Generic cartridges and methods for multiplex nucleic acid detection

Abstract

The field of the present invention generally relates to the detection of nucleic acid targets in a multiplex reaction setting. Specifically, disclosed herein are methods, kits, kits of parts, systems, or components thereof, for performing multiplex PCR detection using a custom gene target panel within a generic detection cartridge. The disclosed methods and kits can be utilized to rapidly design automated multiplex PCR-based detection assays for numerous, i.e., dozens, of personalized and / or customized gene targets, typically including mutations, SNPs, pathogenic sequences, epigenetic lesions, and the like. The general principles underlying the disclosed methods and products are: (1) a panel-agnostic generic detection cartridge preloaded with a generic reporter; and (2) the provision of a target-specific multiplex PCR oligonucleotide pool within the cartridge that, in the presence of target under PCR amplification conditions, specifically reacts with the generic reporter and generates a signal therefrom. As a result, the methods and products disclosed herein greatly simplify the standard diagnostic assay development pipeline and are therefore highly advantageous for bringing custom-selected genetic testing panels to laboratories and patients at a faster rate than previously possible.

Description

Technical Field

[0001] The field of the present invention generally relates to the detection of nucleic acid targets in multiplex reaction settings. In particular, disclosed herein are methods, kits, kits of parts, systems, and their components for performing multiplex PCR detection using a custom gene target panel within a generic detection cartridge. The disclosed methods and kits can typically be utilized to rapidly design automated multiplex PCR-based detection assays for a large number, i.e., dozens, of personalized and / or customized gene targets, including mutations, SNPs, pathogenic sequences, epigenetic lesions, etc. The general principles underlying the disclosed methods and products are: (1) a panel-agnostic generic detection cartridge pre-loaded with a generic reporter; and separately (2) the provision of a target-specific multiplex PCR oligonucleotide pool that can specifically react with the generic reporter within the cartridge and lead to the generation of molecules that can generate a signal in the presence of the target under PCR amplification conditions. As a result, the methods and products disclosed herein greatly simplify the standard diagnostic assay development pipeline and are thus highly advantageous in bringing custom-selected gene test panels to laboratories and patients at a much faster rate than previously possible.

[0002] Background There is a worldwide need for the rapid and straightforward development of personalized diagnostic assays that enable the monitoring of genetically diverse disease states such as infection, organ transplant rejection, or cancer in an individual. Such assays should provide high reliability and remain cost-effective based on patient-specific genetic information. Currently existing methods are too time- and resource-intensive and as a result cannot provide suitable solutions.

[0003] In the field of oncology, since the first few years of the 21st century, we have witnessed a paradigm shift from cancer type-specific diagnosis and treatment to a more personalized approach to diagnosis and pan-cancer therapy that strongly focuses on the underlying genetics and immune responses of the individual (Hanahan and Weinberg, 2011, Cell 144:646-674). Therefore, cancer is now a comprehensive term encompassing many different genetically determined acquired proliferative syndromes, rather than a single uniform disease, and it is now more widely recognized that each cancer type, and even individual cancers, appear to have unique gene mutation profiles (Ciriello et al., 2013, Nat Gen 45:1127-1133). Therefore, despite the continued prevalence of detectable and therapeutically targetable driver mutations, such as those in the KRAS, BRAF, or EGFR genes, in many cancers, a significant number of cancer cases remain, where individually focused trials can greatly benefit patient management and outcomes.

[0004] One way to implement such personalized screening is through next-generation sequencing (NGS). While NGS analysis is gradually becoming more affordable, it remains relatively expensive and requires expert involvement for data interpretation, making it typically out of reach for general practitioners. Furthermore, the waiting time for NGS results remains considerable. Therefore, NGS is largely unsuitable for routine or regular follow-up cancer treatment, and will likely remain so for some time. Given the speed, sensitivity, ease of use, and availability of existing fully automated systems, perhaps the best solution could be provided by qPCR-based detection of selected targets. Moreover, these fully automated systems enable the global deployment of trials, bringing them closer to patients and ensuring access to patients worldwide. While excellent mutation-panel-specific diagnostic trials are readily available to cancer patients from several providers, including Biocartis NV, which possesses a state-of-the-art assay development pipeline, the provision of personalized, custom target-specific cartridges falls below the manufacturer's profit margin. Despite limited funding and return on investment considerations, subjecting new diagnostic cartridges to current development procedures still involves standard development and manufacturing lead times that are too long from an individual perspective. As a result, there is an urgent need to transform cancer follow-up therapy for individuals, particularly for molecular surveillance testing, post-surgery, and minimal residual disease (MRD) monitoring, by developing personalized gene assays much faster and at a lower cost.

[0005] The sudden outbreak of the 2020 global Covid pandemic, which imposed lockdowns on many manufacturers (further contributing to extended timelines and time to market) and severely restricted many patients' access to healthcare and disease monitoring systems, highlighted the unprecedented need for new methods to develop faster and more cost-effective custom qPCR detection-based testing. Particularly in light of the emergence of SARS-CoV-2 as a constantly mutating, entirely new viral pathogen that makes detection difficult, it was especially desirable that such new methods be generally applicable to the detection of sequences from non-human sources and be highly customizable and easily adaptable to include the detection of rapidly changing sequences.

[0006] The inventors believe they have developed such an approach by providing a novel platform that includes a generic, i.e., targeted agnostic general detection cartridge containing a generic reporter, which is compatible with a large multiplex qPCR target-specific and customizable oligonucleotide pool that can be introduced into a cartridge along with a clinical sample to detect the target in the cartridge. The inventors believe that the approach presented herein will find unprecedented applications in, but is not limited to, personalized molecular surveillance studies using liquid biopsies, personalized therapy selection, treatment and / or recurrence monitoring, postoperative follow-up, MRD detection, recurrence monitoring in cases of adjuvant therapy and even recurrence, acquisition of resistance mutations and monitoring of response to treatment, as well as in cell therapies, personalized cancer vaccines and neoantigen-targeted immunotherapies. The methods and products disclosed herein are equally applicable to non-cancer applications, including transplant monitoring or prenatal testing, as well as to the field of infectious diseases, such as the detection of non-human sequences (e.g., to detect viral and bacterial pathogens), the detection of sepsis, microbiome characterization, and many other applications.

[0007] This disclosure provides methods, kits, component kits, systems, and their components for performing multiplex detection of gene targets using a customized gene target panel in a generic detection cartridge for point-of-care (PoC) devices. The general principle underlying the disclosed methods and products is based on providing at least two separate components: (1) A panel-agnostic generic detection cartridge pre-loaded with a generic reporter configured to detect the presence of a generic sequence; and separately (2) A target-specific multiplex PCR oligonucleotide pool that, in the presence of a target under PCR amplification conditions, contains a generic sequence and consequently leads to the generation of a molecule (or even a detectable molecule) that can specifically react with a generic reporter within the cartridge and generate a signal from it.

[0008] The concepts described above are very new in the current implementation of diagnostic assay development. To date, to the best of the applicant's knowledge, only assay-specific sample-to-result diagnostic cartridges exist that are pre-loaded with oligonucleotide reagents specific to assay gene targets that are predefined by the assay gene target, such as appropriate detection reactions and target-therapable mutations. While existing cartridges can be excellently rapid and highly sensitive in detecting their defined gene targets, their design involves considerable effort in terms of optimization, validation, testing, and material costs and latency, for example, target-specific reagents such as oligonucleotide probes or other sometimes very sophisticated amplification and / or detection systems. In addition, once a working cartridge containing a fixed diagnostic panel is developed, it is not easy to further modify or adapt its target-specific reagents, for example, to include additional targets in the panel. For example, any negative interactions between newly introduced oligonucleotides and the original oligonucleotides must first be eliminated so as not to impede the performance of the modified product. Additional problems may arise during selection, or potential incompatibility between dyes or quenchers may occur after the addition of new target-specific reporters, such as probes, to existing and pre-optimized solutions. Problems can arise at multiple levels, and therefore, it must be recognized that even seemingly trivial changes to existing target-specific assays to update or adapt to the needs of specific users are not straightforward and may require considerable product redesign effort.

[0009] The methods and products presented herein, including kits, component kits, cartridges, systems, and components, address some or all of the above shortcomings by providing sample-to-result generic detection cartridges with all necessary sample preparation and amplification reactions pre-loaded, but instead of the diagnostic target-specific reagents of existing assay-specific cartridges, the generic detection cartridges include a general reporter (e.g., a labeled probe) configured to detect the presence of a generic sequence tag. To avoid the effort of design or redesign and reduce development costs as well as waiting times for the synthesis and delivery of target-specific reporters, such generic detection cartridges can be pre-tested, characterized, and produced on a large scale and stockpiled for rapid supply to clients such as hospitals, clinics, or testing centers when needed. The customer can then define and order a desired mix of custom-selected target-specific oligonucleotide subsets compatible with the cartridge. The oligonucleotide subsets may include target-specific primers or primer pairs, but may also include one or more additional oligonucleotides that act as primers or probes, depending on the amplification reaction selected. The compatibility of oligonucleotide subset mixes with generic cartridges equipped with generic reporters will depend on: (i) their ability to perform a single multiplex amplification reaction within the cartridge; and (ii) the composition of each target-specific oligonucleotide subset to generate nucleic acid products containing a generic sequence tag associated with and detectable by a defined generic reporter within the cartridge, in the presence of its target.

[0010] As a result, from the user's perspective, the difference in the concept described above compared to conventional methods is that the user receives two (or more) components, including a generic cartridge and a target-specific oligonucleotide pool, instead of a single package containing a cartridge already pre-loaded with reagents specific to a fixed diagnostic panel. Consequently, instead of inserting only the biological sample into the assay-specific cartridge, the user also inserts the mix of target-specific oligonucleotides; a procedure schematically shown in Figure 1, which constitutes only the minimum additional handling burden compared to current methods.

[0011] Conversely, from an assay design perspective, the generic cartridge-based approach has enormous potential to significantly reduce the time to deliver new assays by at least several months to a year, and in some cases up to several years, according to our estimates. This is because it eliminates the design effort required to standardize nucleic acid isolation reactions and reporter systems for each generic cartridge type and to focus on establishing efficient multiplexing reactions with mixes of target-specific oligonucleotide subsets.

[0012] However, there is a widely established understanding in this field that achieving high levels of multiplexing is difficult. For example, a person skilled in the art who wants to detect, say, 20 different variants in a currently available Biocartis Idylla™ cartridge with five amplification chambers would naturally aim to design five parallel 4plexes (one in each chamber) without even considering performing one or more parallel 20plex reactions in each of the chambers. In particular, if a given system has a certain number of wavelength-specific detection channels linked to the amplification chambers and adapted to capture signals from reporters within them, a person skilled in the art would not likely consider performing multiplex reactions with more targets than the number of detection channels in such amplification chambers.

[0013] Increasing the number of targets in multiplex amplification becomes even less likely when low-copy targets are involved. A prominent example of this is the presence of variants in circulating cell-free DNA (cfDNA) from bodily fluid samples, including plasma or urine. Appropriate monitoring tests should enable the detection of cfDNA at a rate of 1% or less, preferably 0.1%, in patient samples. It is well known to those skilled in the art that developing a singleplex qPCR assay capable of detecting 1% or less of variants is already difficult. Consequently, it will be apparent to those skilled in the art that developing a multiplex qPCR assay capable of detecting 1% of each target variant is even more difficult, as the different primers and probes used in the qPCR reaction may interfere with each other, thereby degrading the performance of the assay.

[0014] Furthermore, the tools commonly available for primer design are still largely based on rudimentary thermodynamic profiling models that predict primer and probe behavior. However, in reality, reaction conditions typically include components not considered in thermodynamic models, such as buffer additives, enzyme-specific behavior related to primer-template mismatch handling, and the effects of PCR ramp rate and cycle time. Consequently, it is widely recognized that available oligonucleotide tools are not particularly suitable for predicting primer and probe behavior, which is especially critical for high-performance assays where less than 1% of variants should be detected by multiplex PCR.

[0015] For the three main reasons mentioned above regarding the widely recognized challenges posed by multiplexing, the inventors believe that no one has yet attempted to develop the generic detection cartridge concept presented herein. However, the inventors have tested the feasibility of the concept presented herein and have created a highly promising generic cartridge prototype that fits with a multiplexing custom-designed gene target panel introduced therein along with a sample. The design principles underlying the generic detection cartridge disclosed herein, as well as the methods, kits, components, and uses based thereon, are generally applicable to all qPCR-based detection techniques, including any random-access sample-to-report device having one or more PCR chambers. Their advantages include significant reductions in design effort, material costs, and order wait times. These and other features and advantages are described below.

[0016] overview This disclosure provides methods, kits, component kits, systems, and their components for performing multiplex detection of gene targets using a customized gene target panel in a generic detection cartridge. The general principle underlying the disclosed methods and products is based on providing at least two separate components: (1) A panel-agnostic generic detection cartridge pre-loaded with a generic reporter configured to detect the presence of a generic sequence; and separately (2) A target-specific multiplex PCR oligonucleotide pool that, in the presence of a target under PCR amplification conditions, contains a generic sequence and consequently leads to the generation of a molecule (further referred to as a detectable nucleic acid product) that can specifically react with a generic reporter within the cartridge and generate a signal from it.

[0017] In particular, in a first general embodiment, a method for detecting multiple gene targets: - To provide a mix of multiple oligonucleotide subsets, each of which subsets is specific to a gene target and which subsets contain a unique generic sequence tag ("unique generic sequence tag" or "UGST"), Each of the subsets is adapted to produce a detectable nucleic acid product containing a unique generic sequence tag under nucleic acid amplification conditions and in the presence of a gene target. To provide a mix; -Separately from the mix, (i) an inlet port for accepting biological samples and / or mixes, (ii) Nucleic acid isolation section located downstream of the inlet port, (iii) Reagents for nucleic acid amplification; (iv) One or more nucleic acid amplification compartments located downstream of the nucleic acid isolation compartment, (v) Multiple generic reporters, each of which contains a generic sequence specific to (complementary to) a unique generic sequence tag contained in one of the detectable nucleic acid products, and which is adapted to generate a signal in the presence of the detectable nucleic acid product; To provide an integrated fluid cartridge that includes, Mixes and biological samples or extracted nucleic acids are inserted into the cartridge by the user. To provide an integrated fluid cartridge Includes, - Manipulating a cartridge after insertion of a biological sample or extracted nucleic acid and mix, comprising performing nucleic acid isolation from the biological sample, followed by multiplex nucleic acid amplification (within the cartridge) including the mix; - When at least one detectable nucleic acid product is generated from the amplification, the signal generated from at least one of several generic reporters contained inside the integrated fluid cartridge is detected. A method is further disclosed that further comprises

[0018] In a further general aspect, a kit or kit of parts, provided as separate components: - A mix of multiple oligonucleotide subsets, each of the subsets being defined as a sequence that is specific for a gene target and does not occur in the genetic information of the organism in which the gene target is detected, and containing a unique generic sequence tag unique to the subset, where each of the subsets is adapted to generate a detectable nucleic acid product containing the unique generic sequence tag under nucleic acid amplification conditions and in the presence of the gene target; A mix of multiple oligonucleotide subsets; and - An integrated fluid cartridge comprising: - An integrated fluid cartridge comprising: (i) An inlet port for receiving a biological sample and / or mix; (ii) A nucleic acid isolation compartment located downstream of the inlet port; (iii) Reagents for nucleic acid amplification; (iv) One or more nucleic acid amplification compartments located downstream of the nucleic acid isolation compartment, and (v) A plurality of generic reporters, each of the plurality of generic reporters containing a generic sequence specific for (and possibly complementary to) one of the unique generic sequence tags and being adapted to generate a signal in the presence of a detectable nucleic acid product containing the unique generic sequence tag An integrated fluid cartridge comprising A kit or kit of parts is disclosed that comprises

[0019] In a further general aspect, a system, or part of a system, the system comprising as separate components: A cartridge engageable with an automated system, the cartridge comprising: a) An inlet port b) Nucleic acid isolation compartment; c) Nucleic acid amplification compartments, preferably more than one nucleic acid amplification compartment, such as two, three, four, five, six, seven or eight or more nucleic acid amplification compartments; Includes, The inlet port is fluidly connected to the nucleic acid isolation compartment, and the nucleic acid isolation compartment is fluidly connected to (one or more) nucleic acid amplification compartments; (One or more) nucleic acid amplification compartments contain a generic reporter molecule, the generic reporter molecule is single-stranded DNA, and: i) The first member of a fluorophore / quencher pair; ii) Stem-loop structure; iii) The second member of a fluorophore / quencher pair; iv) Unique generic sequence tag ("UGST") binding site; v) Polymerase elongation blockers; Includes, cartridges, A mixture of oligonucleotides: a) A pair of target-specific amplification primers configured for amplification of a target nucleic acid, preferably in which at least one member of the target-specific primer pair is allele-specific; b) A mediator probe, extending from 5' to 3': i) A first portion containing UGST, wherein the sequence of UGST is complementary to the UGST binding site of the generic reporter molecule; ii) A second portion that is complementary to the first strand of the target nucleic acid sequence to be amplified; Preferably, polymerase elongation blockers Mediator probes, including A mixture of oligonucleotides containing A system, or a part of a system, including, is disclosed.

[0020] In a more general embodiment, a system, or a part of a system, wherein the system is a separate component: -A cartridge that can be engaged with an automated system: a) Entrance port, b) Nucleic acid isolation compartment; c) Nucleic acid amplification compartments, preferably more than one nucleic acid amplification compartment, such as two, three, four, five, six, seven or eight or more nucleic acid amplification compartments; Includes, The inlet port is fluidly connected to the nucleic acid isolation compartment, and the nucleic acid isolation compartment is fluidly connected to (one or more) nucleic acid amplification compartments; (One or more) nucleic acid amplification compartments contain a generic reporter molecule, the generic reporter molecule is single-stranded DNA, and: i) The first member of a fluorophore / quencher pair; ii) Stem-loop structure; iii) The second member of a fluorophore / quencher pair; iv) Unique generic sequence tag ("UGST") binding site; v) Polymerase elongation blockers; Includes, cartridges, A mixture of oligonucleotides: a) A target-specific amplification primer pair wherein at least one member of the target-specific primer pair comprises a stem-loop structure when bound to a target, preferably at least one member of the target-specific primer pair is allele-specific, and; b) A mediator probe, extending from 5' to 3': i) A first potion containing a unique generic array tag ("UGST"); ii) A second portion complementary to the stem-loop structure or complement of the allele-specific primer; Preferably, polymerase elongation blockers Mediator probes, including A mixture of oligonucleotides containing A system, or a part of a system, including, is disclosed.

[0021] In a more general embodiment, a system, or a part of a system, wherein the system is a separate component: - A device configured to accept a cartridge, The cartridge, a) Entrance port, b) Nucleic acid isolation compartment; c) Nucleic acid amplification compartments, preferably more than one nucleic acid amplification compartment, such as two, three, four, five, six, seven or eight or more nucleic acid amplification compartments; Includes, The inlet port is fluidly connected to the nucleic acid isolation compartment, and the nucleic acid isolation compartment is fluidly connected to (one or more) nucleic acid amplification compartments; (One or more) nucleic acid amplification compartments contain a generic reporter molecule. The generic reporter molecule is single-stranded DNA, and: i) The first member of a fluorophore / quencher pair; ii) Stem-loop structure; iii) The second member of a fluorophore / quencher pair; iv) Unique generic sequence tag ("UGST") binding site; v) Polymerase elongation blockers; including, apparatus, -A mixture of oligonucleotides: a) A target-specific amplification primer pair configured for amplification of a target nucleic acid, wherein at least one member of the target-specific primer pair is allele-specific; b) A mediator probe, extending from 5' to 3': i) A first portion containing UGST, wherein the sequence of UGST is complementary to the UGST binding site of the generic reporter molecule; ii) A second portion that is complementary to the first strand of the target nucleic acid sequence to be amplified; Includes, Preferably, polymerase elongation blockers Mediator probes, including A mixture of oligonucleotides containing A system, or a part of a system, including, is disclosed.

[0022] In a more general embodiment, a system or a part of a system is disclosed, wherein the system is configured to determine the presence or absence of a target sequence, the presence or absence of mutations in the target sequence, specific alleles of the target sequence, pathogens and allogenes.

[0023] In some embodiments, the nucleic acid amplification compartment of the cartridge contains reagents for nucleic acid amplification.

[0024] In some embodiments, the nucleic acid isolation compartment contains nucleic acid extraction / purification reagents or is in fluid contact with a separate compartment containing nucleic acid extraction / purification reagents.

[0025] In some embodiments, the cartridge contains multiple generic reporter molecules within the nucleic acid amplification compartment, each of which has a different UGST binding site.

[0026] In some embodiments, each of the multiple generic reporter molecules contains a different reporter.

[0027] In some embodiments, the oligonucleotide mixture comprises multiple target-specific amplification primer pairs, each primer pair being specific to a different target, preferably with at least one member of each primer pair comprising an allele-specific primer; and multiple mediator probes, wherein i) a first portion of each mediator probe comprises a UGST complementary to the UGST binding site of one generic reporter molecule among multiple generic reporter molecules; and ii) a second portion of each mediator probe comprises a mediator probe complementary to the first strand of different target nucleic acids of multiple target nucleic acid sequences to be amplified.

[0028] In a more general embodiment, a system, or a part of a system, wherein the system is a separate component: - A device configured to accept a cartridge, wherein the cartridge is a) Entrance port, b) Nucleic acid isolation compartment; c) Nucleic acid amplification compartments, preferably more than one nucleic acid amplification compartment, such as two, three, four, five, six, seven or eight or more nucleic acid amplification compartments; Includes, The inlet port is fluidly connected to the nucleic acid isolation compartment, and the nucleic acid isolation compartment is fluidly connected to (one or more) nucleic acid amplification compartments; (One or more) nucleic acid amplification compartments contain a generic reporter molecule, the generic reporter molecule is single-stranded DNA, and: i) The first member of a fluorophore / quencher pair; ii) Stem-loop structure; iii) The second member of a fluorophore / quencher pair; iv) Unique generic sequence tag ("UGST") binding site; v) Polymerase elongation blockers; including, apparatus, A mixture of oligonucleotides: a) A target-specific amplification primer pair wherein at least one member of the target-specific primer pair comprises a stem-loop structure when bound to a target, preferably at least one member of the target-specific primer pair is allele-specific, and; b) A mediator probe, extending from 5' to 3': i) A first potion containing a unique generic array tag ("UGST"); ii) A second portion complementary to the stem-loop structure or complement of the allele-specific primer; Includes, Preferably, polymerase elongation blockers Mediator probes, including A mixture of oligonucleotides containing A system, or a part of a system, including, is disclosed.

[0029] In some embodiments of the systems disclosed herein, the allele-specific primers include ARMS primers.

[0030] In a more general embodiment, a method for detecting a target nucleic acid is disclosed. In some embodiments, the method is: - Amplifying a nucleic acid sample from a target using a target-specific primer pair, preferably in which at least one member of the target-specific primer pair is allele-specific, and the amplification reaction is carried out in the presence of a mediator probe and a generic reporter molecule; a) The mediator probe is positioned from 5' to 3': i) A first potion containing a unique generic array tag ("UGST"); ii) A second portion that is complementary to the first strand of the target nucleic acid; Preferably a polymerase elongation blocker. Includes; b) Generic reporter molecules: i) The first member of a fluorophore / quencher pair; ii) Stem-loop structure; iii) The second member of a fluorophore / quencher pair; iv) UGST binding sites that are complementary to UGST on the mediator probe; v) Polymerase elongation blockers; Includes, A member of a fluorophore / quencher pair is placed via a stem-loop structure to quench the fluorophore. To amplify; - Detecting the signal produced by the fluorophore of a fluorophore / quencher pair in the presence of the target nucleic acid, thereby detecting the presence of the target nucleic acid. This includes. In a further general embodiment, a method for detecting a target nucleic acid is disclosed.

[0031] In some embodiments, a method for detecting a target nucleic acid is: - Amplifying a nucleic acid sample from a target using a target-specific primer pair, At least one member of the target-specific primer pair includes a stem-loop structure when bound to the target, and preferably at least one member of the target-specific primer pair is allele-specific. The amplification reaction was carried out in the presence of a mediator probe and a generic reporter molecule; a) The mediator probe is positioned from 5' to 3': i) A first potion containing a unique generic array tag ("UGST"); ii) A second portion complementary to the stem-loop structure or complement of the allele-specific probe; Preferably a polymerase elongation blocker. Includes; b) Generic reporter molecules: i) The first member of a fluorophore / quencher pair; ii) Stem-loop structure; iii) The second member of a fluorophore / quencher pair; iv) UGST binding sites that are complementary to UGST on the mediator probe; v) Polymerase elongation blockers; Includes, A member of the fluorophore / quencher pair is placed via a stem-loop to quench the fluorophore. To amplify; - Detecting the signal produced by the fluorophore of a fluorophore / quencher pair in the presence of the target nucleic acid, thereby detecting the presence of the target nucleic acid. Includes.

[0032] In some embodiments of the methods disclosed herein, the allele-specific primer includes an ARMS primer.

[0033] In some embodiments, the systems or methods disclosed herein include a reference system comprising a reference target nucleic acid sequence comprising at least a portion of the KIF11 gene sequence, including the non-coding region of the KIF11 gene. In some embodiments, the reference system comprises one or more of SEQ ID NOs: 69-80, preferably: SEQ ID NOs: 69 and 70; SEQ ID NOs: 71 and 72; SEQ ID NOs: 73 and 74; SEQ ID NOs: 75 and 76; SEQ ID NOs: 77 and 78; and a primer pair selected from SEQ ID NOs: 79 and 80; preferably the amplified region is detected by a probe selected from SEQ ID NOs: 81-86, preferably via a generic reporter selected from SEQ ID NOs: 87 and 88.

[0034] In some embodiments, the disclosed systems, methods, kits, and their components provide superior results compared to prior art methods. For example, but not limited to, in some embodiments, the disclosed systems, methods, kits, and components are less expensive (e.g., compared to NGS), easier to use (e.g., compared to any other approach, e.g., qPCR or NGS in a plate-based system), and / or faster to design and commercialize (e.g., compared to any other approach, see above).

[0035] In yet another general embodiment, methods, kits, uses of their components, and each of their embodiments described herein are provided for advantageous applications including, for example, the detection of multiple gene targets, possibly in a sample obtained from a patient. The patient may be a cancer patient, a patient with an infectious disease, a transplant recipient, or a pregnant expectant mother. If the patient is a cancer patient, advantageous uses of the disclosed methods, kits, and components include, but are not limited to, post-NGS analysis patient surveillance, monitoring of response to treatment or therapy, detection or monitoring of minimal residual disease (MRD), post-operative follow-up, or personalized cancer neoantigen-targeted immunotherapy selection.

[0036] In yet another general embodiment, a method for detecting a target nucleic acid from a subject: The process involves amplifying a nucleic acid sample from a target using a target-specific primer pair, At least one member of the target-specific primer pair (preferably allele-specific and) includes a stem-loop structure ("FuseTag") when bound to the target, The amplification reaction was carried out in the presence of the generic reporter molecule (2); a) FuseTag from 5' to 3': i) A first potion containing a unique generic array tag ("UGST"); ii) Stem-loop structure; iii) A second potion that is complementary to the target and preferably allele-specific; Includes, b) The generic reporter molecule (2) is: i) The first member of a fluorophore / quencher pair; ii) Stem-loop structure; iii) The second member of a fluorophore / quencher pair; iv) UGST binding sites that are complementary to UGST on FuseTag; v) Polymerase elongation blockers; Includes, A member of a fluorophore / quencher pair is placed via a stem-loop structure to quench the fluorophore. To amplify; The presence of the target nucleic acid is detected by detecting the signal produced by the fluorophore of a fluorophore / quencher pair in the presence of the target nucleic acid. A method is provided that includes this.

[0037] In another embodiment, a method for quantifying the number of target nucleic acids in a sample relative to the KIF11 nucleic acid in the sample: 1. In a KIF11 amplification reaction, the KIF11 nucleic acid contained in the sample is amplified using a KIF11-specific primer pair, i. Each member of the KIF11-specific primer pair is complementary to—independently of—a KIF11 region located within an exon, intron, or non-coding sequence of the KIF11 gene; ii. The KIF11 amplification reaction is carried out in the presence of a KIF11-detectable probe. Amplifying KIF11 nucleic acid; 2. To detect the signal produced in a KIF11 amplification reaction using a KIF11-detectable probe; 3.2. Quantify the signal; 4. Amplifying a target nucleic acid from a sample using a target-specific primer pair in a targeted amplification reaction; i. The target amplification reaction is carried out in the presence of a target-detectable probe. Amplifying target nucleic acids; and 5. To detect signals produced by target-detectable probes in targeted amplification reactions; Quantifying the signal in 6.5; 7.3. Standardize the quantified signal from 6. to the quantified signal from 7.3., thereby quantifying the number of target nucleic acids in the sample relative to the KIF11 nucleic acids in the sample. Methods including the following are disclosed.

[0038] In a further embodiment, a method for determining the presence of gDNA in a sample: 1. Amplifying the KIF11 nucleic acid contained in a sample using a first and second KIF11-specific primer pair in a KIF11 amplification reaction, a. At least one member of the first KIF11-specific primer pair is complementary to a KIF11 intron or a non-coding sequence of the KIF11 gene; b. An amplification reaction using a first KIF11-specific primer pair is carried out in the presence of a first KIF11-detectable probe; c. Each member of the second KIF11-specific primer pair is located within the KIF11 exon; d. The amplification reaction using a second KIF11-specific primer pair is carried out in the presence of a second KIF11-detectable probe. To amplify; 2. To detect the signals produced by the first and second KIF11-detectable probes in the first and second KIF11 amplification reactions; 3. Quantify the signals from the first and second KIF11 amplification reactions; 4. Standardizing the quantified signal of the first amplification reaction against the quantified signal of the second amplification reaction, thereby determining the presence of gDNA in the sample, preferably determining that the sample is mitochondrial DNA, cDNA, mRNA, rRNA, tRNA, hnRNA, microRNA, lncRNA, cfDNA, cell-free tumor DNA, or siRNA sample. Methods including the following are disclosed.

[0039] In a further embodiment, a method for determining the integrity of nucleic acids in a sample: 1. Amplifying the KIF11 nucleic acid contained in a sample using a first and second KIF11-specific primer pair in a KIF11 amplification reaction, a. Each member of the first KIF11-specific primer pair is complementary to—independently of—a KIF11 region located within an exon, intron, or non-coding sequence of the KIF11 gene; b. An amplification reaction using a first KIF11-specific primer pair is carried out in the presence of a first KIF11-detectable probe; c. Each member of the second KIF11-specific primer pair is complementary to—independently to—an exon, an intron-based KIF11 region, or a non-coding sequence of the KIF11 gene; d. An amplification reaction using a second KIF11-specific primer pair is carried out in the presence of a second KIF11-detectable probe; e. Preferably, the amplicons generated by the amplification reaction of the first and second KIF11-specific primer pairs are located far enough apart to avoid interference, for example, the amplicons are at least 600 base pairs (bp), such as at least 1 kilobase pair, at least 700 bp, 800 bp, 900 bp, or even more. To amplify; 2. Determining a threshold in each of the nucleic acid amplification reactions, ○ Measuring at least one signal whose intensity is related to the amount of nucleic acid sequence amplified in the reaction at multiple different time points during the amplification reaction; ○By determining the number of cycles related to the characteristics of the derivative that represent the threshold, To make a decision; 3. Compare the thresholds (threshold cycle counts) of the first and second KIF11 amplification reactions; Includes, 4. The difference (ΔCq) between the threshold cycle counts of the first and second KIF11 amplification reactions is a measure of the completeness of the genomic DNA; Step 2 is optional. ○ Deriving the amplification curve from the signal measurement; ○Calculate the derivative of the amplification curve and confirm the characteristics of the derivative; ○Calculate the second derivative of the amplification curve. It can include, The features include a positive peak in the second derivative. The method will be disclosed.

[0040] Furthermore, in a further embodiment, a method for determining gDNA fragmentation, The first, second, and third amplification reactions preferably generate three (first, second, and third) KIF11 amplicons of lengths identifiable by PCR; • Determining the Cq values ​​of the first, second, and third amplification reactions; • To compare the Cq of the first, second, and third amplification reactions; Includes, The difference in Cq values ​​between the first, second, and third amplification reactions is an indicator of gDNA fragmentation. The method will be disclosed.

[0041] Furthermore, in a further embodiment, the method for determining gDNA fragmentation can also be used to evaluate the contamination of cell-free DNA or cell-free tumor DNA with more intact genomic DNA derived from leukocytes. • To generate a first KIF11 amplicon ("short") by a first amplification reaction; and • The second amplification reaction generates a second KIF11 amplicon ("long"); • Determining the Cq values ​​of the first and second amplification reactions; • Determining the ΔCq values ​​of the first and second amplification reactions; Includes, ΔCq is an indicator of gDNA fragmentation. The method will be disclosed.

[0042] Furthermore, in a further embodiment, a use is disclosed for amplifying a region of the genomic reference gene KIF11 of a kit including primers and instructions including an amplification protocol and analysis of results, wherein the primers are a primer pair selected from: SEQ ID NOs: 69-80, preferably: SEQ ID NOs: 69 and 70; SEQ ID NOs: 71 and 72; SEQ ID NOs: 73 and 74; SEQ ID NOs: 75 and 76; SEQ ID NOs: 77 and 78; and SEQ ID NOs: 79 and 80; preferably, the amplified region is detected by a probe selected from SEQ ID NOs: 81-86, preferably via a generic reporter selected from SEQ ID NOs: 87 and 88.

[0043] Furthermore, in a further embodiment, a method for quality control of processing, isolation, and amplification processes: • Sample processing; • Nucleic acid isolation; • To generate three KIF11 amplicons of distinctly different lengths through amplification reactions; • Determine the Cq value of each of the KIF11 amplicons; Includes, The Cq value of the KIF11 amplicon is a measure of quality control for processing, isolation, and amplification. The method will be disclosed.

[0044] Furthermore, in a further aspect, it is a kit: (a) At least one probe designed to anneal to the amplicon of the KIF11 gene; (b) Products and reagents required to carry out the annealing reaction; and (c) Instructions for use; Includes, The probe is a probe that is at least in part specific to the amplicons described herein. The kit will be revealed.

[0045] Furthermore, in a further embodiment, a system for the automated processing of biological samples: An enclosure configured to include one or more sample processing modules, each sample processing module configured to hold a removable cartridge as described herein, wherein the system is configured to operate the sample processing module to perform PCR to determine the presence and / or quantity of one or more target genes, and optionally to determine the level of one or more target DNA sequences in the corresponding removable sample cartridge, wherein the processing of the sample in the corresponding removable sample cartridge is: Enclosure: • Providing a sample into the inlet port for receiving the biological sample of the cartridge; and implementing a method including using the cartridge: A means for isolating nucleic acids from a biological sample received in an inlet port, the means being able to enter into fluid communication with the inlet port for receiving the biological sample; Multiple chambers containing reagents and / or buffers for performing PCR, which are fluidly connected to the inlet port for receiving biological samples and located downstream thereof; • Includes a chamber containing the PCR mix; • Containing at least one chamber containing primers for amplifying all or one region of the KIF11 gene; and • Includes a probe for detecting all or a region of the KIF11 gene, The multiple chambers, - Performing nucleic acid amplification within the chamber to detect and / or quantify the target nucleic acid. Includes, The KIF11 gene is used as an internal standard to detect and / or quantify the target nucleic acid. The system will be disclosed.

[0046] In a preferred embodiment, in a method, use, kit, kit of components, system and its components, at least one member of a target-specific primer pair is allele-specific and includes a stem-loop structure when bound to the target.

[0047] Furthermore, in a further embodiment, a kit is disclosed for performing PCR to detect and / or quantify one or more target genes and optionally detect and / or quantify nucleic acids, the kit comprising a cartridge as described herein and optionally a container containing instructions for use. [Brief explanation of the drawing]

[0048] For a thorough understanding, please refer to the following detailed explanation in conjunction with the attached drawings. [Figure 1] This diagram illustrates a typical workflow where all generic reagents (i.e., non-panel-specific reagents) are present inside the generic cartridge, while panel-specific reagents and samples are added by the user through the sample inlet port of the generic cartridge. [Figure 2] This diagram illustrates the reaction mechanism of mediator probe PCR. Extension of the forward (FW) primer by polymerase leads to hydrolysis of the target-specific components of the mediator probe and the release of the free mediator. In the next step, the free mediator can bind to the generic reporter. Note that the fluorophore and quencher of the generic reporter can be exchanged (not shown). Once the free mediator has been extended, a fluorescent signal is generated by substitution of the quencher or fluorophore modification and / or hydrolysis of the quencher or fluorophore-binding nucleotide (not shown). Note that non-hydrolyzable mediator probes and generic reporters cannot be extended by polymerase (indicated by squares); RE primers are reverse primers. [Figure 3A]This figure illustrates the performance of ARMS primers for mutation detection in a 96-well format qPCR instrument in singleplex (i.e., including only the primers required to amplify one target) and multiplex (i.e., including the primers required to amplify multiple targets). Targets are added as synthetic mutant targets (EGFR G719A; EGFR InsFQEA, EGFR L861Q) at various concentrations in the PCR reaction shown in the legend (which always includes 10,000 copies of wild-type genomic DNA, as well as a mixture of oligonucleotides of primers, mediator probes, and generic reporters, and polymerase, dNTPs, and PCR salts). Raw curves are shown; X axis: number of PCR cycles, Y axis: arbitrary fluorescence units. Black squares represent conditions where 10,000 copies of wild-type genomic DNA are present, but no synthetic mutant targets are added. [Figure 3B] This figure illustrates the performance of ARMS primers for mutation detection in a 96-well format qPCR instrument in singleplex (i.e., including only the primers required to amplify one target) and multiplex (i.e., including the primers required to amplify multiple targets). Targets are added as synthetic mutant targets (EGFR G719A; EGFR InsFQEA, EGFR L861Q) at various concentrations in the PCR reaction shown in the legend (which always includes 10,000 copies of wild-type genomic DNA, as well as a mixture of oligonucleotides for primers, mediator probes, and generic reporters, and polymerase, dNTPs, and PCR salts). Cq values ​​are shown; X axis: number of PCR cycles, Y axis: arbitrary fluorescence units. Black squares represent conditions where 10,000 copies of wild-type genomic DNA are present but no synthetic mutant targets are added. [Figure 4]This figure illustrates the performance of ARMS primers in multiplex (i.e., an oligonucleotide mixture containing primers and mediator probes required to amplify multiple targets) in an integrated sample-to-result instrument. Targets are added as synthetic mutant targets at varying concentrations through the sample inlet port, along with the oligonucleotide mixture containing primers, mediator probes, and a pre-quantified formalin-fixed paraffin-embedded (FFPE) clinical sample containing approximately 7,000 copies of genomic DNA per PCR chamber. Generic reporters, polymerases, and dNTPs are spotted in different chambers of the cartridge. Mutants (synthetic targets): 100 (1.4%) - 50 (0.7%) - 10 (0.1%) - 0 (0.0%) copies per PCR. The PCR salt is generic and is the liquefied buffer portion present in one of the reagent containers of the cartridge. RFU = Relative Fluorescence Units, i.e., the signal obtained from the qPCR components of the integrated sample-to-result instrument; [Figure 5] This figure illustrates the performance of an integrated sample-to-result instrument for detecting wild-type genomic DNA in multiplex (i.e., including an oligonucleotide mixture containing primers and mediator probes required to amplify multiple targets). The oligonucleotide mixture, including primers, mediator probes, and either an FFPE clinical sample or extracted DNA, was added through the sample inlet port. A generic reporter, polymerase, and dNTPs were spotted in different chambers of the cartridge. The PCR salt was generic and part of the liquefaction buffer present in one of the reagent containers of the cartridge. The Y-axis represents signal intensity, the X-axis represents cycle count, and A-E represent different PCR compartments. [Figure 6A]This diagram illustrates a concept enabling the discrimination of neighboring markers, where the ARMS primer includes a stem-loop structure whose stem can be arbitrarily composed of a target sequence, and the 3' end of the primer is shown as one design of the ARMS primer containing a stem-loop structure with a sequence specific to the target sequence. The binding site of the mediator probe overlaps (at least partially) with the ARMS primer (indicated as "FW primer containing stem-loop"). When the target is not amplified, the mediator probe cannot bind to the target sequence and therefore cannot produce a signal. The mediator probe also cannot bind to the ARMS primer because it is within the stem-loop structure and therefore inaccessible to the mediator probe. When the target is amplified, the stem structure can be unfolded by polymerase, thereby creating a binding site for the mediator probe. Once bound, free mediators are released as the other primer extends, and these free mediators can bind to the spotted generic reporter and produce a signal. [Figure 6B]This diagram illustrates a concept that enables the discrimination of neighboring markers, where the ARMS primer includes a stem-loop structure in which the stem can be arbitrarily composed from the target sequence, and the stem-loop structure defines the 5' end of the primer, which can be used as a generic terminal tag. Similar to Panel A, the binding site of mediator probe 2 overlaps (at least partially) with that of the ARMS primer (indicated as "G12C primer"). When the target is not amplified, mediator probe 2 cannot bind to the target sequence and therefore cannot produce a signal. Mediator probe 2 also cannot bind to the ARMS primer (G12C primer) because it is within the stem-loop structure and therefore inaccessible to the mediator probe. When the target is amplified, the stem structure can be unfolded by polymerase, thereby creating a binding site for the mediator probe. Once bound, free mediators are released as the other primer extends, and these free mediators can bind to the spotted generic reporter and produce a signal. The presence of mediator probe 1 is optional but can be used as a positive control and / or to increase the options for detecting the target. The mediator probe consists of a first potion containing a unique generic sequence tag ("UGST") complementary to the UGST binding site of the corresponding generic reporter molecule (universal reporter) from 5' to 3', a second potion that is complementary to the target (mediator probe 1) or complementary to the ARMS primer (mediator probe 2), and a polymerase elongation blocker (indicated by a square box).A generic reporter molecule (universal reporter) is a single-stranded DNA molecule and includes: i) a first member of a fluorophore / quencher pair; ii) a stem-loop structure; iii) a second member of a fluorophore / quencher pair; iv) a unique generic sequence tag ("UGST") binding site; and v) a polymerase elongation blocker. [Figure 6C] This diagram illustrates a concept that enables the discrimination of neighboring markers, where the ARMS primer includes a stem-loop structure whose stem can be arbitrarily composed of a target sequence, illustrating the “FuseTag” concept that enables the discrimination of neighboring markers. Compared to panel B, the forward ARMS primer is modified ("FuseTag" primer), and here from 5' to 3': a first portion containing a unique generic sequence tag ("UGST"), which when released is shown as free mediator 2 in Figure 6C; a stem-loop structure; and a second portion that is complementary to (and preferably allele-specific to) the target. When the specific target is not amplified by the FuseTag primer, the unique generic sequence tag (mediator 2) is not released and therefore cannot produce a signal. However, mediator probe 1 can still be hydrolyzed by another forward primer present in the multiplex reaction mixture. [Figure 7A]Figure 6 illustrates the demonstration of distinguishing between KRAS G12C and wild-type background from KRAS G12D according to the concept shown in the diagram, using a multiplex (i.e., including an oligonucleotide pool containing primers and mediator probes required to amplify multiple targets). ARMS primers containing stem-loops to enable selective amplification of KRAS G12C are added along with reverse primers and mediator probes designed to bind to the stem-loop portion of the incorporated KRAS G12C ARMS primers. The mix also contains 10,000 genomic copies of wild-type DNA along with polymerase and all other components required to initiate a functional PCR reaction. Either a synthetic KRAS G12C mutant target (see panel A) or a synthetic KRAS G12D mutant target (see panel A) is added to the mix in a 10-fold titration series (with an estimated input of 5000-500-50-0 copies / PCR). The mix is ​​added to different wells of a 96-well qPCR-compatible plate. PCR curves are shown. Observational data show that stem loops within ARMS primers enable differentiation from KRAS G12D to KRAS G12C and wild-type background down to a minimum of 500 copies in 10,000 genomic copies of wild-type DNA. [Figure 7B]Figure 6 illustrates the demonstration of discriminating between KRAS G12C and wild-type background from KRAS G12D according to the concept shown in the diagram, using a multiplex (i.e., including an oligonucleotide pool containing primers and mediator probes required to amplify multiple targets). ARMS primers containing stem-loops to enable selective amplification of KRAS G12C are added along with reverse primers and mediator probes designed to bind to the stem-loop portion of the incorporated KRAS G12C ARMS primers. The mix also contains 10,000 genomic copies of wild-type DNA along with polymerase and all other components required to initiate a functional PCR reaction. Either a synthetic KRAS G12C mutant target (see left panel B) or a synthetic KRAS G12D mutant target (see right panel B) is added to the mix in a 10-fold titration series (with an estimated input of 5000-500-50-0 copies / PCR). The mix is ​​added to different wells of a 96-well qPCR-compatible plate. Figure 7A shows the Cq values ​​of the PCR curve. The observed data indicate that the stem loop within the ARMS primer enables differentiation of KRAS G12C and wild-type background from KRAS G12D to a minimum of 500 copies or less of wild-type DNA in 10,000 genome copies. [Figure 7C]Figure 6 illustrates the demonstration of discriminating between KRAS G12C and wild-type background from KRAS G12D according to the concept shown in the diagram, using a multiplex (i.e., including an oligonucleotide pool containing primers and mediator probes required to amplify multiple targets). An ARMS primer containing a stem-loop enabling selective amplification of KRAS G12C is added along with a reverse primer and a mediator probe designed to bind to the stem-loop portion of the incorporated KRAS G12C ARMS primer. The mix also contains 10,000 genomic copies of wild-type DNA, along with polymerase and all other components required to initiate a functional PCR reaction. The performance of the same ARMS primer containing a stem-loop for KRAS G12C in a one-piece sample-to-result instrument in the presence of pre-quantified formalin-fixed paraffin-embedded (FFPE) wild-type samples containing approximately 10,000 copies of genomic DNA per PCR chamber and synthetic mutant targets of various copy numbers shown is also illustrated. [Figure 8] This diagram illustrates how the degree of fragmentation can be verified using a KIF11-based QCplex. The right-hand panel shows a good quality sample; in this case, all curves (1, 2, and 3) pass through the same threshold per Cq value. In the middle panel, the curves for the intermediate (2) and longest (3) amplicons are shifted to the right (i.e., towards higher Cq values). This means the sample contains fewer "long" DNA and is therefore fragmented. The left-hand panel represents a highly fragmented sample, as can be seen from the absence of a qPCR result for the longest amplicon (3) and the shift of the intermediate amplicon (2) towards higher Cq values. [Figure 9A]This figure shows that the delta Cq between long and short KIF11 amplicons detected by mediator reaction strongly correlates with the degree of DNA fragmentation in the evaluated FFPE sample. The approximation curve for short KIF11 amplicons is shown by a line with a circle, while the approximation curve for long KIF11 amplicons is shown by a normal line. Panel A shows the following: no fragmentation, high-quality gDNA sample, delta Cq (long-short): 0.2. [Figure 9B] This figure shows that the delta Cq between long and short KIF11 amplicons detected by mediator reaction strongly correlates with the degree of DNA fragmentation in the evaluated FFPE samples. The approximation curve for short KIF11 amplicons is shown by a line with a circle, while the approximation curve for long KIF11 amplicons is shown by a normal line. Panel B shows the following: low fragmentation samples; delta Cq (long-short) 1.2 (left) and 0.9 (right). [Figure 9C] This figure shows that the delta Cq between long and short KIF11 amplicons detected by mediator reaction strongly correlates with the degree of DNA fragmentation in the evaluated FFPE samples. The approximation curve for short KIF11 amplicons is shown by a line with a circle, while the approximation curve for long KIF11 amplicons is shown by a normal line. Panel C shows the following: moderately fragmented samples; delta Cq (long-short) 4.7 (left) and 5.6 (right). [Figure 9D] This figure shows that the delta Cq between long and short KIF11 amplicons detected by mediator reaction strongly correlates with the degree of DNA fragmentation in the evaluated FFPE samples. The approximation curve for short KIF11 amplicons is shown by a line with a circle, while the approximation curve for long KIF11 amplicons is shown by a normal line. Panel D shows the following: highly fragmented samples; delta Cq (long-short) 9.4 (left) and N / A (long amplicon not detected; right). [Figure 10]This figure shows mutation detection using FuseTag primers in singleplex PCR on a 96-well format qPCR instrument for four different targets: top left: BRAF V600E; top right: EGFR E709K; bottom left: EGFR S768I; bottom right: EGFR L861Q; the X-axis shows the number of PCR cycles; the Y-axis shows fluorescence (in arbitrary units). The targets are added as 200 copies of synthetic mutant targets in the PCR reaction (which always includes 2,000 copies of wild-type genomic DNA, as well as a universal reporter, polymerase, dNTPs, and PCR salts). The black line represents the amplification result of 200 copies in a gDNA background, and the gray line represents the case where wild-type genomic DNA is present but no synthetic mutant targets are added. [Figure 11] This figure illustrates mutation detection using FuseTag primers in multiplex PCR on a 96-well format qPCR instrument in a gDNA background. Targets are added as synthetic mutant targets at varying concentrations, along with primers and mediator probes, as well as an oligopool containing 1000 copies of wild-type genomic DNA. A generic reporter, polymerase, dNTPs, and PCR salts (e.g., MgCl2) are added to each reaction along with a liquefaction buffer containing the components necessary to release DNA from the FFPE sample. Triangles represent 500 copies of the BRAF V600E target; black circles represent 100 copies of the target; and diamonds represent 20 copies of the target. Squares represent the negative control, i.e., 0 copies of the target. The X-axis shows the number of PCR cycles; the Y-axis shows fluorescence (in arbitrary units).

[0049] Detailed explanation This disclosure provides methods, kits, component kits, systems, and components thereof for performing multiplex detection of gene targets using a customized gene target panel in a generic detection cartridge.

[0050] It has been shown that using a system, or a part of a system, method, kit, kit of components, and their components, it is possible to detect the presence of one or more target sequences using a multiplex generic (i.e., non-target-specific and therefore inexpensive) fluorescently labeled reporter that can exceed the number of fluorescent signals that can be discriminated in a single PCR reaction chamber in an instrument requiring only a single sample inlet source. In contrast, contemporary techniques are limited by the number of fluorescent signals that can be discriminated in a single PCR reaction (for example, in the case of Idylla® by conventional techniques, six signals can be discriminated because there is no distinction as to what goes into each of the different PCR chambers (only one sample inlet exists)). This technique allows the use of generic fluorescently labeled reporters while simultaneously leveraging the multiplex capability across all PCR reaction chambers available in the instrument.

[0051] In a first general embodiment, a method for detecting multiple gene targets: providing a mix of multiple oligonucleotide subsets, each of which subsets is specific to a gene target and contains a unique generic sequence tag in the subset, To provide a mix of multiple oligonucleotide subsets, each of which subsets is adapted to produce a detectable nucleic acid product containing a unique generic sequence tag under nucleic acid amplification conditions and in the presence of a gene target; apart from the mix, (i) an inlet port for receiving a biological sample and / or the mix; (ii) a nucleic acid isolation compartment located downstream of the inlet port; (iii) reagents for nucleic acid amplification; (iv) one or more nucleic acid amplification compartments located downstream of a nucleic acid extraction chamber; and (v) a plurality of generic reporters, each of which contains a generic sequence specific to the unique generic sequence tag contained in one of the detectable nucleic acid products. A method is provided to provide an integrated fluid cartridge containing a plurality of generic reporters adapted to generate a signal in the presence of a mix and a biological sample or isolated nucleic acid, into which a user inserts the mix and the cartridge, and to operate the cartridge after insertion of the biological sample and the mix, which includes performing nucleic acid isolation from the biological sample, followed by multiplex nucleic acid amplification (inside the cartridge) of the mix; and to further include detecting a signal generated from at least one of the plurality of generic reporters contained inside the integrated fluid cartridge, if at least one detectable nucleic acid product is generated from the amplification.

[0052] As used herein, the term “gene target” (and as used herein interchangeably with “target nucleic acid” unless specifically required by context) refers to any gene, transcript, general nucleic acid, or any fragment or form of any of the above that can be targeted for detection or investigation by a diagnostic assay. Examples of gene targets include, but are not limited to, genes (sometimes called “target genes”), gene variants, specific mutations or short nucleotide polymorphisms (SNPs) within a gene, allelic types, or gene variants. As used herein, the term “variant” may refer to any gene variant, i.e., any genetic feature that is known or expected to differ across gene samples. As used herein, the term “variant” may be interpreted as a type of “gene target” and may refer to a specific mutation, SNPs, or gene rearrangement including replication and deletion. Gene rearrangements, replication, and deletions may affect small regions, such as regions of one or a few base pairs (bp), or large regions, such as large chromosomal deletions that extend over multiple kilobase pairs (kbp). In this context, the term “variant” typically refers to a known genetic difference between a tissue requiring monitoring and a normal tissue in a subject, and can be treated as synonymous with the terms “specific allele,” “mutation,” “SNP,” and “variant,” according to their standard meanings as used in the fields of molecular biology and biotechnology. When an allele-specific primer pair member is referred to herein, the member binds to the specific allele under appropriate conditions but does not bind to any other allele of the gene. Similarly, when an allele-specific probe is referred to herein, the probe binds to and / or detects the specific allele of the gene under appropriate conditions but does not bind to and / or detect any other allele.

[0053] As used herein, the term “oligonucleotide” refers to relatively short, or oligomeric, nucleic acids, typically less than 200 nucleotides ("nt"). Oligonucleotides are often synthetic and can contain various modifications, such as modified bases, or can be conjugated into various molecules, such as different functionalities. As used herein, the term “oligonucleotide subset” should be interpreted as a functionally associated group of oligonucleotides that are specific to a “gene target.” Oligonucleotides in an oligonucleotide subset will typically hybridize, depending on the application, in or around a sequence that covers or is adjacent to a gene target, perhaps to enable amplification or detection of the gene target. A typical target-specific oligonucleotide subset will also include at least one primer, possibly a primer pair, and possibly an oligonucleotide probe that is specific to the gene target, such as a gene variant or mediator probe. Generally, the term “oligonucleotide mixture” will be used herein to represent a target-specific amplification primer pair ("oligonucleotide subset") and probe, preferably a target-specific probe, and more preferably a mediator probe. As used herein, the term "multiple" in "multiple oligonucleotide subset," "multiple gene targets," or "multiple oligonucleotide mixture" should be understood to refer to more than one, for example, multiple gene targets. In the context of multiplex amplification or multiplex PCR, the term "multiple" usually refers to more than one, such as within the range of 2, 3, 4, 5, 6, 7, 8, 9, 10, or multiples of 10. The term "mix of multiple oligonucleotide subsets" should then be interpreted as a composition, possibly a solution, or at least a partially dried form thereof (e.g., a lyophilized form) containing multiple oligonucleotide subsets mixed together. In some contexts, the term "mix" may also refer to a "panel" or "set" containing multiple oligonucleotide subsets.

[0054] As used herein, the terms “nucleic acid” and its synonym “polynucleotide” are given the common sense in the art and refer to polymers of primarily ribonucleotides or primarily deoxyribonucleotides linked to one another by phosphodiester bonds between nucleotide monomers. (Deoxy)nucleotides are phosphorylated forms of (deoxy)nucleosides, most commonly adenosine, guanosine, cytidine, thymidine, or uridine. These nucleosides consist of a pentose sugar, which is ribose or deoxyribose, and a nitrogenous base ("nucleic acid base," or simply "base"), which is one of adenine, guanine (a purine), cytosine, thymine, or uracil (a pyrimidine). The sequence in which these bases (or their nucleosides, or the latter nucleotides) follow each other in a nucleic acid chain is called the “nucleic acid sequence” and is conventionally indicated as the so-called 5'-to-3'-direction, referring to the chemical orientation of the nucleic acid chain. The "5'" derives from a reference to the 5' carbon of the first (deoxy)ribose ring where the nucleic acid sequence reading begins, and the "3'" derives from the 3' carbon of the last (deoxy)ribose ring where the nucleic acid sequence reading ends. A nucleic acid sequence could be, for example, ATATGCC, which should be interpreted herein as referring to the 5'-ATATGCC-3' nucleic acid sequence. Under the same convention, the latter sequence would be complementary to the sequence 5'-GGCATAT-3', or simply GGCATAT. Nucleic acids include, but are not limited to, genomic DNA, mitochondrial DNA or methylated DNA, cDNA, mRNA, rRNA, tRNA, hnRNA, microRNA, lncRNA, siRNA, and various modified versions thereof, as well as DNA and RNA. Nucleic acids can most commonly be obtained from natural sources, such as biological samples obtained from different types of organisms. On the other hand, nucleic acids can also be synthesized, recombined, or otherwise produced by any of the known artificial methods (e.g., PCR).

[0055] When used herein, the term “separately” in the specific context of a mix of multiple oligonucleotide subsets provided separately from a cartridge, or a cartridge provided separately from the mix, should be understood to mean that there is no physical connection between the mix and the cartridge until the moment the user inserts the mix or a portion of it into the cartridge. For example, the mix may be provided in liquid form inside a vial or tube or any other container. In such an example, the user would more likely open such a container and pour its contents, including the mix, into the cartridge, or transfer them, for example, by pipette. Alternatively, the mix may be provided into the cartridge spotted and / or absorbed onto a solid medium such as cellulose, or onto a piece of parchment, and bound to the medium. Other alternatives also exist, including beads, soluble tablets, or capsules. In any of these events, the mix in the context of the present invention is not physically contained therein until simultaneously or later transferred into the cartridge by the user before the biological material or isolated nucleic acid is also provided into the cartridge. In possible examples, the mix and cartridge may even be provided at different times, for example, when they are sold or shipped to the user on different days.

[0056] When used herein, the term “biological sample,” or simply “sample,” is intended to include a variety of specimens or solutions of a biological source containing nucleic acids and / or cellular material, whether it is freshly obtained from an organism (i.e., a fresh tissue sample) or preserved by any method known in the art (e.g., frozen or FFPE sample). Examples of biological samples include: cell cultures such as mammalian cells (but also eukaryotic microbial cultures), body fluids, body fluid precipitates, wash specimens, fine-needle aspirations, biopsy specimens, tissue specimens, cancer cells, other types of cells obtained from a patient, cells or in vitro cultured cells from tissues of an individual being tested and / or treated for disease or infection, or forensic specimens. Non-limiting examples of body fluid specimens include whole blood, bone marrow, cerebrospinal fluid (CSF), ascites, pleural fluid, lymph, serum, plasma, urine, chyle, feces, ejaculate, sputum, nipple aspirates, saliva, swab specimens, wash or perfusion fluids and / or brush specimens. In one embodiment, the sample is a mitochondrial DNA, cDNA, mRNA, rRNA, tRNA, hnRNA, microRNA, lncRNA, cfDNA, cell-free tumor DNA, or siRNA sample.

[0057] As used herein, the terms “liquid biopsy” or “liquid biopsy sample” are understood to refer to any non-tissue specimen, in particular a fluid sample obtained from a subject. Liquid biopsy sources include, but are not limited to, blood, plasma, serum, urine, cerebrospinal fluid (CSF), amniotic fluid, saliva, sweat, tears, breast milk, semen, feces, pleural fluid, ascites, or other fluids. Analysis of nucleic acids in liquid biopsy samples can minimize the need for costly, invasive, and often painful tissue and / or tumor biopsies, enabling the monitoring of dynamic disease or other physiological conditions. For example, in cancer patients, cell-free tumor DNA or RNA extracted from a liquid biopsy may be used in some cases to detect mutations, translocations, or copy number changes, and the expression of specific cancer markers. Blood (as well as plasma, serum, or whole blood) is the most commonly described fluid used in the analysis of liquid biopsy samples in humans. In cancer patients, blood is a source of circulating tumor cells (CTCs), as well as cell-free DNA (cfDNA) and cell-free RNA (cfRNA), including circulating tumor DNA (ctDNA) and circulating tumor RNA (ctRNA), respectively, released by tumor tissue. These can be used to detect mutations present in the patient's tumor. DNA may or may not be methylated. However, it is noteworthy that ctDNA contains only a small fraction of the cfDNA present in blood, highlighting the importance of maximizing the sample volume for nucleic acid analysis to detect rare mutations. Furthermore, cfDNA is always of low quality, fragmented to the approximate size of nucleosomes (140 bp–170 bp). Consequently, for certain cancer types, including kidney, prostate, and upper and lower urothelial carcinoma, alternative liquid biopsy approaches such as urine may be a richer source of tumor-derived material. Urine also has other unique advantages, such as ease of collection (no trained medical staff required), lack of patient discomfort (improved patient compliance), and potentially fewer contaminating proteins compared to blood.

[0058] Furthermore, as used herein, the term “nucleic acid isolation” should be interpreted as any form of releasing nucleic acids from biological material to make them available for amplification. Within the scope of this disclosure, the term may encompass any procedure involving the liquefaction of a biological sample or any nucleic acid extraction or purification on a solid support such as silica.

[0059] The term “polymerase chain reaction” or “PCR” should be understood to refer to a common laboratory nucleic acid amplification technique that relies on thermal cycling as well as the use of at least primers, typically primer pairs, and DNA polymerase. “Quantitative PCR” or simply “qPCR” is used herein to define a PCR-based experimental technique used to amplify and possibly simultaneously detect or quantify a target DNA molecule. In contrast to standard PCR, where the reaction product is detected at its end, i.e., after thermal cycling is complete, a key feature of qPCR is that the DNA product is detected during thermal cycling while the reaction proceeds in “real time”; hence, qPCR is also known as “real-time PCR.” Currently, many different types of qPCR exist. For example, qPCR can be used to quantify the number of messenger RNAs when starting from a reverse transcription (RT) step, and is thus called reverse transcriptase qPCR or RT-qPCR. Where used herein, the term “quantitative PCR” or simply “qPCR” is used preferentially over the term “real-time PCR” or “RT-PCR” to avoid confusion with reverse transcription PCR (often abbreviated as RT-PCR). Most qPCRs use one of the following most common methods for detecting fluorescence-involved product amplification in real time: (a) intercalation of a nonspecific fluorescent dye with any double-stranded DNA; (b) fluorescence is generated by a nucleic acid-binding fluorescent dye upon binding to double-stranded DNA; (c) fluorophores are released by probe digestion during primer extension; or (d) fluorescence is emitted by a fluorescent dye bound to a probe that fluoresces after binding to the target during nucleic acid synthesis. Fluorescence emitted from the reaction mixture is monitored in real time as the amplification reaction occurs, although the fluorescence is too weak to be distinguished from the background during the initial amplification cycles. Fluorescent signals generated during thermal cycling are detected by appropriate optical detection systems and tracked from the moment the background threshold is exceeded until the reaction plateaus.The copy number of a target sequence can be estimated using either relative or absolute quantification methods, typically by analyzing the shape of the resulting amplification curve (standard curve method), by comparison with a standard reference, or by determining when the signal exceeds a certain threshold (often called the Ct value, but sometimes also called the Cp value or Cq value). In relative quantification, the target nucleic acid level estimated in a given sample using Ct or standard curve analysis is expressed in comparison to the value obtained for the same target in another reference sample, e.g., an untreated control sample. Conversely, in absolute quantification, the qPCR signal can be related to the input copy number using a standard curve, or it can be calculated according to more recent digital PCR methods. Currently, the first method is more prevalent and forms the basis for estimating the amount of target DNA by comparing the obtained value with a pre-made standard curve. These and other qPCR quantification methods are widely known in the art, and their calculations can vary more or more depending on the given application and qPCR system.

[0060] Fluorescence is by far the most commonly used method, but any measurable characteristic can be used in qPCR.

[0061] As used herein, the “quantification cycle” or “Cq” value of an amplification reaction is defined as the number of partial cycles required for fluorescence to reach a threshold, indicating the position of the amplification curve relative to the cycle axis. Since Cq is directly related to the starting concentration of the target, and the difference in Cq values ​​is related to the starting concentration ratio, the Cq value is inversely related to the amount of target nucleic acid present in the sample and is associated with the number of target copies in the sample. Lower Cq values ​​(typically below 29 cycles) indicate a large amount of target nucleic acid. Higher Cq values ​​(above 38 cycles) mean a smaller amount of target nucleic acid.

[0062] ΔCq is calculated in various methods, kits, component kits, systems, or components disclosed herein and is a log ratio of concentrations, i.e., the log of the concentration of the target nucleic acid standardized to the concentration of a reference nucleic acid, such as the KIF11 gene or region thereof. In some embodiments, ΔCq is calculated between the threshold cycle numbers (Cq) of the first and second, and possibly third, KIF11 amplification reactions as a measure of the presence (completeness or contamination) of genomic DNA.

[0063] The KIF11 amplicon was found to be exceptionally useful in double ΔCq analysis of qPCR results developed by Livak and Schmittgen (2001 Methods 25:402-8) or in analyses based on standard curve methods for relative quantification, such as those developed by Pfaffl (2004 Quantification strategies in real-time PCR. In MW Pfaffl, AZ of quantitative PCR. La Jolla, CA, USA: International University Line).

[0064] Generally, the Cq result of qPCR measurement is primarily dependent on the starting concentration of the target nucleic acid, especially within the same experiment. Therefore, qPCR results can be reported as ΔCq and double ΔCq values, representing the gene expression ratio and inter-experimental multiplier change, respectively. Thus, ΔCq=0 indicates that the starting concentrations of the target nucleic acid and the reference gene are the same. For example, the values ​​of ΔCq and double ΔCq for experiments that meet pre-defined requirements such as nucleic acid integrity, gDNA contamination, gDNA fragmentation, and process control can be established by those skilled in the art according to specific needs.

[0065] In amplification reactions, the quantification of the output signal, such as fluorescence, produced by a detectable probe, often involves a process of mapping input values ​​from a larger set to output values ​​from a smaller set, often by a finite number of elements, such as rounding and truncation. This can be carried out by any means known to those skilled in the art, preferably through digital signal processing using dedicated software.

[0066] Standardization refers to the process of adjusting values ​​measured at different scales, such as the signal produced by a target nucleic acid in an amplification reaction, to a conceptually common scale, such as the signal obtained by a reference probe, e.g., KIF11, in the amplification reaction. For example, the value obtained by the target nucleic acid in the amplification reaction is adjusted to the value obtained by the KIF11 gene amplification reaction, preferably the target nucleic acid and the KIF11 gene are amplified in the same amplification reaction and / or obtained from the same sample.

[0067] The threshold represents the number of amplification cycles or amplification time required for a detectable signal, such as a fluorescent signal, representing a positive PCR result, to exceed a baseline threshold level ("background noise"). The threshold may be a threshold cycle number in a thermal cycling amplification reaction, or it may be a time value (e.g., amplification time) in an isothermal nucleic acid amplification reaction. The threshold can be determined by any means known to those skilled in the art. For quantitative PCR, the threshold cycle value is determined for each target nucleic acid amplified in the test and calibration samples. It is important that the method used to determine the threshold provides a reproducible value. Such reproducibility can be achieved by positioning the threshold in the logarithmic period of the amplification curve. Preferably, the threshold is derived from the first or second derivative of the amplification curve.

[0068] The threshold can be determined by calculating the number of cycles or time value associated with the positive peak of the first derivative of the amplification curve. The threshold (e.g., the threshold number of cycles in thermal cycling amplification or the time value in isothermal amplification) can also be expressed by the position of the peak of the quadratic curve. The threshold for nucleic acid sequence amplification can be determined by: (i) deriving the amplification curve of the nucleic acid sequence from the measured signal; (ii) calculating the derivative of the amplification curve; (iii) examining the characteristics of the derivative, e.g., the first and / or second derivative; and (iv) determining the threshold associated with the characteristics of the derivative.

[0069] As used herein, the term “primer” refers to an oligonucleotide, whether naturally occurring or synthetically produced, such as in a purified restriction digest, which can act as a starting point for nucleic acid sequence synthesis when placed under conditions that induce the synthesis of a primer extension product complementary to the nucleic acid chain, i.e., in a suitable buffer (where “buffer” includes pH, ionic strength, cofactors, etc.) and in the presence of different nucleotide triphosphates and polymerases at a suitable temperature. One or more nucleotides of the primer can be modified, for example, by the addition of a methyl group, biotin or digoxigenin moiety, a fluorescent tag, or by using a radionucleotide or a detectable universal marker, in which case the primer can act as a probe. As used herein, the terms “variant-specific primer” or “allele-specific primer,” used interchangeably, refer to a primer that specifically binds to a variant sequence. Similarly, the term “variant-specific primer pair” refers to a pair of primers intended to produce an amplicon only in the presence of a variant in a PCR reaction. A variant-specific primer pair may include, for example, a variant-specific primer such that an amplicon is generated only when a variant is present and the variant-specific primer has a suitable binding site. Alternatively, a variant-specific primer pair may include two primers that bind only in the correct orientation and at the appropriate distance when a variant is present. For example, if the variant is a deletion, the distance between the primers in the primer pair may be too large in the absence of the deletion to generate an amplicon in the PCR reaction. In the presence of the deletion, the primers bound to the target of the variant-specific primer pair have the appropriate distance for amplicon generation. The same concept can be applied to gene rearrangement. One type of variant-specific primer is the so-called "ARMS-primer".ARMS stands for Amplification Refractory Mutation System, where PCR is frequently applied to identify point mutations or polymorphisms, and DNA is amplified by allele-specific primers. ARMS PCR uses a pair of primers, including ARMS primers and usually general PCR primers. ARMS primers typically have the following spatial characteristics: (1) typically about 20-40 bp in length; (2) the nucleotide at the 3' end of the primer is usually complementary to the target nucleotide, i.e., G for C, or C for G, and T for A, or A for T. A mismatch at this position can dramatically reduce amplification. A:G, G:A, and C:C mismatches have the worst effect, while other mismatches have varying degrees of effect. For example, in a mutation with an AT substitution, the ARMS primer for the mutant allele should have the nucleotide most complementary to nucleotide T, i.e., A. Primers for normal alleles at the same position should be complementary to nucleotide A, i.e., should have T; (3.) possibly one or more additional mismatches in one of the last 5-10 nucleotides of the ARMS primer, which may further improve its specificity. In some embodiments, allele-specific primers such as ARMS primers contain a stem-loop structure when hybridized to the target nucleic acid sequence (see, e.g., Figure 6). The term “amplicon” refers to the result of the production of one or more copies of a gene fragment or target sequence (amplification of a gene fragment or target sequence), which can be produced by any means known to those skilled in the art, such as by PCR. In this context, amplification reaction refers to the production of one or more copies of a gene target or target nucleic acid. When used herein, the term amplicon encompasses the term “PCR product”.

[0070] The methods, kits, kits of parts, systems, or components disclosed relate to cartridges for automated systems, possibly PoC systems or instruments. As used herein, the term “cartridge” should be understood as a self-contained assembly of chambers and / or channels, formed as a single object that can be transferred or moved as a single fitting into or outside a larger apparatus suitable for receiving or connecting to such a cartridge. The cartridge and its apparatus can be considered to form an automated system (or even an automated platform). In some embodiments, the system further includes one or more reaction components, such as oligonucleotide mixtures, reporter molecules, and reagents for amplification reactions (“PCR mix”), such as buffers, salts, enzymes, etc. Some components of the cartridge may be tightly connected, while others may be flexibly connected to and movable relative to other components of the cartridge. Similarly, as used herein, the term “fluid cartridge” shall be understood as a cartridge containing at least one chamber or channel suitable for treating, processing, discharging, or analyzing a liquid, preferably a fluid. An example of such a cartridge is described in International Publication No. 2007004103. Advantageously, a fluid cartridge may be a microfluidic cartridge. Generally, as used herein, the terms “fluid” or sometimes “microfluidic” refer to systems and arrangements dealing with the behavior, control, and manipulation of fluids that are small in at least one or two dimensions (e.g., width and height or channel), typically geometrically restricted to a sub-millimeter scale. Such small amounts of fluid are moved, mixed, separated, or otherwise processed at a microscale requiring small size and low energy consumption. Microfluidic systems include structures such as micropneumatic systems (pressure sources, liquid pumps, microvalves, etc.) and microfluidic structures for handling micro, nano-, and picoliter volumes (microfluidic channels, etc.).In this context, exemplary and highly suitable fluid systems are described in European Patent No. 1896180, European Patent No. 1904234, and European Patent No. 2419705. Accordingly, the term “chamber” should be understood as any functionally defined compartment of any geometric shape within a fluid or microfluidic assembly, defined by at least one wall and including the means necessary to carry out the function attributed to that compartment. In line with these intentions, “amplification chamber” should be understood as a compartment within a (micro)fluidic assembly that is suitable for implementation and is specifically provided within the assembly for carrying out the amplification of nucleic acids. Examples of amplification chambers include PCR chambers and qPCR chambers. Accordingly, in alternative embodiments, such cartridges may include oligonucleotide generic probes. The terms “chamber” and “compartment,” including multiple versions, are used interchangeably herein unless specifically required by context.

[0071] The term “probe” generally refers to any measurable properties of the probe that change when the probe interacts with an analyte, and thus allow for the study of the interaction between the probe and the analyte. The probe is preferably a nucleic acid having a tag, for example, by radiolabeling or chemical labeling, and more preferably by fluorescent labeling. The measurable properties of a target-detectable probe will change when the target is amplified, for example, while producing a measurable signal, and similarly, the measurable properties of a KIF11-detectable probe will change when KIF11 is amplified, for example, while producing a measurable signal. Preferably, the signal produced by the target-detectable probe will differ from the signal produced by the KIF11-detectable probe, allowing for signal differentiation. As used herein, the term “generic reporter” (which is interchangeable with the term “generic reporter molecule” herein) should be interpreted as any oligonucleotide probe capable of producing a measurable signal or signal change as a result of its hybridization with a unique generic sequence tag that is at least partially, preferably substantially complementary to at least its components. In a simplified form, a generic reporter can be interpreted as a labeling probe specific to a generic sequence tag. In some embodiments, the generic reporter comprises a single-stranded DNA molecule containing the following elements: a first member of a fluorophore / quencher pair; a stem-loop structure; a second member of the fluorophore / quencher pair; a UGST binding site complementary to the UGST of the mediator probe; and a polymerase elongation blocker, where the member of the fluorophore / quencher pair is placed via the stem-loop to quench the fluorophore in the absence of UGST binding and elongation of the mediator probe. Exemplary, non-limiting generic reporter molecules are shown in Figures 2 and 6.

[0072] As used herein, the term “mediator probe” refers to a single-stranded DNA sequence comprising: a first portion containing the following elements between 5' and 3': a unique generic sequence tag ("UGST"); and a second portion containing a sequence complementary to the first strand of the target nucleic acid, or a sequence complementary to a portion of an allele-specific primer (or a complement to a portion of an allele-specific primer). For example, in some embodiments, an allele-specific primer includes a stem-loop structure when hybridized to its target. Upon amplification, the stem-loop sequence of the primer becomes a portion of the amplified sequence (one or more) (see, for example, Figure 6). In some embodiments, the UGST of the mediator probe is complementary to all or part of such a stem-loop sequence or its complement.

[0073] As used herein, the term “stem-loop,” also known as “hairpin,” specifically refers to intramolecular base pairing that occurs in single-stranded nucleic acids such as primers and probes, when two normally complementary regions of the same strand in a nucleotide sequence, when read in opposite directions, base-pair to form a double helix ending in an unpaired loop. The structure is a stem-loop or hairpin loop.

[0074] The term “generic sequence tag” should be understood as a sequence, typically within the length range of an oligonucleotide, that is either absent or present in negligibly small amounts in the genetic information of the organism in which the gene target is detected. Examples of possible unique sequence tags, but not limited to these, include, nuromers, scrambled synthetic sequences, sequences from different / phylogenetically distant organisms, and unique molecular identifiers. In the context of a generic sequence tag being “unique” to a “(oligonucleotide) subset (from a mix),” it should be understood that strictly one generic sequence tag corresponds to strictly one “oligonucleotide subset” that is specific to one “gene target.” As used herein, “oligonucleotide subset” includes, but is not limited to, one or more amplification primers and one or more probes, such as mediator probes, that are specific to a target.

[0075] As used herein, the term “detectable nucleic acid product” refers to a product or byproduct of an amplification reaction of a gene target with an oligonucleotide subset specific to the target. A detectable nucleic acid product should be understood to be detectable by including a “unique generic sequence tag” (“UGST”) that can be detected by a generic reporter, such as a probe having a label. An example of a detectable nucleic acid product may be an amplicon produced by a primer or primer pair that incorporates a generic sequence tag sequence and forms a subset portion from a multiple oligonucleotide subset. An alternative detectable nucleic acid product may be a cleaved or otherwise released portion of an amplicon, primer, or probe, the released portion incorporating the generic sequence tag into its sequence. A particular example of such a detectable nucleic acid product is a first portion of a mediator probe that is released by cleavage (e.g., by the 5'-3' exonuclease activity of a polymerase) and contains a generic sequence tag, such as a unique generic sequence tag.

[0076] In short, a method is disclosed in which the user inserts not only a biological sample into a cartridge, but also a separately provided mix of multiple custom target-specific oligonucleotide subsets. This is in contrast to the procedure with existing assay-specific cartridges in which the target-specific oligonucleotides are provided inside the cartridge. However, in the examples disclosed herein, in order to enable multiplex nucleic acid amplification within the cartridge, the mix of target-specific oligonucleotide subsets must be provided by the user into the cartridge, equal to the sample which may optionally contain the target gene defined by the mix. In examples in which at least one detectable nucleic acid product is generated from the amplification, a signal is generated and can be detected by at least one of several generic reporters contained within the cartridge.

[0077] The approach presented herein enables extremely rapid custom assay design, thereby allowing gene-targeted panels to be fully defined by the customer. Achieving this flexibility to design a panel fully defined by the client and then transfer it into a standard panel-specific cartridge is simply not possible under the realities of the current production pipelines of diagnostic cartridge manufacturers. The considerable effort and cost invested in bringing a standard panel-specific cartridge to market for a relatively small number of uses by a single user would not only be unprofitable, but would probably not even yield a return on the investment. However, within the scope of the disclosed concept, where the main investment is spent on generic detection cartridges and customized panel design can be rationalized relatively easily afterward, perhaps with the help of machine learning algorithms, it is becoming feasible to implement personalized panels for monitoring tumor patients. For example, in an interesting example of the disclosed method, at least one, preferably more, multiple oligonucleotide subsets are specific to gene targets identified in a next-generation sequencing (NGS) analysis performed on the sample provided in the cartridge from the individual from which the biological sample was obtained. In such an example, for instance, a tumor sample from a patient is first analyzed by NGS to determine significant tumor-associated lesions. Based on the results, a custom panel of target-specific oligonucleotide subsets containing oligonucleotide reagents specific to the selected identified lesions and genes can be designed and produced relatively quickly. From this point onward, the status of the selected lesions and genes can be easily and cost-effectively monitored using generic detection cartridges, custom-designed panels, and samples from the patient, such as blood or plasma. Thus, the patient's tumor status and response are monitored and monitored at the molecular level for any potential situations requiring changes in medical procedures or treatments after surgery or a series of procedures, without the need to repeat NGS analysis.

[0078] In the following examples of the disclosed methods, kits, component kits, systems, and components that are compatible with the previous examples, multiple generic reporters are immobilized within an integrated fluid cartridge, preferably by immobilization within one or more nucleic acid amplification compartments. Depending on the design of a given generic cartridge, immobilization can be carried out by covalent or affinity interactions as known in the art and may be useful in generic cartridges based on monolithic or etched chip-like structures with controlled liquid flow channels. An alternative option is to provide reagents in a matrix-containing spot solution, which can then be dried or lyophilized to a glassy state or similar to immobilize the reagents as well as protect them and stabilize their shelf life. Upon contact with an aqueous solution, such dried matrix is ​​hydrated and releases the reagents trapped therein. Accordingly, in the examples of the disclosed methods, systems, components, kits, and component kits, multiple generic reporters are immobilized in a spot solution.

[0079] If such immobilization is performed inside one or more amplification chambers, other reagents may naturally be included in the spot mix along with the generic reporter, which may include, for example, dNTPs and / or enzymes such as polymerase and reverse transcriptase. Depending on stability considerations, one or more spots containing different reagents may be placed in selected compartments.

[0080] In other possible examples that conform to any of the above examples of methods, kits, component kits, systems, and components disclosed, a spot solution, such as a spot solution containing a methylation-sensitive enzyme, is provided. In line with these intentions, a methylation-sensitive restriction enzyme (MSRE) or a methylation-dependent restriction enzyme (MDRE) can be added to the spot solution. When a mixture containing a mix of isolated DNA and multiple oligonucleotide subsets enters a PCR chamber, the MSRE / MDRE digests the unmethylated / methylated recognition sites when incubated at a certain temperature (20–50°C, typically 30–37°C). In the next step, the mixture of DNA, target-specific oligonucleotide subset, and MSRE / MDRE is heated to a temperature between 50–110°C, typically between 70–99°C, to inactivate the MSRE / MDRE. Simultaneously, optionally, a hot-start PCR enzyme can be activated, followed by a typical qPCR cycling protocol. Using this approach, any methylation signature can be easily detected within the same generic detection cartridge.

[0081] Spotting generic reporters and / or other reagents is simple and positively correlates with long shelf life. Depending on the reagent type, different spotting solution compositions or immobilization methods can be used. The location of spotting or immobilization of reagents such as MSREs / MDREs can also be determined within different compartments or channels depending on the given cartridge infrastructure, heater location, etc. For example, they can be located upstream of the amplification chamber.

[0082] The choice of placing different reagents and performing processes within a given compartment will generally depend on the internal design of the particular generic cartridge. In the following possible examples of the disclosed methods, kits, kits of components, systems, and components that conform to any of the preceding examples, multiplex nucleic acid amplification and signal generation from at least one of several generic reporters are carried out within one or more amplification compartments. Such arrangements not only place the detectable nucleic acid product generated during amplification in close proximity to the generic reporter, but also allow for real-time monitoring of the reaction and target detection. Other arrangements using flow cells and separating amplification and signal detection can also be envisioned instead.

[0083] In other possible examples of the methods, kits, kits of components, systems, and components disclosed herein, nucleic acids isolated from a biological sample and a mix of multiple oligonucleotide subsets can be moved into at least two different amplification compartments within an integrated fluid cartridge. This is beneficial in at least two examples: firstly, when repetitions such as duplicates or triplicates of the same reaction are considered, e.g., for barely detectable low copy number targets; or secondly, in the case of automated systems relating to amplification chambers and having a defined or fixed number of wavelength-specific detection channels adapted to capture signals from reporters provided therein. In the latter example, by separating the nucleic acid and oligonucleotide pool mixture between two or more amplification compartments, more targets can be detected from a multiplex reaction by providing or spotting different generic reporters in different amplification compartments, even though different generic reporters can be conjugated with detectable dyes within the same channel. Thus, in a possible example of this example, different generic reporters are provided in different amplification compartments within the same cartridge.

[0084] Furthermore, in one example of the above example, the signal generated in the presence of a detectable nucleic acid product can be generated from a luminescent dye, and presumably here, different amplification compartments containing different generic reporters contain a set of the same or at least partially overlapping luminescent dyes. In line with these considerations, if we imagine a generic detection cartridge having three amplification compartments, each monitored within four different channels (e.g., red, yellow, green, and blue), the nucleic acid and oligonucleotide pool mixture would be separated among the three compartments. In each of the three compartments, the same multiplex amplification would occur, and within each compartment, four different generic reporters could signal the presence of a detected nucleic acid product amplified within four different channels (red, yellow, green, and blue). Since each of the four different generic reporters per compartment may be specific to a different target from the multiplex, 12 different targets from the multiplex could be associated with 12 different detection events across the three amplification compartments, each enabling detection within four channels. In possible embodiments of the last two examples, multiplex nucleic acid amplification including a mix can be performed simultaneously in each of the two or more amplification compartments for time considerations.

[0085] In another embodiment, methods, kits, kits of components, systems, and components are disclosed, wherein one or more of a multiple oligonucleotide subset comprises at least primers containing a unique generic sequence tag, and the detectable nucleic acid product is an amplicon containing the unique generic sequence tag. In this simple embodiment, the oligonucleotide subset may simply comprise a target-specific primer pair in which one of the primers per pair contains, for example, a stem-loop structure, a generic sequence tag detectable by a generic reporter in a generic detection cartridge. In this example, the detectable nucleic acid product is the amplicon itself, which is produced as part of the multiplex amplification.

[0086] In a completely different alternative embodiment of the disclosed method, kits, kits of components, systems, and components are provided in which one or more subsets of multiple oligonucleotides comprise at least one primer and at least one mediator probe, the mediator probe comprises a unique generic sequence tag, and the detectable nucleic acid product is a cleaved free mediator comprising the unique generic sequence tag. This embodiment is based on the mediator reaction principle disclosed in European Patent No. 2776585 by Albert Ludwig University of Freiburg and schematically shown in Figure 2. It has been found to work very efficiently in the prototype cartridge described in the following Examples section. The inventors have further modified the original principle by combining it with ARMS primers, which has resulted in improved sensitivity. Thus, in some embodiments of the latter example, at least one primer is an ARMS primer.

[0087] To better distinguish closely located mutations, certain target-specific ARMS primers have been further modified to include a stem-loop structure whose stem can be arbitrarily composed of the target sequence. The inventors have further modified a mediator probe so that it at least partially overlaps with the stem-loop modified ARMS primer. The concept is shown in Figure 6. In upper panel A, the 5' end of the ARMS primer contains a sequence complementary to the target sequence, whereas in lower panel B, the 5' end of the ARMS primer terminates with a stem-loop-stem structure that acts as a generic terminal tag, which has the advantage of a streamlined primer design. The 3' component of the stem of the ARMS primer may have a sequence derived from the target sequence or may be a different sequence. When the target is not amplified, the mediator probe cannot bind to the target sequence and therefore cannot produce a signal. The mediator probe also cannot bind to its complementary sequence in the ARMS primer because it is located within a stem-loop structure and is therefore inaccessible to the mediator probe. When the target is amplified, the stem structure can be unfolded by polymerase, thereby creating a binding site for the mediator probe. Once bound, the free mediator is released when the other primer extends, and the free mediator can bind to the spotted generic reporter and generate a signal. In line with the above, in the specific embodiments of the last two examples, the ARMS primer comprises a stem-loop structure, and preferably, the mediator probe sequence further at least partially overlaps with the sequence contained in the stem-loop structure.

[0088] In another example, a method is provided for the isolation of nucleic acids from a biological sample, presumably carried out in the presence of a mix of multiple oligonucleotide subsets, possibly within a nucleic acid extraction compartment. In this particular example, the user can add the mix of multiple oligonucleotide subsets together with the biological sample in a cartridge, which saves time. Surprisingly, the inventors observed that this works very well with a nucleic acid isolation protocol including a liquefaction protocol in an Idylla®-based generic cartridge prototype. An alternative involves first providing the mix of multiple oligonucleotide subsets and pumping it more deeply into the internal cartridge space, then, once the mix has reached the desired compartment in the cartridge, adding the biological sample, and only those initiating the nucleic acid isolation protocol. Another alternative may include two inlet ports in the cartridge, one for the mix and one for the biological sample.

[0089] In some examples, methods, uses, kits, component kits, systems, and components are provided for biological samples that are solid tissue samples, possibly fixed solid tissue samples, preferably formalin-fixed paraffin-embedded (FFPE) samples. In such examples, nucleic acid isolation may or may consist of liquefaction of the solid tissue sample.

[0090] In alternative examples, methods, uses, kits, component kits, systems, and components are provided for biological samples that are liquid biopsy samples. In certain embodiments of the above examples, methods, uses, kits, component kits, systems, and components are provided for nucleic acid isolation that includes cell-free nucleic acid isolation. In further embodiments of the latter embodiments, nucleic acid isolation may or may consist of nucleic acid extraction, preferably nucleic acid extraction on a solid carrier.

[0091] In further examples, methods, uses, kits, kits of parts, systems, and components are provided in which at least a portion of an oligonucleotide subset from a mix of multiple oligonucleotide subsets is designed by a computer implementation method including machine learning or artificial intelligence.

[0092] Control is crucial for the robustness of any experiment and any decisions based on such experiments, especially when using highly sensitive techniques such as amplification of target genes. However, it is important to note that the success of amplification of a control gene only provides indirect information about the integrity of the target gene itself. Therefore, the selection of appropriate control genes must be carried out wisely. At least one requirement is that the control gene is not prone to changes such as somatic mutations or copy number changes across a wide range of diseases, including cancer. Also, in a typical setting, the effectiveness of treatment options in various disease situations is evaluated by following changes in the target gene, which requires standardization of data against a reference gene. Therefore, a crucial prerequisite in the selection of reference genes is that they should not be affected by the treatment. To date, the most commonly used reference genes are housekeeping genes (HKGs). However, standardization of data against random HKGs can lead to errors in calculating the standardization factors used to compare treatment conditions, and thus mask biological differences between samples. In fact, selecting an appropriate reference gene is difficult unless the copy number, stability of the reference gene, and the relationship between the target gene and the disease in question are known. In this specification, we have confirmed that the kinesin family member 11 gene ("KIF11"; NCBI Entrez Gene:3832) is genomically very stable with respect to copy number variations, somatic mutations, and SNPs in virtually all cancer types for which publicly available data are available. KIF11 has been identified as a very promising generic control that is likely to provide consistent performance across all comprehensive populations.

[0093] Since the inventors have confirmed that KIF11 is a remarkably stable genomic region in most cancer types, the KIF11 gene or region can be used as a universally suitable genomic reference target (reference gene) as a substitute or addition to housekeeping reference genes used simultaneously in a wide range of methods such as PCR, LCR, NGS, and CGH, as described, for example, in Lemma et al. (Identification and Validation of Housekeeping Genes for Gene Expression Analysis of Cancer Stem Cells, PLOS ONE | DOI:10.1371 / journal.pone.0149481 February 19, 2016). The use of such a universally suitable reference gene is particularly useful in multiple gene panels because it allows the use of only a single reference gene when evaluating the presence of mutations in different genes, in contrast to using stable regions of different genes that serve as reference genes to assess the presence of mutations in corresponding genes, which then requires the use of more reagents and a larger space in the multiplex area of ​​the test instrument.

[0094] In some examples, methods, uses, kits, component kits, systems, or components are provided in which a mix of multiple oligonucleotide subsets includes subsets specific to a region within the KIF11 gene. In possible embodiments thereof, the region within the KIF11 gene is used as a genomic reference gene, or the KIF11 amplicon generated by the subset is used as a genomic reference gene. Preferred embodiments of this example relate to the human KIF11 gene, but may also relate to other mammalian homologs. In certain examples, the region within KIF11 is located in either exons 6, 8, 18, 21, intron 6, the exon 21-intron 21 boundary, or a non-coding region of the KIF11 gene. In further embodiments, methods, uses, kits, kits of components, systems, or components are provided in which one or more of a multiple oligonucleotide subset comprises primers specific to a region within the human KIF11 gene, preferably at least two of the multiple oligonucleotide subsets comprises primers specific to different regions in the human KIF11 gene, and the primers are designed to produce two KIF11 amplicons of distinctly different lengths. As used herein, the term “KIF11 amplicon” refers to an amplicon of the KIF gene or KIF11 region, where the amplicon is the result of an amplification reaction of the KIF11 gene or KIF11 region, respectively. “KIF11 region” refers to a fragment or portion of the KIF11 gene. The KIF11 gene refers to an open reading frame and includes the 5' and 3' untranslated regions (UTRs) and regulatory sequences at the 5' and 3' positions. Therefore, the term "non-coding region of the KIF11 gene" refers to the 5' and 3' untranslated regions (UTRs) as well as the regulatory sequences at the 5' and 3' positions.

[0095] Since we have confirmed that KIF11 is a remarkably stable genomic region in most cancer types, the use of KIF11 as a genomic reference target or housekeeping gene reference for any DNA or RNA amplification reaction, particularly in cancer, regardless of the methods, uses, kits, component kits, systems, or components disclosed herein is generally disclosed herein. In specific examples, regions within KIF11 are located within any of the following exons 6, 8, 18, 21, intron 6, the exon 21-intron 21 boundary, or non-coding regions of the KIF11 gene and are also disclosed herein as suitable genomic reference target regions.

[0096] In one embodiment, KIF11 or the KIF11 region can be used as a reference gene to assess the integrity of gDNA. Genomic DNA (gDNA) integrity plays a crucial role in defining gDNA quality and can affect the application of downstream molecules such as PCR, comparative genomic hybridization (CGH), or whole-genome sequencing approaches. Several factors affect gDNA integrity, primarily due to preanalytical procedures such as sample DNA storage, repeated freeze-thaw cycles, retention in tubes, evaporation, and / or denaturation. Additional factors such as humidity, temperature, and temperature variability, the persistence of nucleases and other chemical agents, and other suboptimal conditions that may occur during transport and gDNA extraction can also impair gDNA integrity. GDNA integrity is a determinant of experimental robustness and reproducibility, particularly to avoid false negatives. High-quality gDNA, such as gDNA with uncompromised integrity, refers to gDNA that is essentially pure, intact, double-stranded, highly concentrated, and / or uncontaminated, but at least suitable for the intended experimental procedure based on the gDNA. The methods provided herein using KIF11 are particularly suitable for determining gDNA fragmentation. As used herein, “absence of gDNA fragmentation” refers to gDNA fragmentation that is undetectable by the methods of the present invention, including non-gDNA fragmentation, such as intact gDNA.

[0097] The regions within the KIF11 gene that generate the KIF11 amplicon, the positions of the members of the KIF11-specific primer pair (e.g., forward and reverse primers), and the length of the KIF11 amplicon used in this invention can be determined according to the needs of those skilled in the art. For example, if the amplicon length of (one or more) gDNA targets is the approximate length of a short KIF11 amplicon, this short KIF11 amplicon can be included as a positive control. If the amplicon length of (one or more) targets is variable, those skilled in the art may choose to include both a "short" KIF11 amplicon and a "long" KIF11 amplicon, such as two KIF11 amplicons of distinguishable lengths, for example, amplicons between 50 and 140 bases and between 141 and 280 bases, respectively. Alternatively, a person skilled in the art may choose to include two distinct KIF11 amplicons of different lengths, where the first KIF11 amplicon is generated from a KIF11 region located entirely within the coding region of the KIF11 gene, and the second amplicon is generated from a KIF11 region located within the non-coding region.

[0098] Those skilled in the art are well familiar with designing primers and PCR conditions suitable for generating the KIF11 amplicons of the present invention, including KIF11 amplicons of distinctly different lengths. Preferably, the target KIF11 amplicon length is between 50 and 280 bases, preferably between 60 and 250 bases, such as 62 bases, 89 bases, 98 bases, 136 bases, 204 bases, or even 280 bases. When designing primers suitable for generating KIF11 amplicons, those skilled in the art may consider the following: Preferably, stem-loop secondary structures with low -ΔG values ​​are avoided in the amplicon. Preferably, the amplicon is located within a structurally stable region. Preferably, palindromic sequences are avoided in the amplicon. Preferably, G:C-rich regions are avoided in the amplicon, for example, with a target G:C content of approximately 50%. Preferably, repeating regions are avoided in the amplicon. Preferably, the target region is intended to span an intron-exon boundary or a target region within a non-coding region. Surprisingly, intron sequences such as the intron 6 region and the non-coding sequence 3' of the KIF11 coding sequence have been found to be exceptionally stable. In possible embodiments, the region within the human KIF11 gene is located in either exons 6, 8, 18, 21, the exon 21-intron 21 boundary, or within intron 6 or a non-coding sequence of the KIF11 gene. Preferred subsets of forward and reverse primers are listed in Table 5.

[0099] When analyzing cell-free DNA or cell-free tumor DNA, it is crucial to avoid gDNA contamination, especially if the assay in use does not distinguish between gDNA and cDNA sequences. Appropriate plasma sample preparation and storage are recommended to avoid gDNA contamination. However, this sample preparation may not be 100% effective and may be susceptible to assay errors or operator errors. Determining the presence of the KIF11 amplicon, as described herein, has been found to be very convenient for assessing the absence of gDNA (with good integrity) in order to ensure quality control that gDNA is absent or present at very low levels. Furthermore, when analyzing the expression levels of certain targets or determining RNA levels using proxies, it is crucial to avoid gDNA contamination, especially if the assay in use does not distinguish between gDNA and cDNA sequences. Enzymatic treatment of samples with DNase I is recommended to eliminate gDNA contamination. However, this enzymatic treatment may not be 100% effective and may be susceptible to assay errors or operator errors. To quality control gDNA removal, determining the presence of the KIF11 amplicon, as described herein, has been found to be very convenient for assessing the absence of gDNA.

[0100] In another embodiment, a kit, a kit of parts, a system, or components thereof, provided as separate components: - A mix of multiple oligonucleotide subsets, each of which subsets is specific to a gene target and contains a unique generic sequence tag within the subset. Each of the subsets is adapted to produce a detectable nucleic acid product containing a unique generic sequence tag under nucleic acid amplification conditions and in the presence of a gene target. A mix of multiple oligonucleotide subsets; and -It is an integrated fluid cartridge: (i) an inlet port for accepting biological samples; possibly a second inlet port for accepting a mix; (ii) A nucleic acid isolation compartment located downstream of the inlet port, which presumably contains at least reagents for the nucleic acid isolation compartment, such as a liquefaction buffer, inside the cartridge, or is arranged to be fluidly connected to it; (iii) Reagents for nucleic acid amplification; (iv) One or more nucleic acid amplification compartments located downstream of the nucleic acid isolation compartment (e.g., nucleic acid extraction chamber), and (v) Multiple generic reporters, each of which contains a generic sequence specific to one of the unique generic sequence tags (unique to the oligonucleotide subset and contained in one of the detectable nucleic acid products), and is adapted to generate a signal in the presence of a detectable nucleic acid product containing the unique generic sequence tag. Includes an integrated fluid cartridge Kits, kits of parts, systems, or components thereof, including the above, are disclosed.

[0101] A polymerase elongation blocker refers to an oligonucleotide (e.g., a primer) that is not complementary to the target sequence, and therefore does not base-pair, cannot be elongated by the enzyme, and / or inhibits polymerase during elongation without being involved as a primer itself, by being made unextendable by adding a base to the 3' end of the oligonucleotide. Various polymerase elongation blockers such as 3SpC3 are exemplified in the Examples section, but any polymerase elongation blocker known in the art, such as 3'-spacer C3, 3'-phosphate, 3'-ddC, or 3'-reverse end can be used.

[0102] In some examples, kits, component kits, systems, or components thereof are provided in which multiple types of generic reporters are fixed inside a cartridge.

[0103] In further examples, kits, kits of components, systems, or components thereof are provided, in which an oligonucleotide subset from a mix of multiple oligonucleotide subsets comprises at least a primer and a mediator probe, preferably the primer being an ARMS primer. In some embodiments, further examples are provided in which the ARMS primer comprises a stem-loop structure, and the mediator probe sequence at least partially overlaps with the sequence contained in the stem-loop structure.

[0104] In several other examples, kits, kits of components, systems, or components thereof are provided, in which one or more of a multiple oligonucleotide subset comprises primers specific to a region within the human KIF11 gene, preferably at least two of the multiple oligonucleotide subsets comprises primers specific to different regions within the human KIF11 gene, and the primers are designed to produce two KIF11 amplicons of distinctly different lengths. In a possible embodiment, the region within the human KIF11 gene is located in either exon 6, 8, 18, 21, or within the exon 21-intron 21 boundary.

[0105] Further disclosures include the use of disclosed methods, kits, component kits, systems, or components thereof for detecting multiple gene targets, possibly in samples from cancer patients, possibly as part of post-NGS analysis patient surveillance or in minimal residual disease monitoring.

[0106] Examples 1. Identification of genomic reference loci applicable to generics. The objective of this application was to provide a generic platform capable of handling a wide variety of custom-designed panels for highly variable and possibly genomically unstable diseases and targets. Therefore, we first attempted to identify robust genomic reference loci or genes. Neoplastic diseases, in particular, involve frequent genomic rearrangements, and in our extensive oncology practice, we test many different housekeeping loci, considering that for each assay, the reference gene or group of loci best selects the function of the tumor type. However, given the general nature of the desired applications herein, we undertook a screening of loci that are minimally affected across a wide range of cancer types by different genetic variations, including point mutations and copy number variations. Our extensive literature and in silico analysis of our and others' data enabled us to rank the top 100 potentially oncologically "stable" genes. These top-ranked potential reference loci are presented in a public web portal. https: / / www.cbioportal.org Further analysis of somatic mutations was performed using The Cancer Genome Atlas (TCGA) database, which was accessed via [a specific method / platform].

[0107] The analysis allowed for further narrowing of the selection to a smaller number of targets, and as a result, the inventors confirmed and extensively validated that KIF11 appears to be an extremely promising genomic reference gene that is only very rarely affected by somatic mutations or copy number variations in tumor samples tested or screened to date. The suitability of KIF11 as a “stable” pan-cancer reference control was also confirmed in a wide variety of tumors in the 228 studies included in TCGA at the time of the inventors' analysis, with only a few exceptions: 2–7% of prostate cancers (TCGA reported amplification or deletion in the studies) and 9% of nerve sheath tumors (amplification). The latter analysis appears to support the remarkable suitability of KIF11 as a genomic reference gene, which has not been previously reported, when used as a generically stable regulatory locus for a wide variety of genomically unstable samples such as tumor samples. Increased KIF11 expression has been widely reported in many different cancer types, and it has even been suggested that it is an oncogene or potential tumor marker in different cancer types. Therefore, our findings are all the more surprising (Daigo et al., 2018 Int J Oncol 52:155-165; Pei et al. 2017 Oncol Lett 6618-6626). https: / / doi.org / 10.3892 / ol.2017.7053 (Imai et al. 2016 Pathobiology 84:16-24). Despite its reported transcriptional overexpression, based on our investigation and data obtained by TCGA, the KIF11 genomic locus appears exceptionally stable, and additional research would be recommended to eliminate the risk of genomic stability of KIF11 as a regulatory region of choice only when designing assays for prostate cancer or nerve sheath tumors.

[0108] To further confirm our findings, we extensively tested the stability of selected KIF11 regions in a wide range of samples and online databases. In particular, for common gene NPs, we focused especially on intron 6, exons 6 and 21, the intron-exon boundary surrounding the latter, and the two longest exons, 8 and 18, which can accommodate long amplicons (243 bp and 280 bp, respectively).

[0109] In conclusion, KIF11 appears to be genomically very stable with respect to copy number variations and somatic mutations in virtually all cancer types for which publicly available data are available, as well as to the SNPs at the exon and exon 21-intron 21 boundaries examined. As a result, KIF11 appears to be a very promising generic control that is likely to provide consistent performance across a wide range of populations.

[0110] 2. Selection of multiplex amplification reactions including generic reporters Several approaches were evaluated for the selection of proof-of-principle nucleic acid amplification methods, including generic reporters. Examples include, but are not limited to, anisotropic loop hairpin primers described in Biocartis NV's International Publication 2020 / 165180, e.g., possible combinations of PASS primers described in European Patent No. 2753717, European Patent No. 2817421, and European Patent No. 1948822 with SpeeDx Ltd's MNAzyme technology, or mediator-probe reactions described in European Patent No. 2776585 by Albert Ludwig University, Freiburg, schematically shown in Figure 2.

[0111] With the desired goal of performing high-target-number multiplexes potentially containing dozens or more target-specific primer pairs and possibly probes in mind, we developed an assay based on a combination of ARMS primers and mediator probe reactions. To date, mediator probe reactions have simply been used in combination with standard primers, i.e., primers that are not mutant-selective or variant-selective and that amplify nucleic acid regions regardless of whether the target mutation / SNP is present within them. As a result, to date, if there is interest in variant-specific detection, the mediator probe would be designed to partially overlap with the target mutation or SNP. However, we found that the above approach requires extensive optimization of the mediator probes to ensure robust allele selectivity. In particular, this would generally not allow for achieving a sensitivity of less than 1% with respect to the detection of SNPs, making it unsuitable for a highly sensitive mutation detection assay. Therefore, we modified the mediator probe reaction-based assay so that ARMS primers are used for selective amplification of mutant-target molecules and the mediator probe does not overlap with the target mutation. ARMS primers may include wobble and / or loops, which the inventors hypothesize may allow for fine-tuning of sensitivity / specificity results in complex multiplex settings, for example, for more challenging targets.

[0112] Figure 3 shows results demonstrating the strong discriminative power of the approach presented herein, illustrating the performance of a 3plex assay combining the use of ARMS primers and mediator probes for highly sensitive detection of SNPs and indels within the EGFR gene. The results show that the titration series of synthetic mutant targets was robustly amplified and clearly detectable against a 10,000-copy wild-type genome background. The 3plex assay was able to detect at least 10 mutant target copies, with sufficient discrimination from the blank limit (LOB, shown as a black square in the figure) for some of the targets of the 3plex assay.

[0113] 3. Configuration of the prototype generic cartridge A generic cartridge prototype was prepared based on a fluid sample processing cartridge owned by Biocartis NV and compatible with their automated molecular testing system, Idylla®. The standard cartridge is manufactured as a single disposable entity containing a sample inlet port, as well as multiple internal compartments for reagents and waste, which are connected to a fluid pathway for sample processing and nucleic acid isolation according to the selected method. The fluid pathway ends in five independent nucleic acid PCR amplification chambers configured to accept a portion of nucleic acid, such as one isolated in the upstream section of the fluid pathway, with amplification reagents pre-loaded. The amplification chambers are equipped with transparent walls that allow for the detection of signals generated during nucleic acid amplification, such as PCR, which is performed using a luminescent dye that signals the presence of the gene target of interest. Once the sample is provided in the cartridge and the cartridge is fed into the Idylla® system, the entire sample-to-result processing program, including the disruption of the biological sample, nucleic acid isolation by either liquefaction or solid-phase extraction column, followed by targeted nucleic acid amplification, and signal detection, is fully automated and organized within the system.

[0114] To create a generic cartridge prototype for the first proof of principle, reagents and buffers optimized for liquefying immobilized solid tissue samples (e.g., FFPE samples) were loaded into an Idylla® cartridge as described in European Patent No. 2958997 in the name of Biocartis NV. Next, spot solutions of five uniquely labeled generic reporters were spotted together with PCR reagents including dNTPs, Taq polymerase, etc., and dried in one of the cartridge's five amplification chambers according to the manufacturer's protocol. Mg 2+ The buffers required for PCR were combined into a buffer used for liquefaction.

[0115] Apart from the generic cartridge prototype, a mix of 61 target-specific oligonucleotide subsets was designed as follows: Each of the 61 target-specific oligonucleotide subsets in the mix contained one forward primer, one reverse primer, and one mediator probe. With the exception of the subset specific to KIF11 as a positive control target, the forward primers were typically designed as allele-selective ARMS primers, i.e., 61 primers that specifically bind to one allele of the target. In this example, one specific allele is considered a target even if it relates to the same gene as another allele targeted by another subset in the mix. As a result, different target-allele-specific subsets can share oligonucleotides with the same sequence, as in this example. That is, the reverse primers and mediator probes in each subset were mostly non-allele-selective and were designed to bind to regions within the gene closer to the target allele. In this experiment, the mix of 61 target-specific oligonucleotide subsets contained 61 forward primers with different sequences (defining 61 gene targets, including different genes and different alleles within the same gene), 9 different reverse primers, and 9 different mediator probes. This was because several reverse primers and mediator probes were designed to constitute specific subsets for different allele targets within the same gene, where different alleles are distinguished by the specificity of the ARMS forward primers. The cartridge layout is shown in columns 3-5 of Table 1 below, indicating the positions where the 9 specific targets can be detected. The sequences of the oligonucleotides are shown in Table 2. The concentrations in the mix can be obtained from Biocartis NV upon request, but can be determined by those skilled in the art.

[0116] [Table 1]

[0117] [Table 2-1] [Table 2-2]

[0118] Each mediator sequence (or "free mediator" containing or consisting of a unique generic sequence tag) that is strictly complementary to one of the generic reporter probes when spotted in the amplification chamber of the generic cartridge prototype (i.e., complementary to the unique generic sequence tag binding site of the generic reporter molecule) was covalently bound to a target-specific sequence to generate one of nine mediator probes together. The mediator sequences can be designed, for example, as 10-30 nucleotides long nuomers (i.e., scramble sequences that do not occur in the human genome), but non-nuomers can also be considered. The lengths of the mediator sequences can vary, but they are considered acceptable if they are predicted to give a specific signal by interacting with their corresponding generic reporter and not cross-react with other generic reporters. An example of such mediator sequences can be found in Wadle et al. (2015, Biomolecular Detection and Quantification 7:1-8, Real-time PCR probe optimization using design of experiments approach). The mediator sequences used herein can also be obtained from Biocartis upon request.

[0119] To verify whether the 61plex reaction via mediator is suitable for generating target-specific signals in generic cartridge prototypes, the following components were added together to the lysis chamber of each prototype cartridge via its sample inlet port: (i) A mix of 61 target-specific oligonucleotide subsets, including primer pairs and mediator probes, 27 µl; (ii) Mock sample material selected from FFPE slices, genomic DNA extracts, or milliQ water corresponding to a no-sample control; (iii) A synthetic target containing the target mutation (10, 50, or 100 copies per PCR chamber) or no synthetic target (negative control).

[0120] Next, the generic prototype cartridge was inserted into the Idylla™ device and fully automated tests were initiated. During these tests, the Idylla™ platform performs pumping of the sample preparation buffer into the lysis chamber and applies heating and high-intensity focused ultrasound (HiFU) treatment to the contents of the lysis chamber to obtain a homogeneous lysate. In the next step, the lysate is heat-inactivated by slow pumping through a heated zone, followed by transfer of a portion of the heat-inactivated lysate containing a mix of nucleic acid targets and a subset of target-specific oligonucleotides into one of five parallel amplification chambers. At this point, a PCR cycling protocol adapted for a 61-plex containing the oligonucleotide mix is initiated, which, in the settings presented herein, results in the generation of free mediators if the appropriate target gene or allele is present in a given lysate portion. The free mediators hybridize to their respective complementary generic reporter probes when spotted in the amplification chamber. The hybridization event results in the generation of a fluorescence signal, which is measured during the annealing / extension step, followed by standard post-processing to correct the data for offset and drift and for determination of the first cycle in which amplification can be detected. Decision trees with two simple parameters were used to exclude curves that had a fluorescence signal that was too weak (see that the ratio of the signal at the plateau to the signal at the baseline should be greater than 0.1), or that had a Cq outside the expected range (see 15 < Cq < 36).

[0121] To test sensitivity, the above procedure was performed using the synthetic target HER2 ex20ins in a titration series of 0-10-50-100 copies per PCR chamber (corresponding to 0.0-0.1-0.7-1.4% mutant copies when used in samples containing 7000 genomic copies per PCR chamber). In both cases, the synthetic target was mixed with high-background clinical samples containing either 7000 genomic copies or 1000 copies of genomic DNA per PCR chamber, which had been previously confirmed to be negative for the target mutation. Exemplary amplification curves are shown in Figures 4 and 5.

[0122] Table 3 lists the sequences of the amplification primers, mediator probes, and generic reporters used in the above examples.

[0123] [Table 3]

[0124] Firstly, as illustrated by HER2 ex20ins in Figure 4, the data unexpectedly demonstrate that the methodology presented herein is capable of detecting at least 10 copies / PCR of the marker. Thus, the method is remarkably sensitive given the complexity of multiplexing. Furthermore, since no false positives were detected across the more than 50 prototype cartridges tested, it can be concluded that the method also meets stringent specificity criteria.

[0125] Furthermore, the data demonstrates that it is possible to detect a specific marker within a particular chamber even if the marker is amplified across all amplification chambers in parallel, identical multiplex reactions with primer pairs and mediator probes provided with the biological sample. For example, EGFR G719A is detected in channel 2 from chamber A but not in other chambers; EGFR S768I is detected in channel 2 from chamber B but not in other chambers. Similarly, EGFR L858R is detected in channel 1 from chamber C but not in other chambers; KRAS G12C is detected in channel 1 from chamber D but not in other chambers. The data further demonstrates that it is possible to distinguish between two variants within the same chamber: namely, C797S is detected in channel 4 from chamber B but not in other channels from chamber B; and EGFR S768I is detected in channel 2 from chamber B but not in other channels from chamber B. Finally, and importantly, the data demonstrates that it is possible to detect multiple markers simultaneously within a single amplification chamber using different generic reporters. To illustrate this, the targets discussed herein were consistently detected in all amplification chambers in combination with a positive control genome reference gene used as a sample processing control.

[0126] 4. Development of improved primer-probe systems for multiplex amplification In some cases, adjacent mutations can lead to different clinical effects, making differentiation potentially valuable. One example of this is the need to differentiate KRAS G12C from other KRAS G12 or G13 mutations when a mutation assay is desired to detect KRAS mutant tumors that may respond to specific KRAS G12C-targeted therapy. Conventionally, in a sample-to-result platform with multiple chambers, this is handled by spotting ARMS primers for KRAS G12C amplification in different chambers from ARMS primers for other KRAS G12 or G13 mutations. However, when working in a situation where all target-specific oligonucleotides are added together through the sample inlet port, it is not possible to maintain spatial separation between specific ARMS primers. Therefore, we further modified the ARMS primers to include a stem-loop structure in which the stem may be optionally composed of the target sequence. The inventors further modified the mediator probe so that, while the mediator probe at least partially overlaps with the modified ARMS primer, the probe is always downstream of the primer used for amplification in a standard mediator probe design. While a conceptual scenario where the mediator probe's binding site is entirely within the ARMS primer is shown in Figure 6, it is conceivable that a portion of the mediator probe's binding site is outside the ARMS primer. The 3' component of the ARMS primer stem may have a sequence derived from the target sequence or a different sequence. When the target is not amplified, the mediator probe cannot bind to the target sequence and therefore cannot produce a signal. The mediator probe also cannot bind to its complementary sequence within the ARMS primer because it is within a stem-loop configuration and therefore inaccessible to the mediator probe. When the target is amplified, the stem structure can be unfolded by polymerase, thereby creating a binding site for the mediator probe.Once bound, free mediators are released as other primers extend, and these free mediators can bind to the spotted generic reporter and generate a signal.

[0127] It will be apparent to those skilled in the art that the use of two or more mediator probes significantly increases detectability. For example, in the case of two mediator probes, four different conditions can be distinguished from one another, for example. [Table 4]

[0128] The results are shown in Figure 7, demonstrating the robustness and universal utility of the concept; that is, the concept is not limited to the generic cartridge of the present invention, but can be widely used in multiplex amplification reactions.

[0129] 5. Development of FuseTag In further development of the primer-probe system described in Example 4, the inventors embarked on simplifying the system to further improve robustness and simultaneously reduce cost. This was achieved by combining the stem-loop primer and mediator probe 2 into a single "FuseTag" primer, thus reducing the number of components compared to the improved primer-probe system in Example 4. Furthermore, the FuseTag primer does not contain modifications such as costly polymerase elongation blockers (indicated by square boxes in the figure), which enables rapid synthesis and repeatability. FuseTag allows for the discrimination of adjacent markers when working with the FLEX Generic Detection cartridge.

[0130] Figure 6C illustrates the “FuseTag” concept, which enables the discrimination of neighboring markers. The modified forward ARMS primer (“FuseTag” primer) comprises a first portion containing a unique generic sequence tag (“UGST”), shown as free mediator 2 in Figure 6C when released, from 5' to 3'; a stem-loop structure; and a second portion complementary to (and preferably allele-specific to) the target. When the specific target is not amplified by the FuseTag primer, the unique generic sequence tag (mediator 2) is not released and therefore cannot produce a signal. However, mediator probe 1 can still be hydrolyzed by another forward primer present in the multiplex reaction mixture.

[0131] Extension of the FuseTag primer by polymerase leads to hydrolysis of the target-specific components of mediator probe 1 and the release of free mediator 1. In the subsequent PCR cycle, extension of the reverse (RE) primer by polymerase leads to hydrolysis of the double-stranded portion of the FuseTag primer, resulting in the release of a unique generic sequence tag (free mediator 2).

[0132] Both free mediator 1 and free mediator 2 can bind to their corresponding universal reporters. Note that the fluorophore and quencher can be exchanged (not shown in Figure 6C). Once the free mediator is extended, a fluorescent signal is generated by substitution of the quencher or fluorophore modification and / or hydrolysis of the quencher or fluorophore-binding nucleotide (not shown). Note that non-hydrolyzable mediator probes and generic reporters cannot be extended by polymerase (indicated by squares).

[0133] The concept was first tested in a singleplex setup. Figure 10 shows the performance of FuseTag primers for mutation detection using singleplex PCR (i.e., including only the primers required to amplify one target) in a 96-well format qPCR instrument for four different targets. The targets were added as 200 copies of synthetic mutant targets in a PCR reaction that always included 2,000 copies of wild-type genomic DNA, as well as a universal reporter, polymerase, dNTPs, and PCR salts (including MgCl2). Four different combinations were tested, including different targets: BRAF V600E, EGFR E709K, EGFR S768I, and EGFR L861Q. The sequences of the various FuseTag primers are shown in Table 4.

[0134] [Table 5]

[0135] The amplification results for 200 copies in a gDNA background are shown by the black line in Figure 10. The control reaction, in which wild-type genome DNA was present but the synthetic mutant target was not added, is shown by the gray line. The results show target specificity that surpasses the negative control.

[0136] The experiments were repeated using KRAS G12C, two variants of EGFR C797S, two variants of EGFR T790M, and EGFR G719S. All of them yielded essentially the same results, namely, target specificity exceeding the negative control (data not shown).

[0137] In conclusion, the results provide evidence of the general feasibility of the "FuseTag" concept as a generic tag that can be used as a target independently.

[0138] To further demonstrate the universal applicability of the "FuseTag" concept, its performance in mutation detection using multiplex PCR was investigated. In particular, a mixture containing an oligonucleotide pool with primers and mediator probes required to amplify multiple targets in a 96-well format qPCR instrument was tested. The targets were identified by the primers and mediator probes, as well as 10 3 The synthetic mutant targets were added at various concentrations along with a mixture containing copies of wild-type genome DNA. A generic reporter, polymerase, dNTPs, and PCR salts (including MgCl2) were added to each reaction along with a liquefaction buffer containing the components that would be needed to release DNA from the FFPE sample. The "FuseTag" primer FEB_FMG_BRAF_T2_F041 is shown in Table 4 (other components are not shown).

[0139] Figure 11 shows the amplification results for 500 (triangles), 100 (black circles), and 20 (diamond) copies in a gDNA background. The control reaction, in which wild-type genomic DNA was present but no synthetic mutant target was added (0 copies), is indicated by a black square. The results show target specificity that surpasses the negative control in the context of multiplexing.

[0140] In conclusion, the results demonstrate the effectiveness and specificity of the "FuseTag" concept across a wide range of applications.

[0141] 6. Development of Quality Control (QC) Amplification Using KIF11 for DNA Fragmentation Evaluation For certain applications, such as determining nucleic acid fragmentation or evaluating contamination of genomic DNA from plasma with short cell-free DNA from plasma in genomic DNA from leukocytes, the inventors initially envisioned a quality control triplex based on three different housekeeping genes, including ABCB, RNaseP, and TFRC. However, finding that these and other tested genes did not exhibit satisfactory levels of stability, and after confirming that KIF11 is surprisingly a stable genomic region free of mutations, the inventors redeveloped the initial triplex fragmentation assay based on different exons of KIF11. Switching to a single stable region was hypothesized to be beneficial in avoiding the copy number variability that can occur when using several different reference genes. Therefore, using different exons of KIF11, primers and probes were developed for the amplification and detection of three distinctly different amplicons of lengths: 62 bp, 98 bp, and 136 bp, respectively. While the cycling conditions inside the Idylla® cartridge are available from Biocartis upon request, the cycling conditions can be readily determined by those skilled in the art. The QC triplex was tested on samples with varying degrees of DNA fragmentation, including DNA from FFPE samples, nucleosomal DNA from blood, and intact genomic DNA. As shown in Figure 8, the KIF11-based QC triplex provides a reliable indicator of the degree of DNA fragmentation and, consequently, the quality, present in a given sample.

[0142] Table 5 lists the sequences of the amplification primers, mediator probes, and generic reporters used in the above examples.

[0143] [Table 6]

[0144] 7. Determination of DNA fragmentation using KIF11 in mediator-based qPCR The next step was the development of DNA fragmentation-controlled qPCR amplification based on KIF11 and its detection by mediator reaction reading, adapted for the prototype generic cartridge described above. To do this, short (82 bp) and long (204 bp) PCR amplicons targeting different exons of KIF11 were designed along with two corresponding mediator probes. Each mediator probe was designed to target its specific generic reporter, conjugated with a different luminescent dye placed in channel 5 and channel 1, respectively. Both reporters were provided within the same amplification chamber of each prototype cartridge so that the qPCR signals associated with the two amplicons and mediators could be detected in the same reaction. The determination of the Cq difference (delta Cq) between the long amplicon qPCR curve and the short amplicon qPCR curve was used as a measure of DNA fragmentation. The performance of the "KIF11 DNA Fragmentation Duplex" was determined against a set of clinical FFPE samples with pre-determined DNA fragmentation levels using an orthogonal ddPCR-based method. As a positive control, the assay was also tested on high-quality genomic DNA (gDNA) (Promega) derived from leukocytes, which were expected not to be fragmented. The results are shown in Figure 9, where lines with circles represent short amplicons and normal lines represent long amplicons. The threshold (horizontal line) was used to determine the Cq value. The results clearly show that the delta Cq value between long and short KIF11 amplicons when detected by the mediator reaction strongly correlates with the degree of DNA fragmentation in the evaluated FFPE samples, and that the fragmentation duplex developed herein is readily usable in generic cartridges of the concept presented herein. Such a duplex is not only very well suited to providing a positive amplification control for personalized oligonucleotide panels, but thanks to its ability to detect fragmented samples, it can also provide an indicator of the reliability of the final result in terms of sample quality function.Next, since extensive fragmentation in FFPE samples correlates with deamination artifacts, both of which are caused by formalin fixation, this KIF11 duplex assay may also be suitable for predicting deamination artifacts. Finally, for cfDNA-based assays from plasma, such as those targeting circulating fetal, tumor, or organ donor DNA target panels, the fragmentation assays presented herein may also be suitable, for example, for providing information on genomic DNA contamination from leukocytes in plasma samples.

[0145] In conclusion, the inventors hereby provide a practical proof-of-principle demonstration of a very unusual approach to fast-tract and highly versatile diagnostic assay and product development. The inventors believe that the concepts presented herein have enormous potential to open up a whole new field for rapid, customized, and individualized molecular testing techniques, as they eliminate the need for customized cartridge design, manufacturing, and QC release. In particular, the methods, kits, component kits, and uses disclosed herein can significantly reduce the development costs and time to bring new assays to patients. Such development acceleration is especially urgent now, as demonstrated by the sudden outbreak of the 2020 global pandemic, which halted and delayed the production of essential medical and diagnostic products worldwide and delayed access to many people seeking treatment decisions and adequate medical care.

Claims

1. A method for quantifying the number of target nucleic acids in a sample relative to the KIF11 nucleic acid in the sample, the following:

1. Amplifying the KIF11 nucleic acid contained in the sample using a KIF11-specific primer pair in a KIF11 amplification reaction, i. Each member of the KIF11-specific primer pair is complementary to—independently to—an exon, an intron-based KIF11 region, or a non-coding sequence of the KIF11 gene; ii. The KIF11 amplification reaction is carried out in the presence of a KIF11-detectable probe. Amplifying KIF11 nucleic acid; 2. To detect the signal produced by the KIF11-detectable probe in the KIF11 amplification reaction; 3.

2. Quantifying the aforementioned signal; 4. Amplifying the target nucleic acid from the sample using a target-specific primer pair in a target amplification reaction; The target amplification reaction is carried out in the presence of a target-detectable probe. Amplifying target nucleic acids; and 5. To detect the signal produced by the target-detectable probe in the target amplification reaction; 6.

5. Quantifying the aforementioned signal; 7.

3. Standardize the quantified signal from 6. with respect to the quantified signal from 7.3., thereby quantifying the number of target nucleic acids in the sample relative to the KIF11 nucleic acids in the sample. Methods that include...

2. The method according to claim 1, wherein the KIF11 region is located in any of exon 6, exon 8, exon 18, exon 21, intron 6, the exon 21-intron 21 boundary, or a non-coding region of the KIF11 gene.

3. The KIF11-specific primer pair is as follows: i. Sequence IDs 69 and 70; ii. Sequence IDs 71 and 72; iii. Sequence IDs 73 and 74; iv. Sequence IDs 75 and 76; v. Sequence IDs 77 and 78; and vi. A primer pair selected from Sequence IDs 79 and 80, and / or The method according to claim 1 or 2, wherein the probe capable of detecting the KIF11 is selected from sequence numbers 81 to 88.

4. The method according to any one of claims 1 to 3, wherein the target nucleic acid and the KIF11 nucleic acid are amplified in the same amplification reaction and / or obtained from the same sample.

5. A method for determining the presence of gDNA in a sample:

1. Amplifying the KIF11 nucleic acid contained in the sample using a first and second KIF11-specific primer pair in a KIF11 amplification reaction, a. At least one member of the first KIF11-specific primer pair is complementary to a KIF11 intron or a non-coding sequence of the KIF11 gene; b. The amplification reaction using the first KIF11-specific primer pair is carried out in the presence of the first KIF11-detectable probe; c. Each member of the second KIF11-specific primer pair is located within the KIF11 exon; d. The amplification reaction using the second KIF11-specific primer pair is carried out in the presence of a second KIF11-detectable probe. Amplification; 2. To detect the signals produced by the first and second KIF11-detectable probes in the first and second KIF11 amplification reactions; 3. To quantify the signals of the first and second KIF11 amplification reactions; 4. Standardizing the quantified signal of the first amplification reaction with respect to the quantified signal of the second amplification reaction, thereby determining the presence of gDNA in the sample, and determining that the sample is a mitochondrial DNA, cDNA, mRNA, rRNA, tRNA, hnRNA, microRNA, lncRNA, cfDNA, cell-free tumor DNA, or siRNA sample. Methods that include...

6. A method for determining the integrity of nucleic acids in a sample:

1. Amplifying the KIF11 nucleic acid contained in the sample using a first and second KIF11-specific primer pair in a KIF11 amplification reaction, a. Each member of the first KIF11-specific primer pair is complementary to—independently to—an exon, an intron-based KIF11 region, or a non-coding sequence of the KIF11 gene; b. The amplification reaction using the first KIF11-specific primer pair is carried out in the presence of the first KIF11-detectable probe; c. Each member of the second KIF11-specific primer pair is complementary to—independently to—an exon, an intron-located KIF11 region, or the non-coding sequence of the KIF11 gene; d. The amplification reaction using the second KIF11-specific primer pair is carried out in the presence of a second KIF11-detectable probe; 2. Determining a threshold in each of the nucleic acid amplification reactions, i. Measuring at least one signal whose intensity is related to the amount of nucleic acid sequence amplified in the reaction at multiple different time points during the amplification reaction; and ii. By determining the number of cycles related to the characteristics of the derivative that represent the threshold, To make a decision; 3. To compare the thresholds (threshold cycle counts) of the first and second KIF11 amplification reactions; Includes, 4. The difference (ΔCq) between the threshold cycle counts of the first and second KIF11 amplification reactions is a measure of the integrity of the nucleic acids in the sample. method.

7. A method for determining gDNA fragmentation, i. To generate three (first, second, and third) KIF11 amplicons of distinguishable lengths by first, second, and third amplification reactions; ii. Determining the Cq values ​​of the first, second, and third amplification reactions; iii. Comparing the Cq values ​​of the first, second, and third amplification reactions; Includes, The difference in Cq values ​​between the first, second, and third amplification reactions is an indicator of gDNA fragmentation, or i. Producing a first KIF11 amplicon ("short") by a first amplification reaction; and ii. To generate a second KIF11 amplicon ("long") through a second amplification reaction; iii. Determining the Cq values ​​of the first and second amplification reactions; iv. Determining the ΔCq values ​​of the first and second amplification reactions; Includes, The aforementioned ΔCq is an indicator of gDNA fragmentation. method.

8. A method for quality control of processing, isolation, and amplification processes, which includes: i. Sample processing; ii. Nucleic acid isolation; iii. To generate three KIF11 amplicons of distinctly different lengths by amplification reactions; iv. Determine the Cq value of each of the KIF11 amplicons; Includes, The Cq value of the KIF11 amplicon is a measure of quality control for the processing, isolation, and amplification. method.

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