, such as a dye, for instance SYBR® or EvaGreen®, rather than sequence-specific labelled probes with the same label as in the embodiment described above. The sequence-unspecific label is preferably in the form of a non-specific fluorescent dye that intercalates with any dsDNA, including the amplified target nucleic acid fragments.

[0093] The various embodiments described in the foregoing for quantifying the target nucleic acid fragments could alternatively be used to detect the target nucleic acid fragments but not necessarily quantify the target nucleic acid fragments. The invention also relates to a kit for detecting target nucleic acid molecules in a sample. The kit comprises beads configured to fragment target nucleic acid molecules in the sample into multiple target nucleic acid fragments. The kit also comprises multiple primer pairs each comprising a unique forward primer and a unique reverse primer, and multiple labelled probes. Each primer pair of the multiple primer pairs is designed to amplify a respective target nucleic acid fragment. Each labelled probe of the multiple labelled probes is complementary or identical to a sequence of a respective target nucleic acid fragment of the multiple target nucleic acid fragments. The multiple labelled probes have a same label.

[0094] The kit is preferably used to perform the method for detecting target nucleic acid molecules in a sample as disclosed herein.

[0095] In an embodiment, detecting the target nucleic acid fragments comprises individually detecting a respective target nucleic acid fragment of the multiple target nucleic acid fragments in the processed sample or in the aliquot thereof.

[0096] In another embodiment, detecting the target nucleic acid fragments comprises detecting the multiple target nucleic acid fragments in the processed sample or in the aliquot thereof by fragment-specific quantification assays using a same detectable label to obtain a combined quantity of the multiple target nucleic acid fragments in the processed sample or in the aliquot thereof.

[0097] In an embodiment, detecting the target nucleic acid fragments comprises detecting the target nucleic acid fragments using a polymerase chain reaction process, preferably qPCR or dPCR.

[0098] In an embodiment, detecting the target nucleic acid fragments comprises detecting the target nucleic acid fragments using a sequencing process, preferably NGS or third generation sequencing.

[0099] In an embodiment, detecting the target nucleic acid fragments comprises amplifying a respective target nucleic acid fragment of the multiple nucleic acid target fragments in the processed sample or in the aliquot thereof in a respective PCR assay, and detecting the amplified target nucleic acid fragments.

[0100] In an embodiment, detecting the target nucleic acid fragments comprises contacting the processed sample or the aliquot thereof with multiple primer pairs each comprising a unique forward primer and a unique reverse primer, and multiple labelled probes. Each primer pair of the multiple primer pairs is designed to amplify a respective target nucleic acid fragment. Each labelled probe of the multiple labelled probes is complementary or identical to a sequence of a respective target nucleic acid fragment of the multiple target nucleic acid fragments. The multiple labelled probes have a same label. The detection also comprises amplifying the multiple target nucleic acid fragments by PCR amplification on the processed sample or the aliquot thereof, detecting labels bound to the amplified target nucleic acid fragments, and detecting the target nucleic acid fragments based on the detected labels bound to the amplified target nucleic acid fragments.

[0101] In an embodiment, detecting the target nucleic acid fragments comprises contacting the processed sample or the aliquot thereof with multiple primer pairs each comprising a unique forward primer and a unique reverse primer, and a sequence-unspecific label. Each primer pair of the multiple primer pairs is designed to amplify a respective target nucleic acid fragment. The detection also comprises amplifying the multiple target nucleic acid fragments by performing PCR amplification on the processed sample or the aliquot thereof, detecting sequence-unspecific labels bound to the amplified target nucleic acid fragments, and detecting the target nucleic acid fragments based on the detected sequence-unspecific labels bound to the amplified target nucleic acid fragments.

[0102] The above-described embodiments enable detection and optionally quantification of target nucleic acid molecules in applications where the processed sample or the aliquot thereof would contain very few copies of the target nucleic acid molecule when using prior art bioanalysis processes. In fact, the embodiments can be used in situations in which the processed sample or the aliquot thereof would contain only a single target nucleic acid molecule when using prior art bioanalysis processes.

[0103] The target nucleic acid molecules in the sample can come from various sources. As an example, the target nucleic acid molecule could come from a pathogen, such as a virus, bacteria, amoeba, fungi, etc. and the bioanalysis process can thereby be used for pathogen detection and diagnosis.

[0104] Another example of target nucleic acid molecules is a vector, drug, drug candidate, a metabolite of a drug or where the concentration or amount of the target nucleic acid molecules is affected by and thereby representative of the presence or activity of a drug. Examples of such drugs are advanced therapies and medical products (ATMP), which may be in the form of nucleic acid molecules, vectors, or nucleic acid molecule containing cells.

[0105] The present method can also be used for detection and optionally quantification of multiple sets of target nucleic acid molecules in the sample. In such an embodiment, step S3 in Fig. 3 comprises detecting, for each set of the multiple sets of target nucleic acid molecules, the target nucleic acid fragments in the processed sample or in the aliquot thereof using a set-specific dye. In such an embodiment, the setspecific dye used for one set of the multiple sets of target nucleic acid molecules is different than setspecific dye or dyes (i.e. , dye(s)) use for remaining set or sets (i.e., set(s)) of the multiple sets of target nucleic acid molecules.

[0106] This embodiment thereby uses set-specific dyes to detect the target nucleic acid fragments present in the sample. By having different such dyes for different sets of target nucleic acid molecules the present invention can be used to detect the presence of and optionally quantify different target nucleic acid molecules in the sample separately or relative to each other.

[0107] The pre-processing of the sample prior to the pre-analysis operation(s) by fragmenting any target nucleic acid molecules in the sample into multiple target fragments improves the sensitivity and reproducibility of bioanalysis processes and in particular when analyzing samples expected to contain minute amounts of target nucleic acid molecules. The pre-processing of the sample further improves the precision of the detection and quantification of the target nucleic acid molecules. Higher precision means that small differences between analyzed samples can be interpreted as being significant due to the improved precision of the invention.

[0108] In addition to improving sensitivity and reproducibility, the approach in many embodiments increases the specificity of the assay compared to the state of the art. When more than one target nucleic acid fragment with a unique sequence is detected the risk that it is a false positive signal is lower than when a single target sequence is measured.

[0109] The embodiments are in particular suitable for detecting target nucleic acid molecules in samples containing minute amounts of such target nucleic acid molecules, and also for their quantification. As an example the sample to processed according to the embodiments for the purpose of detecting and optionally quantifying any target nucleic acid molecules contained therein preferably comprises, or is at least expected to comprise, no more than 100 target nucleic acid molecules, preferably no more than 90 target nucleic acid molecules, no more than 80 target nucleic acid molecules, no more than 70 target nucleic acid molecules, no more than 60 target nucleic acid molecules, no more than 50 target nucleic acid molecules, no more than 40 target nucleic acid molecules, no more than 30 target nucleic acid molecules, no more than 20 target nucleic acid molecules or no more than 10 target nucleic acid molecules prior to the fragmentation step or operation. The embodiments can also be applied to samples containing fewer target nucleic acid molecules, such as at most 9, 8, 7, 6, 5, 4, 3, 2 or even one target nucleic acid molecule.

[0110] EXAMPLES

[0111] EXAMPLE 1

[0112] Commercial human genomic DNA (Roche, Cat. 11691112001) of approximately 200 ng / pl was diluted to approximately 12 ng / pl. This primary sample was divided into two aliquots, of which one was fragmented using a Bioruptor® Pico sonication device (Diagenode SA, Cat. B01060010) to an average fragment length of 400 bp. The non-fragmented and fragmented aliquots were then diluted for either direct analysis by real-time polymerase chain reaction (qPCR) or for extraction followed by qPCR analysis. In total, four samples constructed from the primary sample were subject to analysis:

[0113] 1 . Non-fragmented direct qPCR;

[0114] 2. Non-fragment extraction followed by qPCR;

[0115] 3. Fragmented direct qPCR; and

[0116] 4. Fragmented extraction followed by qPCR.

[0117] Eight SYBR® based qPCR assays targeting the P53 gene were designed and validated based on sequencing primers received from Sahlgrenska Center for Cancer Research (Gbteborg University), see Table 1. The eight assays were pre-mixed and used together in an 8-plex qPCR of 10 pl with 2 pl template (except for two reactions of each non-fragmented and fragmented post extraction sample to increase the detection level). Reactions consisted of 200 nM of each primer and 1 x of SsoAdvanced Universal SYBR® Green Supermix (Biorad, Cat. 1725270) run in a cfx384 Real-Time System (Biorad). The cycling protocol was 3 min pre-heating to 95°C followed by 4 cycles of 10 sec at 95°C, 20 sec at 60°C, and 20 sec at 72°C, followed by a melt curve analysis between 65°C and 95°C.

[0118] Table 1 - sequencing primers

[0119]

[0120] *TP53_nF and TP53_nR (n=1 ...8) represent forward (F) and reverse (R) sequencing primer no. n targeting the P53 gene.

[0121] The pre-extracted sample was analyzed at five concentrations prepared by two-fold dilutions with four replicates per concentration. Assuming targets for all eight assays were amplifiable, the estimated number of target molecules per qPCR were 30, 15, 7.5, 3.8 and 1.9 copies. A qPCR was considered positive if the Cq-value was below the average of the eight No Template Control (NTC) replicates minus two standard deviations. With this setup all samples, except for that having the lowest concentration, were positive. For the lowest concentrated sample two of the four qPCR replicates were positive.

[0122] The three intermediate concentrations of the pre-extracted sample were subjected to an extraction procedure using the QIAmp Mi n El ute ccfDNA Mini Kit (Qiagen, Cat. 55204) excluding the magnetic beads steps (1 - 5 in Kit Manual). For step 6, 2 l of the extracted DNA described above was added to 200 pl Bead Elution Buffer and another 300 pl Buffer ACB was added. From step 7, the Kit Manual was followed and double eluted in 20 pl water, where the eluate is reapplied to the column for re-elution to maximize yield as described in the Kit Manual.

[0123] The post-extraction qPCRs were performed the same way as the pre-extraction qPCRs described above with the following exception: only three concentrations from the extraction were used. In addition, two replicates of each of the non-fragmented and fragmented samples of the highest concentration of the post-extracted sample were run with 4 pl template in a 10 pl reaction to increase the total amount of targets in in the qPCR. Table 2. Number of positive reactions and total number of reactions in qPCR measurements of the nonfragmented and fragmented samples, respectively, run either pre- or post-extraction

[0124] ‘Negative results may be due to inhibition caused by high template addition

[0125] This Example used extraction as an example of a pre-analysis operation incurring losses of molecules. The losses caused by the extraction meant that the prior art bioanalysis process (“Non-fragmented”) was not capable of detecting any target DNA molecules following extraction (Post-extraction, Nonfragmented) whereas target DNA molecules could be detected at all concentrations prior to extraction (Pre-extraction, Non-fragmented). However, fragmenting the target DNA molecules according to the invention prior to the extraction improved the sensitivity and enabled detection of target DNA fragments (Post-extraction, Fragmented).

[0126] EXAMPLE 2

[0127] Bakers yeast cells were suspended in TE buffer and diluted 10 or 100 times in TE buffer. Each of the three concentrations was divided into four 200 pl aliquots referred to as subsamples. 0.1 g of 0.1 mm silica glass beads (Merck Life Science, Cat. No. BMSD113101) were added to two of the subsamples. One of each subsample with beads and without beads were then treated with either 3 minutes of vortexing at high speed (6000 rpm) or pipetting up and down 50 times through a 200 pl pipette tip.

[0128] After the treatment, DNA was extracted from 100 pl of each sample using Qiagen DNeasy blood & tissue kit (Cat. No. 69504) with the recommended protocol for cells and eluted in 200 pl AE buffer provided with the kit. Ten qPCR assays (Table 3) were designed for Saccharomyces cerevisiae Chromosome IX amplifying sequences separated by at least 1000 base pairs. All ten qPCR assays were probe-based using FAM reporter dye. All ten primer pairs and separately all ten probes were pre-mixed before the qPCR. The qPCR was run in a 384 well plate using Qiagen Multiplex PCR kit (Cat. No. 206143) in 10 pl reactions with 2 pl template in 4 replicates on a Biorad Opus cfx384 instrument. The temperature program consisted of 15 min at 95°C, and then 40 cycles of 15 sec at 95°C and 45 sec at 60°C.

[0129] Table 3 - Primers and probes for qPCR assays

[0130] Fig. 6 compares the samples vortexed in the presence (circles) and absence (triangles) of silica glass beads. Vortexing the cells using silica glass beads resulted in a 5 Cq earlier signal increase, corresponding to a 30-fold difference. Fig. 7 correspondingly compares the samples pipetted 50 times in the presence (circles) and absence (triangles) of silica glass beads. Pipetting the sample in the presence of silica glass beads resulted in a 3 Cq earlier signal increase as compared to not using silica glass beads.

[0131] Fig. 8 compares the samples vortexed (circles) with those pipetted 50 times (tringles) in the presence of silica glass beads. Vortexing resulted in 2 Cq earlier signal increase, corresponding to a 4-fold difference, In this setting vortexing was more efficient than pipetting before the extraction, but pipetting with beads was still 3 Cq better than vortexing without beads.

[0132] The samples vortexed in the presence and absence of silica glass beads were extracted, further diluted 100 times and run in qPCR as described above this time in 16 replicates. Table 4 shows the Cq-values for those samples. All 16 replicates of the sample vortexed in the presence of silica beads were positive, while only six replicates for the sample vortexed in the absence of beads were positive. This further emphasized the increase in sensitivity when using silica beads to enhance the fragmentation.

[0133] Table 4 - Cq values

[0134] Samples vortexed or pipetted with beads were further analyzed in digital PCR (dPCR). Two reaction mixes were prepared with either one of the assays in Table 3 or all ten assays. 9 pl of template for each sample was mixed with 36 pl of a reaction mix consisting of 54 pl 4x QI Acuity Probe master mix (Qiagen, Cat. No. 250101), 17.3 pl 10 pM primer mix, 8.64 pl 10 pM Probe / Probe mix and 116.9 pl water. 40 pl of these reactions were added to a QIAcuity 8 well, 26k plate (Qiagen, Cat No. 250031) and run in a Qiagen QIAcuity One instrument with a temperature program consisting of 2 min at 95°C, and then 40 cycles of 15 sec at 95°C and 45 sec at 60°C.

[0135] If the DNA is intact the number of positive partitions should be the same using one or ten assays since the whole target molecule is in the same partition (stronger signal may develop per partition, but the number of positive partitions stays the same). If the DNA is fragmented, each fragment should end up in a different partition leading to many more positive partitions. As seen in Figs. 9 and 10, and Table 5, the number of positive partitions increased dramatically when using ten assays instead of one assay and the sample was subject to fragmentation (Vortex / beads or Pippett / beads). The results show that fragmentation resulted in at least 10-fold increase in the number of targets, evidencing the fragments behaved as independent detectable molecules. In fact the estimated increase was larger than 10, which could be due to copy number variation, overloading of the chip in combination of not having digested it with restriction enzyme to small fragments prior to loading, which is common practice to obtain clean data, but could not be used here, as the purpose was to demonstrate sufficient fragmentation had already happened during the bead treatment before extraction.

[0136] Table 5 - results of dPCR assays

[0137] EXAMPLE 3

[0138] Four Probe based digital PCR (dPCR) assays were designed to detect a 752 bp sequence corresponding to a gene fragment of Mycobacterium tuberculosis (TB). The probe of each assay had a different reporter dye (Table 6). A synthetic gene fragment gBIock was designed for the 752 bp TB sequence and ordered from Integrated DNA Technologies. A restriction enzyme, Bsrl, was chosen so that it cut the between the four assays.

[0139] Table 6 - Primers and probes for dPCR assays

[0140] Approximately 1 *108copies of the gBIock were treated for 1 hour at 65°C, then 20 minutes at 80°C, either with or without the restriction enzyme present. An aliquot of this sample was diluted to 1 *103copies / pl and was used as template in subsequent dPCR. A reaction mix was prepared with all four assays, 800 nM of each primer and 400 nM of each probe, and 1 x of Q I Acuity Probe PCR kit (Qiagen, Cat. No. 250101). For each sample, 9 pl of the template was mixed with 36 pl of reaction mix and was then loaded on to QI Acuity Nanoplate 26k 8-well (Qiagen, Cat. No. 250031) that was sealed and loaded into a QIAcuity One, 5plex System. The PCR was run according to the following protocol, 2 min at 95°C followed by 40 cycles of 15 sec at 95°C and 45 sec at 60°C. The plate was then read in the green, yellow, orange and crimson channels.

[0141] The dPCR assays were analyzed pairwise to see if the signal from the different dyes came from the same partition or from a different partition. If the gBIock was intact the signal would come from the same partition. However, if the gBIock had been fragmented it would be unlikely that the signal would come from the same partition. As can be seen in Table 7, without the restriction enzyme present in the incubation, about 80% of the signal came from the same wells, while if the restriction enzyme was present only about 3% of the signals came from the same partitions.

[0142] Table 7 - Assay cross-reaction

[0143] The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible. The scope of the present invention is, however, defined by the appended claims.

Claims

CLAIMS1 . A method for detecting target nucleic acid molecules in a sample, the method comprising: fragmenting target nucleic acid molecules in the sample into multiple target nucleic acid fragments; subjecting the sample to at least one pre-analysis operation comprising extraction and / or purification of nucleic acid molecules to get a processed sample or an aliquot thereof comprising target nucleic acid fragments; detecting the target nucleic acid fragments in the processed sample or in the aliquot thereof; and concluding the target nucleic acid molecules were present in the processed sample based on the detected target nucleic acid fragments2. The method according to claim 1 , wherein pre-analysis operation additionally comprises at least one pre-analysis operation selected from the group consisting of precipitation of the sample, and exposing the sample to a target molecule compromising condition.

3. The method according to claim 1 or 2, wherein detecting the target nucleic acid fragments comprises individually detecting a respective target nucleic acid fragment of the multiple target nucleic acid fragments in the processed sample or in the aliquot thereof.

4. The method according to claim 1 or 2, wherein detecting the target nucleic acid fragments comprises detecting the multiple target nucleic acid fragments in the processed sample or in the aliquot thereof by fragment-specific quantification assays using a same detectable label to obtain a combined quantity of the multiple target nucleic acid fragments in the processed sample or in the aliquot thereof.

5. The method according to any one of claims 1 to 4, wherein detecting the target nucleic acid fragments comprises detecting the target nucleic acid fragments using a polymerase chain reaction process, preferably real-time polymerase chain reaction, qPCR, or digital polymerase chain reaction, dPCR.

6. The method according to any one of claims 1 to 4, wherein detecting the target nucleic acid fragments comprises detecting the target nucleic acid fragments using a sequencing process, preferably next-generation sequencing, NGS or third generation sequencing.

7. The method according to any one of claims 1 to 6, wherein detecting the target nucleic acid fragments comprises:amplifying a respective target nucleic acid fragment of the multiple nucleic acid target fragments in the processed sample or in the aliquot thereof in a respective polymerase chain reaction, PCR assay; and detecting the amplified target nucleic acid fragments.

8. The method according to any one of claims 1 to 6, wherein detecting the target nucleic acid fragments comprises: contacting the processed sample or the aliquot thereof with multiple primer pairs each comprising a unique forward primer and a unique reverse primer, and multiple labelled probes, wherein each primer pair of the multiple primer pairs is designed to amplify a respective target nucleic acid fragment; each labelled probe of the multiple labelled probes is complementary or identical to a sequence of a respective target nucleic acid fragment of the multiple target nucleic acid fragments; and the multiple labelled probes have a same label; amplifying the multiple target nucleic acid fragments by performing polymerase chain reaction, PCR, amplification on the processed sample or the aliquot thereof; detecting labels bound to the amplified target nucleic acid fragments; and detecting the target nucleic acid fragments based on the detected labels bound to the amplified target nucleic acid fragments.

9. The method according to any one of claims 1 to 6, wherein detecting the target nucleic acid fragments comprises: contacting the processed sample or the aliquot thereof with multiple primer pairs each comprising a unique forward primer and a unique reverse primer, and a sequence-unspecific label, wherein each primer pair of the multiple primer pairs is designed to amplify a respective target nucleic acid fragment; amplifying the multiple target nucleic acid fragments by performing polymerase chain reaction, PCR, amplification on the processed sample or the aliquot thereof; detecting sequence-unspecific labels bound to the amplified target nucleic acid fragments; and detecting the target nucleic acid fragments based on the detected sequence-unspecific labels bound to the amplified target nucleic acid fragments.

10. The method according to claim 1 or 2, wherein detecting the target nucleic acid fragments comprises quantifying the target nucleic acid fragments in the processed sample or in the aliquot thereof; andconcluding the target nucleic acid molecules were present comprises quantifying the target nucleic acid molecules in the processed sample based on the quantified target nucleic acid fragments.11 . The method according to claim 10, wherein quantifying the target nucleic acid fragments comprises individually quantifying a respective target nucleic acid fragment of the multiple target nucleic acid fragments in the processed sample or in the aliquot thereof; and quantifying the target nucleic acid molecules comprises quantifying the target nucleic acid molecules in the processed sample based on the individually quantified target nucleic acid fragments.

12. The method according to claim 10, wherein quantifying the target nucleic acid fragments comprises quantifying the multiple target nucleic acid fragments in the processed sample or in the aliquot thereof by fragment-specific quantification assays using a same detectable label to obtain a combined quantity of the multiple target nucleic acid fragments in the processed sample or in the aliquot thereof; and quantifying the target nucleic acid molecules comprises quantifying the target nucleic acid molecules in the processed sample based on the combined quantity of the multiple target nucleic acid fragments.

13. The method according to any one of claims 10 to 12, wherein quantifying the target nucleic acid fragments comprises quantifying the target nucleic acid fragments using a polymerase chain reaction process, preferably real-time polymerase chain reaction, qPCR, or digital polymerase chain reaction, dPCR.

14. The method according to any one of claims 10 to 12, wherein quantifying the target nucleic acid fragments comprises quantifying the target nucleic acid fragments using a sequencing process, preferably next-generation sequencing, NGS or third generation sequencing.

15. The method according to any one of claims 10 to 14, wherein quantifying the target nucleic acid fragments comprises: amplifying a respective target nucleic acid fragment of the multiple nucleic acid target fragments in the processed sample or in the aliquot thereof in a respective polymerase chain reaction, PCR assay; and quantifying the amplified target nucleic acid fragments.

16. The method according to any one of claims 10 to 14, wherein quantifying the target nucleic acid fragments comprises: contacting the processed sample or the aliquot thereof with multiple primer pairs each comprising a unique forward primer and a unique reverse primer, and multiple labelled probes, wherein each primer pair of the multiple primer pairs is designed to amplify a respective target nucleic acid fragment; each labelled probe of the multiple labelled probes is complementary or identical to a sequence of a respective target nucleic acid fragment of the multiple target nucleic acid fragments; and the multiple labelled probes have a same label; amplifying the multiple target nucleic acid fragments by performing polymerase chain reaction, PCR, amplification on the processed sample or the aliquot thereof; detecting labels bound to the amplified target nucleic acid fragments; and quantifying the target nucleic acid fragments based on the detected labels bound to the amplified target nucleic acid fragments.

17. The method according to any one of claims 10 to 14, wherein quantifying the target nucleic acid fragments comprises: contacting the processed sample or the aliquot thereof with multiple primer pairs each comprising a unique forward primer and a unique reverse primer, and a sequence-unspecific label, wherein each primer pair of the multiple primer pairs is designed to amplify a respective target nucleic acid fragment; amplifying the multiple target nucleic acid fragments by performing polymerase chain reaction, PCR, amplification on the processed sample or the aliquot thereof; detecting sequence-unspecific labels bound to the amplified target nucleic acid fragments; and quantifying the target nucleic acid fragments based on the detected sequence-unspecific labels bound to the amplified target nucleic acid fragments.

18. The method according to any one of claims 1 to 17, wherein fragmenting the target nucleic acid molecules comprises subjecting the sample to a physical fragmentation process selected from the group consisting of sonication, radiation, shearing forces, bead beating, heat, acidic pH and basic pH.

19. The method according to claim 18, wherein fragmenting the target nucleic acid molecules comprises: contacting the sample with beads; andinducing movement of the beads within the sample.

20. The method according to any one of claims 1 to 19, wherein the target nucleic acid molecules are target deoxyribonucleic acid, DNA, molecules; and fragmenting the target nucleic acid molecules comprises contacting the sample with at least one endonuclease, preferably at least one restriction endonuclease, to fragment the target DNA molecules in the sample into multiple target DNA fragments.21 . The method according to any one of claims 1 to 20, wherein the target nucleic acid molecules are target double-stranded deoxyribonucleic acid, dsDNA, molecules; fragmenting the target nucleic acid molecules comprises denaturing the target dsDNA molecules into multiple target single-stranded DNA, ssDNA, molecules; and subjecting the sample to the at least one pre-analysis operation and quantifying the target fragments are performed under denaturing conditions.

22. The method according to any one of claims 1 to 21 , wherein the target nucleic acid molecules are target double-stranded deoxyribonucleic acid, dsDNA, molecules; fragmenting the target nucleic acid molecules comprises: contacting the sample with at least one nicking endonuclease to fragment at least one strand of the target dsDNA molecules; and denaturing the target dsDNA molecules into multiple single-stranded target fragments.

23. The method according to any one of claims 1 to 19 wherein the target nucleic acid molecules are target ribonucleic acid, RNA, molecules; and fragmenting the target nucleic acid molecules comprises contacting the sample with at least one endoribonuclease to fragment the target RNA molecules in the sample into multiple target RNA fragments.

24. The method according to claim 23, wherein fragmenting the target nucleic acid molecules comprises: contacting the sample with at least deoxyribonucleic acid, DNA, oligonucleotide complementary to a respective portion of the target RNA molecules; andcontacting the sample with ribonuclease H.

25. The method according to any one of the claims 1 to 24, wherein the sample comprises multiple sets of target nucleic acid molecules; detecting the target nucleic acid fragments comprises detecting, for each set of the multiple sets of target nucleic acid molecules, the target nucleic acid fragments in the processed sample or in the aliquot thereof using a set-specific dye; and a set-specific dye used for one set of the multiple sets of target nucleic acid molecules is different than set-specific dye(s) used for remaining set(s) of the multiple sets of target nucleic acid molecules.

26. The method according to any one of claims 1 to 25, wherein the sample is not subject to any extraction and / or purification of nucleic acid molecules prior to fragmenting the target nucleic acid molecules in the sample into the multiple target nucleic acid fragments.

27. The method according to any one of claims 1 to 26, wherein the sample is a body fluid sample or a body tissue sample.

28. The method according to claim 27, wherein the sample is a body fluid sample selected from the group consisting of a blood sample, a blood plasma sample, a blood serum sample, a saliva sample, a cerebrospinal fluid sample, a urine sample, a sputum sample, a semen sample, a synovial fluid sample, and an amniotic fluid sample, preferably selected from the group consisting of a blood sample, a blood plasma sample, and a blood serum sample.

29. The method according to any one of claims 1 to 28, wherein the at least one pre-analysis operation has a combined nucleic acid molecule equal to or less than 50 %.

30. A kit for detecting target nucleic acid molecules in a sample, the kit comprises: beads configured to fragment target nucleic acid molecules in the sample into multiple target nucleic acid fragments; multiple primer pairs each comprising a unique forward primer and a unique reverse primer; and multiple labelled probes, wherein each primer pair of the multiple primer pairs is designed to amplify a respective target nucleic acid fragment;each labelled probe of the multiple labelled probes is complementary or identical to a sequence of a respective target nucleic acid fragment of the multiple target nucleic acid fragments; and the multiple labelled probes have a same label.

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