Use of complementary single-stranded fluorescent reporter complexes in PCR.

Abstract

This application relates to multiplex digital polymerase chain reaction (PCR) assays, methods, and systems in which target sequences are detected using a mediator probe and a complementary single-stranded fluorescent reporter complex, preferably with a combination of at least two colors (color combination approach). The method of the present invention differs from prior art methods in the universal reporter complex used to generate fluorescent signals in the PCR assay. The method of the present invention is simple to perform and efficient for accurately determining the concentration of multiple target sequences (typically more than 10) in a complex sample.

Description

[Technical Field]

[0001] The present application relates to multiplex digital polymerase chain reaction assays (dPCR assays), methods, and systems (color combination approach) in which target sequences are characterized by a combination of at least two colors. The methods of the present invention differ from prior art methods in the analytical steps implemented to process the fluorescence recordings of the dPCR assays. In particular, these methods are characterized by the fact that only two categories of compartments should be considered: 1) the total number of "all negative" compartments, which have low fluorescence levels for all fluorophore types, and 2) the number of compartments showing high fluorescence levels for all (but only one) fluorophore types corresponding to the color combination encoding the target sequence. This method is simple to implement and efficient for accurately determining the concentration of a large number of target sequences (usually more than 10) in a complex sample. [Background technology]

[0002] Digital polymerase chain reaction (dPCR) is a powerful and sensitive method that can be used to detect rare mutations in nucleic acid samples. This emerging technology provides quantitative information about nucleic acids with unparalleled precision. Applications range from clinical specialties such as oncology for genotyping and monitoring of lung cancer, areas with high research activity, organ transplantation, microbiology, virology, or non-invasive prenatal testing, to environmental research or health and safety monitoring of food and feed products.

[0003] In many fields, whether in biology or medicine, there is a need to extract as much information as possible from one given sample, ideally from a single experiment. This need for multiplexed analysis can be driven by several reasons, such as the rare availability of samples or efforts to reduce the cost of analysis. Therefore, there is a need for digital PCR to detect more than one or two targets at once.

[0004] Currently, most available commercial platforms offer only two-color detection, but multiplexing strategies are being developed that involve manipulating probe and primer concentrations to generate populations of different fluorescence amplitudes for a given detection channel [1, 2].

[0005] A multiplexing strategy that ensures robust results is to use multiple separate detection channels, assign one target per detection channel, and take advantage of the many fluorophores available for fluorescent nucleic acid detection. This is the "one color, one target" approach. For example, a dPCR assay using allele-specific fluorescent TaqMan® probes requires a dPCR instrument with an R detection channel to detect R somatic mutations in biomarker genes. In such a dPCR duplex assay configured on a dPCR instrument with two detection channels, a first probe that recognizes a first nucleic acid sequence (e.g., a wild-type allele) and a second probe that recognizes a second nucleic acid sequence (e.g., a specific mutant allele) are used. Upon hybridization of the first or second probe to the amplification product in the dPCR compartment, the probe releases its fluorophore through the exonuclease or endonuclease activity of the DNA polymerase. The fluorophore emitted from the first probe is detected through a first fluorescence detection channel that is separate from the second fluorescence detection channel that detects the fluorophore emitted from the second probe. Nevertheless, this "one color, one target" approach is limited to detecting R or fewer target sequences using a dPCR instrument with R detection channels [3].

[0006] One approach to increasing the level of multiplexing has been described in the literature and is known as "intensity-based multiplexing" ([4]). This assay combines two or more TaqMan® probe types that share the same fluorophore type but have different target sequences for at least one of the fluorophore types used in the assay. Two or more TaqMan® probe types that share the same fluorophore type are combined in the assay at two distinct concentrations. When using only two probe types per fluorophore type, one probe type has a low concentration ("lo" type) and one probe type has a high concentration ("hi" type). As a result, after PCR, if a compartment contains a "lo" type target, the fluorescence level of the associated fluorophore type in the compartment will exceed the set positive threshold, but at a lower level than if the compartment contains a "hi" type target. After PCR, assuming all targets are present in the sample, for each fluorophore type for which intensity-based multiplexing was performed, there will be a "lo" positive population of the first droplet, which is the negative population of droplets; a "hi" positive population of the second droplet with a higher measured fluorescence level; and possibly a "lo+hi" positive population of the third droplet with an even higher measured fluorescence level (which contains both the "lo" and "hi" targets so that the fluorescence signals are additive). Using this approach, different targets are distinguished by utilizing the measured fluorescence intensities of the compartments in addition to an initial binary classification of droplets according to a set positivity threshold.

[0007] This approach can be completely unreliable in the presence of inhibitors or low-quality nucleic acids that may differentially affect multiple targets, resulting in smearing, substitutions, or fusions of compartment populations not observed for assay-calibrated controls [5].

[0008] Another drawback of Lindner et al.'s [4] intensity-based approach is the complexity of droplet classification and data analysis. Indeed, using this approach, multiple thresholds must be set for each fluorophore type to distinguish between multiple populations of positive compartments, all of which have different fluorescence intensities. For example, when using a two-level intensity-based approach, three thresholds are required for each fluorophore type used to properly classify the compartment populations and properly detect and quantify the target of interest: one threshold for "lo" positive compartments, one threshold for "hi" positive compartments, and one threshold for "lo+hi" positive compartments.

[0009] Given this shortcoming, there remains a need for higher levels of multiplexing with fewer thresholds and simpler analysis of the data.

[0010] In this context, Marras et al. ([6]) proposed a real-time PCR method involving color-coding each target using two or more colors (the "color-combination approach"). However, in this method, the authors tested only samples containing one target sequence (Figure 1 in Marras et al.). The authors acknowledged that when multiple targets are present in a sample, the use of combinatorially labeled hybridization probes leads to results that are difficult to interpret. This limitation is due to the fact that Marras et al. used real-time PCR, which means that when multiple targets are present in a sample, they are simultaneously amplified in the same reaction, resulting in competing parallel PCR reactions and modified fluorescent signals.

[0011] This limitation can be circumvented by using digital PCR (dPCR). In digital PCR, when multiple targets are present in a sample, they are separated into different compartments during the partitioning step, thereby significantly reducing the likelihood of competing PCR reactions. However, the implementation of color combinations in digital PCR has not yet been demonstrated.

[0012] Nevertheless, co-encapsulation (i.e., compartments containing at least two different target sequences) still exists in digital PCR, with a subset of compartments containing more than one type of target sequence. When using a "one color, one target" approach, it is easy to deduce which target sequences are present in compartments that are positive for multiple fluorophore types, since each color encodes a different type of target sequence. However, this task becomes significantly more difficult when at least one target sequence is color-coded using two or more fluorophore types. Compartments that are positive for more fluorophore types than the number of fluorophore types used for color coding are ambiguous regarding the content of the target sequence. Processing such ambiguous compartments and deconvolving the measured fluorescent signals is complicated. This complexity is further increased by the fact that commercially available dPCR instruments now offer five, six, or more fluorescent detection channels. For example, dual color coding on a six-channel dPCR instrument allows for simultaneous monitoring and analysis of 15 different targets in the same sample.

[0013] In this regard, data interpretation of results generated by combinatorially labeled hybridization probes specific for multiple targets remains a challenge, especially when dPCR instruments with more than three fluorescence detection channels are used.

[0014] Therefore, there remains a need to identify color combination approaches that would enable accurate and reproducible quantification of the presence and concentration of multiple (typically more than 10) target sequences in complex samples by using dPCR devices containing more than three detection channels.

[0015] There is also a need to use universal reporter probes rather than direct specific probes such as TaqMan® probes. This will reduce the design and use of fluorescently labeled probes, thereby reducing the cost and complexity of experimental design. Because linear probes are known to produce undesirable side effects (Huang et al., PNAS 2022), to date, universal reporter probes are usually molecular beacon probes that can include an extended 3' end that allows complementary mediators to hybridize.

[0016] However, in practice, their hairpin structure can make it difficult to generate molecular beacon molecules with 3D constraints. Furthermore, molecular beacon reporter molecules must be synthesized by grafting a fluorophore type and a quencher onto the same chain, which can be difficult for some fluorophore types and is more expensive than reporter molecules with only a fluorophore or a quencher. For these reasons, we sought an alternative universal reporter that is more stable and easier to manufacture than molecular beacons, which have 3D structural constraints due to their loop shape.

[0017] Therefore, the objects of the present invention are as follows: - providing a dPCR method that is simple to perform, efficient for accurately determining the presence and concentration of multiple target sequences in a sample, and applicable to a set of biologically meaningful sample sources; and / or - To provide a universal reporter that is more stable and / or easier to manufacture and / or easier to use than molecular beacons. Detailed Description of the Invention

[0018] The present application provides methods for the detection and quantification of a target sequence or multiple target sequences in a nucleic acid sample using PCR assays, particularly multiplex dPCR assays, including both direct and indirect probes.

[0019] In particular, the present invention is directed to an in vitro PCR method for detecting the presence and / or quantifying at least one nucleic acid target sequence (TSi) in a biological sample containing nucleic acid molecules, the method comprising: A) contacting the sample with a set containing, for each TSi, a direct probe or an indirect probing system carrying a fluorophore group (described below); B) amplifying said at least one nucleic acid target in the presence of an enzyme having nuclease activity; C) detecting or measuring the fluorescence intensity for each fluorophore group; D) Optionally, processing the data collected in step C) to quantify the concentration of at least one TSi in the biological sample.

[0020] This method can be used to detect different mutated target sequences. This method can also be used to assess microsatellite instability (MSI) and / or detect genome editing products.

[0021] In a particular embodiment, the method of the present invention comprises the main steps of a conventional dPCR assay, namely: a) contacting the sample with a fluorophore-bearing probe capable of directly or indirectly binding to a target sequence (TS); b) separating the sample into a set of compartments; c) exponentially amplifying the TS in the presence of a PCR reaction mix; d) measuring the fluorescence intensity of each fluorophore type for each compartment. definition

[0022] As used herein, the term "digital PCR" refers to a PCR assay in which a sample is separated into multiple compartments, with a PCR reaction occurring in each compartment. Signals from each compartment are detected, allowing for quantitative quantification of nucleic acids through statistical analysis. Currently available commercial digital PCR platforms rely on two distinct approaches. The first was chamber digital PCR, which relied on a 2D array of microchambers to partition the sample [7]. After filling with PCR mix, the microchambers were thermally cycled on a flat-block thermocycler and then imaged using fluorescence to reveal amplified positive compartments. The second approach, droplet digital PCR, instead partitions the sample into a bulk emulsion of tiny droplets using platform-specific consumables. The emulsion is transferred to a PCR tube or plate for thermal cycling. After PCR amplification, data acquisition is performed in a process similar to flow cytometry, whereby droplets are read fluorescently one by one as they pass in front of a single laser excitation source [8]. The naica® system combines the use of a 2D array format with droplet compartments to perform digital PCR using a hybrid approach called "Crystal digital PCR™." First, the sample is partitioned into a 2D monolayer array of monodisperse droplets, called droplet crystals. These droplet crystals are transferred to a fluorescence microscope, thermally cycled on a flat-block thermocycler, and imaged to reveal amplification compartments. The entire process takes place within a specifically designed microfluidic chip, such as a sapphire or ruby ​​chip, and requires the use of two devices: i) the naica® Geode, which performs sample partitioning and droplet crystal thermal cycling, and ii) the naica® Prism, an automated fluorescence microscope equipped with three or six separate fluorescence channels ([9]). Another approach for digital PCR performs fluorescence imaging at multiple steps during PCR amplification, and compartments are classified based on the resulting fluorescence curves. This is a hybrid between digital PCR and real-time PCR.All known dPCR devices can be used to carry out the method of the present invention, in particular chamber digital PCR, droplet digital PCR, Crystal Digital PCR™, BEAMing (beads, emulsion, amplification and amplification)-based digital PCR and microfluidic chip-based digital PCR.

[0023] As used herein, the terms "partition" or "partitioned" refer to the separation of a sample into multiple portions, or "compartments." Typically, a sample is divided into at least 500 compartments. The compartments are generally physical, such that the sample in one compartment does not mix, or substantially does not mix, with the sample in an adjacent compartment. The compartments can be solid or fluid. In some embodiments, the compartments are solid compartments, e.g., microwells. In some embodiments, the compartments are fluid compartments, e.g., droplets. In some embodiments, the fluid compartments (e.g., droplets) are the result of a mixture of immiscible fluids (e.g., water and oil). In some embodiments, the fluid compartments (e.g., droplets) are aqueous droplets surrounded by an immiscible carrier fluid (e.g., oil). As used herein, "substantially all compartments" refers to at least approximately any one of 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more of the total number of compartments. The compartments described herein can be in any suitable format. Microwell plates, capillaries, oil emulsions, and arrays of miniaturized chambers with nucleic acid binding surfaces can be used to divide samples. In preferred embodiments, the compartments of the methods of the present invention are droplets. In preferred embodiments, the naica® dPCR platform is used to perform the division step of the methods of the present invention. In some embodiments, the Sapphire chip of the naica® dPCR platform is used to divide samples. Typically, the Sapphire chip contains four microchambers, each with a two-dimensional monolayer of droplets. In some embodiments, the Ruby chip of the naica® dPCR platform is used to divide samples. Typically, the Ruby chip contains 16 microchambers, each with a two-dimensional monolayer of droplets. In some embodiments, data from dPCR reactions in droplets from different microchambers of the Sapphire chip are combined to provide quantification of the target sequence the method is designed to detect.

[0024] In a preferred embodiment, the sample is divided into a sufficient number of compartments so that at least the majority of compartments have no more than 1-5 target regions or template molecules thereof (e.g., about 1, 2, 3, 4, or 5 target regions or amplification products thereof). Specifically, most compartments have two or more target regions or amplification products, but each has fewer than three target regions or amplification products.

[0025] Generally, the compartments can contain excess enzymes, probes and primers so that each mixed compartment is more likely to successfully amplify any target region present within it.

[0026] In a preferred embodiment, the volumes of all compartments are constant. The method of the present invention can also be performed on compartments whose volumes are not constant. In another embodiment, the distribution of the volumes of the compartments around the mean volume is known before performing the method of the present invention, and the obtained results are corrected to take into account this distribution around the mean. In another embodiment, the volumes of the compartments are measured while performing the method, for example by using fluorescence measurements obtained on the compartments, and the obtained results are corrected to take into account the measured compartment volumes.

[0027] Steps a) and b) of the method of the present invention can be performed in any order. Thus, the sample can be divided first, and then the detection reagent (e.g., probe, enzyme, etc.) is incorporated into the divided sample. Alternatively, in a preferred embodiment, the sample and the detection reagent (e.g., probe, enzyme, etc.) can be contacted first, and then the sample can be divided. Preferably, in the method of the present invention, the sample is divided immediately after mixing the reagents together so that substantially all or most of the reaction (e.g., DNA amplification, DNA cleavage, etc.) occurs after division. In other cases, the reagents are mixed under conditions, such as at a temperature at which the reaction proceeds slowly or not at all. In this condition, the sample is then divided, and the reaction is finally initiated and allowed to proceed, for example, by adjusting the temperature. Other triggers, such as optical or chemical triggers, can be used.

[0028] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to polymers of nucleotides of any length. These include DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Polynucleotides may contain, for example, locked nucleic acids (LNAs), minor groove binders (MGBs), methylated nucleotides and their analogs, or any Tm enhancer blockers or baits that minimize off-target nucleic acid selection. Nucleic acids may be single-stranded, double-stranded, or in a more highly condensed hybridization form and may contain chemical modifications. The terms "polynucleotide" or "nucleic acid" may also be used to refer to sequences encoded by a nucleic acid, including sense strand (i.e., coding strand) and antisense strand (i.e., non-coding strand) sequences in a double-stranded nucleic acid molecule.

[0029] As used herein, the term "target sequence" refers to a unique genomic location that defines the location of an individual nucleic acid sequence of interest, comprising one or more consecutive nucleotides. In some embodiments, the target sequence is a single nucleotide location of interest. In some embodiments, the target sequence is at least about 2, 3, 5, 10, 15, 20, or 25 consecutive nucleotides. A gene may contain multiple target sequences of interest. "Target sequence" may refer to either the sense or antisense strand sequence of a target region. A target sequence may be any target nucleotide sequence of interest, for example, a sequence or variant sequence represented in the genome of a target pathogen.

[0030] In the context of the present invention, a "target sequence" is preferably a mutant sequence, as defined below, which is present in low abundance in a sample. In some embodiments, the target sequence is not a wild-type sequence.

[0031] As used herein, the terms "mutated sequence" and "variant sequence," which are used interchangeably herein, refer to any sequence change in a sequence of interest compared to a reference sequence. Quantifying their concentrations in a sample may be the objective of the methods of the present invention. Mutational sequences include, but are not limited to, insertions, deletions, and substitutions, including single nucleotide changes and alterations of two or more nucleotides in a sequence. Mutational sequences may be located at mutational hotspots, microsatellite sequence loci, or within site-specific genome editing reagents. These may be SNVs or SNPs. A "mutational hotspot" refers to a locus known to be prone to naturally occurring mutations, for example, in disease tissues or disease states. As used herein, the term "single nucleotide variant" or "SNV" for short refers to a single nucleotide change at a specific position in a genomic sequence. When alternative alleles occur in a population at appreciable frequencies (at least 1% of the population), SNVs are also known as "single nucleotide polymorphisms" or "SNPs." As used herein, a "microsatellite sequence locus" refers to a region of genomic DNA containing one to seven short repeat sequence elements, e.g., one to five, or one to four base pairs in length. Each sequence repeated at least once within a microsatellite locus is referred to herein as a "repeat unit." Each microsatellite locus typically contains at least seven repeat units, e.g., at least ten repeat units, or at least 20 repeat units. A "site-specific genome editing reagent" refers to a component or set of components that can be used for site-specific genome editing. Generally, such a reagent contains a targeting module and a nuclease module.

[0032] The term "wild-type sequence" as used herein refers to a sequence of interest, e.g., a sequence corresponding to a dominant allele of a gene, or a sequence to which one wishes to compare the unmodified sequence of a locus. This is generally a sequence that is more abundant than a mutant sequence in a sample being tested.

[0033] As used herein, the term "polymerase chain reaction" or "PCR" refers to a method in which a specific segment of a target double-stranded DNA is amplified. PCR is well known to those skilled in the art. Exemplary PCR reaction conditions typically include either two-step or three-step cycles. A two-step cycle includes a denaturation step followed by a hybridization / extension step. A three-step cycle includes a denaturation step followed by a hybridization step, followed by a separate extension step. Also contemplated herein are polymerase chain reaction assays performed without thermal cycling, including, but not limited to, rolling-circle amplification (RCA), isothermal PCR, and loop-mediated isothermal amplification (LAMP).

[0034] A "primer" is a generally short, single-stranded polynucleotide that generally has a free 3'-OH group and hybridizes to a target sequence to bind to the target nucleic acid and subsequently promote polymerization of a polynucleotide complementary to the target nucleic acid. Primers can be of various lengths and are often less than 50 nucleotides in length, e.g., 12-30 nucleotides in length. Primers can be DNA, RNA, or chimeras of DNA and RNA portions. In some cases, primers can contain one or more modified or non-natural nucleotide bases. In the context of the present invention, each section may contain multiple primer sets corresponding to multiple target sequences. In some embodiments, each primer set includes a forward primer and a reverse primer for amplifying a target sequence. In some embodiments, the forward primer and reverse primer are oligonucleotide primers that anneal to opposite strands of a nucleic acid molecule and flank the target sequence. The primer set enables the production of amplification products specific to the target fragment during a PCR reaction.

[0035] As used herein, a "tag" is a relatively short oligonucleotide containing 8 to 25 nucleotides, preferably 8 to 18 nucleotides, more preferably 10 to 16 nucleotides, and even more preferably 12 to 14 nucleotides. In the context of the present invention, it may also be referred to as a "flap" sequence. This sequence is contained in the 5' region of the mediator probe and is therefore a signature of the target sequence, or a portion thereof. After cleavage by a nuclease enzyme, when the 3' region of the mediator probe is bound to the target sequence, it can hybridize with a tag complementary sequence (TCS) located on a reporter molecule. This latter hybridization causes modification of the signal carried by the reporter molecule, so that the presence of the target sequence in a sample can be detected / quantified. The nucleotide sequence of the tag is usually optimized by conventional means so that its melting temperature in the TCS is lower than that of the 3'-terminal probe region of the mediator probe (allowing hybridization on the TSi).

[0036] Thus, a "tag complementary sequence" or "TCS" corresponds to an oligonucleotide implemented with a CSSFR m-reporter of the present invention, preferably in its 3' region, the sequence of which is partially or entirely complementary to that of a tag oligonucleotide as defined above. As a tag itself, a TCS typically comprises 8-25 nucleotides, preferably 8-18 nucleotides, more preferably 10-16 nucleotides, and even more preferably 12-14 nucleotides.

[0037] For example, a nucleic acid sequence is "complementary" to another nucleic acid when at least two consecutive bases of a first nucleic acid or primer combine in antiparallel binding or hybridize with at least a subsequence of the second nucleic acid to form a duplex. In some embodiments, complementary refers to the preference for hydrogen bond base pairing between the nucleotide bases G, A, T, C, and U, such that in DNA, A pairs with T and G pairs with C, and in RNA, G pairs with C and A pairs with U when two given polynucleotides or nucleotide sequences are annealed to each other.

[0038] A first nucleic acid sequence that "corresponds to" a second nucleic acid sequence is a sequence that is identical to or complementary to the second nucleic acid sequence or a portion of the second nucleic acid sequence, and includes the second nucleic acid sequence or its complementary sequence. When the second nucleic acid sequence has a unique feature, such as a mutation, a nucleic acid sequence that "corresponds to" the second nucleic acid sequence includes a sequence that has the unique feature or its complement.

[0039] As used herein, "hybridization" and "annealing" refer to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonding between the bases of nucleotide residues. Hydrogen bonding can occur through Watson-Crick base pairing, Hoogsteen bonding, or any other sequence-specific manner. A nucleic acid, or a portion thereof, "hybridizes" to another nucleic acid under conditions such that nonspecific hybridization is minimized in a physiological buffer (e.g., pH 6-9, 25-150 mM chloride salt) at a defined temperature. In some embodiments, the defined temperature at which specific hybridization occurs is room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is greater than room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is at least about 37°C, 40°C, 42°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C. In some embodiments, the defined temperature at which specific hybridization occurs is 37°C, 40°C, 42°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C, or is about 37°C, about 40°C, about 42°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, or about 80°C.

[0040] In the context of oligonucleotide hybridization, the melting temperature (T M ) is defined as the temperature at which an oligonucleotide molecule is half single-stranded (and thus "melted") and half double-stranded (i.e., annealed to its complementary strand). M T varies depending on the length of the hybridized oligonucleotide molecule and its specific nucleotide sequence. M The calculation of T also takes into account experimental conditions including primer / probe concentration, target oligo, and salt / ionic strength. M The temperature should be designed to match the temperatures used during PCR cycling. MMethods and calculators that allow determining T are well known in the art. In the context of the present invention, M The values ​​can be used to optimize the [tag / TCS] oligonucleotides as described below.

[0041] Each compartment may contain a polymerase, an enzyme that performs template-directed synthesis of polynucleotides, e.g., DNA and / or RNA. The term "polymerase" encompasses both full-length polypeptides and domains with polymerase activity. DNA polymerases are well known to those skilled in the art, but are not limited to those derived from Pyrococcus furiosus ( Pyrococcus furiosus ), Thermococcus littoralis ( Thermococcus litoralis ) and Terumotoga maritima ( Thermotoga maritime ), or modified versions thereof. Further examples of commercially available polymerase enzymes include, but are not limited to, Klenow fragment (New England Biolabs Inc.), Taq DNA polymerase (QIAGEN), 9°WM DNA polymerase (New England Biolabs Inc.), Deep Vent™ DNA polymerase (New England Biolabs Inc.), Manta DNA polymerase (Enzymatics), Bst DNA polymerase (New England Biolabs Inc.), and Phi29 DNA polymerase (New England Biolabs Inc.). In some embodiments, the polymerase is a DNA-dependent polymerase. In some embodiments, the polymerase is an RNA-dependent polymerase, such as a reverse transcriptase.

[0042] As used herein, "amplification" generally refers to the process of generating two or more copies of a desired sequence. This can be performed using thermal cycling or by any polymerase chain reaction assay performed without any known means, such as rolling circle amplification (RCA), isothermal PCR, and loop-mediated isothermal amplification (LAMP). Components of an amplification reaction can include, but are not limited to, primers, polynucleotide templates, polymerase, nucleotides, dNTPs, and the like. An "amplification product" refers to a nucleic acid fragment formed as the product of a PCR amplification reaction that is a copy of a portion of a specific target nucleic acid, such as a target fragment containing a target region. Although an amplification product is generally double-stranded DNA, reference can be made to its individual strands.

[0043] The droplets support PCR amplification of one or more template molecules using uniform assay chemistry and workflow similar to those widely used in real-time PCR applications (

[10] ). Once droplets are generated, they can be transferred onto a PCR plate, and emulsion PCR reactions can be run in a thermal cycler using a classical PCR program. Alternatively, droplets generated on the naica® system's Sapphire or Ruby chips can be subjected to thermal cycling using a classical PCR program. Thermal cycling is carried out to the endpoint.

[0044] To overcome the technical challenges associated with amplifying low-complexity sequences such as microsatellite sequences, the annealing temperature and / or extension time of the amplification step can be increased. For example, a typical annealing temperature is 55°C, and for microsatellite locus detection, the annealing temperature can be increased by an amount of 3-15°C.

[0045] As used herein, "specific" when used in the context of a primer specific to a target nucleic acid, or a probe specific to a target nucleic acid, or a tag specific to a TCS, refers to a level of complementarity between the primer / probe and the target, or between the tag and the TCS, such that an annealing temperature exists at which the primer / probe or tag preferentially anneals and mediates amplification and fluorescent detection of the target nucleic acid or TCS, and does not anneal, or mediates amplification and fluorescent detection of non-target sequences / non-TCS present in the sample.

[0046] As used herein, the term "probe" refers to a molecule (e.g., a protein, a nucleic acid, an aptamer, etc.) that specifically interacts with or specifically binds to a target polynucleotide. Non-limiting examples of molecules that specifically covalently bind to or specifically bind to a target polynucleotide include nucleic acids (e.g., oligonucleotides), proteins (e.g., antibodies, transcription factors, zinc finger proteins, non-antibody protein scaffolds, etc.), and aptamers. Generally, probes are labeled with a detectable label. The probe can indicate the presence or level of a target polynucleotide by either an increase or decrease in signal from the detectable label. In some embodiments, the probe detects a target polynucleotide in an amplification reaction that is digested by the 5' to 3' exonuclease or endonuclease activity of a DNA-dependent DNA polymerase. In the context of the present invention, the probe is preferably a nucleic acid probe. Furthermore, the probe sequence can incorporate modified bases such as locked nucleic acid bases (LNA® bases), MGBs, or other Tm enhancers, or can incorporate means to minimize off-targeting. This probe can be a mediator probe (not carrying any label) or a labeled probe, examples of which are provided below.

[0047] As used herein, "color combination," in the context of digital PCR experiments, refers to the concept of using more than one fluorophore type for at least one target. The method of the present invention requires that at least one target sequence (TS) be characterized by at least two different fluorophore types (or "colors"). Therefore, in contrast to prior art dPCR methods in which each TS is bound to only one fluorophore type / color, this is hereinafter referred to as the "color combination approach" or "color combination method."

[0048] As used herein, the term "fluorophore type" corresponds to the chemical structure of a given fluorophore moiety. Several different fluorophore types can be used in the methods of the invention, such as FAM, VIC, Yakima Yellow®, HEX, ROX, Cy®5, ATTO®550, ATTO®700, etc., as well as several different quencher types, such as TAMRA™, BHQ™1, BHQ™2, BHQ™3, etc.

[0049] The detection device or detection unit of a digital PCR system uses one or more fluorescence detection channels. A "fluorescence detection channel" typically combines an excitation channel that illuminates a sample with light filtered to contain only a narrow band of wavelengths (excitation wavelengths) and an emission channel that collects light emitted by the sample only within a narrow band of wavelengths (emission wavelengths) distinct from the excitation wavelengths. Commercially available digital PCR systems have detection devices or detection units that acquire data (point detectors, PMTs, or 2D image sensors) from one, two, three, four, five, six, or more different fluorescence detection channels. For simplicity, the verb "image" is used regardless of whether the collected data is from a point detector or an image sensor. In all cases, each fluorescence detection channel utilizes a distinct pair of excitation and emission wavelengths. In the methods of the present invention, the detection device or detection unit is preferably capable of acquiring data (point detectors, PMTs, or 2D image sensors) from at least five, or even six, or more different fluorescence detection channels.

[0050] For example, the Prism 6 instrument, a Naica System detector manufactured by Stilla Technologies, has six distinct fluorescence detection channels: "Blue," "Teal," "Green," "Yellow," "Red," and "Infrared" channels, which combine excitation channels with bandwidths of 450-490 nm, 509-519 nm, 533-557 nm, 564-586 nm, 625-643 nm, and 645-695 nm, respectively, with emission channels with bandwidths of 505-535 nm, 530-551 nm, 568-593 nm, 600-640 nm, 655-685 nm, and 708-753 nm.

[0051] When imaging fluorophore moieties using a detection unit with multiple detection channels, the light emitted by the fluorophore moiety is primarily detected in one of the detection channels. For example, a FAM fluorophore moiety is primarily detected in the "blue" detection channel of a Prism 6 instrument. However, light emitted by the fluorophore moiety may also be detected in other detection channels, a phenomenon known as "fluorescence spillover." For example, a FAM fluorophore moiety is also detected in the "teal" detection channel of a Prism 6 instrument, but with a lower signal intensity than the "blue" detection channel. The distribution of light detected from a given fluorophore type across all fluorescence detection channels is referred to as the fluorescence signature of that fluorophore type.

[0052] In the methods of the present invention, the PCR data collection step is preferably performed using an optical detector with multiple detection channels, such as a six-color detection system (e.g., Stilla's naica® Prism 6 system or Qiagen's Qiacuity 5-colors system). In a given fluorescence detection unit (such as Prism 6), different fluorophore types have different fluorescence signatures.

[0053] The term "sample" as used herein refers to a sample that may be subjected to the methods described herein, with or without prior processing, such as nucleic acid extraction, fragmentation, dilution / concentration, or other pretreatment. The sample may be a biological sample or may be obtained by processing or manipulating a biological sample, such as a biological fluid or biological tissue. The biological sample preferably contains, for example, tumor tissue, scattered cells, feces, blood cells, plasma, serum, lymph nodes, urine, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, pancreatic juice, gastric juice, amniotic fluid, cerebrospinal fluid, or serous fluid. Alternatively, it may be an environmental sample, such as sewage.

[0054] In some embodiments, the sample at the time of collection is ready to be loaded into a digital PCR device for analysis. This is usually the case when the amount of the target sequence is very low in the sample, for example, this is the case for mutant sequences in tumor samples.

[0055] However, in preferred embodiments, the concentration of nucleic acid molecules present in a sample must be adjusted, for example, by diluting the sample or by concentrating the sample (e.g., by dialysis or by lyophilization and reconstitution) to provide a concentration suitable for dPCR. Diluting the sample is particularly important to reduce the amount of sequences present in high abundance in the sample (e.g., WT sequences). In some embodiments, the method is performed using a first sample, and the concentration of nucleic acid molecules in the sample is adjusted based on the count of compartments that each generate a positive signal via three or more detection channels; if (e.g., when) the count is greater than a predetermined value, the adjustment is to decrease the concentration of nucleic acid molecules in the sample by diluting the sample; or if (e.g., when) the count is less than a predetermined value, the adjustment is to increase the concentration of nucleic acid molecules in the sample by concentrating the sample. In some embodiments, the dilution factor or concentration factor is based on the count of compartments that each generate a positive signal via three or more detection channels. In some embodiments, the concentration of nucleic acid molecules in the sample is adjusted based on the estimated concentration of wild-type sequences, mutant sequences, or specific allelic sequences at one or more of the multiple target regions in the sample. In some embodiments, the method is repeated with the sample diluted to one or more concentrations to provide optimal concentrations for accurate quantitation of different genotypes (e.g., wild-type sequences, mutant sequences, and / or allelic sequences) at different target regions.

[0056] In a preferred embodiment, the concentration of the sample is adjusted so that the maximum concentration of target sequence before dividing the sample is at least about 50, 75, 100, 150, 200, 250, 500, 1000, 2000, 5000, or more copies per μL, preferably at least about 150-500 copies per μL.

[0057] The method of the present invention preferably includes a step of calculating a "fluorescence correction matrix" or "spillover correction matrix" using a reference sample. This step is performed before running samples in a digital PCR experiment combining different fluorophore types. The importance of this step is well known in the art. This is due to the fact that the fluorescence detection unit does not directly measure the fluorescence levels of individual fluorophore types. In fact, the fluorescence detection unit measures the light level received by each fluorescence detection channel from each compartment. The light level in one fluorescence detection channel is the sum of the fluorescence signals from all fluorophore types, and each fluorophore has a distinct contribution defined by its fluorescence signature. Usually, one fluorophore type is the main contributor to the fluorescence signal detected in a given fluorescence detection channel. Nevertheless, other fluorophore types can contribute to the total signal. For example, when FAM, Yakima Yellow®, and ATTO® 550 fluorophore types are used in conjunction on a Prism 6 instrument, Yakima Yellow® is the primary contributor to the signal detected in the "teal" channel, but the FAM and ATTO® 550 fluorophores also have secondary signals that add to Yakima Yellow® in the "teal" channel. Overall, the fluorescence signal in a single fluorescence detection channel is therefore the sum of the fluorescence signal intensity for each fluorophore type combined in the experiment and the fluorescence signature of that fluorophore in that channel. Add "background fluorescence" to any additional signal from the background.

[0058] In an experiment, when using n separate fluorescence detection channels and M different fluorophore types, n signals are acquired for each compartment, each being the sum of M contributors (plus "background fluorescence") from the M fluorophore types. In practice, the useful information is the signal intensity I(m) from each fluorophore type. Mathematically, this translates into n linear equations with M unknown variables. This mathematical problem is i) When there are n or fewer fluorophore types combined in an experiment (M≦n), ii) When all fluorophore types have distinct fluorescent signatures can only be solved by

[0059] As a result, only fluorophore types with distinct fluorescence signatures on a given fluorescence detection unit should be combined in one digital PCR experiment or assay. In this case, a "fluorescence correction matrix" or "spillover correction matrix" can be applied to convert n signals from n fluorescence detection channels into M fluorescence levels from M different fluorophore types. A "fluorescence correction matrix" is an n × (n + 1) matrix, where "+ 1" is related to the estimated known "background fluorescence."

[0060] Before running samples in a digital PCR experiment combining different fluorophore types, a fluorescence compensation matrix is ​​preferably calculated using a reference sample. Typically, a "no template control (NTC)" sample and a "single-color control" sample are used to estimate the fluorescence compensation matrix. The NTC sample allows for an estimation of "background fluorescence," while the "single-color control" samples, each containing only one fluorophore type, are used to estimate the fluorescence signature of each fluorophore type across all fluorescence detection channels. While such control experiments allow for the estimation of the fluorescence compensation matrix, the estimation can be imperfect, such that the conversion of n signals from n fluorescence detection channels to M signals from M fluorophore types is also imperfect, resulting in uncertainties and errors in the measurements of fluorophore signal intensities. More complex algorithms can be used to analyze the data, taking into account such possible signal uncertainties.

[0061] In the following sections, unless otherwise stated: - no more than n fluorophore types are combined in an experiment (M≦n); - all fluorophore types have distinct fluorescent signatures that are distinct from each other, - It is recommended that spillover corrections be estimated from control samples.

[0062] In some embodiments, it is possible to use more than n fluorophore types, but only if the fluorophore types can be grouped into n sets of fluorophore types, where within each set, different fluorophore types have similar fluorescent properties and can be treated indistinguishably while performing the methods of the invention. For example, FAM / AlexaFluor488 / Atto495 can be treated as such a set of fluorophore types.

[0063] As a result, for each compartment, the fluorescence signal intensities from the M fluorophore types are preferably measured using the n signals from the n fluorescence detection channels by applying a pre-generated fluorescence compensation matrix.

[0064] Reference herein to "about" a value or parameter includes (and describes) a variation that is directed to the value or parameter itself. For example, a description that refers to "about X" includes the description "X." For example, a value of about X can be within (i.e., ±) 10%, 5%, 2%, 1% or less of X. The present invention's color combination method

[0065] In a first aspect, the present invention relates to a multiplex digital PCR (dPCR) method for detecting and / or quantifying the presence of a set of at least five different nucleic acid target sequences (TS) in a biological sample containing nucleic acid molecules, the method comprising: a) contacting the sample with a fluorophore-bearing probe capable of binding directly or indirectly to a TS, wherein each target sequence i (TSi) is i characterized by different fluorophore types, i is greater than or equal to 2, and b) separating the sample into a set of compartments; c) exponentially amplifying the TS in the presence of a PCR reaction mix; d) measuring the fluorescence intensity of each fluorophore type for each compartment; e) for each fluorophore type, - compartments with a fluorescent signal below the positive threshold for the fluorophore type; - counting compartments having a fluorescent signal higher than the positive threshold for the fluorophore type, N0 = total number of compartments with fluorescent signals below the positivity threshold for all fluorophore types, N i = k characterized by TSi ithe total number of compartments with fluorescent signals higher than the positive threshold for only one fluorophore type, and determining f) processing the data collected in step e) to quantify the concentrations of at least five TSi in the biological sample.

[0066] In some embodiments, the positivity threshold for each fluorophore type is predefined. In some embodiments, the positivity threshold is set while performing the method. In some embodiments, the positivity threshold is predefined and adjusted while performing the method.

[0067] In a preferred embodiment, the positivity threshold for each fluorophore type is defined and / or adjusted to be above the cluster of compartments with the lowest fluorescence intensity, which corresponds to the "all negative cluster," and below any cluster of compartments with a higher level of fluorescence intensity than the "all negative cluster."

[0068] In the method of the present invention, the number of fluorophore types used to characterize each target sequence (TS) is designated by the letter "k". More precisely, in the method of the present invention, each target sequence i (TSi) is designated by k i characterized by different fluorophore types, i is greater than or equal to 2.

[0069] In a preferred embodiment, k i is the same for each and every TS detected in an experiment. For example, each and every TS in an experiment is characterized by two (and only two) fluorophore types. For example, each and every TS in an experiment is characterized by three (and only three) fluorophore types.

[0070] In this embodiment, at most k i (and k i Only compartments with fluorescent signals above the positive threshold for k fluorophore types (k or less) should be considered in the methods of the present invention.i +1 (or k i +2, etc.) should not be considered in the methods of the present invention. i K i = 3 fluorophore types, compartments with fluorescent signals higher than the positive threshold for 4, 5 etc. fluorophore types should not be considered in the methods of the invention.

[0071] More precisely, in the method of the present invention, i Characterize k i Only compartments with a fluorescent signal higher than the positivity threshold for a fluorophore type should be considered as "positive compartments." All other compartments should be considered as "negative compartments" or excluded from the analysis. For example, if a TS is characterized by three fluorophore types, FAM, Yakima Yellow®, and ATTO® 550, only compartments with a fluorescent signal higher than the positivity threshold for only FAM, Yakima Yellow®, and ATTO® 550 are retained as "positive" for this TS. Compartments that are negative for all fluorophore types are retained as "negative." All compartments positive for only a subset of the FAM, Yakima Yellow®, and ATTO® 550 fluorophores (e.g., FAM only, FAM and Yakima Yellow® only, Yakima Yellow® and ATTO® 550 only, FAM, Yakima Yellow®, Cy® 5, and ATTO® 550, etc.) can be retained as "negative" for this TS. These also cannot be considered in the analysis and are ignored. All other compartments that are positive for any fluorophore type other than FAM, Yakima Yellow® and ATTO® 550 are not considered in the analysis and are ignored for this TS.

[0072] In another embodiment, k i will be different for each TS detected in an experiment. For example, some TSs are characterized by two (and only two) fluorophore types, while others are characterized by three (or four) fluorophore types.

[0073] Even in this case, k i Target sequence TS characterized by fluorophore types i Regarding k i Compartments with a fluorescent signal higher than the positive threshold for the +1 fluorophore type should not be considered in the methods of the present invention. For example, the target sequence TS i K i = 3 fluorophore types, compartments with fluorescent signals higher than the positive threshold for 4 fluorophore types are considered to be TS i should not be taken into consideration in the method of the present invention. j K j = 2 fluorophore types, compartments with fluorescent signals higher than the positive threshold for 3 fluorophore types are considered to be TS j should not be taken into consideration in the method of the present invention.

[0074] In the context of the present invention, as proposed above, the total number of different channels or fluorophore types or wavelengths or signals that can be unambiguously detected by the dPCR device used in the proposed method is designated by the letter "n".

[0075] Preferably, the number n of different fluorescence detection channels available for analysis in the method of the present invention is 4 or more, preferably 5 or more, more preferably 6 or 7. It may be greater than 7.

[0076] In the context of the present invention, the total number of different fluorophore types carried by all probes used in an assay (i.e., the total number of different fluorophore types combined in an experiment) is designated as the letter "M".

[0077] In a preferred embodiment, M is less than or equal to n, as explained above.

[0078] The method of the present invention is distinguished from prior art methods in large part by its reliance in step e) on a binary "negative or positive" classification of a compartment for each fluorophore type based on the compartment's measured fluorescence level for that fluorophore type. Thus, in the method of the present invention, the exact fluorescence intensity value of a compartment does not matter, as long as it is above or below a positive threshold. In the present invention, this threshold is used only to distinguish between positive and negative compartments.

[0079] As used herein, a "positivity threshold" corresponds to the intensity value that distinguishes between zones exhibiting high intensity of one fluorophore type and zones exhibiting low intensity of the same fluorophore type.

[0080] In a preferred embodiment, each fluorophore type is defined separately on a 1D plot view, which can also be defined on a 2D plot view if this is more suitable to those skilled in the art.

[0081] Once this positivity threshold is set for each fluorophore type, a number "N0" corresponding to the total number of compartments with fluorescent signals below the positivity threshold for all fluorophore types; ·TS i characterized by k i It is easy to determine the number "Ni", which corresponds to the total number of compartments that have a fluorescent signal higher than the positive threshold only for one fluorophore type.

[0082] Based on these numbers, according to the classical Poisson law, at least 5 TSs in the biological sample are identified.i Concentration C i It is possible to quantify. For example, the following formula:

number

[0083] In a particular embodiment, step e) of the method of the invention further comprises the determination of "N1", which corresponds to the total number of compartments having a fluorescent signal higher than the positive threshold for only one fluorophore type. This number N1 can be determined in two different cases: 1) When abundant sequences (e.g., WT) are detected in a single color (see details in this embodiment and Example 4 below).

[0084] In this first case, N1 is used as the "N" in calculating the concentration of the abundant sequence in the sample. i " should be considered as such. 2) The sequence is not detected in a single color in the assay.

[0085] In this second case, N1 is the TS in the sample. i can be considered for calculation of the concentration of , which can be treated in the analysis as a "negative compartment" (number "N0"). In this latter case, for each target sequence i, the concentration C is calculated using the following formula:

number

[0086] In another embodiment, N1 is excluded from the analysis and treated as an "artifact section" or "noise section." In this case, for each target sequence i, the concentration C i is the following formula:

number

[0087] More generally, step e) of the method of the present invention involves calculating a number of compartments from 1 to k corresponding to the total number of compartments having a fluorescent signal higher than the positive threshold for j fluorophore types. j (1 <k<k j ) in the sample. If there are no sequences detected in j colors in the assay, then N1 is the total number of TS j can be considered for calculation of the concentration of , which can be treated in the analysis as a "negative compartment" (number "N0"). In this latter case, for each target sequence i, the concentration C i is the following formula:

number

[0088] Or N j can be excluded from the analysis and treated as an "artifact section" or "noise section." In this case, for each target sequence i, the concentration C is calculated using the following formula:

number

[0089] For a dPCR experiment using n distinct fluorescence detection channels, and therefore color coding with a maximum of M≦n distinct fluorophore types, the number of different targets that can be measured independently using the color combination approach according to the invention is given by the following equation, where k denotes the number of fluorophore types used to encode each one of the targets:

number

[0090] As an example, using a dPCR instrument with 6 different detection channels, if the color combination method of the present invention uses k=2 different fluorophore types for each target, the total number of distinct targets that can be detected and quantified is

number

number

[0091] If a dPCR instrument is equipped with seven different detection channels, and the color combination method of the present invention uses k=2 different fluorophore types for each target, the total number of distinct targets that can be detected and quantified is

number

number

[0092] Thus, the method of the present invention makes it possible to detect and / or quantify the presence of at least 10, preferably at least 15, more preferably at least 20, and even more preferably at least 30 different TSs in a biological sample.

[0093] In certain embodiments, the methods of the present invention allow for the generation of another set of target sequences (referred to herein as TS e It is also possible to detect and / or quantify the TS, which are referred to as TSs, in this case using a "one color-one sequence approach," i.e., e is characterized (encoded) by only one fluorophore type.

[0094] Two cases must be distinguished: either this single fluorophore type is used to characterize the set of TSi (i.e., used in the color combination approach according to the present invention), or this single fluorophore type is not utilized to characterize the set of TSi.

[0095] In the first case, step a) of the method of the invention comprises: k for detecting a first set of at least five target sequences TSi i Use different fluorophore types and use these k i One and only one of the fluorophore types is characterized by only one fluorophore type. e Another target sequence is TS e In other words, in this embodiment, one fluorophore type is used in combination with several other fluorophore types to detect one or more TSi, as well as the sequence TSi. e Used as a single color to detect

[0096] In this case, the method of the present invention allows the detection of at least five different nucleic acid target sequences (TSi) and one nucleic acid target sequence (TS e ) can be detected and / or quantified by the following steps: a) contacting the sample with a fluorophore-bearing probe capable of directly or indirectly binding to a TS, wherein each of at least five target sequences i (TSi) is selected from k i characterized by different fluorophore types, k i is 2 or more, and one array TS e is characterized by only one fluorophore type, this latter fluorophore type being one of the fluorophore types used to detect the at least one TSi; b) separating the sample into a set of compartments; c) exponentially amplifying the TS in the presence of a PCR reaction mix; d) measuring the fluorescence intensity of each fluorophore type for each compartment; e) for each fluorophore type, - compartments with a fluorescent signal below the positive threshold for a fluorophore type, - counting compartments with a fluorescent signal higher than a positive threshold for a fluorophore type, N0 = total number of compartments with fluorescent signals below the positivity threshold for all fluorophore types, N i = k characterized by TSi i the total number of compartments with fluorescent signals higher than the positive threshold for only one fluorophore type, determining a N e =TS e determining the total number of compartments having a fluorescent signal higher than the positive threshold only for the fluorophore type used to characterize f) at least five TSi and TS in the biological sample e and processing the data collected in step e) to quantify the concentration of

[0097] In this embodiment, at most k i (and k iOnly compartments with fluorescent signals above the positive threshold for k fluorophore types (k or less) should be considered in the methods of the present invention. i +1 (or k i +2, etc.) should not be considered in the methods of the present invention. i K i = 3 fluorophore types, compartments with fluorescent signals higher than the positive threshold for 4, 5 etc. fluorophore types should not be considered in the methods of the invention.

[0098] In this embodiment, -the following formula:

number

number

number

[0099] In the second case, the method of the invention allows the identification, in a biological sample containing nucleic acid molecules, of two different sets of TS: - one set of at least five TSi according to the color combination method of the invention, as defined above and below, - one or more target sequences TS characterized by fluorophore type e This makes it possible to detect the presence and / or quantify one set of TS containing the fluorophore type, where the fluorophore type is not used to characterize the set of TSi.

[0100] In this case, step a) of the method of the invention comprises: a) contacting the sample with fluorophore-bearing probes capable of binding directly or indirectly to two sets of TSs, wherein for the first set of target sequences, each target sequence i (TSi) is k i characterized by different fluorophore types, k i is greater than or equal to 2, and the second set of target sequences is defined as being characterized by other fluorophore types not used to characterize the first subset of TSi.

[0101] The other steps of the method of the present invention remain unchanged.

[0102] In particular, C i The calculation of is done using the number N as defined above. i and N0 and the formula:

number

[0103] One of the first steps of the method of the invention consists of contacting the sample with a fluorophore-bearing probe that is capable of binding directly or indirectly to a TS.

[0104] One skilled in the art can use any probe that can be coupled to a fluorophore and that binds to the TS either directly or via another molecule (usually unlabeled, such as a mediator probe).

[0105] An example of a fluorophore-bearing probe that can indirectly bind to a TS is the TaqMan® probe.

[0106] Examples of fluorophore-bearing probes that can indirectly bind to TS are the molecular beacons described herein or complementary single-stranded fluorescent reporters (CSSFRs). These reporter molecules are usually bound to a mediator probe that can directly bind to TS. The mediator probe can also be bound to a reporter molecule.

[0107] In the color combination analysis of the present invention, several detection systems can be used. In particular, the following direct and indirect probes can be used: TaqMan® Probes

[0108] As used herein, a "TaqMan® probe" consists of an oligonucleotide sequence, referred to as the "probe sequence" or "target sequence," to which a fluorophore moiety and a quencher are covalently attached. Typically, the fluorophore moiety is attached to the 5' end of the probe, and the quencher is covalently attached to the 3' end of the probe.

[0109] Each TaqMan® probe consists of a sequence complementary to a target sequence, a type of fluorophore, and a type of quencher. All TaqMan® probe types combined in an experiment differ in at least one respect: either the target sequence or the type of fluorophore used.

[0110] In one embodiment, data for the methods of the invention are generated using at least 2, 4, 6, 8, 10, or 20 TaqMan® probes, each carrying a different fluorophore type, the combination of which characterizes the target sequence in a unique manner.

[0111] The color combinations of the present methods require the use of two or more different TaqMan® probes for each target sequence. For each target sequence, the two or more different TaqMan® probes used must have different fluorophore types and may have different nucleotide sequences or quenchers.

[0112] For a given target, if two or more different TaqMan® probes used have different, non-overlapping nucleotide sequences, the TaqMan® probes are said to be "non-competing." If two or more TaqMan® probes share the same nucleotide sequence, the TaqMan® probes are said to be "competing."

[0113] In a particularly preferred embodiment, the method of the present invention uses at least two competitive TaqMan® probes, i.e., at least two TaqMan® probes carrying different fluorophore types and containing the same nucleotide sequence complementary to the same target sequence.

[0114] When using "competing" TaqMan® probes, the concentrations of the two probes in the reaction mix may need to be adjusted so that both probes are cleaved at the same rate during PCR. Indeed, if the two probes are at the same concentration and one competing TaqMan® probe binds more preferentially to the target sequence during PCR, that TaqMan® probe will be preferentially cleaved in each cycle of the PCR reaction. This can cause an increase in the fluorescence level of only the "more preferential" TaqMan® probe, masking the signal from the other probes. By reducing the concentration of the "more preferential" probe relative to the "other probes," the effective binding efficiency of the two probes in each PCR cycle is balanced. As a result, the fluorescence levels of both fluorescent probe types can be detected.

[0115] In one embodiment of the present invention, the use of competitive TaqMan® probes to perform color combination involves, in the first step, designing an assay using a single target sequence instead of a large number of target sequences. This first step is used to verify whether the fluorescent signals in the positive zones are sufficiently separated from the fluorescent signals in the negative zones for both colors encoding the single target sequence. This verification is performed using the fluorescent probe types separately and also using a mixture of both fluorescent probe types. When using a mixture of both fluorescent probe types, if at least one of the two fluorescent signals from the two colors encoding the single target sequence is not sufficiently separated, the probe concentrations of the color mixture can be adjusted so that the fluorescent signals from both fluorescent probe types used to encode the single target sequence are sufficiently separated.

[0116] Example 1 below discloses the use of such TaqMan® probes in the methods of the present invention. Other direct probes

[0117] Alternative direct probes have been described in the art and can be used in addition to or instead of TaqMan® probes in the methods of the present invention. For example, molecular beacon hybridization probes or probe pairs can be used as disclosed in Marras S. et al., 2019. In addition, so-called "yin-yang" probes, as disclosed by Li Q. et al. (2022), can be used. These probes are double-stranded probes composed of two long, complementary oligonucleotides that specifically hybridize to a target sequence. The positive strand is labeled with a fluorophore, and the negative strand is labeled with a quencher. In the presence of the target sequence, the negative strand is displaced by the target, and the fluorophore becomes fluorescent. Therefore, these double-stranded targets interact directly with the target and do not contain a complementary labeled sequence that interacts with a mediator probe, as described below. Mediator Probe

[0118] In other embodiments, instead of TaqMan® probes, data for the methods of the present invention are generated by using fluorophore-bearing probes that indirectly detect target sequences. In this embodiment, the fluorophore-bearing probes are referred to as "fluorescent detection molecules." These molecules do not themselves bind to the target sequence. However, they contain tag-complementary sequences that are complementary to sequences present on the mediator probe, which is capable of directly binding to the TS. Thus, such embodiments include mediator probes that are capable of directly binding to the target sequence.

[0119] The methods of the present invention can use competitive or non-competitive mediator probes.

[0120] In a particularly preferred embodiment, the method of the present invention uses at least two competitive mediator probes, i.e., at least two mediator probes carrying different fluorophore types and containing the exact same nucleotide sequence complementary to the same target sequence.

[0121] In the methods of the invention, for a given target, it is also possible to use two or more "non-competing" mediator probes that have different (overlapping or non-overlapping) nucleotide sequences that are complementary to the target sequence.

[0122] In certain embodiments, a set of at least two mediator probe types and at least two fluorescent detection molecules are provided for each target sequence TS. i It is designed for.

[0123] In the "color combination" method of the present invention, each fluorescent detector molecule is bound to a distinct fluorophore type such that each target sequence is preferably bound to at least two fluorophore types.

[0124] More precisely, each target sequence TS i For , the set contains at least two mediator probes, each mediator probe being Target sequence TS i a 3'-terminal probe region that is complementary to a 5'-terminal mediator region containing at least one tag sequence (tag); A biological cleavage site located between the mediator region and the probe region, which allows an enzyme with exonuclease or endonuclease activity to mediate cleavage of the two regions during the amplification process of the target sequence. In each set, the 3'-terminal probe regions of at least two mediator probes are TS. iThe 5'-terminal mediator regions of at least two mediator probes contain different tag sequences that, upon cleavage, can activate different signals (e.g., bind to different fluorophore-bearing probes).

[0125] In addition, each target sequence TS i For example, the set contains at least two fluorescent detector molecules, the molecules comprising at least: a single sequence (tag complementary sequence - TCS) that is complementary to and hybridizes to at least one of the tag sequences (tags) located in the 5' terminal mediator region of one of the mediator probes of the set; and containing a quencher group whose fluorescence is modified at a tag complementary sequence (TCS) on the detection molecule as a result of hybridization or extension of the tag sequence of the 5'-terminal mediator region, and a fluorophore, upon cleavage; In each set, at least two fluorescent detector molecules carry different fluorophore types, so that the target sequence TS i are consequently characterized by at least two different tags, at least two different TCSs, and at least two different fluorophore types.

[0126] Thus, in a preferred embodiment, the mediator probe is capable of binding directly to a TS and, upon binding during an amplification reaction, is capable of releasing a tag that is complementary to a tag complementary sequence (TCS) located on a fluorescent detector molecule that carries a quencher group and a fluorophore, the fluorescence of which, when released on the TCS on the detector molecule, is modified as a result of hybridization or extension of the tag sequence of the mediator probe.

[0127] In practice, when a compartment contains the target sequence of interest, the PCR reaction causes an increase in the fluorescence intensity of at least two fluorophore types carried by fluorescent detector molecules, or "reporters." Based on the measured fluorescence intensity of each fluorophore type, each compartment can be classified as negative or positive using TaqMan® probes and a methodology similar to that described above. Fluorescent reporter

[0128] In the method of the present invention, the fluorescent detector molecule can be any universal reporter that classically binds to the mediator probe. In particular, it is possible to use a linear or beacon universal reporter that contains a tag-complementary sequence, a fluorophore type, and a quencher. If the fluorescent detector molecule contains a loop, the tag-complementary sequence TCS can be located inside the loop (

[11] ) or outside the loop (see EP 2776585).

[0129] In a preferred embodiment, the fluorescent detector molecule used in the method of the present invention is any reporter probe carrying at least a fluorophore type, a quencher and a TCS, such as molecular beacons, Scorpions, HybProbes, Hybeacons, Biomers and any other tools that have been successfully established to be used as universal reporters (see, for example, https: / / www.biomers.net / en / Products / DNA / Mediator_Probe_PCR.html).

[0130] In particularly preferred embodiments, the fluorescent detector molecules used in the methods of the present invention are molecular beacons or complementary single-stranded fluorescent detector reporter (CSSFR) molecules, as described in more detail herein. Molecular Beacon Molecules

[0131] Molecular beacon molecules are well known in the art and are described, for example, in European Patent No. 2776585

[11] . These molecules are specifically designed to display a nucleotide hairpin structure, to which a quencher and a fluorophore are attached.

[0132] In these reporter molecules, the tag complementary sequence (TCS) can be located either within the hairpin structure or outside the hairpin structure.

[0133] We successfully tested our method using four molecular beacon reporters to measure the concentrations of six target sequences (Figures 5-6, Example 2A). In this example, the TCS was placed in the hairpin structure of the molecular beacon (Figures 5-6). Complementary single-stranded fluorescent reporter (CSSFR)

[0134] In practice, their hairpin structure can make it difficult to generate molecular beacon molecules with 3D constraints. Furthermore, molecular beacon reporter molecules must be synthesized by grafting a fluorophore type and a quencher onto the same chain, which can be difficult for some fluorophore types and is more expensive than reporter molecules with only a fluorophore or a quencher. For these reasons, we proposed using CSSFR as an alternative mediator probe.

[0135] The complementary single-stranded fluorescent reporter (CSSFR) complex was prepared using the following oligonucleotides (see Figure 7): - a primary reporter oligonucleotide molecule (m-reporter), i) a sequence that is complementary to the tag sequence carried by the corresponding mediator probe ("tag complementary sequence", TCS); ii) at its 5' end, a reporter molecule, which may be either a fluorophore or a quencher group; a complementary reporter oligonucleotide molecule (c-reporter), i) a sequence complementary to the 5' end sequence of the m-reporter; ii) an oligonucleotide containing at its 3' or 5' end a reporter molecule which may be a fluorophore group if the m-reporter has a quencher group, or a quencher group if the m-reporter has a fluorophore group.

[0136] In another embodiment, the c-reporter can be modified at both the 3' and 5' ends to add one quencher and one fluorophore group, or two fluorophore groups, or two quencher groups.

[0137] In this system, the fluorescence of the fluorophore group is modified on the tag complementary sequence (TCS) on the m-reporter as a result of hybridization or extension of the tag sequence of the 5'-terminal mediator region upon cleavage from the mediator probe.

[0138] These conjugates are described in detail below.

[0139] We successfully tested our method using four CSSFR molecules to measure the concentrations of six target sequences (FIG. 7, Example 2B).

[0140] There are several advantages to using universal reporters, especially CSSFR, instead of TaqMan® probes: First, the "color combination" multiplexing approach can be implemented using only a small number of universal reporters. When using M detection channels, only M universal reporters are required to implement color combination. This is true regardless of the number of fluorophore types combined for each target.

[0141] For example, by using six detection channels and two fluorophore types per target sequence, only six CSSFR types are needed to detect up to 15 targets, and for each target sequence, only two different mediator probes should be designed, each conjugated to a different CSSFR.

[0142] The following six CSSFR molecules are used: CSSFR#1-FAM: Contains TCS#1 and fluorophore-type FAM CSSFR#2-YY: Contains TCS#2 and fluorophore type YY CSSFR#3-ATTO550: Contains TCS#3 and fluorophore-type ATTO550 CSSFR#4-ROX: Contains TCS#4 and fluorophore-type ROX CSSFR#5-Cy5: Contains TCS#5 and fluorophore-type Cy5 CSSFR#6-ATTO700: Contains TCS#6 and fluorophore-type ATTO700

[0143] When detecting the target sequence TS1, Mediator probe #1: Contains TS1-specific sequence + tag #1 (which triggers CSSFR#1) Mediator probe #2: Contains TS1-specific sequence as #1 + tag #2 (which triggers CSSFR #2) Use.

[0144] When detecting the target sequence TS2, Mediator probe #3: Contains TS2-specific sequence + tag #1 (which triggers CSSFR #1) Mediator probe #4: Contains TS2-specific sequence as #3 + tag #3 (which triggers CSSFR #3) Use.

[0145] When detecting the target sequence TS3, Mediator probe #5: Contains TS3-specific sequence + tag #1 (which triggers CSSFR #1) Mediator probe #6: Contains TS3-specific sequence as #5 + tag #4 (which triggers CSSFR #4) Use.

[0146] When detecting the target sequence TS4, Mediator probe #7: Contains TS4-specific sequence + tag #1 (which triggers CSSFR #1) Mediator probe #8: Contains the TS4-specific sequence as #7 + tag #5 (which triggers CSSFR #5) ·… Use.

[0147] The table below compares the number of TaqMan® probe types and CSSFR types required to reach a given multiplexing level for different numbers of detection channels using two color combinations per target sequence.

[0148] [Table 1]

[0149] Another advantage of using a universal reporter instead of a TaqMan® probe is reduced assay development time and cost. Indeed, fluorescently labeled oligonucleotides, such as TaqMan® probes or fluorescent reporter molecules, typically take longer to manufacture than naked oligonucleotide sequences, such as PCR primers or mediator probes. While PCR primers or mediator probes can typically be ordered and delivered in less than a week, fluorescently labeled oligonucleotides can take more than a month to be delivered from a typical oligonucleotide manufacturer. Similarly, the cost of fluorescently labeled oligonucleotides is typically 5-10 times higher than the cost of an equivalent primer sequence. Therefore, the fewer fluorescently labeled molecules required, the better in terms of cost and development time.

[0150] Another advantage of using a universal reporter is that the same reporter can be used across different assays targeting different types of sequences or mutations. These assays differ from each other by the type of mediator probe with a different 3' target-specific sequence, but share the same set of reporter tag sequences. Sharing a reporter across different assays allows for larger orders of fluorescently labeled reporter oligonucleotides, further reducing costs through economies of scale.

[0151] Finally, compared to TaqMan®-based approaches, another advantage of the universal reporter approach for "color combination" multiplexing is that the ratio of reporter and compensation matrix needs to be optimized only once and does not change during assay development and optimization of the mediator probe sequence. Indeed, as mentioned in the section on TaqMan® probes, the concentration ratio between two competing TaqMan® probes needs to be adjusted for each probe sequence. As a result, the ratio may need to be adjusted each time the probe sequence is changed during the assay development process. Furthermore, changes in the TaqMan® probe sequence may also lead to modifications of the compensation matrix, which must be readjusted during the assay development process. Such adjustments require the collection of additional experimental data and may be redundant when using more than three detection channels. In contrast, when using reporters for "color combination" according to the present invention, the sequence does not change during the assay development process. Assay development typically involves optimization of primer and target-specific mediator probe sequences, none of which are fluorescently labeled. PCR using CSSFR reporter

[0152] In a second aspect (which may be combined with the first aspect or considered separately therefrom), the present invention is also directed to an in vitro PCR method for detecting the presence and / or quantifying at least one (preferably at least three, more preferably at least five) nucleic acid target sequences (TSi) in a biological sample comprising nucleic acid molecules, said method comprising: A) Sample and for each TSi, i) at least one mediator probe, a 3'-terminal probe region that is complementary to the target sequence (TSi); a 5'-terminal mediator region containing one tag sequence; - a biological cleavage site located between the mediator region and the probe region, whereby an enzyme having nuclease activity can mediate cleavage of the two regions during the amplification process of the nucleic acid target sequence (TSi), a mediator probe; ii) contacting at least one complementary single-stranded fluorescent reporter (CSSFR) complex of the present invention with a set containing: B) amplifying the at least one nucleic acid target in the presence of an enzyme having nuclease activity, thereby cleaving the 5'-terminal mediator region of the mediator probe at the cleavage site when the 3'-terminal probe region hybridizes to its complementary target sequence, so that the tag sequence contained in the cleaved 5'-terminal mediator region of the mediator probe hybridizes to the tag-complementary sequence TCS present on the CSFFR molecule, stimulating one or more fluorophores present in the sample, and detecting changes in one or more signals emitted from the one or more fluorophores; C) detecting or measuring the fluorescence intensity for each fluorophore group; D) optionally processing the data collected in step c) to quantify the concentration of at least one TSi in the biological sample.

[0153] The 3'-terminal probe region complementary to the target sequence typically contains 13-30 nucleotides, preferably 15-25 nucleotides. In addition, the 3' end of the mediator probe can be blocked with a phosphate group or other common blockers (C3 / C6 spacer, dideoxynucleotide, phosphate, inverted base, 3' amino) to avoid undesired amplification on this side of the molecule. Blocking steps are recommended by many oligonucleotide manufacturers; see, for example, https: / / www.biomers.net / en / Products / DNA / Real-time_PCR / PCR_Blocker.html.

[0154] Preferably, the cleavage site between the 5'-terminal mediator region and the 3'-probe region is located immediately after the first hybridized base of the 3'-probe region, as proposed in

[15] .

[0155] As used herein, the term "CSSFR complex" of the present invention refers to - a primary reporter oligonucleotide molecule (m-reporter) containing a set of mediator probes that bind to TSi and at its 5' end at least one sequence ("tag complementary sequence", TCS) that is complementary to a tag sequence carried by a reporter molecule, which may be either a fluorophore or a quencher group; a sequence which is complementary at its 3' and / or 5' end and / or between its 3' and 5' end to the 5' end sequence of the m-reporter; a quencher group or a fluorophore group if the m-reporter has at least one fluorophore group, or a bimolecular nucleotide complex comprising a complementary reporter oligonucleotide molecule (c-reporter), which contains a quencher group when the m-reporter has a fluorophore group; The fluorescence of the fluorophore group of the c-reporter molecule or the m-reporter molecule changes as a result of hybridization or extension of the tag sequence on the m-reporter to which the 5'-terminal mediator region cleaved from the mediator probe is attached on the tag complementary sequence (TCS).

[0156] The m-reporter molecule in the CSSFR complex is a linear nucleotide molecule containing at least 30 nucleotides, more precisely it usually comprises 30 to 80 nucleotides, preferably 40 to 50 nucleotides.

[0157] The m-reporter molecules of the CSSFR reporters of the present invention contain at least one, preferably at least two, and more preferably three or more tag complementary sequences (TCSs) that are partially or completely complementary to the tag sequences located on the corresponding mediator probes. In the context of the present invention, these tag complementary sequences (TCSs) should be relatively short, typically comprising 8-25 nucleotides, preferably 8-18 nucleotides, more preferably 10-16 nucleotides, and even more preferably 12-14 nucleotides. As disclosed above, these TCSs are oligonucleotides having a sequence identical to the sequence of at least one tag contained in at least one mediator probe of the set.

[0158] The 3' end of the m-reporter molecule is preferably blocked by a phosphate group or other common blockers (C3 / C6 spacer, dideoxynucleotide, phosphate, inverted base, 3' amino) to avoid unwanted amplification on this side.

[0159] The c-reporter molecule in the CSSFR complex of the present invention is relatively short, typically comprising 8 to 25 nucleotides, preferably 8 to 18 nucleotides, more preferably 10 to 16 nucleotides, and even more preferably 12 to 14 nucleotides.

[0160] When carrying a quencher compatible with some fluorescent dyes, the c-reporter molecule can be used in a CSSFR complex containing a different m-reporter molecule. For example, the quencher BHQ1 can be used to quench blue and till to obtain a fluorescent dye, and then a c-reporter carrying BHQ1 can be used to hybridize an m-reporter carrying blue and till to obtain a fluorescent dye (see Example 2).

[0161] In this reporter complex, the m-reporter molecule preferably contains at least two different tag complementary sequences (TCS), preferably multiple different tag complementary sequences, as disclosed in

[16] .

[0162] In this reporter complex, the complementary reporter oligonucleotide molecule (c-reporter) is short, typically containing 8-25 nucleotides, preferably 8-18 nucleotides, more preferably 10-16 nucleotides, even more preferably 12-14 nucleotides (usually 15-20 nucleotides), and preferably about 18 nucleotides.

[0163] The method of the present invention is preferably a multiplex method that allows for the detection and / or quantification of the presence of at least two, at least three, at least four, at least five, and more preferably at least ten nucleic acid target sequences (TSi) in a sample of interest. More preferably, it is a multiplex digital PCR (dPCR) method. Even more preferably, it is the multiplex digital PCR method described in the first embodiment.

[0164] In this method, it is possible to distinguish between multiple target sequences using a single color label and measuring different intensity levels of this color (this embodiment of intensity-based multiplexing is known in the art, see, e.g.,

[17] ).

[0165] Preferably, however, the method of the invention uses at least two colors of labels for some (if not all) of the target sequences, in other words, the method of the invention preferably involves the use of at least two different labeled reporter complexes for at least one TSi of several target sequences whose presence is to be detected by the method of the invention (not excluding the use of only one color for some other TSi).

[0166] In the methods of the present invention, each TSi can feature a specific fluorophore or combination of fluorophores that is different from the fluorophores or combinations of fluorophores of each of the other target sequences.

[0167] Nevertheless, when a target sequence is characterized by two or more fluorophores, it is possible in the method of the invention to use sets that bind to different target sequences TSi and TSj containing CSSFR complexes carrying the same fluorophore group and even the same CSSFR complex.

[0168] In the method of the present invention, it is possible to detect, for example, six TSi with four differently labeled CSSFR complexes whose m-reporter principal molecules carry two different TCSs (see Example 2B.1).

[0169] In the method of the present invention, it is possible to detect, for example, 12 or 15 TSi with six CSSFR molecules carrying different TCSs and six different fluorescent dyes (see Examples 2B.2 and 2B.3).

[0170] As shown in Example 5, in the method of the present invention, it is advantageous to adjust the concentration of the main reporter oligonucleotide molecule (m-reporter) in the reporter complex so that the final concentration is 0.05 to 2 μM, preferably 0.1 to 0.5 μM, and more preferably 0.1 to 0.25 μM.

[0171] In addition, for each target sequence TSi, the melting temperature T of the tag sequence TAGi hybridized on the corresponding TCSi of the CSSFR molecule is determined. M is the T of the 3'-end probe region of the mediator probe hybridized on the target sequence TSi. M It is advantageous to design the tag sequence so that the resulting T of [TAGi / TCSi] hybridization is lower than M T of [mediator probe / TSi] hybridization M It should be 3°C lower, preferably 5°C lower, and more preferably 10°C lower than the Methods that include two different tags per TSi

[0172] In a first embodiment, the method of the present invention involves the use of at least two mediator probes and at least two reporter complexes per target sequence to be detected, in which the TS to be detected is characterized by a unique combination of at least two different fluorophore types, preferably exactly two fluorophore types.

[0173] In this case, as shown in Figure 17A, the set thus includes, for TSi of at least one (preferably each) of several target sequences: - at least two mediator probes as defined above, each containing a 3'-terminal probe region complementary to the target sequence TSi and a 5'-terminal mediator region containing one tag sequence, wherein the tag sequences of the at least two mediator probes are different and are complementary to and hybridize with at least one TCS of at least one reporter complex of the set; - at least two reporter complexes as described above, wherein each m-reporter molecule contains one or more different tag complementary sequences (TCS) that are complementary to and hybridize with a tag sequence located in the 5'-terminal mediator region of one of the set's minimum two mediator probes, Each of the at least two reporter complexes is selected such that at least one (preferably each) TSi is k i characterized by different fluorophore types, k i are labeled differently so that there are two or more.

[0174] Preferably, at least one of the reporter complexes in the set contains at least two or more different tag complementary sequences (TCSs) that are complementary to and hybridize to at least two or more different tag sequences located in the 5'-terminal mediator regions of at least two or more mediator probes specific for two or more different target sequences. This embodiment is highlighted in Figure 17B. The presence of several TCSs on a reporter molecule has also been described in the art (

[16] ).

[0175] In a particular embodiment, the method of the invention is a method for detecting at least two target sequences TSi and TSj, comprising: -TSi is a set of two mediator probes as defined above, the 3' portions of which are specific for the target sequence TSi and the 5' portions of which contain the first tag sequence TAGil or the second tag sequence TAGi2, respectively; two CSSFR complexes as defined above, each containing a TCS specific for TAGi1 or TAGi2, respectively, and the complexes being differentially labeled; Contains -TSj is the set two mediator probes as defined above, the 3' portions of which are specific for the target sequence TSj and the 5' portions of which contain a first tag sequence TAGj1 or a second tag sequence TAGj2, respectively; - two CSSFR complexes as defined above, each containing a TCS specific for TAGj1 and TAGj2, which complexes are differentially recognized;

[0176] In this method, if two target sequences TSi and TSj are characterized by one common fluorophore group, one of the two tags TAGi and TAGj can be exactly or partially identical. However, in this case, the other TAGi or TAGj must be different. In other words, all TAGi and TAGj cannot have the same nucleotide sequence.

[0177] In this particular embodiment, the two sets specific for two different target sequences, TSi and TSj, contain at least one CSSFR complex carrying the same fluorophore group, as highlighted in Figure 17B.

[0178] In other words, two sets specific for two different target sequences, TSi and TSj, can contain at least one CSSFR complex in common. This embodiment is highlighted in Figure 17B.

[0179] In a preferred embodiment, the set used in the method of the present invention contains, for each TSi, at least two reporter complexes as defined above, each of the at least two reporter complexes being such that each TSi is k i characterized by different fluorophore types, k i are labeled differently so that there are two or more.

[0180] In another preferred embodiment, the set used in the method of the invention comprises, for some TSi, at least two reporter complexes as defined above, each of the at least two reporter complexes being such that each TSi is k i characterized by different fluorophore types, k iThe TSj contains two reporter complexes, which are differentially labeled such that the number of reporter complexes is 2 or more, and for some TSj, only one reporter complex as defined above, such that the TSj is detected in only one color. Methods containing one tag per TSi

[0181] In a second embodiment, the method of the present invention involves the use of only one mediator probe to detect a specific TSi, in which case the mediator probe contains a tag that can hybridize to the TCS present on at least two different labeled CSSFR reporters such that the TSi is detected by a resulting color combination.

[0182] In this case, the method of the invention provides that the set comprises, for at least one (preferably each) TSi of several target sequences: i) one mediator probe as defined above, containing a 3'-terminal probe region that is complementary to the target sequence TSi and a 5'-terminal mediator region that contains one tag sequence; ii) at least two reporter complexes as defined above, wherein each m-reporter molecule contains an identical tag complement sequence (TCS) that is complementary to and hybridizes to a tag sequence located in the 5'-terminal mediator region of a mediator probe of the set; Each of the at least two reporter complexes is selected such that at least one (preferably each) TSi is k i characterized by different fluorophore types, k i are labeled differently, so that there are two or more.

[0183] This embodiment is illustrated in Figure 18A.

[0184] In this method, at least one, and preferably each, of the m-reporter molecules contained in the at least two reporter complexes bound to each TSi contains at least two or more different tag complementary sequences (TCSs) that are complementary to and hybridize to at least two or more different tag sequences located in the 5'-terminal mediator regions of at least two or more mediator probes specific for two or more different target sequences. This is illustrated in Figure 18B. The presence of several TCSs on a reporter molecule has also been described in the art (

[16] ).

[0185] In this context, a method according to the invention aimed at detecting at least two target sequences TSi and TSj therefore comprises: -The set includes: one mediator probe, the 3' portion of which is specific for the target sequence TSi and the 5' portion of which contains the tag sequence TAGi; two CSSFR complexes, each containing a TCS specific for a TAGi, the complexes being differentially labeled; Contains -The set includes: only one mediator probe, the 3' portion of which is specific for the target sequence TSj and the 5' portion of which contains the tag sequence TAGj; Two CSSFR complexes, each containing a TCS specific for TAGj, are differentially labeled.

[0186] In a particular embodiment of the method of the invention, the two sets specific for two different target sequences TSi and TSj contain at least one CSSFR complex carrying the same fluorophore group.

[0187] In other words, two sets specific for two different target sequences TSi and TSj can contain one or more CSSFR complexes in common.

[0188] Sets designed according to the above two specific embodiments, namely: a set for detecting a target sequence TSi, one mediator probe, the 3' portion of which is specific for the target sequence TSi and the 5' portion of which contains the tag sequence TAGi; two CSSFR complexes as defined above, each containing a TCS specific for a TAGi, the complexes being differentially labeled; and in the same manner, a set for detecting a target sequence TSj, two mediator probes, the 3' portions of which are specific to the target sequence TSj and the 5' portions of which contain a first tag sequence TAGj1 or a second tag sequence TAGj2, respectively; It is possible to carry out the method of the invention using a set containing two CSSFR complexes as defined above, each containing a TCS specific for TAGj1 and TAGj2, said complexes being differentially labeled. Detecting TSi with only one color

[0189] In certain situations (see below for WT sequences), it may be useful and preferable to detect a particular target sequence with only one color. The methods of the present invention also encompass this case and may involve the use of one mediator probe specific for only one CSSFR to detect one particular TSi.

[0190] Consequently, in a preferred embodiment of the method of the present invention (illustrated in FIG. 7), at least one of the TSi is So that the TSi can be detected in only one color, only one mediator probe as defined above, the 3' portion of which is specific for the target sequence TSi and the 5' portion of which contains TAGi; - Only one CSSFR complex as defined above containing a TCS and fluorophore group specific for the TAGi is detected in the set containing

[0191] As suggested in FIG. 17C, the method of the present invention, when used to detect at least two target sequences TSi and TSj, can be used to detect, for example, a set for detecting TSi, two mediator probes as defined above, the 3' portions of which are specific for the target sequence TSi and the 5' portions of which contain the first tag sequence TAGil or the second tag sequence TAGi2, respectively; a set containing two CSSFR complexes as defined above, each containing a TCS specific for TAGi1 or TAGi2, respectively, the complexes being differentially labeled; - a set for TSj, So that the TSj can be detected in only one color, only one mediator probe as defined above, the 3' portion of which is specific for the target sequence TSj and the 5' portion of which contains TAGj; and a set containing only one CSSFR complex containing a TAGj and a TCS specific for a fluorophore group, as defined above; may include:

[0192] Alternatively, and as suggested in FIG. 18C, the method of the present invention, when used to detect at least two target sequences TSi and TSj, So that the TSi can be detected in only one color, a set for detecting TSi, only one mediator probe, the 3' portion of which is specific for the target sequence TSi and the 5' portion of which contains the tag sequence TAGj; a set containing only one CSSFR complex containing a TCS and a fluorophore group specific for the TAGi; - a set for detecting TSj, only one mediator probe, the 3' portion of which is specific for the target sequence TSj and the 5' portion of which contains TAGi; a set containing two CSSFR complexes, each containing a TCS specific for a TAGi, the complexes being differentially labeled.

[0193] In a preferred embodiment, the method of the second aspect using the CSSFR reporter of the present invention contains the same steps and embodiments (particularly the thresholding steps and calculations) as described for the color combination method of the present invention, as disclosed in the first aspect of the present invention. Rich WT on its own channel, only one color.

[0194] As further detailed in the statistical section below, in certain embodiments, the invention provides that a given first target sequence whose abundance is significantly higher than the abundance of other target species in the analyzed sample (typically the abundant target species is wild-type) is color-coded using a first set of fluorophore types, and all other target sequences are color-coded using two or more fluorophore types each selected from a second set of fluorophore types, all different from the first set.

[0195] Thus, in preferred embodiments of the invention, no more than one fluorophore type is dedicated to the most abundant target sequence in a sample (typically the abundant target sequence is wild type).

[0196] In this case, because a unique fluorophore binds to a specific sequence of interest, n-1 fluorophore types remain that can be used to detect other target sequences. For example, if the number of fluorescence detection channels, n, in a dPCR instrument is 6, the method of the present invention can bind a unique combination of two of the five fluorophore types to each TS. i This is carried out by assigning the WT sequence to the TS sequence. i different from

number

number

[0197] In prior art approaches to detecting fluorescent dyes useful in digital PCR, fluorophores are assigned to "channels," which in a fluorescence reader actually correspond to excitation sources, e.g., LEDs and filters, coupled to emission filters. These emission / excitation filter pairs are designed according to the emission and excitation spectra of commonly used fluorophores, such as FAM, HEX, ROX, Cy® dyes, Atto™ dyes, and Yakima Yellow. The fluorescence reader described in WO 2022 / 063845, for example, describes six emission LED and filter / excitation filter pairs, which reveal six usable channels.

[0198] In one embodiment of the present invention, additional encoding channels are made available through the use of Large Stokes Shift Dyes (LSSDs) and by accordingly separating the prior art emission / excitation pairs (

[12] ,

[13] ). In other words, while the prior art typically refers to channels containing fixed emission / excitation filter pairs, the use of LSSDs in this embodiment of the present invention requires that the fluorescence reader be able to excite the LSSD in a first channel while detecting its fluorescent emission in a remote, separate channel. This approach means that the fluorescence detection hardware can select excitation and emission hardware independently of each other, rather than as a fixed pair.

[0199] In a preferred embodiment of the invention, at least one of the fluorophore types is one of the large Stokes shift dyes (LSSDs).

[0200] Thus, in this embodiment of the invention, color combinations may be performed using one or more LSSDs in addition to the commonly used fluorophores of the prior art.

number

[0201] Those skilled in the art know how to select LSSDs for a particular application on a particular system, taking into consideration such factors as their spectral separation from existing fluorophores based on their fluorescent properties, as well as charges carried by the LSSD molecules that may interfere with DNA or the chemistry used by a particular dPCR instrument.

[0202] In a non-limiting example, the inventors have successfully implemented the following experimental protocol using LSSD, for example:

[0203] [Table 2]

[0204] Any of these LSSDs (DyLight™-LS-515, DyLight™-LS510, DY-521-XL) can be used in the methods of the present invention, as well as any other LSSD that can be conjugated to an oligonucleotide sequence, such as a TaqMan® probe.

[0205] In a more preferred embodiment, in the methods of the invention, no more than one fluorophore type is dedicated to the most abundant species in the sample, and at least one of the fluorophore types is one of the giant Stokes shift dyes (LSSD). Statistical Analysis of the Data Processing Protocol of the Present Invention

[0206] When detecting target sequences using color combinations, compartments containing multiple targets (coencapsulation) are ambiguous and must be discarded from the analysis. For example, an assay is set up to detect target 1 using FAM & Cy3, target 2 using FAM & Cy5, and target 3 using Cy3 & Cy5 fluorophores. In one such example, compartments positive for FAM, Cy3, and Cy5 could be the result of coencapsulation of targets 1 and 2, or targets 1 and 3, or targets 2 and 3, or targets 1, 2, and 3. As a result, when compartments are experimentally classified as FAM, Cy3, and Cy5 positive, it is impossible to predict which target sequences are present in the compartment. Such triple-positive compartments are positive, but ambiguous regarding their contents.

[0207] The consequence of discarding ambiguous compartments is a loss of assay sensitivity.

[0208] The loss in sensitivity is directly proportional to the number of all droplets that are excluded relative to the maximum number of droplets that can be analyzed.

[0209] During the analysis of a given target i, all compartments that are positive for any other target j are excluded, so for that target i, the loss in sensitivity is:

number

[0210] Furthermore, the error in digital PCR results is

number

[14] ), so that the error (loss of precision) in the measurement for a given target i is

number

[0211] From the above, the loss of sensitivity and precision will vary depending only on experimental factors:

number

[0212] This is shown in Figure 3, which provides the sensitivity for target i as a function of the total concentration of the other targets in the assay using a sapphire chip (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France). Based on this graph, we can see that the sensitivity drops off sharply between 100 cp / μL and 1000 cp / μL.

[0213] In fact, droplets with multiple target sequences are discarded from the analysis, as their contents are not analyzed. This loss in analyzed droplets directly impacts assay sensitivity. The more co-encapsulation, the greater the sensitivity impact. Figure 3 shows a model of the encapsulation event for different background gene concentrations and the impact of background gene concentration levels on sensitivity. For background levels below 100 cp / μL, the loss in sensitivity is less than 10%, increasing rapidly at larger background levels.

[0214] According to the present invention, when the analyzed samples are of the liquid biopsy type, 80% of the samples have a wild-type ctDNA concentration in the PCR mix that is less than 200 cp / μL and a 10-fold lower mutant ctDNA concentration. As a result, according to the present invention, using a color combination to detect wild-type DNA is expected to result in a loss of sensitivity of up to 20% in 80% of cases. For this reason, the color combination method according to the present invention works very well for rare event detection applications, where the expected concentrations are low and therefore result in only a small loss of sensitivity.

[0215] The color combinations according to the present invention also work very well in assays in which only one target is detected in a given sample, i.e., where the presence of one given target is most often associated with the absence of all other targets. Indeed, in this case, Σ j≠i vc j = 0, and there is no loss of sensitivity. Relative Sensitivity Calculations for the Methods of the Invention in the Context of Mutant Allele Fraction Determination

[0216] The color combination approach of the present invention affects the absolute sensitivity of the assay. Assuming an assay and digital PCR system with perfect analytical sensitivity, if a detectable target is present in a compartment, the compartment is always called positive, and the limit of detection (LOD) at the 95% confidence level of the assay for each target i using the color combination approach is given by:

number

[0217] This means that the target average concentration is

number

[0218] For some applications, particularly liquid biopsies, the most important measure of sensitivity is not absolute sensitivity but sensitivity relative to a reference template, e.g., wild-type (WT) DNA.

[0219] In such cases, the measure of interest is the Mutant Allelic Fraction (MAF):

number

[0220] The minimum detectable concentration of the mutant target was calculated above.

number

[0221] Assuming the WT DNA concentration is significantly higher than the concentration of any other target in the assay, the lowest MAF measurable using the color combination is:

number

[0222] The next step can be written as follows:

number

[0223] Figure 4 shows the function f(x)=e x The plot of / x is shown. x=vc WT = 1 gives the lowest possible MAF measurement, from which the MAF min = 3e / N.

[0224] By comparison, in the context of prior art approaches where one fluorophore type is used for each target sequence in the assay design, the lowest possible MAF measurement is at the highest WT concentration, i.e., saturating.

number

number

[0225] Thus, when using the method according to the invention with a digital system capable of generating N=20,000 compartments, the lowest MAF measurable in a given assay is ln(20000)*e=27 times higher than when using the simple "one color=one target" assay of the prior art.

[0226] To understand this, using the method of the present invention, the lowest measurable MAF is 0.03% for sapphire tips (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) and 0.05% for opal tips (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France). These numbers remain acceptable, considering that typically reported MAFs with clinical utility exceed 0.01%.

[0227] Or, given x=vc wt About MAF min By comparing the "one color = one target" prior art approach with the "color combination" method of the present invention,

number

[0228] Thus, as the WT concentration increases, the loss in relative sensitivity increases.

[0229] For example, in a sapphire chip (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France), C WT = 1000 cp / μL (corresponding to x = 0.62), the relative sensitivity loss is e 0.62 = 1.86, and the minimum achievable MAF is 0.44% with the color combination method according to the present invention compared to 0.24% with the simple assay design of the prior art.

[0230] Statistical analysis also shows that more than 15 additional targets can be detected using only one of the colors used in the color combination without a significant decrease in performance.

[0231] In this approach, we use only one fluorophore type to target the first target sequence, TS. e Detect TS e The concentration of e All other target sequences are referred to as TS. i is detected using two fluorophore types.

[0232] Concentration C e TS e This can be measured by counting the compartments that are all negative or only positive for the fluorophore types used in TS. This means that all compartments that are positive for two or more fluorophore types are counted as TS. e This means that the concentration of TS is excluded from the measurement. e The loss of sensitivity for

number

[0233] TS e The sum of the concentrations of all targets other than the target detected using two different fluorophore types is the main parameter driving the loss of sensitivity.

[0234] For example, if one target sequence, such as a wild-type sequence, is expected to have a high abundance, a separate and distinct fluorophore type is used, while other fluorophore types and detection channels are used to detect such abundant target sequences (e.g., FAM / blue channel), and a color combination is used to detect low abundance target sequences (such as mutant target sequences). In this approach, the sensitivity of the assay for low abundance target sequences is not affected or reduced by the presence of the isolated, abundant target sequence. Generating a Fluorescence Correction Matrix in the Context of a Target Sequence Encoded Using the Methods of the Invention

[0235] In a preferred embodiment, the method of the present invention comprises, prior to step a), a step of generating a fluorescence compensation matrix as described above, which is then applied to measure, for each compartment, the fluorescence levels or signal intensities from the M fluorophore types using n signals from the n fluorescence detection channels.

[0236] In particular, for each fluorescent probe used in the assay, a correction matrix is ​​constructed in which the fluorescence parameters are defined for each fluorescence channel.

[0237] In prior art methods, the correction matrix was constructed by collecting fluorescence data from samples made from single-color controls for all fluorescent probes present in the assay. In this case, the single-color control for a given fluorescent probe consisted of a mixture of all primers and all fluorescent probes used in the assay plus a DNA template of the target sequence detected by the fluorescent probe of interest.

[0238] In contrast, in the methods of the present invention, where target sequences are coded using a combination of colors, a different setup is required for generating the fluorescence compensation matrix: in this embodiment of the method of the present invention, the mixture of primers and fluorescent probes is in the reaction mixture as in prior art methods, except that one of the fluorescent probes used for the color coding of the target sequence under test is missing. For example, if TS1 is detected by the following two probes: one green and one yellow, the mixture of the green single-color control does not contain the yellow probe of target TS1 and is therefore used as a reference for the compensation matrix, ensuring that TS1 is detected by only one probe of interest (green). Computer implementation of the data analysis method of the present invention Each of the steps d) and / or e) and / or f) of the method according to the invention described above is not performed in a purely abstract or purely intellectual way, but involves the use of technical means.

[0239] Typically, step d) of the method according to the invention described above should be carried out by a photodetector (to measure the fluorescence intensity of each fluorophore type for each compartment) and at least one computer, one central processing unit or computing unit, one analog electronic circuit (preferably dedicated), one digital electronic circuit (preferably dedicated), and / or one microprocessor (preferably dedicated). Furthermore, software means are useful for setting the positive threshold intensity for each fluorophore type.

[0240] Typically, each of e) and / or f) of the method according to the invention described above should be implemented by at least one computer, one central processing unit or arithmetic unit, one analog electronic circuit (preferably dedicated), one digital electronic circuit (preferably dedicated), and / or one microprocessor (preferably dedicated), and / or software means.

[0241] In another aspect, the present invention relates to a computer program comprising instructions which, when executed by a computer, carry out steps e) and f) of the method according to the invention (preferably also carrying out at least part of step d) of the method according to the invention, in particular setting a positive threshold intensity for each fluorophore type).

[0242] In another aspect, the present invention relates to a computer program product comprising instructions which, when executed by a computer, cause a computer to perform steps e) and f) of the method according to the invention (preferably also to perform at least part of step d) of the method according to the invention, in particular setting a positive threshold intensity for each fluorophore type).

[0243] In another aspect, the present invention relates to a computer-readable storage medium comprising instructions which, when executed by a computer, cause the computer to perform steps e) and f) of the method according to the invention (preferably also to perform at least part of step d) of the method according to the invention, in particular setting a positive threshold intensity for each fluorophore type). Advantages over prior art dPCR assays

[0244] As mentioned above, the "intensity-based multiplexing" of Lindner et al., 2021 ([4]) combines two or more TaqMan® probe types that share the same fluorophore type but have different target sequences for at least one of the fluorophore types used in the assay. Two or more TaqMan® probe types that share the same fluorophore type are combined in the assay at two distinct concentrations. By using only two probe types per fluorophore type, one probe type has a low concentration ("lo" type) and one probe type has a high concentration ("hi" type). As a result, after PCR, if a compartment contains a "lo" type target, the fluorescence level of the associated fluorophore type in the compartment will exceed the set positive threshold, but at a lower level than if the compartment contains a "hi" type target. After PCR, assuming all targets are present in the sample, for each fluorophore type for which intensity-based multiplexing was performed, there will be a "lo" positive population of the first droplet, which is the negative population of droplets; a "hi" positive population of the second droplet with a higher measured fluorescence level; and possibly a "lo+hi" positive population of the third droplet with an even higher measured fluorescence level (which contains both the "lo" and "hi" targets so that the fluorescence signals are additive). Using this approach, different targets are distinguished by utilizing the measured fluorescence intensities of the compartments in addition to an initial binary classification of droplets according to a set positivity threshold.

[0245] Assuming two levels of fluorescence intensity are used for each M=N fluorescence detection channel / fluorophore type, the maximum number of detectable targets, i.e., the maximum level of multiplexing, is up to 2N. Using six detection channels, this yields a maximum of 12 targets.

[0246] Compared to the "color combination" approach of the present invention, this results in the maximum level of multiplexing with intensity-based multiplexing decreasing immediately, and this decreasing rapidly and significantly, as the number of fluorescent channels increases beyond 5. For example, with n=10 fluorescent detection channels, the color combination approach of the present invention allows for the detection of up to 45 targets, while the intensity-based multiplexing of Lindner et al. allows for less than half the maximum of 20 targets.

[0247] [Table 3]

[0248] Another drawback of Lindner et al.'s intensity-based approach, compared to the color combination of the present invention, is the complexity of droplet classification and data analysis. Using the color combination of the present invention, only one positive threshold needs to be set for each fluorophore type used in the experiment. Using the intensity-based approach, multiple thresholds must be set for each fluorophore type to distinguish between multiple populations of positive compartments, all of which have different fluorescence intensities. When using a two-level intensity-based approach, three thresholds are required for each fluorophore type used to properly classify the compartment populations and properly detect and quantify the target of interest: one threshold for the "lo" positive compartment, one threshold for the "hi" positive threshold, and one threshold for the "lo+hi" positive compartment.

[0249] The table below compares the threshold numbers for both approaches for different numbers of fluorescence detection channels.

[0250] [Table 4]

[0251] This table highlights that the color combination approach of the present invention allows for higher levels of multiplexing and simpler data analysis with fewer thresholds.

[0252] Furthermore, when using intensity-based multiplexing, classification of compartments using a positivity threshold often fails due to interactions between simultaneous PCR amplification of targets bound to one fluorophore type and PCR amplification of targets bound to another fluorophore type. For example, during PCR amplification, if a compartment contains a target bound to a "hi"-FAM TaqMan® probe type and also contains a target bound to a "hi"-Cy5 TaqMan® probe type, both targets will be simultaneously amplified. This simultaneous amplification can reduce the efficiency of one or both PCR reactions, resulting in the measured fluorescence intensity from the "hi"-FAM TaqMan® probe type at the end of PCR amplification being lower than expected, comparable to the expected fluorescence intensity of the "lo"-FAM TaqMan® probe. When analyzing data using the above positivity threshold, compartments will be incorrectly classified as containing the "lo"-FAM target instead of the "hi"-FAM target, leading to erroneous detection and quantification. This occurs especially when two or more TaqMan® probe types "compete" for the same set of PCR primers during PCR amplification. To circumvent this problem, more complex approaches to compartment classification have been used. They rely on drawing multiple multidimensional polygonal regions around all compartments containing a given target of interest. This polygonal classification approach remains practical for two or three fluorescence detection channels (2D or 3D polygons), but becomes very cumbersome for larger numbers of detection channels.

[0253] Another drawback of the intensity-based multiplexing approach is that compartment classification is not robust to the phenomenon of "rain" in digital PCR.

[0254] Ideally, in digital PCR, all compartments containing the same target (or multiple targets) of interest are always amplified with high reaction efficiency and have the same measured fluorescence level. For a well-designed assay, there is a clear difference in the measured fluorescence level between the negative and positive compartments. Furthermore, the measured fluorescence level of the compartment does not correspond to either the negative or positive compartment, so it cannot exist between the levels of the negative and positive compartments. However, experimentally, compartments with intermediate levels of fluorescence exist. These are usually compartments that contain targeted nucleic acid molecules, but where the PCR reaction occurs at reduced efficiency, so that the endpoint fluorescence level is below the normal fluorescence level for the positive compartment. These compartments with intermediate levels of fluorescence intensity are called "rain."

[0255] There are multiple causes of "Rain" -Poor assay design, - partial malfunction of the digital PCR system or digital PCR consumables used, Thermal malfunction during PCR, Fluorescence readout errors, Abnormal compartment volume, Dust particles, partial malfunction, PCR inhibitors contained in the sample, - Sample-induced decrease in PCR efficiency.

[0256] Given the number of sources of rain, some degree of rain is inevitable when performing digital PCR experiments.

[0257] When using a simple "one color-one target" approach in digital PCR, if the fluorescence level exceeds a set positive threshold, the rain is treated as a positive compartment or is excluded from the analysis. However, when using an "intensity-based multiplexing" approach, rain poses an additional problem of misclassification. In fact, rain arising from a compartment that should have a "hi" level of fluorescence intensity may end up having a fluorescence intensity corresponding to a compartment within the "lo" level of fluorescence intensity. In such a case, instead of being classified as containing a target corresponding to a "hi" level of fluorescence intensity, the compartment is misclassified as containing a target corresponding to a "lo" level. Such misclassification of rain compartments is unavoidable when using an "intensity-based multiplexing" approach and can result in false positive results and further errors in the quantification of target nucleic acids.

[0258] When using the "color combination" approach according to the present invention, there is only one set of positive thresholds for each fluorophore type so that the rain can be processed in the same manner as when using the simple "one color-one target" approach.

[0259] In conclusion, compared to "intensity-based multiplexing", the "color combination" approach described herein has the following advantages: - an increase in the multiplexing level when the number of detection channels exceeds 5, -Simplification of division classification, - A significant reduction in the number of positive thresholds required to classify compartments, -Improved robustness against rain. CSSFR complex of the present invention and kit containing the same

[0260] The present invention also relates to the CSSFR complexes of the invention themselves, as described above.

[0261] As used herein, the term "CSSFR complex" means - a primary reporter oligonucleotide molecule (m-reporter) containing at least one sequence ("tag complementary sequence", TCS) complementary to the tag sequence carried by the mediator probe of the set that binds to the TSi and, at its 5' end, a reporter molecule, which may be either a fluorophore or a quencher group; a sequence which is complementary at its 3' and / or 5' end and / or between its 3' and 5' end to the 5' end sequence of the m-reporter; a quencher group or a fluorophore group if the m-reporter has at least one fluorophore group, or a bimolecular nucleotide complex comprising a complementary reporter oligonucleotide molecule (c-reporter), which contains a quencher group when the m-reporter has a fluorophore group; The fluorescence of the fluorophore group of the c-reporter molecule or the m-reporter molecule changes as a result of hybridization or extension of the tag sequence on the m-reporter to which the 5'-terminal mediator region cleaved from the mediator probe is attached on the tag complementary sequence (TCS).

[0262] The m-reporter molecule in the CSSFR complex is a linear nucleotide molecule containing at least 30 nucleotides, more precisely it usually comprises 30 to 80 nucleotides, preferably 40 to 50 nucleotides.

[0263] The m-reporter molecules of the CSSFR reporters of the present invention contain at least one, preferably at least two, more preferably three or more tag complementary sequences (TCSs) that are partially or completely complementary to the tag sequences located on the corresponding mediator probes. In the context of the present invention, these tag complementary sequences (TCSs) should be relatively short, i.e., typically comprising 8-25 nucleotides, preferably 8-18 nucleotides, more preferably 10-16 nucleotides, and even more preferably 12-14 nucleotides.

[0264] The 3' end of the m-reporter molecule can be preferably blocked with a phosphate group or other common blockers (C3 / C6 spacer, dideoxynucleotide, phosphate, inverted base, 3' amino) to avoid unwanted amplification on this side of the molecule. Blocking steps are recommended by many oligonucleotide manufacturers, see for example https: / / www.biomers.net / en / Products / DNA / Real-time_PCR / PCR_Blocker.html.

[0265] The c-reporter molecule in the CSSFR complex of the present invention is relatively short, typically comprising 8 to 25 nucleotides, preferably 8 to 18 nucleotides, more preferably 10 to 16 nucleotides, and even more preferably 12 to 14 nucleotides.

[0266] When carrying a quencher compatible with some fluorescent dyes, the c-reporter molecule can be used in a CSSFR complex containing a different m-reporter molecule. For example, the quencher BHQ1 can be used to quench blue and till, so that a c-reporter carrying BHQ1 can be used to hybridize an m-reporter carrying blue and till (see Example 2).

[0267] In this reporter complex, the m-reporter molecule preferably contains at least two different tag complementary sequences (TCS), preferably multiple different tag complementary sequences, as disclosed in

[16] .

[0268] In this reporter complex, the complementary reporter oligonucleotide molecule (c-reporter) is short, typically containing 8-25 nucleotides, preferably 8-18 nucleotides, more preferably 10-16 nucleotides, even more preferably 12-14 nucleotides (usually 15-20 nucleotides), and preferably about 18 nucleotides.

[0269] In another aspect, the present invention also relates to the in vitro use of at least one complementary single-stranded fluorescent reporter (CSSFR) complex as defined above in a PCR method (preferably a dPCR method, more preferably a dPCR method as described above) for detecting and / or quantifying the presence of at least one, preferably at least two, more preferably at least five nucleic acid target sequences (TSi) in a biological sample containing nucleic acid molecules. As shown in Examples 2.B.2 and 2.B.3, the CSSFR complex of the present invention can be advantageously used to reliably and rapidly highlight and quantify the presence of 12 or 15 different nucleic acid target sequences (TSi).

[0270] In another aspect, the present invention relates to a kit comprising at least two, preferably at least three, more preferably at least four complementary single-stranded fluorescent reporter (CSSFR) complexes as defined above, wherein the sequences of the m-reporter molecules of these universal reporters are advantageously designed to detect at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TSs using a minimum number of fluorophore groups.

[0271] Preferably, each CSSFR conjugate in the kit carries a different fluorophore group.

[0272] The CSSFR complexes contained in the kit can be used to detect one or more TSs. When they carry different TCSs, they can be used to detect several TSs. Some complexes can also be used to detect only one TS.

[0273] The kit of the present invention may further comprise a mediator probe designed to detect TS by two mediator probes carrying two different tags specifically recognized by two differently labeled CSSFR complexes according to the method of the present invention. In this case, the kit further comprises at least two mediator probes whose 3'-terminal probe regions are complementary to the target sequence TSi and whose 5'-terminal mediator regions contain two different tag sequences, the tag sequences being complementary to and hybridizing to tag-complementary sequences carried by the m-reporter oligonucleotide molecules of at least two complementary single-stranded fluorescent reporter (CSSFR) complexes of the kit.

[0274] Alternatively, or in addition, the kit of the present invention may further comprise a mediator probe designed so that TS is detected by only one mediator probe carrying a tag specifically recognized by two differently labeled CSSFR complexes according to the method of the present invention. In this case, the kit further comprises at least one mediator probe whose 3'-terminal probe region is complementary to the target sequence TSi and whose 5'-terminal region contains a tag sequence that is complementary to and hybridizes to at least two tag-complementary sequences carried by the m-reporter oligonucleotide molecules of at least two complementary single-stranded fluorescent reporter (CSSFR) complexes of the kit.

[0275] The kits of the present invention can include any of the mediator probes described above.

[0276] The kits of the present invention may also contain at least one CSSFR complex that allows for the detection of two, three or more target sequences (by including an appropriately selected TCS in the m-reporter molecule).

[0277] The kit of the present invention comprises at least one CSSFR complex that allows for the detection of only one target sequence.

[0278] The kits of the invention may further comprise any buffers or molecular tools that can be used to carry out the methods of the invention, in particular: (a) amplification reagents for amplifying nucleic acid targets, and / or (b) a nuclease.

[0279] Finally, instructions for proper use of the kit to detect several target sequences may be included. [Brief explanation of the drawings]

[0280] [Figure 1] Data from an experiment detecting six target sequences at a concentration of 100 cps / μL, with color combinations using two fluorophore types per target sequence and a total of four fluorophore types across all six target sequences (Example 1). Units on the x- and y-axes are Arbitrary Fluorescence Units (AFU), which reflect the fluorescence intensity of the fluorophore types. [Figure 2] a) Dilution series of a single target sequence detected by color combinations without other target sequences. b) Dilution series of a single target sequence detected by color combinations with a background of five other target sequences. c) Confidence intervals on a target sequence (PhiX) at 100 cp / μL with increasing background levels of another target sequence (Lambda). [Figure 3] Assay sensitivity of target with increasing background levels. [Figure 4] This is a plot of the function f(x)=ex / x. [Figure 5] The principle of combining a mediator probe with a molecular beacon to be used as a universal reporter is detailed. [Figure 6] The principle of the combination mediator probe with a molecular beacon as a universal reporter (A: target sequence 1, B: target sequence 2) is shown. [Figure 7]The principle of combining a mediator probe with CSSFR to be used as a universal reporter is detailed. [Figure 8] 1D thresholding in a universal reporter experiment using linear reporters. The units on the x-axis are droplet index, which is stacked for eight samples, and the units on the y-axis are arbitrary fluorescence units (AFU), which reflect the fluorescence intensity of the fluorophore type. [Figure 9] Data from an experiment detecting three target sequences at a concentration of 100 cps / μL, with color combinations using three fluorophore types per target sequence (Example 3). Units on the x and y axes are Arbitrary Fluorescence Units (AFU), which reflect the fluorescence intensity of the fluorophore types. [Figure 10] Data are shown from a typical experiment (see Example 4) detecting 11 target sequences at a concentration of 100 cps / μL (excluding ESR1 E380WT, which was used at approximately 200 cps / μL, and ALB, whose concentration was approximately 160 cps / μL), using a total of five fluorophore types and color combinations, with one gene (del19ref) visualized with only one fluorophore type (Cy3 / green). The units on the x- and y-axes are arbitrary fluorescence units (AFU), reflecting the fluorescence intensity of the fluorophore types. [Figure 11] Figure 1 shows 1D thresholding for a universal reporter experiment using a linear reporter, as described in Example B.2. The units on the x-axis are droplet index stacked for 16 conditions, and the units on the y-axis are arbitrary fluorescence units (AFU), which reflect the fluorescence intensity of the fluorophore type. The figure shows that the best resolution in the six color channels is obtained with primer concentrations between 0.25 and 0.5 μM and mediator probe concentrations between 0.5 and 1 μM. [Figure 12]The concentrations of the primers and mediator probes correspond to the concentrations of the target sequences disclosed in Example B.2. The results obtained for the best conditions are obtained with primer concentrations between 0.25 and 0.5 μM and mediator probe concentrations between 0.5 and 1 μM. [Figure 13] This figure shows 1D thresholding for a universal reporter experiment using a linear reporter, as described in Example B.3. The units on the x-axis are droplet indexes stacked for 16 conditions, and the units on the y-axis are arbitrary fluorescence units (AFU), which reflect the fluorescence intensity of the fluorophore type. The exposure time is doubled for all channels, with the blue channel fixed at 250 ms. This figure shows that the best separation in the six color channels can be obtained for primer concentrations between 0.25 and 0.5 μM and mediator probe concentrations between 0.5 and 1 μM. [Figure 14] The concentrations of the target sequences are as disclosed in Example B.3, depending on the concentrations of the primers and mediator probe. The results obtained for the best conditions are obtained with primer concentrations between 0.25 and 0.5 μM and mediator probe concentrations between 0.5 and 1 μM. [Figure 15] Design optimization of c-reporter and tag sequence lengths. This figure shows that the best resolution is obtained with the shortest c-reporter and shortest tag sequences tested. [Figure 16] Design optimization of m-reporter concentration. This figure shows that the best separation is obtained with m-reporter concentrations between 0.1 and 0.25 μM. [Figure 17]This figure details the principle of combining a mediator probe and a CSSFR used as a universal reporter when the target sequence is characterized by one or two TAG sequences whose complementary sequences (TCSs) are carried by one or two CSSFR complexes. (A) The TSi is characterized by two colors (blue and green), and the set detecting the TSi contains: two mediator probes specific for the TSi (3' portion), each containing a TAGi1 and a TAGi2 (5' portion), and two CSSFR complexes, one containing a TCS specific for TAGi1 and one containing a TCS specific for TAGi2, with the two complexes being differentially labeled (blue / green). (B) As in (A), the TSi is characterized by two colors, but one CSSFR complex contains a TCS shared with the set detecting another target sequence. (C) In a PCR method, at least one of the target sequences is detected by a single color. Thus, in this example, TSi is detected with a set containing: one mediator probe specific for TSi (3' portion) and containing TAGi (5' portion), one CSSFR complex containing a TCS specific for TAGi and a fluorescent dye group (green), and TSj is detected with a set containing: two different mediator probes specific for TSj (3' portion) and containing TAGj1 and TAGj2 (5' portions), two CSSFR complexes, one containing a TCS specific for TAGj1 and one containing a TCS specific for TAGj2, the two complexes being differently labeled (pink / red). [Figure 18]The principle of combining a mediator probe with one or two CSSFRs used as universal reporters is detailed below. (A) The TSi is characterized by two colors (blue and green), and the set used to detect the TSi contains: one mediator probe specific for the TSi (3' portion) and containing a TAGi (5' portion); two CSSFR complexes, each containing a TCS specific for the TAGi and differentially labeled (blue / green); (B) Three target sequences TS1, TS2, and TS3 are detected by three different mediator probes containing TAG1, TAG2, and TAG3, respectively, which are recognized by CSSFRs carrying several target sequences in a combined order. As a result, each TS is finally characterized by two colors (blue and green for TS1, blue and red for TS2, and green and red for TS3). (C) Two target sequences TSi and TSj are detected. TSi is detected with one color (green), and TSj is detected with two colors (blue and red). The set for detecting TSi contains one mediator probe specific for -TSi (3' portion) and containing TAGi (5' portion), and one CSSFR complex containing a TCS specific for -TAGi and a fluorescent dye group (green). The set for detecting TSj contains one mediator probe specific for -TSj (3' portion) and containing TAGj (5' portion), and two CSSFR complexes that are differently labeled (blue / red), each containing a TCS specific for -TAGj. [Example]

[0281] Example 1: Use of two TaqMan® probes per target sequence to perform the color combination method of the present invention: 6 target sequences - 2 colors per target sequence - 4 colors total

[0282] Materials and Methods Forward and reverse primers for different target sequences (see table below) are used at a concentration of 0.5 μM.

[0283] Two probes with different fluorophores and quenchers are used for each target sequence. The combinations of target sequences, dye-containing probes, and concentrations used for these probes are detailed in the table.

[0284] The fluorophore type / color combinations applied per target sequence were: ESR1 L536P (infrared and yellow), PhiX174 (infrared and teal), PBR322 (red and infrared), ALB (red and teal), Lambda (yellow and teal), and puc18 (yellow and red).

[0285] [Table 5]

[0286] These sequences are shown as SEQ ID NOs: 1 to 24 in the sequence listing.

[0287] Supplier details are as follows: -Kaneka Eurogentec SA-5 Rue Bois Saint-Jean,4102 Seraing,Belgium, -IDT-Integrated DNA Technologies,Inc.-1710 Commercial Park-Coralville,Iowa 52241-USA.

[0288] The target sequence for the assay is double-stranded synthetic DNA corresponding to the amplification product ("gBlocks™" fragment from Integrated DNA Technologies, Inc.), used at a concentration of approximately 100 cps / μL.

[0289] To perform PCR, naica® Multiplex PCR Mix containing fluorescein (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) was added to the reaction. 4% DMSO (vol / vol) was included in the reaction to increase component stability. 7 μL of the reaction was loaded onto an Opal tip (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France). Negative controls (no DNA) and single-color controls were included to control the reaction and establish matrix correction.

[0290] The division and PCR cycles are carried out in a Geode device (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France).

[0291] The PCR cycle program included an initial step of denaturation at 95°C for 3 minutes, followed by 60 cycles of denaturation and hybridization-extension at 95°C for 15 seconds and 62°C for 30 seconds, respectively.

[0292] The chip is then released (returned to atmospheric pressure) at 25° C. and 5 mbar / s. The chip was imaged using a Prism6 imager (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) using the following exposure times: 125ms for blue, 350ms for teal, 125ms for green, 150ms for yellow, 500ms for red, and 500ms for infrared.

[0293] Data are analyzed using Crystal Miner software (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France). For matrix correction, single-color controls are used.

[0294] A positivity threshold is set to distinguish negative and positive droplets from the rest of the droplets. This is typically done in the 1D dot plot view.

[0295] In the next step, a population is defined for each target sequence using the population editor in the Crystal Miner software. For example, for the Phix174 target sequence, positive droplets are designated as those that are positive for infrared and teal, and droplets that do not show fluorescence are counted as negative droplets. Droplets that show any other color combination are excluded from the analysis. In this way, the number of positive droplets for a target sequence can be determined, and the concentration of that target sequence in the sample can be estimated, among other features.

[0296] Figure 1 shows data from a typical experiment using a total of four fluorophore types to detect six target sequences at a concentration of 100 cps / μL, whereas the color combination method of the present invention uses two fluorophore types per target sequence.

[0297] result A series of experiments were performed using the above protocol to assess the dynamic range accessible when detecting multiple target sequences coded with two fluorophore types / colors.

[0298] We observed good linearity between predicted and actual concentrations from 0.1 to 10,000 cp / μL both when only one target sequence was spiked into the sample (Figure 2a) and when six target sequences were detected together (Figure 2b). In the latter case, one target sequence was varied in concentration from 0.1 to 10,000 cp / μL, while the other five target sequences remained constant at 100 cp / μL. Furthermore, the measured detection limits are comparable to those routinely obtained with prior art methods utilizing a single color per target sequence, despite significant background concentrations.

[0299] In a further experiment, the confidence level for detection of a fixed target sequence was investigated using varying amounts of background target sequence (Figure 2c), where a fixed concentration of 100 cp / µL of target sequence (PhiX) was detected in the presence of increasing levels of background target sequence (Lambda), ranging from 0.1 cp / µL up to 10,000 cp / µL.

[0300] The confidence interval at a fixed concentration of PhiX is seen to plateau at approximately 10% for concentrations of the background target sequence Lambda up to 2000 cp / μL, as expected, and then increase sharply for higher concentrations of the background target sequence.

[0301] This effect is believed to result from co-encapsulation events being discarded from analysis according to the methods of the present invention; high background target sequence concentrations result in a higher number of co-encapsulation events, thereby resulting in fewer droplets being analyzed according to the methods of the present invention. A direct consequence is that when background target sequence concentrations (which may be, e.g., wild-type) become excessively elevated, the confidence level decreases at a fixed target sequence concentration.

[0302] As previously mentioned, whether this result is a practical limitation of the method of the present invention varies greatly depending on the application, particularly as exemplified in the section entitled "Statistical Analysis" of the data processing protocol of the present invention. For the detection of rare events in liquid biopsies, this effect is not a factor because the expected concentration is low. In situations where a reference target sequence or a highly enriched target sequence is being detected, one fluorophore type can be used in isolation to detect the highly enriched target sequence and overcome the effects of co-encapsulation events.

[0303] Example 2: Use of two mediator probes (MPs) triggering two universal reporters (URs) for each target sequence to perform the color combination method of the present invention This approach involves the use of a non-fluorescent probe (mediator probe, MP) composed of a sequence specific for the target and an artificial sequence in the 5' region called a "flap" that is not complementary to the target. During extension, the polymerase cleaves the probe after the first base hybridizes to the target, thereby releasing the flap sequence. This flap sequence then hybridizes to a universal reporter, which can be, for example, a molecular beacon (Example 2A below) or a linear reporter (Example 2B below). A. Use of Molecular Beacons as Universal Reporters

[0304] The use of molecular beacons as universal reporters is shown schematically in FIG.

[0305] In this example, for each target sequence, two mediator probes compete to activate two molecular beacons that act as universal reporters, as depicted in Figure 6. A total of six encoded target sequences were tracked using four different fluorescence channels.

[0306] Materials and Methods For each target sequence, double-stranded synthetic DNA (gBlocks™ fragments from Integrated DNA Technologies, Inc.) corresponding to the amplification product was used.

[0307] For color combination experiments, mixtures containing an estimated concentration of each DNA fragment of 1000 cp / μL (10×) were prepared.

[0308] The color combinations applied for each target sequence are: ESR1 L536P (green and red), PhiX174 (red and teal), PBR322 (green and yellow), ALB (red and yellow), Lambda (green and teal), and puc18 (yellow and teal).

[0309] All oligonucleotides were purchased from Kaneka Eurogentec SA (5 Rue Bois Saint-Jean, 4102 Seraing, Belgium). In this strategy, a blocker was added to prevent hybridization of uncleaved mediator probes to molecular beacons, which would result in an increase in fluorescence background. In the preparation process, primers, probes, molecular beacons, and blockers corresponding to six target sequences were assembled in stock solutions specific to each reagent type. The composition of the reagent mixture and the sequences of the oligonucleotides were as follows: The primer mix uses the primer sequences listed in the table in Example 1.A above at a concentration of 5 μM for each primer. Mediator probes were prepared using the following components, all containing phosphate 3' modifications (no 5' modifications), mixed at 5 μM for each mediator probe:

[0310] [Table 6]

[0311] These sequences are listed as SEQ ID NOs: 25 to 36 in the sequence listing.

[0312] A blocker mix was prepared using the following components, all containing phosphate 3' modifications (no 5' modifications), mixed at 5 μM for each blocker:

[0313] [Table 7]

[0314] These sequences are listed as SEQ ID NOs: 37 to 48 in the sequence listing.

[0315] A molecular beacon mix was prepared using the following components, mixed at 5 μM for each molecular beacon:

[0316] [Table 8]

[0317] These sequences are listed as SEQ ID NOs: 49 to 52 in the sequence listing.

[0318] PCR sample preparation Experiments were performed at an approximate concentration of 100 cp / μL for the six target sequences. A no-template negative control (NTC, no-template control) was performed. Two replicates were performed for each condition.

[0319] In addition to the DNA template mixture and oligonucleotide mixture described above, naica® Multiplex PCR Mix (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) was used.

[0320] The detailed mixture preparation for each of the two experimental conditions was as follows:

[0321] [Table 9]

[0322] Chip loading and dPCR execution PCR samples were loaded onto Sapphire chips (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) and processed in the Geode instrument according to standard procedures. The usual Sapphire split and release program was used with the PCR cycling program described below: Initial denaturation: 95℃ for 3 minutes 60 cycles: 95°C for 15 seconds, then 62°C for 30 seconds.

[0323] After dPCR runs, chips were scanned using a Prism6 reader and the standard Sapphire scanning template (ScanningTemplate_Prism6_SapphireChip_naica-multiplex-PCR-MIX_Taqman_v1.4).

[0324] Data analysis Data analysis was performed using Crystal Miner software. To generate the compensation matrix, a single-color control consisting of a mix of universal reporters combined with one single mediator probe was used to activate only one color.

[0325] A 1D threshold was set for each color channel to define negative and positive populations. Color assignments for each target sequence were then configured in the population editor section of the software. For example, PhiX174 was defined as a positive population in the teal and red channels and negative in all other channels. After editing the populations, the results were exported and the quantification reviewed. The experimentally measured concentrations were in good agreement with the theoretical concentration of 100 cp / μL for each of the six targets.

[0326] [Table 10] B. Use of Linear Reporters as Universal Reporters B.1 6 target sequences / 4 colors

[0327] The use of a linear reporter as a universal reporter is shown schematically in FIG.

[0328] In this example, for each target sequence, two mediator probes compete to activate two linear reporters that act as universal reporters. A total of six encoded target sequences were tracked using four different fluorescence channels.

[0329] Materials and Methods For each target sequence, double-stranded synthetic DNA corresponding to the amplification product (gBlocks™ fragment manufactured by Integrated DNA Technologies, Inc., SEQ ID NOs: 152 to 166) was used.

[0330] For color combination experiments, mixtures containing an estimated concentration of each DNA fragment of 1000 cp / μL (10×) were prepared.

[0331] The color combinations applied for each target sequence are: ESR1 L536P (infrared and red), PhiX174 (red and teal), PBR322 (infrared and yellow), ALB (red and yellow), Lambda (infrared and teal), and puc18 (yellow and teal).

[0332] All oligonucleotides were purchased from Kaneka Eurogentec SA (5 Rue Bois Saint-Jean, 4102 Seraing, Belgium). In the preparation process, primers, probes, and linear reporters (including an m-reporter strand with a flap sequence and a binding site for a fluorophore, and a c-reporter strand complementary to the m-reporter strand containing a quencher) corresponding to six target sequences were assembled in a stock solution specific to each reagent type. The composition of the reagent mixture and the sequences of the oligonucleotides were as follows: The primer mix uses the primer sequences listed in the table in Example 1.A above at a concentration of 5 μM for each primer. Mediator probes were prepared using the following components, all containing phosphate 3' modifications (no 5' modifications), mixed at 5 μM for each mediator probe:

[0333] [Table 11]

[0334] These sequences are listed as SEQ ID NOs: 53 to 64 in the sequence listing.

[0335] The m-reporter strand mix was prepared using the following components, containing the fluorophores shown as 5'-modified and phosphate 3'-modified, mixed at 5 μM for each m-reporter strand:

[0336] [Table 12]

[0337] These sequences are listed as SEQ ID NOs: 65 to 68 in the sequence listing.

[0338] The c-reporter strand mix was prepared using the following components but containing the quencher shown as a 3' modification (no 5' modification) and mixed at 5 μM for each c-reporter strand:

[0339] [Table 13]

[0340] These sequences are listed as SEQ ID NOs: 69 to 72 in the sequence listing.

[0341] PCR sample preparation Experiments were performed at two linear reporter A-strand concentrations (0.25 μM and 0.1 μM) with an approximate concentration of 100 cp / μL for the six targets. For both linear reporter concentrations, a negative control (NTC, no template control) without template was also performed. Two replicates were performed for each condition.

[0342] In addition to the DNA template mixture and oligonucleotide mixture described above, naica® Multiplex PCR Mix (Stilla Technology, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) was used.

[0343] The detailed mixture preparation for each of the four experimental conditions was as follows:

[0344] [Table 14]

[0345] [Table 15]

[0346] Chip loading and dPCR execution PCR samples were loaded onto Sapphire chips (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) and processed in the Geode instrument according to standard procedures. The usual Sapphire split and release program was used with the PCR cycling program described below: Initial denaturation: 95℃ for 3 minutes 60 cycles: 95°C for 15 seconds, then 58°C for 30 seconds

[0347] After dPCR runs, chips were scanned using a Prism6 reader and the standard Sapphire scanning template (ScanningTemplate_Prism6_SapphireChip_naica-multiplex-PCR-MIX_Taqman_v1.4).

[0348] Data analysis Data analysis was performed using Crystal Miner software. To generate the compensation matrix, a single-color control consisting of a mix of universal reporters combined with one single mediator probe was used to activate only one color.

[0349] To define the negative and positive populations, a 1D threshold was set for each color channel, as illustrated in FIG.

[0350] Next, color assignments for each target sequence were set in the population editor section of the software. For example, PhiX174 was defined as a positive population in the teal and red channels and negative for all other channels. After editing the populations, the results were exported and the quantification was reviewed. The experimentally measured concentrations were in good agreement with the theoretical concentration of 100 cp / μL for each of the six targets.

[0351] [Table 16] B.2 12 target sequences / 6 colors

[0352] In this example, two competing mediator probes were used to activate two linear reporters that act as universal reporters, and 10 targets were detected with a color combination. Two additional targets were detected with a single color (blue or red) using a single mediator probe to activate a single universal reporter. Red was also used in combination with another color to detect other targets. Blue was only used to detect one target (TSN).

[0353] Overall, 12 targets were detected in this example, and the color attributes for each target are shown in the table below:

[0354] [Table 17]

[0355] Materials and Methods For each target sequence, double-stranded synthetic DNA (gBlocks™ fragment manufactured by Integrated DNA Technologies, Inc.) corresponding to the amplification product was used (SEQ ID NOs: 152 to 166).

[0356] For color combination experiments, mixtures containing an estimated concentration of each DNA fragment of 3000 cp / μL (30×) were prepared.

[0357] All oligonucleotides were purchased from Kaneka Eurogentec SA (5 Rue Bois Saint-Jean, 4102 Seraing, Belgium). In the preparation process, primers, probes, and linear reporters (including an m-reporter with a flap sequence and a binding site for a fluorophore, and another c-reporter strand that is complementary to the m-reporter strand and contains a quencher) corresponding to 12 target sequences were assembled in a stock solution specific for each reagent type. The composition of the reagent mixture and the sequences of the oligonucleotides were as follows: The primer mix uses the primer sequences listed in the table below at a concentration of 2.5 μM for each primer.

[0358] [Table 18]

[0359] A mediator probe mix was prepared using the following components, all containing phosphate 3' modifications (no 5' modifications), mixed at 5 μM for each mediator probe ({X} designates LNA):

[0360] [Table 19]

[0361] The m-reporter strand mix was prepared using the following components, containing the fluorophores shown as 5'-modified and phosphate 3'-modified, mixed at 5 μM for each m-reporter strand:

[0362] [Table 20]

[0363] The c-reporter strand mix was prepared using the following components, but containing the quencher shown as a 3' modification (no 5' modification), mixed at 10 μM for each c-reporter strand:

[0364] [Table 21]

[0365] PCR sample preparation Experiments were performed individually using four primer mix concentrations combined with two mediator probe concentrations with approximate concentrations of the 12 targets at 100 cp / μL. For each condition, a no-template negative control (NTC, no template control) was performed. Two replicates were performed for the positive and negative controls.

[0366] In addition to the DNA template mixture and oligonucleotide mixture described above, naica® PCR mix (Stilla Technology, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) was used.

[0367] The detailed mixture preparation for each of the 16 experimental conditions was as follows:

[0368] [Table 22]

[0369] [Table 23]

[0370] [Table 24]

[0371] [Table 25]

[0372] Chip loading and dPCR execution PCR samples were loaded onto Ruby Chip Consumables (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) and processed in the Geode instrument according to standard procedures. The usual Ruby split and release program was used, along with the PCR cycling program described below: Initial denaturation: 95℃ for 3 minutes 60 cycles: 95°C for 15 seconds, then 58°C for 60 seconds.

[0373] After the dPCR run, the chip was scanned using a Prism 6 reader using the following exposure times: 500ms for blue, 800ms for teal, 250ms for green, 350ms for yellow, 1000ms for red, and 1000ms for infrared.

[0374] Data analysis Data analysis was performed using Crystal Miner software. To generate the compensation matrix, a single-color control consisting of a mix of universal reporters combined with one single mediator probe was used to activate only one color.

[0375] To define the negative and positive populations, a 1D threshold was set for each color channel, as illustrated in Figure 11. The best conditions allowing for setting 1D thresholds in six color channels were obtained using 0.5 μM primer and 0.5 μM mediator.

[0376] Color assignments for each target sequence were then configured in the population editor section of the software. For example, PhiX174 was defined as a positive population in the teal and red channels and negative for all other channels. After editing the populations, the results were exported and the quantification reviewed. The experimentally measured concentrations were in good agreement with the theoretical concentration of 100 cp / μL for each of the 12 targets. See Figure 12. B.3 15 target sequences / 6 colors

[0377] In this example, two competing mediator probes were used to activate two linear reporters that act as universal reporters, and 15 targets were detected with color combinations. The color attributes for each target are shown in the table below:

[0378] [Table 26]

[0379] Materials and Methods For each target sequence, double-stranded synthetic DNA corresponding to the amplification product (gBlocks™ fragment manufactured by Integrated DNA Technologies, Inc., SEQ ID NOs: 152 to 166) was used.

[0380] For color combination experiments, mixtures containing an estimated concentration of each DNA fragment of 3000 cp / μL (30×) were prepared.

[0381] All oligonucleotides were purchased from Kaneka Eurogentec SA (5 Rue Bois Saint-Jean, 4102 Seraing, Belgium). In the preparation process, primers, probes, and linear reporters (including an m-reporter strand with a flap sequence and a binding site for a fluorophore, and another c-reporter strand that is complementary to the m-reporter and contains a quencher) corresponding to 15 target sequences were assembled in a stock solution specific for each reagent type. The composition of the reagent mixture and the sequences of the oligonucleotides were as follows: The primer mix uses the primer sequences listed in Example B.2 and the primers listed in the table below, at a concentration of 2.5 μM for each primer.

[0382] [Table 27]

[0383] A mediator probe mix was prepared using the sequences listed in Example B.2 and the following elements, all containing phosphate 3' modifications (no 5' modifications), mixed at 5 μM for each mediator probe ({X} designates LNA):

[0384] [Table 28]

[0385] The m- and c-reporter strand mixes were prepared at identical concentrations and with the same sequences as described in Example B.2.

[0386] PCR sample preparation Similar to B.2, experiments were performed with four primer mix concentrations combined with two mediator probe concentrations with approximate concentrations of 15 targets at 100 cp / μL. For each condition, a no-template negative control (NTC, no template control) was performed. Two replicates were performed for the positive and negative controls.

[0387] In addition to the DNA template mixture and oligonucleotide mixture described above, naica® PCR mix (Stilla Technology, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) was used.

[0388] The detailed mixture preparation for each of the 16 experimental conditions was as follows:

[0389] [Table 29]

[0390] [Table 30]

[0391] [Table 31]

[0392] [Table 32]

[0393] Chip loading and dPCR execution PCR samples were loaded onto Ruby Chip Consumables (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) and processed in the Geode instrument according to standard procedures. The usual Ruby split and release program was used, along with the PCR cycling program described below: Initial denaturation: 95℃ for 3 minutes 60 cycles: 95°C for 15 seconds, then 58°C for 60 seconds.

[0394] After the dPCR run, the chip was scanned using a Prism 6 reader using the following exposure times: 500ms for blue, 800ms for teal, 250ms for green, 350ms for yellow, 1000ms for red, and 1000ms for infrared.

[0395] Data analysis Data analysis was performed using Crystal Miner software. The same correction matrix as in Example B.2 was used.

[0396] To define the negative and positive populations, a 1D threshold was set for each color channel, as illustrated in Figure 13. The best conditions allowing for setting 1D thresholds in six color channels were obtained using 0.5 μM primer and 0.5 μM mediator.

[0397] Next, color assignments for each target sequence were set in the population editor section of the software. For example, PhiX174 was defined as a positive population in the teal and red channels and negative for all other channels. After editing the populations, the results were exported and the quantification was reviewed. The experimentally measured concentrations were in good agreement with the theoretical concentration of 100 cp / μL for each of the 15 targets. See Figure 14.

[0398] Example 3: Use of three TaqMan® probes bearing different colors for each target sequence to perform the color combination method of the present invention Materials and Methods Forward and reverse primers for different target sequences (see table below) are used at a concentration of 0.5 μM.

[0399] Three probes with different fluorophores and quenchers are used for each target sequence at a concentration of 0.25 μM. The color combinations applied for each target sequence are as follows: ESR1 L536P (green, yellow, and infrared), PhiX174 (yellow, red, and infrared), and ALB (yellow, red, and teal).

[0400] [Table 33]

[0401] Supplier details are as follows: Kaneka Eurogentec SA-5 Rue Bois Saint-Jean,4102 Seraing,Belgium, IDT-Integrated DNA Technologies,Inc.-1710 Commercial Park-Coralville,Iowa 52241-USA.

[0402] The target sequence for the assay is double-stranded synthetic DNA corresponding to the amplification product ("gBlocks™" fragment from Integrated DNA Technologies, Inc. - 1710 Commercial Park - Coralville, Iowa 52241 - USA, SEQ ID NOs: 152-166), used at the following concentrations: ESR1 L536P: 450cps / μL, PhiX 174: 450cps / μL, ALB 600cps / μL.

[0403] To perform PCR, naica® Multiplex PCR Mix containing fluorescein (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) was added to the reaction. 4% DMSO (vol / vol) was included in the reaction to increase component stability. 7 μL of the reaction was loaded onto an Opal tip (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France). Negative controls (no DNA) and single-color controls were included to control the reaction and establish matrix correction.

[0404] The division and PCR cycles are carried out in a Geode device (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France).

[0405] The PCR cycle program included an initial step of denaturation at 95°C for 3 minutes, followed by 60 cycles of denaturation and hybridization-extension at 95°C for 15 seconds and 62°C for 30 seconds, respectively.

[0406] The chip is then released (returned to atmospheric pressure) at 25° C. and 5 mbar / s. The chip was imaged using a Prism6 imager (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) using the following exposure times: 125ms for blue, 400ms for teal, 125ms for green, 175ms for yellow, 500ms for red, and 500ms for infrared.

[0407] Data are analyzed using Crystal Miner software (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France). For matrix correction, single-color controls are used. A threshold is set to isolate negative droplets from the rest. This is typically done in the 1D dot plot view. In the next step, the population editor in the Crystal Miner software is used to define populations for each target sequence. For example, for the Phix174 target sequence, positive droplets are designated as those that are positive for infrared, red, and yellow, and droplets that do not show fluorescence are counted as negative droplets. Droplets that show any other color combination are excluded from the analysis (Figure 9).

[0408] result By finding the number of positive droplets for each target sequence according to the methods of the present invention, it is possible to estimate, among other features, the concentration of that target sequence in the sample.

[0409] After compiling the populations, the results were exported and the quantification reviewed. The experimentally measured concentrations were in good agreement with the theoretical concentrations for each of the three targets.

[0410] [Table 34]

[0411] Example 4: Use of two TaqMan® probes per target to perform a color combination in combination with a single TaqMan® probe to detect the final target: 10 target sequences - two colors per target sequence - and one gene in a single color

[0412] Materials and Methods Forward and reverse primers for different target sequences (see table below) are used at a concentration of 0.5 μM.

[0413] Two probes with different fluorophores and quenchers are used for each target sequence except for one. The primers, probes with dyes and quenchers, and concentrations used are listed in the table below.

[0414] The color combinations applied for each target sequence are as follows: ESR1 L536P (blue and teal), PhiX174 (infrared and yellow), PBR322 (blue and infrared), ALB (blue and green), Lambda (yellow and teal), puc18 (infrared and teal), TP53 R282W (teal and green), TP53 R248WT (yellow and green), MRM1 (infrared and green), ESR1 E380WT (blue and yellow), and Del19ref (green).

[0415] [Table 35] JPEG2025538399000067.jpg171132

[0416] Supplier details are as follows: -Kaneka Eurogentec SA-5 Rue Bois Saint-Jean,4102 Seraing,Belgium, -IDT-Integrated DNA Technologies,Inc.-1710 Commercial Park-Coralville,Iowa 52241-USA.

[0417] The target sequences for the assays are double-stranded synthetic DNAs corresponding to the amplification products ("gBlocks™" fragments from Integrated DNA Technologies, Inc., 1710 Commercial Park, Coralville, Iowa 52241, USA, SEQ ID NOs: 152-166) used at an approximate concentration of 100 cps / μL, except for ESR1 E380WT, which has a concentration of approximately 200 cps / μL, and ALB, which has a concentration of approximately 160 cps / μL.

[0418] To perform PCR, fluorescein-free naica® PCR mix (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) was added to the reaction. 4% DMSO (vol / vol) was included in the reaction to increase component stability. 7 μL of the reaction was loaded onto an Opal tip (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France). Negative controls (no DNA) and single-color controls were included to control the reaction and establish matrix correction.

[0419] The division and PCR cycles are carried out in a Geode device (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France).

[0420] The PCR cycle program included an initial step of denaturation at 95°C for 3 minutes, followed by 60 cycles of denaturation and hybridization-extension at 95°C for 15 seconds and 62°C for 30 seconds, respectively.

[0421] The chip is then released (returned to atmospheric pressure) at 25° C. and 5 mbar / s. The chip was imaged using a Prism6 imager (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) using the following exposure times: 125ms for blue, 350ms for teal, 125ms for green, 150ms for yellow, 500ms for red, and 500ms for infrared.

[0422] Data are analyzed using Crystal Miner software (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France). For matrix correction, single-color controls are used.

[0423] Set a threshold to isolate negative droplets from the rest. This is usually done in the 1D dot plot view.

[0424] In the next step, the population editor in the Crystal Miner software was used to define populations for each target sequence. For example, for the Phix174 target sequence, positive droplets were designated as those positive for infrared and yellow. Furthermore, droplets that showed no fluorescence were counted as negative droplets. Droplets showing any other color combination were excluded from the analysis (Figure 10).

[0425] result In this way, it is possible to retrieve the number of positive droplets for a target sequence and estimate, among other features, the concentration of that target sequence in the sample.

[0426] Example 5: Mediator probe / CSSFR structure optimization Effect of Ac-reporter length and Tm of tag sequence In this example, the effects of the length of the c-reporter and the length of the tag sequence included in the mediator probe were investigated. One target (PhiX174) was detected in a single color using a single mediator probe to activate a teal universal reporter. Four different mediator probes were tested with increasing tag sequence lengths (13, 15, 18, and 21 nucleotides) and increasing Tm values ​​when bound to the m-reporter. For each mediator probe, two different c-reporters were evaluated, each 18 or 24 nucleotides in length.

[0427] Materials and Methods For the target sequence, double-stranded synthetic DNA corresponding to the PhiX174 amplification product (gBlocks™ fragment from Integrated DNA Technologies, Inc., SEQ ID NOs: 152-166) was used at an estimated concentration of 1000 cps / μL (10x).

[0428] The Tm values ​​of the tag sequence and c-reporter when bound to the m-reporter were evaluated using the IDT online tool (https: / / eu.idtdna.com / calc / analyzer) for the following parameters: Oligo concentration: 0.5 μM ·Na + Concentration: 50mM, Magnesium ++ Concentration: 5mM, ·dNTP concentration: 0.2 μM.

[0429] All oligonucleotides were purchased from Kaneka Eurogentec SA (5 Rue Bois Saint-Jean, 4102 Seraing, Belgium). In the preparation process, primers, probes, and linear reporters (including an m-reporter strand with a flap sequence and a binding site for a fluorophore, and another c-reporter strand complementary to the m-reporter strand containing a quencher) were assembled in a stock solution specific for each reagent type. The composition of the reagent mixture and the sequences of the oligonucleotides were as follows: The primer mix uses the primer sequences listed in the table below at a concentration of 5 μM for each primer.

[0430] [Table 36]

[0431] Each mediator probe was individually prepared at a concentration of 5 μM. All contained a phosphate 3' modification (no 5' modification). The sequences are listed below:

[0432] [Table 37]

[0433] A teal-labeled m-reporter strand mix (teal_m-reporter) was prepared at 5 μM. The sequences are described in the previous examples. Each c-reporter strand was prepared individually at 5 μM and contained a BHQ1 quencher at the 3′ position. The sequences are listed below:

[0434] [Table 38]

[0435] PCR sample preparation Experiments were performed individually for each mediator probe in combination with both c-reporters. Targets were at an approximate concentration of 100 cp / μL. For mediator probes with 15-nucleotide tags, a no-template negative control (NTC, no-template control) was also performed.

[0436] In addition to the DNA template mixture and oligonucleotide mixture described above, naica® Multiplex PCR Mix (Stilla Technology, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) was used.

[0437] The detailed mix preparation for each condition was as follows:

[0438] [Table 39]

[0439] [Table 40]

[0440] [Table 41]

[0441] Chip loading and dPCR execution PCR samples were loaded onto Sapphire chips (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) and processed in the Geode instrument according to standard procedures. The usual Sapphire split and release program was used with the PCR cycling program described below: Initial denaturation: 95℃ for 3 minutes 45 cycles: 95°C for 15 seconds, then 58°C for 30 seconds.

[0442] After dPCR runs, chips were scanned using a Prism6 reader and the standard Sapphire scanning template (ScanningTemplate_Prism6_SapphireChip_naica-multiplex-PCR-MIX_Taqman_v1.4).

[0443] Data analysis Data analysis was performed using Crystal Miner software. To define the negative and positive populations, a 1D threshold was set on the teal channel, as illustrated in Figure 15. After 1D thresholding, the results were exported and the quantification and separability scores were considered. The results are shown below:

[0444] [Table 42]

[0445] Regarding the length of the c-reporter, the separation between positive and negative populations was very poor, making it impossible to set a threshold for the longest c-reporter. The quantification results were inconsistent with the target concentrations for mediator probes with the longest tag sequences (21 and 18 nucleotides), with separation scores below 5 for all conditions. In contrast, using shorter c-reporters resulted in better separation, allowing thresholds to be set for three of the four mediator probes tested. The quantification obtained for these three mediator probes was close to the target concentrations. These results may be explained by potential competition between the c-reporter and the complementary strand of the m-reporter formed by extension of the released tag sequence. The greater the length and Tm of the c-reporter, the greater the competition.

[0446] Considering the length of the tag sequence, the resolution is not good for the longest tag sequence (21 nt), while the threshold can be easily set for the other three mediator probes with the smallest c-reporters. The best resolution score is obtained by the mediator probe with the smallest tag sequence. In fact, the resolution score is higher than 6 for mediator probes with tag sequences of 13 and 15 nucleotides. This can be explained by the characteristics of the Tm of the tag sequence when bound to the m-reporter relative to the Tm of the sequence-specific portion of the mediator probe when bound to the target. In fact, the mediator probe must first hybridize to the target to release the tag sequence during extension, which is later hybridized to the m-reporter to generate a signal. Therefore, the Tm of the specific portion of the sequence must naturally be higher than the Tm of the tag sequence. In this model, the Tm of the specific portion of the sequence when bound to the target was estimated at 69.2 °C. The Tm difference between the probe-specific portion and the tag sequence is shown below:

[0447] [Table 43]

[0448] A Tm difference of 4.4°C does not appear to be sufficient to achieve effective tag release, however, good results were observed with a specific probe that had a Tm 9.6°C higher than the mediator probe tag sequence. Effect of Bm-reporter concentration

[0449] In this example, the effect of m-reporter concentration was investigated. Increasing concentrations of teal m-reporter (0.1-0.75 μM) were tested. One target (PhiX174) is detected in a single color using one mediator probe to activate the teal universal reporter. The three successful mediator probes described in Example 5A were tested in combination with four different teal m-reporter concentrations.

[0450] Materials and Methods For the target sequence, double-stranded synthetic DNA corresponding to the PhiX amplification product (gBlocks™ fragment from Integrated DNA Technologies, Inc., SEQ ID NOs: 152-166) was used at an estimated concentration of 1000 cps / μL (10x).

[0451] All oligonucleotides were purchased from Kaneka Eurogentec SA (5 Rue Bois Saint-Jean, 4102 Seraing, Belgium). In the preparation process, primers, probes, and linear reporters (including an m-reporter with a flap sequence and a binding site for a fluorophore, and a c-reporter strand complementary to the m-reporter strand containing a quencher) were assembled in a stock solution specific for each reagent type. The composition of the reagent mixture and the sequences of the oligonucleotides were as follows: The primer mix uses the primer sequences described in Example 5A at a concentration of 10 μM for each primer. Mediator probes with tag sequences of 13, 15 or 18 nucleotides were individually prepared at a concentration of 5 μM. The sequences are described in Example B.2. A teal-labeled m-reporter strand mix (teal_m-reporter) was prepared at 5 μM. The sequences are described in the previous examples. The c-reporter strand of 18 nucleotides (teal_c-reporter_v3) was prepared individually at 5 μM. The sequence is listed below:

[0452] PCR sample preparation Experiments were performed individually for each mediator probe with increasing concentrations of teal m-reporter. The m-reporter / c-reporter ratio was kept constant for each condition, with the c-reporter concentration 1.5-fold higher than the m-reporter concentration. Targets were at an approximate concentration of 100 cp / μL.

[0453] In addition to the DNA template mixture and oligonucleotide mixture described above, naica® Multiplex PCR Mix (Stilla Technology, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) was used. The detailed mix preparation for each mediator probe tested was as follows:

[0454] [Table 44]

[0455] Chip loading and dPCR execution PCR samples were loaded onto Sapphire chips (Stilla Technologies, 1, Mail du Professeur Georges Mathe - 94800 Villejuif, France) and processed in the Geode instrument according to standard procedures. The usual Sapphire split and release program was used with the PCR cycling program described below: Initial denaturation: 95℃ for 3 minutes 45 cycles: 95°C for 15 seconds, then 58°C for 30 seconds.

[0456] After dPCR runs, chips were scanned using a Prism6 reader and the standard Sapphire scanning template (ScanningTemplate_Prism6_SapphireChip_naica-multiplex-PCR-MIX_Taqman_v1.4). Data analysis

[0457] Data analysis was performed using Crystal Miner software. To define the negative and positive populations, a 1D threshold was set on the teal channel, as illustrated in Figure 16. After 1D thresholding, the results were exported and the quantification and separability scores were considered. The results are shown below:

[0458] [Table 45]

[0459] For the three mediator probes tested, the separation between positive and negative populations was very poor at high m-reporter concentrations. At the highest m-reporter concentrations, a threshold could not be set, and the quantification results did not match the target concentration. In contrast, lowering the m-reporter concentration improved the results, allowing for accurate threshold setting and therefore accurate quantification. The best results were obtained with mediator probes with 18- and 15-nucleotide tag sequences coupled to the lowest m-reporter concentrations (0.1 μM and 0.25 μM).

[0460] References JPEG2025538399000078.jpg222164JPEG2025538399000079.jpg81164

Claims

1. 1. An in vitro PCR method for detecting and / or quantifying the presence of at least one nucleic acid target sequence (TSi) in a biological sample containing nucleic acid molecules, said method comprising: A) For the sample and each TSi, i) at least one mediator probe, a 3'-terminal probe region that is complementary to said target sequence (TSi); - a 5'-terminal mediator region containing one tag sequence; a biological cleavage site located between the mediator region and the probe region, whereby an enzyme with nuclease activity can mediate the cleavage of the two regions during the amplification process of the nucleic acid target sequence (TSi); a mediator probe comprising: ii) at least one complementary single-stranded fluorescent reporter (CSSFR) complex, said reporter complex comprising: - a primary reporter oligonucleotide molecule (m-reporter) containing at least one sequence ("tag complementary sequence", TCS) complementary to the tag sequence carried by the set of mediator probes that bind to the TSi and at its 5' end by a reporter molecule, which can be either a fluorophore or a quencher group; a sequence complementary to the 5'-terminal sequence of said m-reporter, and a quencher group, or a fluorophore group if the m-reporter has at least one fluorophore group, or a complementary reporter oligonucleotide molecule (c-reporter) containing a quencher group when the m-reporter has a fluorophore group; at least one complementary single-stranded fluorescent reporter complex comprising: contacting a set containing the fluorescence of the fluorophore group of the c-reporter or m-reporter molecule is modified on the tag complementary sequence (TCS) on the m-reporter as a result of hybridization of the binding tag sequence of the 5' terminal mediator region, or extension thereof, upon cleavage from the mediator probe; B) amplifying said at least one nucleic acid target in the presence of an enzyme having nuclease activity; C) detecting or measuring the fluorescence intensity of each fluorophore group; D) optionally processing the data collected in step C) to quantify the concentration of said at least one TSi in said biological sample; A method comprising:

2. The set comprises, for at least one TSi of a number of target sequences: at least two mediator probes, each containing a 3'-terminal probe region complementary to said target sequence TSi and a 5'-terminal mediator region containing a tag sequence, wherein said tag sequences of said at least two mediator probes are different and are complementary to and hybridize with at least one TCS of at least one reporter complex of said set; - at least two reporter complexes as defined in claim 1, wherein each m-reporter molecule contains one or more different tag complementary sequences (TCS) that are complementary to and hybridize with a tag sequence located in the 5'-terminal mediator region of one of the minimum two mediator probes of the set; wherein each of the at least two reporter complexes comprises at least one TSi ｉ are differentially labeled as characterized by different fluorophore types, ｉ The method of claim 1 , wherein is 2 or greater.

3. 3. The method of claim 2, wherein at least one, and preferably each, of the reporter complexes in the set contains at least two or more different tag complementary sequences (TCS).

4. 4. A method according to claim 2 or 3 for detecting at least two target sequences TSi and TSj, comprising: - the set for TSi is - two mediator probes, the 3' portions of which are specific for said target sequence TSi and the 5' portions of which contain a first tag sequence TAGil or a second tag sequence TAGi2, respectively; - two CSSFR complexes, each containing a TCS specific for TAGi1 or TAGi2, respectively, said complexes being differentially labeled; Contains - the set for TSj is - two mediator probes, the 3' portions of which are specific for said target sequence TSj and the 5' portions of which contain a first tag sequence TAGj1 or a second tag sequence TAGj2, respectively; - two CSSFR complexes, each containing a TCS specific for TAGj1 and TAGj2, said complexes being recognized differently; A method comprising:

5. The method according to any one of claims 2 to 4, wherein the two sets specific for two different target sequences TSi and TSj contain at least one CSSFR complex carrying the same fluorophore group.

6. The method according to any one of claims 2 to 5, wherein the two sets specific for two different target sequences TSi and TSj contain at least one CSSFR complex in common.

7. The set contains, for each TSi, at least two reporter complexes as defined in claim 1, each of the at least two reporter complexes being k ｉ are differentially labeled as characterized by different fluorophore types, ｉ The method according to any one of claims 1 to 6, wherein is 2 or more.

8. The set contains, for some TSi, at least two reporter complexes as defined in claim 1, each of the at least two reporter complexes being k ｉ are differentially labeled as characterized by different fluorophore types, ｉ is two or more, and for some TSj, contains only one reporter complex as defined in claim 1, so that said TSj are detected in only one color.

9. At least one of the TSi is - only one mediator probe, the 3' portion of which is specific for said target sequence TSi and the 5' portion of which contains TAGi; Only one CSSFR complex containing a TCS and a fluorophore group specific for the TAGi, 9. The method according to claim 1, wherein the TSi is detected with a set containing:

10. A method according to any one of claims 1 to 6 and 8 to 9 for detecting at least two target sequences TSi and TSj, comprising: - the set for TSi is - two mediator probes, the 3' portions of which are specific for said target sequence TSi and the 5' portions of which contain a first tag sequence TAGil or a second tag sequence TAGi2, respectively; - two CSSFR complexes, each containing a TCS specific for TAGi1 or TAGi2, respectively, said complexes being differentially labeled; Contains - the set for TSj is - only one mediator probe, the 3' portion of which is specific for said target sequence TSj and the 5' portion of which contains TAGj; Only one CSSFR complex containing a TCS specific for the TAGj and fluorophore group, so that said TSj can be detected in only one color.

11. The set comprises, for at least one TSi of a number of target sequences: i) one mediator probe containing a 3'-terminal probe region complementary to the target sequence TSi and a 5'-terminal mediator region containing a tag sequence; ii) at least two reporter complexes as defined in claim 1, wherein each m-reporter molecule contains a tag complementary sequence (TCS) that is complementary to and hybridizes to the tag sequence located in the 5'-terminal mediator region of the mediator probe of the set; wherein each of the at least two reporter complexes comprises at least one TSi ｉ are differentially labeled to feature different fluorophore types, ｉ The method of claim 1 , wherein is 2 or greater.

12. The method of claim 11, wherein at least one, preferably each, of the m-reporter molecules contained in the at least two reporter complexes that bind to each TSi contains at least two or more different tag complement sequences (TCS).

13. 13. A method according to any one of claims 11 to 12 for detecting at least two target sequences TSi and TSj, comprising: - the set for TSi is one mediator probe, the 3' portion of which is specific for said target sequence TSi and the 5' portion of which contains the tag sequence TAGi; - two CSSFR complexes, each containing a TCS specific for a TAGi, said complexes being differentially labeled; Contains - the set for TSj is one mediator probe, the 3' portion of which is specific for said target sequence TSj and the 5' portion of which contains the tag sequence TAGj; - two CSSFR complexes, each containing a TCS specific for TAGj, said complexes being differentially labeled; A method comprising:

14. The method according to any one of claims 11 to 13, wherein the two sets specific for two different target sequences TSi and TSj contain at least one CSSFR complex carrying the same fluorophore group.

15. The method according to any one of claims 11 to 14, wherein the two sets specific for two different target sequences TSi and TSj contain at least one CSSFR complex in common.

16. At least one of the TSi is - only one mediator probe, the 3' portion of which is specific for said target sequence TSi and the 5' portion of which contains TAGi; Only one CSSFR complex containing a TCS and a fluorophore group specific for the TAGi, 16. The method according to claim 11, wherein the TSi is detected with a set containing:

17. 17. A method according to any one of claims 11 to 16 for detecting at least two target sequences TSi and TSj, comprising: - the set for TSi is - only one mediator probe, the 3' portion of which is specific for said target sequence TSi and the 5' portion of which contains TAGj; Only one CSSFR complex containing a TCS and a fluorophore group specific for the TAGi, so that the TSi can be detected in only one color, - the set for TSj is one mediator probe, the 3' portion of which is specific for said target sequence TSi and the 5' portion of which contains the tag sequence TAGi; - two CSSFR complexes, each containing a TCS specific for a TAGi, said complexes being differentially labeled; A method comprising:

18. 18. A method according to any one of claims 1 to 17 for detecting at least two target sequences TSi and TSj, comprising: - the set for TSi is one mediator probe, the 3' portion of which is specific for said target sequence TSi and the 5' portion of which contains the tag sequence TAGi; - two CSSFR complexes, each containing a TCS specific for a TAGi, said complexes being differentially labeled; Contains - the set for TSj is - two mediator probes, the 3' portions of which are specific for said target sequence TSj and the 5' portions of which contain a first tag sequence TAGj1 or a second tag sequence TAGj2, respectively; - two CSSFR complexes, each containing a TCS specific for TAGj1 and TAGj2, said complexes being differentially labeled; A method comprising:

19. The method of claims 1 to 18, wherein each TSi is characterized by a specific fluorophore or combination of fluorophores that differs from the fluorophores or combinations of fluorophores of each of the other target sequences.

20. 20. The method according to any one of claims 1 to 19, wherein at least two sets binding to two different target sequences TSi and TSj contain CSSFR complexes carrying the same fluorophore group, preferably the same CSSFR complexes.

21. The method according to any of claims 1 to 20, wherein six TSi are detected with four differently labeled CSSFR complexes whose m-reporter principal molecules carry two different TCSs.

22. The method according to any one of claims 1 to 20, wherein 12 or 15 TSi are detected with 6 CSSFR molecules having different TCSs and 6 different fluorescent dyes.

23. The method according to any of claims 1 to 22, wherein the size of the complementary reporter oligonucleotide molecule (c-reporter) and the tag length of the reporter complex is short, preferably about 18 nt.

24. The method of any one of claims 1 to 23, wherein the concentration of the primary reporter oligonucleotide molecule (m-reporter) in the reporter complex is 0.1 to 0.25 µM.

25. For each target sequence TSi, the melting temperature T of the tag sequence TAGi hybridized on the corresponding TCSi of the CSSFR molecule Ｍ is T of the 3'-end probe region of the mediator probe hybridized on the target sequence TSi Ｍ The method of any one of claims 1 to 24, wherein the

26. The following steps: - between steps A) and B), separating said biological sample into a set of compartments each containing, on average, 1 to 3 copies of said analyte, - during step B), exponentially amplifying said at least one nucleic acid target in the presence of an enzyme with nuclease activity, - during step C), measuring for each compartment the fluorescence intensity of each fluorophore group, - optionally during step D) processing the data collected in step C) in order to quantify the concentration of at least one TSi in said biological sample, The method according to any one of claims 1 to 25, which is a dPCR method comprising:

27. For each fluorophore type, - compartments with a fluorescent signal below the positive threshold for said fluorophore type, - compartments with a fluorescent signal higher than the positive threshold for said fluorophore type and step e) counting the number of times ... ・N ０ = the total number of compartments with fluorescent signals below the positive threshold for all the fluorophore types, ・N ｉ = k characterizing TSi ｉ the total number of compartments having a fluorescent signal higher than the positive threshold for only one fluorophore type; 27. The method of claim 26, wherein the step of determining

28. 28. The method of claim 27, further comprising step f) of processing the data collected in step e) to quantify the concentration of at least one TSi in the biological sample.

29. For each target sequence TSi, the calculation of its concentration Ci in step f) is carried out according to Poisson's law, preferably according to the formula [Equation 1] 29. The method of claim 28, wherein v is the volume of a compartment and d is the dilution factor used to dilute the biological sample into a microfluidic well in which dPCR is performed.

30. Step e) determines N corresponding to the total number of compartments having a fluorescent signal higher than the positive threshold for only one fluorophore type. １ Further comprising determining C ｉ But the formula: [Equation 2] The method according to any one of claims 27 to 29, wherein in step f) the calculation is performed using

31. 31. The method of any of claims 26 to 30, wherein step C) further comprises setting the positivity threshold intensity for each fluorophore type, wherein the positivity threshold is defined for each fluorophore type separately, preferably on a 1D plot view, and corresponds to an intensity value that distinguishes between compartments exhibiting one fluorophore type at high intensity and those exhibiting the same fluorophore type at low intensity.

32. k ｉ is constant for all target sequences i, and k ｉ The method of any of claims 26 to 31, wherein compartments having a fluorescent signal higher than the positive threshold for +1 fluorophore type are not considered.

33. A complementary single-stranded fluorescent reporter (CSSFR) complex, the reporter complex comprising: - a primary reporter oligonucleotide molecule (m-reporter) containing at least one sequence ("tag complementary sequence", TCS) complementary to the tag sequence carried by the set of mediator probes that bind to the TSi and at its 5' end by a reporter molecule, which can be either a fluorophore or a quencher group; a sequence complementary to the 5'-terminal sequence of said m-reporter, and a quencher group, or a fluorophore group if the m-reporter has at least one fluorophore group, or a quencher group when the m-reporter has a fluorophore group a complementary reporter oligonucleotide molecule (c-reporter) containing Contains A complementary single-stranded fluorescent reporter complex in which the fluorescence of the fluorophore group of the c-reporter molecule is modified as a result of hybridization or extension of the tag sequence of the 5'-terminal mediator region cleaved from the mediator probe on the tag complementary sequence (TCS) on the m-reporter.

34. 34. The reporter complex of claim 33, wherein the m-reporter molecule contains at least two or more different tag complement sequences (TCS).

35. The reporter complex according to any one of claims 33 to 34, wherein the complementary reporter oligonucleotide molecule (c-reporter) of the reporter complex is short in size.

36. A kit comprising at least two, preferably at least three, more preferably at least four complementary single-stranded fluorescent reporter (CSSFR) complexes as defined in claims 33-35.

37. 37. The kit of claim 36, wherein the m-reporter molecule comprises at least one complementary single-stranded fluorescent reporter (CSSFR) complex containing at least two or more different tag complementary sequences (TCS).

38. 38. The kit of any one of claims 36 to 37, further comprising at least two mediator probes whose 3'-terminal probe regions are complementary to the target sequence TSi and whose 5'-terminal mediator regions contain two different tag sequences, wherein the tag sequences are complementary to and hybridize with tag-complementary sequences carried by the m-reporter oligonucleotide molecules of at least two complementary single-stranded fluorescent reporter (CSSFR) complexes of the kit.

39. 39. The kit of any one of claims 36 to 38, further comprising at least one mediator probe, the 3'-terminal probe region of which is complementary to the target sequence TSi and the 5'-terminal region of which contains a tag sequence that is complementary to and hybridizes to at least two tag-complementary sequences carried by the m-reporter oligonucleotide molecules of at least two complementary single-stranded fluorescent reporter (CSSFR) complexes of the kit.

40. A kit according to any one of claims 36 to 39, comprising at least one mediator probe according to claim 38 and at least one mediator probe as defined in claim 39.

41. 41. A kit according to any one of claims 36 to 40, comprising at least one CSSFR complex which allows for the detection of two, three or more target sequences.

42. 42. The kit according to any one of claims 36 to 41, comprising at least one CSSFR complex that allows for the detection of only one target sequence.

43. (a) amplification reagents for amplifying nucleic acid targets, and / or (b) a nuclease; The kit of any one of claims 36 to 42, further comprising:

44. 35. Use of at least one complementary single-stranded fluorescent reporter (CSSFR) complex as defined in any of claims 32 to 34 in a PCR method for detecting the presence and / or quantifying at least one nucleic acid target sequence in a biological sample containing nucleic acid molecules.

45. 35. Use of at least six complementary single-stranded fluorescent reporter (CSSFR) complexes as defined in any of claims 32 to 34 in a PCR method for detecting the presence and / or quantifying at least twelve nucleic acid target sequences in a biological sample containing nucleic acid molecules.

46. 35. Use of at least six complementary single-stranded fluorescent reporter (CSSFR) complexes as defined in any of claims 32 to 34 in a PCR method for detecting the presence and / or quantifying at least 15 nucleic acid target sequences.

47. 47. The use of claim 45 or 46, wherein each of the TSi is characterized by a unique combination of exactly two different fluorophore types.

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