WO2025122063A1 - Methods of detecting target nucleic acids - Google Patents

Abstract

The present disclosure relates to methods of detecting and/or quantifying one or more target nucleic acid molecules in a sample, comprising: a) contacting the sample with a reaction mix comprising: i) reagents for amplifying the target nucleic acid molecules; ii) an Argonaute (Ago) enzyme, one or more guide single stranded uncleic acid molecules, and one or more reporter nucleic acid molecules; and iii) a pyrophosphatase (PPase) for regenerating Mg²⁺ in the reaction mix; b) partitioning the mixture of the sample and the reaction mix into a plurality of compartments; c) amplifying the one or more target uncleic acid molecules in each compartment to obtain amplified target nucleic acid molecules; and d) detecting a signal in each compartment based on cleavage of the reporter nucleic acid molecules by the Ago enzyme in the presence of the amplified target nucleic acid molecules and guide single stranded nucleic acid molecules.

Description

METHODS OF DETECTING TARGET NUCLEIC ACIDS

Technical field

The present invention relates to the field of molecular' biology. More specifically, it relates to methods of detecting one or more target nucleic acid molecules, and compositions and kits for performing the methods.

Background

Nucleic acid detection and quantification technologies are widely used for a variety of applications, from biomedical research to clinical diagnostics to environmental monitoring. The most popular method, RT-qPCR, offers advantages in speed and sensitivity, but requires precise thermal cycling and high PCR efficiency. Digital PCR (dPCR) solves this problem by subdividing the PCR reaction into thousands of independent reactions, and is more tolerant to inhibition than qPCR. However, digital PCR requires a longer reaction time. Rapid isothermal amplification methods, such as recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP), detect nucleic acids with high sensitivity, but can suffer from nonspecific amplification and arc often not quantitative. CRISPR/Cas-bascd biosensing, or CRISPR/Cas-based biosensing coupled with isothermal amplification, has been used to detect a variety of viruses as well as cancer genes and for quantification applications. However, due to the collateral cleavage nature of Cas protein, it is difficult to detect several targets in a multiplex reaction. Thus, an improved method for rapid, sensitive, and quantitative detection of multiple nucleic acid targets in a single multiplex reaction is still desired. Accordingly, it is generally desirable to overcome or ameliorate one or more of the above-mentioned difficulties.

Summary

Disclosed herein is a method of detecting and/or quantifying one or more target nucleic acid molecules in a sample, the method comprising: a) contacting the sample with a reaction mix comprising: i) reagents for amplifying the one or more target nucleic acid molecules; ii) an Argonaute (Ago) enzyme, one or more guide single stranded nucleic acid molecules and one or more reporter nucleic acid molecules for detecting amplified target nucleic acid; and iii) a pyrophosphatase (PPase) for regenerating Mg²⁺ in the reaction mix; b) partitioning the mixture of the sample and the reaction mix into a plurality of compartments; c) amplifying the one or more target nucleic acid molecules in each compartment under suitable conditions to obtain amplified target nucleic acid molecules; and d) detecting a signal in each compartment based on cleavage of the reporter nucleic acid molecules by the Argonaute enzyme in the presence of the amplified target nucleic acid and guide single stranded nucleic acid molecules, thereby allowing detection and/or quantification of the one or more target nucleic acid molecules in the sample.

Disclosed herein is a method of detecting and/or quantifying one or more target nucleic acid molecules in a sample, the method comprising: a) preparing a plurality of compartmentalised reaction volumes, each reaction volume comprising a volume of the sample and a reaction mix comprising: i) reagents for amplifying the target nucleic acid molecules; ii) an Argonaute (Ago) enzyme; iii) one or more guide single stranded nucleic acid molecules capable of hybridizing to the target nucleic acid molecules; iv) one or more reporter nucleic acid molecules capable of generating a detectable signal upon cleavage by the Ago enzyme; and v) a pyrophosphatase (PPase) for regenerating Mg²⁺ in the reaction mix; b) incubating the compartments under suitable conditions for i) amplifying the one or more target nucleic acid molecules to obtain amplified target nucleic acid molecules, and ii) cleavage of the one or more reporter nucleic acid molecules by the Ago enzyme in the presence of the amplified target nucleic acid molecules and guide single stranded nucleic acid molecules; and c) detecting a signal generated by cleavage of the reporter nucleic acid molecules in each compartment.

Disclosed herein is a reaction mix comprising: i) reagents for amplifying one or more target nucleic acid molecules; ii) an Argonaute (Ago) enzyme, one or more guide single stranded nucleic acid molecules and one or more reporter nucleic acid molecules for detecting amplified target nucleic acid; and iii) a pyrophosphatase (PPase) for regenerating Mg²⁺ in the reaction mix. Disclosed herein is a kit comprising a reaction mix as defined herein.

Brief description of the drawings

Embodiments of the present invention are hereafter described, by way of non-limiting example only, with reference to the accompanying drawings in which:

Figure 1. The Multiplex Omni-purpose Technology in One-Pot via Isothermal amplification and Argonaute (MOTOPIA) for single molecular level nucleic acid detection and quantification, a) Mechanism and workflow of MOTOPIA. Sample DNA/RNA or lysate can be used as input for MOTOPIA. During the reaction, the isothermal amplification method first amplifies the target with DNA polymerase, followed by Argonaute- based detection. The amplified target DNA will be cut by Argonaute protein through base pairing with designed guide DNAs, generating a DNA product with a 5’ phosphate as a new guide DNA. This new guide DNA will then activate Argonaute to cleave the sequence-specific DNA probe, which is fluorescently quenched. When the probe is cut, a fluorescent signal is generated, allowing for the multiplex detection of several targets in one reaction by assigning different fluorescent signals to different probes, b) MOTOPIA used for rapid quantification of vector copy number (VCN) in CAR-T cells, c) MOTOPIA specifically monitors CAR-T cell expansion in patient blood after treatment, d) MOTOPIA detects replication-competent lentivirus (RCL) and high-risk adventitious viruses during CAR-T cell manufacturing, e) Schematic of digital absolute quantification. The digital quantification separates the reaction into thousands of individual partitions and quantifies the target by counting the positive partitions using Poisson distribution.

Figure 2. Development of the one-pot LAMP-Ago assay, a) Illustration of the Mg²⁺ recycling strategy enabling the one-pot reaction. During DNA amplification, dNTP incorporation consumes Mg²⁺ and forms magnesium pyrophosphate precipitate (left). To counter this, pyrophosphatase (PPase) is introduced to react with magnesium pyrophosphate, regenerating Mg²⁺ and maintaining levels for Ago activity (right), b-e) Comparison of LAMP-Ago reactions under various conditions: normal (b), increased Mg²⁺ (c), decreased dNTP (d), and the addition of PPase (e). LAMP-based amplification was monitored using the SYTO 82 orange fluorescent nucleic acid stain, while Ago- based detection was monitored with a FAM-labeled, fluorescently quenched DNA probe, f) Effect of Ago on the sensitivity and speed of the one-pot LAMP- Ago reaction. Reactions were performed at various Ago concentrations with PPase present. The y-axis represents the maximum reaction rate of Ago cleavage before reaching reaction saturation. Data arc presented as mean ± s.d. (n = 3). g) The fluorescent of LAMP- Ago reaction at different temperatures. The one-pot LAMP-Ago assay was first subjected to 65°C for LAMP, followed by 95°C for Ago reaction. Real-time fluorescence (FAM) was measured during the Ago reaction at 95°C for 30 min, with readings at 37°C every 10 min interval. Data are mean ± s.d. (n = 4). h) Ago cleavage on the DNA probe at various loop target DNA amplicon concentrations, i-j) End-point fluorescence after Ago cleavage. Ago-only reactions were conducted at 95 °C for 20 min with different concentrations of target linear (i) or loop (j) DNA amplicons, followed by fluorescence measurement at 95 °C or 37°C. k) Continuous fluorescence versus temperature plots. The fluorescence of FAM dye, FAM labelled DNA and FAM-quencher double labelled DNA were measured with temperature decreasing from 95°C to 37°C. Data are mean ± s.d. (n = 3).

Figure 3. Sensitivity of different Argonaute proteins for nucleic acid detection. Different concentrations of the target were tested with different Argonaute proteins. The end-point fluorescence level was recorded after a three-hour reaction at each protein’ s optimum reaction temperature.

Figure 4. Ion requirement of Argonaute-based detection. Argonaute was tested with varying concentrations of Mn²⁺ or Mg²⁺, both with and without guide DNA.

Figure 5. Effect of PPase concentration on one-pot LAMP-Ago reaction. Various target concentrations were evaluated in the LAMP-Ago one -pot reaction, with differing concentrations of PPase incorporated. LAMP-bascd amplification was monitored using the SYTO 82 orange fluorescent nucleic acid stain, while Ago-based detection was monitored with a FAM-labeled, fluorescently quenched DNA probe. The grey curve represents the negative control, indicating 0 aM target concentration. Figure 6. Effect of pfAgo concentration on one-pot LAMP-Ago reaction. Various target concentrations were evaluated in the LAMP-Ago one-pot reaction, with differing concentrations of pfAgo incorporated. LAMP-based amplification was monitored using the SYTO 82 orange fluorescent nucleic acid stain, while Ago-based detection was monitored with a FAM-labeled, fluorescently quenched DNA probe. The grey curve represents the negative control, indicating 0 aM target concentration.

Figure 7. LAMP-Ago reaction at different temperatures. The one-pot LAMP-Ago assay was first subjected to 65°C for LAMP, followed by 95°C for Ago reaction. Realtime fluorescence HEX (a), ROX (b) and Cy5 (c) was measured during the Ago reaction at 95°C for 30 min, with readings at 37°C every 10 min. Data are mean ± s.d. (n = 4).

Figure 8. Effect of target DNA concentrations on Ago cleavage.

Figure 9. Continuous fluorescence versus temperature plots. The fluorescence of fluorescent dye, fluorescent labelled DNA and fluorescent-quencher double labelled DNA were measured with temperature decreasing from 95°C to 37°C. Data are mean ± s.d. (n = 3).

Figure 10. Effect of 95°C pre-treatment and swarm primer on LAMP reaction, a) Schematic of LAMP swarm primers, b) LAMP reaction kinetic curves with different treatments. LAMP-based amplification was monitored using the SYTO 82 orange fluorescent nucleic acid stain.

Figure 11. Development of an MOTOPIA-based assay for multiplex virus detection, a) Working principle of MOTOPIA-based virus detection panel. Primers, guide DNA, and probe DNA with different fluorescent colors were designed for the MOTOPIA-based virus detection reaction targeting RCL (FAM), human control (HEX), human adenovirus (ROX), and herpes simplex virus (CY5). b) Specificity of MOTOPIA-based multiplex virus detection, c-f) Sensitivity of MOTOPIA-based multiplex virus detection, c) RCL. d) human control, e) human adenovirus, f) herpes simplex virus. Figure 12. Development of a digital MOTOPIA-based assay for vector copy number (VCN) quantification, a) Working principle of dMOTOPTA-based quantification. The reaction is partitioned into numerous small compartments, each functioning independently. Isothermal amplification is employed to amplify the target within each partition. The target DNA is cleaved by Argonaute, initiating cleavage of the complementary DNA probe. Partitions containing the specific target exhibit corresponding fluorescence signals when either vector (FAM) or human (HEX) sequences are present. Quantification of the target’s copy number is achieved by counting partitions with positive fluorescence signals using Poisson distribution, b) Duplex quantification of VCN and human reference DNA using dMOTOPIA. Varied concentrations of vector DNA and human DNA sequences are introduced to the reaction, followed by dMOTOPIA quantification, c) Linear' relationship of input DNA amount and dMOTOPIA copy number result for duplex VCN and human DNA quantification, d) Comparison of dMOTOPIA and dPCR’s quantification of VCN on WHO standards.

Figure 13. Validation and Comparison of dMOTOPIA-based VCN Quantification for CAR-T Cell Analysis, a) Experimental workflow for validation. Lentiviral- mediated transduction of human T cells with varying multiplicities of infection (MOI) was performed using lentivirus encoding CAR. Evaluation of CAR-T cells involved utilizing digital PCR and dMOTOPIA to quantify extracted DNA from approximately 1 million cells, along with applying dMOTOPIA to assess lysate from 0.01 million directly-lysed cells. Transduction efficiency was determined by flow cytometry. In the duplex dMOTOPIA assay, the integrated vector sequence is measured using FAM fluorescence, while the human reference gene is measured using ROX fluorescence, b) Comparison of dMOTOPIA with dPCR. The left panel illustrates dMOTOPIA results obtained from extracted DNA, while the right panel presents dMOTOPIA results from cell lysate. Comparison with digital PCR findings is shown, c) Comparison of VCN result obtained via dMOTOPIA and dPCR, alongside the corresponding transduction efficiency data from flow cytometry, d) Impact of MOI and collection time on VCN. Investigation of VCN results derived from dMOTOPIA and digital PCR, in relation to varying MOIs and collection days during the CAR-T cell manufacturing process, e-f) Correlation of dMOTOPIA with dPCR for VCN quantification in patient CAR-positive or CAR-negative T cells. dMOTOPIA was performed on either extracted DNA (e) or cell lysate (f), while dPCR was conducted using extracted DNA. Each dot represents a patient, with CAR-positive T cells or CAR-negative T cells manufactured using different bioreactors.

Figure 14. Monitoring of CAR-T cell expansion using dMOTOPIA in patient blood, a) Patient information. Patients included those with multiple myeloma (MM) following BCMA-directed CAR-T cell therapy (n=3, lentiviral vector) and those with diffuse large B-cell lymphoma (DLBCL) following axi-cel treatment (n=4, retroviral vector), b) Experimental design for blood sample collection. Peripheral blood samples were collected from patients at different days post-treatment, c) Experimental workflow for benchmarking dMOTOPIA against flow cytometry. Peripheral blood mononuclear' cells (PBMCs) were extracted from patient blood post-CAR-T treatment and cryopreserved for analysis. Approximately 1 mL was used for flow cytometry, while around 20 pL was used for dMOTOPIA analysis on cell lysate, d) CAR-T cell expansion monitoring after CAR-T therapy using dMOTOPIA and flow cytometry, e) Correlation between CAR transgene copies quantified by dMOTOPIA and CAR-T cell numbers determined by flow cytometry. 96 blood samples from 7 patients at different time points were included.

Figure 15. dMOTOPIA-based quantification of retroviral vector, a) Scatter plot of dMOTOPIA on different concentration of retroviral DNA. b) Linear relationship between the input DNA amount and the dMOTOPIA copy number result for retroviral vector quantification, c) Linear relationship between the input VCN and dMOTOPIA VCN result for retroviral vector copy number quantification. Retroviral vector DNA were mixed with human DNA at different ratio, result in VCN at different level.

Fig. 16. Validation of the MOTOPIA-based assay for multi-virus target CAR-T cell analysis, a) Experimental workflow for benchmarking MOTOPIA against qPCR. Different concentrations of human adenovirus (hAdV), herpes simplex virus (HSV), or RCL plasmid containing lentivirus VSV-G were spiked into CAR-T cell samples at 1 million cells/mL. 200 pL of CAR-T cells were evaluated using qPCR, while 40 pL of CAR-T cells were analyzed using MOTOPIA in a DNA extraction-free manner, b-d) Benchmarking MOTOPIA against qPCR for virus detection. The dashed lines represent the cut-off fluorescent value for MOTOPTA (threshold=mean blank + 1.65*s.d. blank) or the cut-off Ct value for qPCR (Ct=39). Data are presented as mean ± s.d. (n = 3). e- g) ROC curves comparing MOTOPIA and qPCR for different virus detection.

Fig. 17. Validation of the MOTOPIA-based assay for point-of-care virus detection, a) Experimental workflow for benchmarking MOTOPIA point-of-care (POC) detection against qPCR. Different concentrations of herpes simplex virus (HSV) or plasmid containing lentivirus VSV-G were spiked into CAR-T cell samples at 1 million cells/mL. 200 pL of CAR-T cells were evaluated using qPCR, while 50 uL of CAR-T cells were analyzed using MOTOPIA in a DNA extraction-free manner, b-d) Benchmarking POC detection against qPCR for duplex vims detection of CAR-T cell containing RCL only (b), HSV only (c) and RCL+HS V (d). The green (RCL) and orange (HSV) dashed lines represent the cut-off fluorescent value for MOTOPIA (threshold = mean blank + 1.65*s.d. blank), while the grey dashed lines represent the cut-off Ct value for qPCR (Ct = 39). Data are presented as mean ± s.d. (n

3). e-f) ROC curves comparing MOTOPIA POC detection and qPCR for RCL (e) or HSV (f).

Detailed description

The present specification teaches methods for detecting and quantifying one or more target nucleic acid molecules in a sample.

Without being bound by theory, the present specification describes a method for multiplex nucleic acid detection and quantification, which is named MOTOPIA (Multiplex Omni-purpose Technology in One-Pot via Isothermal amplification and Argonaute) herein. The one-pot approach combines isothermal amplification with Argonaute-based detection. The amplified target nucleic acid (e.g., target DNA) is cleaved by Argonaute through base pairing with designed guide single-stranded nucleic acids (e.g., single-stranded guide DNAs), generating a cleavage product with a 5’ phosphate as a new guide nucleic acid. This new guide nucleic acid activates Argonaute to cleave a sequence-specific nucleic acid reporter, which is initially fluorescently quenched. When the reporter is cleaved, a fluorescent signal is generated, allowing for the detection of the nucleic acid. The use of different reporters that can generate different fluorescent signals in a single reaction volume allows for multiplex detection of several targets in one reaction volume (Fig. la). A pyrophosphatase enzyme (PPase) was introduced to enhance the efficiency of the one-pot reaction (Fig. 2a-e) by releasing divalent cations (e.g., Mg²⁺, Mn²⁺) bound by pyrophosphate in the mixture, which are required by the Ago enzyme. For quantification purposes, the reaction can be carried out in numerous small compartments. Isothermal amplification is first used to amplify the target within each compartment before Ago-mediated cleavage of the nucleic acid reporter. The number of copies of each target nucleic acid can be determined by counting the partitions with positive fluorescence signals using Poisson distribution.

Disclosed herein is a method of detecting one or more target nucleic acid molecules in a sample, the method comprising: a) contacting the sample with a reaction mix comprising: i) a reagents for amplifying the one or more target nucleic acid molecules; ii) an Argonaute (Ago) enzyme, one or more guide single stranded nucleic acid molecules and one or more reporter nucleic acid molecules for detecting amplified target nucleic acid; and iii) a pyrophosphatase (PPase) for regenerating Mg²⁺ in the reaction mix; b) amplifying the one or more target nucleic acid molecules under suitable conditions to obtain amplified target nucleic acid molecules; and c) detecting a signal based on cleavage of the reporter nucleic acid molecules by the Argonaute (Ago) enzyme in the presence of the amplified target nucleic acid molecules and guide single stranded nucleic acid molecules, thereby allowing detection of one or more target nucleic acid molecules in the sample.

Also disclosed herein is a method of detecting and/or quantifying one or more target nucleic acid molecules in a sample, the method comprising: a) contacting the sample with a reaction mix comprising: i) a reagents for amplifying the one or more target nucleic acid molecules; ii) an Argonaute (Ago) enzyme, one or more guide single stranded nucleic acid molecules and one or more reporter nucleic acid molecules for detecting amplified target nucleic acid; and iii) a pyrophosphatase (PPase) for regenerating Mg²⁺ in the reaction mix; b) partitioning the mixture of the sample and the reaction mix into a plurality of compartments; c) amplifying the one or more target nucleic acid molecules in each compartment under suitable conditions to obtain amplified target nucleic acid molecules; and d) detecting a signal in each compartment based on cleavage of the reporter nucleic acid molecules by the Argonaute (Ago) enzyme in the presence of the amplified target nucleic acid molecules and guide single stranded nucleic acid molecules.

Disclosed herein is a a method of detecting and/or quantifying one or more target nucleic acid molecules in a sample, the method comprising: a) preparing a plurality of compartmentalised reaction volumes, each reaction volume comprising a volume of the sample and a reaction mix comprising: i) reagents for amplifying the target nucleic acid molecules; ii) an Argonaute (Ago) enzyme; iii) one or more guide single stranded nucleic acid molecules capable of hybridizing to the target nucleic acid molecules; iv) one or more reporter nucleic acid molecules capable of generating a detectable signal upon cleavage by the Ago enzyme; and v) a pyrophosphatase (PPase) for regenerating Mg²⁺ in the reaction mix; b) incubating the compartments under suitable conditions for i) amplifying the one or more target nucleic acid molecules to obtain amplified target nucleic acid molecules, and ii) cleavage of the one or more reporter nucleic acid molecules by the Ago enzyme in the presence of the amplified target nucleic acid molecules and guide single stranded nucleic acid molecules; and c) detecting a signal generated by cleavage of the reporter nucleic acid molecules in each compartment.

The methods herein offer various advantages over other methods of nucleic acid detection and quantification, such as quantitative PCR (qPCR), digital PCR (dPCR), and CRTSPR/Cas-based detection. For example, unlike CRISPR/Cas-based methods, which may require multiple Cas enzymes, methods herein use a single Ago endonuclease, and do not require the presence of a PAM sequence on the target nucleic acids. Furthermore, methods of this disclosure are sensitive enough to detect nucleic acid targets in cell lysates, which removes the need to prepare nucleic acid extracts from samples, thus greatly simplifying sample processing and reducing the amount of sample required for testing. This is particularly advantageous when samples arc limited or targets are present at very low concentrations, as is the case, for example, when testing for contaminant DNA or transduced genes during biomanufacturing processes. Detection using methods herein is also rapid, with turn-around of less than a day. The methods as defined herein may be used in cell therapy quality control (such as for detecting viral vectors or adventitious viruses and quantifying vector copy number), as well as for pathogen detection and quantification, gene expression analysis, rare mutant detection, detection of copy number variations, sequencing library quantification, SNP or point mutation detection, and other areas where nucleic acid detection or quantification is needed. Furthermore, these methods can be used for safety and release testing in the pharmaceutical, nutraceutical, environmental, and food industries. Additionally, they can be used for pathogen diagnostics for public health and biosecurity purposes.

The inventors show that M0T0P1A can be used for quality control testing at various stages in CAR-T manufacturing and cell therapy. For quantification of vector copy number (VCN), the MOTOPIA assay produced results faster and with similar accuracy to dPCR even though it used 98% less cell samples. For monitoring CAR-T cell expansion in vivo, MOTOPIA was twice as fast and required 50-fold less sample compared to flow cytometry. For multiplex detection of viral vectors and adventitious viruses, MOTOPIA achieved sensitivity down to 0.21 pfu/pL, with double the reaction speed and 5-fold reduction in sample use compared to qPCR. Additionally, the inventors also show that MOTOPIA can be readily adapted for point-of-care testing when used with a portable instrument.

As used herein, the term “sample” is used in its broadest sense and includes specimens and cultures obtained from any source, including both biological and environmental sources. Samples include tissues, cells, body fluids and isolates thereof etc., isolated from a subject, as well as tissues, cells and fluids etc. present within a subject (i.e., the sample is in vivo). Samples also include cell and tissue cultures from cell lines or subject-derived specimens. Examples of samples include: whole blood, blood fluids (e.g., serum and plasm), lymph and cystic fluids, sputum, stool (or fecal), tears, mucus, hair, skin, ascitic fluid, cystic fluid, urine, nipple exudates, nipple aspirates, sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, archival samples, explants and primary and/or transformed cell cultures derived from patient tissues, etc. Samples may be undergo processing before analyzing it for a target nucleic acid. In one embodiment, the sample is a cell lysate sample or a nucleic acid sample. The nucleic acid sample may be a substantially pure or a semi-purified preparation of nucleic acids. The nucleic acid sample may be prepared from a cell lysate.

As used herein, the term “nucleic acid”, and equivalent terms such as “polynucleotide”, refer to a polymeric form of nucleotides of any length, such as ribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs), that comprise purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The nucleic acid may be double- stranded or singlestranded. References to single-stranded nucleic acids include references to the sense or antisense strands. The backbone of the polynucleotide can comprise sugars and phosphate groups, as may typically be found in RNA or DNA, or modified or substituted sugar or phosphate groups. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. The terms nucleoside, nucleotide, deoxynucleoside and deoxynucleotide generally include complements, fragments and variants of the nucleoside, nucleotide, deoxynucleoside and deoxy nucleotide, or analogs thereof.

It would be understood that nucleic acids used in the present invention may be structurally and/or chemically modified to, for example, prolong their activity in samples potentially containing nucleases, during performance of the disclosed methods, or to improve shelf-life in a kit. Thus, primers, guide nucleic acids, reporter nucleic acids and any other probes may be chemically modified. In some embodiments, said structural and/or chemical modifications include the addition of tags (such as a fluorescent tag, biotin, or a 5’ tail), the addition of phosphorothioatc (PS) bonds, 2’-O- methyl modifications and/or phosphoramidite C3 Spacers during synthesis.

The term “target nucleic acid” includes any polynucleotide that may be detected or analyzed by a method as defined herein. The target nucleic acid may be naturally- occurring or synthetic. A target nucleic acid may be present in a sample obtained using any methods known in the art. The target nucleic acid may contain DNA, RNA, or a combination thereof, where the polynucleotide contains any combination of deoxyribo- and/or ribonucleotides. Polynucleotides may be single stranded or double stranded, or contain portions of both double stranded or single stranded sequence. Polynucleotides may contain any combination of nucleotides or bases, including, for example, uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguaninc and any nucleotide derivative thereof.

As used herein, the term “nucleotide” may include nucleotides and nucleosides, as well as nucleoside and nucleotide analogs, and modified nucleotides, including both synthetic and naturally occurring species. Polynucleotides may be any suitable polynucleotide, including but not limited to cDNA, mitochondrial DNA (mtDNA), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), nuclear RNA (nRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small Cajal body-specific RNA (scaRNA), microRNA (miRNA), double stranded (dsRNA), ribozyme, riboswitch or viral RNA. Polynucleotides may be contained within any suitable vector, such as a plasmid, cosmid, fragment, chromosome, or genome.

The terms “detecting”, “determining”, “measuring”, “evaluating”, “assessing” and “assaying” arc used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and qualitative determinations. Assessing may be relative or absolute. The method as defined herein may comprise measuring or visualising the levels of one or more polynucleotide analytes in a sample.

Target nucleic acids

Methods herein may be used for any purpose for which detection of viral, bacterial or other nucleic acids is desirable, including diagnostic and prognostic applications, such as in laboratory and clinical settings.

In some embodiments, the target nucleic acid is a nucleic acid that is endogenous to a cell. Alternatively, the target nucleic acid can be a nucleic acid introduced to or expressed in the cell by infection of the cell with a pathogen, for example, a viral or bacterial genomic RNA or DNA, a plasmid, a viral or bacterial mRNA, or the like.

In some embodiments, the target nucleic acid is diagnostic for a disease state. The disease state can be an infection, an organ disease, a blood disease, an immune system disease, a cancer, a brain and nervous system disease, an endocrine disease, a pregnancy- or childbirth-related disease, an inherited disease, or an environmentally- acquired disease, cancer, or a fungal infection, a bacterial infection, a parasite infection, or a viral infection. Thus, in some embodiments, the method is useful for detecting a nucleic acid (e.g., DNA or RNA) from a bacterium, fungus, virus (e.g., a doublestranded DNA virus, a single- stranded DNA virus, a double-stranded RNA virus, a positive sense RNA virus, a negative sense RNA virus, a retrovirus, etc.), or parasite. In some embodiments, the methods may be used for detection of a nucleic acid for genotyping.

In some embodiments, the target nucleic acid is associated with a pathogen, including pathogenic bacteria such as E. faecalis, E. faecium, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus (e.g., MRSA), E. coli O157:H7, Borrelia burgdorferi, Helicobacter pylori, Ehrlichia chaffeensis, Clostridium difficile, Vibrio cholerae 0139, Salmonella enterica, Bartonella henselae, Streptococcus pyogenes, Chlamydia pneumoniae, Clostridium botulinum, Corynebacterium amycolatum, Klebsiella pneumonia, Vibrio vulnificus, and Parachlamydia.

In one embodiment, the target nucleic acid molecule is a viral nucleic acid, such as viral genomic RNA or DNA. The viral nucleic acid may be DNA or RNA.

Exemplary viruses that can be detected include, without limitation, Myoviridac, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae (including human herpes virus, and Varicella zoster virus), Malocohcrpcsviridac, Lipothrixviridac, Rudiviridac, Adenoviridae, Ampullaviridae, Ascoviridae, Asfarviridae (including African swine fever virus), Baculoviridae, Cicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Maseilleviridae, Mimiviridae, Nudiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae (including Simian virus 40, JC virus, BK virus), Poxviridae (including Cowpox and smallpox), Sphaerolipoviridae, Tectiviridae, Turriviridae, Dinodnavirus, Salterpro virus, Rhizidovirus, a Coronaviridae virus, a Picomaviridae virus, a Caliciviridae virus, a Flaviviridae virus, a Togaviridae virus, a Bomaviridae, a Filoviridae, a Paramyxoviridac, a Pncumoviridac, a Rhabdoviridac, an Arcnaviridac, a Bunyaviridac, an Orthomyxoviridae, or a Deltavirus. In some embodiments, the virus is coronavirus (e.g., SARS-Cov-2), SARS, Poliovirus, Rhinovirus, Hepatitis A, Norwalk virus, Yellow fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Zika virus, Rubella virus, Ross River virus, Sindbis virus, Chikungunya virus, Borna disease virus, Ebola virus, Marburg virus, Measles virus, Mumps virus, Nipah virus, Hendra virus, Newcastle disease virus, Human respiratory syncytial virus, Rabies vims, Lassa vims, Hantavirus, Crimean-Congo hemorrhagic fever virus, Influenza, or Hepatitis D vims.

The target nucleic acid may also be a vector derived from a virus, bacterium or fungus, such as a plasmid vector or a viral vector. As used herein, a “viral vector” refers to a recombinant virus or a derivative thereof which is capable of introducing genetic material into a host cell by means of transduction or non-propagative infection. The vector may be a gene delivery vector, antisense delivery vector or gene therapy vector.

In some embodiments, the target nucleic acid is a double-stranded DNA (dsDNA) molecule. In some embodiments, the target nucleic acid is a double-stranded RNA (dsRNA) molecule. In some embodiments, the target nucleic acid is a single-stranded DNA (ssDNA) molecule. In some embodiments, the target nucleic acid is a singlestranded RNA (ssRNA) molecule.

In one embodiment, the one or more target nucleic acid molecules is a viral nucleic acid, wherein the virus is selected from the group consisting of a lentivirus, a retrovirus, an adenovirus (c.g., a human adenovirus), and a herpes simplex virus (HSV). In one embodiment, the target nucleic acid molecule comprises or is complementary to a nucleic acid sequence selected from the group consisting of TTCTACAGATGGAGTG (SEQ ID NO: 101), GGCCTCAACGCCTTCT (SEQ ID NO: 102), CGCGCGGYCACGTCGT (SEQ ID NO: 103), TGTGATAGCAATAGGG (SEQ ID NO: 104) and GTCGCCGCCCCTCGCC (SEQ ID NO: 105).

Nucleic acid amplification

The target nucleic acid molecule may be amplified by any amplification technique that is well known in the art, using reagents known in the art for the amplification technique. The reagents for amplification will generally include a polymerase, nucleotides (e.g. dNTPs), one or more sets of primers, and a cation such as Mg²*, Mn²*, Fe²*, Co²⁺, NP , Cu²⁺. Zn²⁺ or Ca²⁺. Where the target nucleic acid is an RNA, the reagents may further include a reverse transcriptase.

Amplifying the target nucleic acid molecule may, for example, comprise performing loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), recombinase- aided amplification (RAA), exonuclease Ill-assisted signal amplification (EASA), exponential amplification reaction (EXPAR), hybridization chain reaction (HCR), helicase-dependent amplification (HDA), isothermal circular strand displacement polymerization (1CSDP), multiple displacement amplification (MDA), nucleic acid sequence-based amplification (NASBA), primase- based whole genome amplification (pWGA), rolling circle amplification (RCA), stranddisplacement amplification (SDA), whole genome amplification (WGA) or polymerase chain reaction (PCR).

In one embodiment, amplifying the one or more target nucleic acid molecules comprises performing an isothermal amplification reaction. In one embodiment, the isothermal amplification reaction is performed at a temperature between 60°C to 70°C, such as at a temperature of about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, or about 70°C.

In one embodiment, the amplification reaction is loop-mediated isothermal amplification (LAMP) or reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP). LAMP or RT-LAMP may be performed at a temperature between 60°C to 70°C. A heat-stable polymerase and/or reverse transcriptase which retains enzyme activity at temperatures of about 60°C or above may be used. An exemplary polymerase for use in LAMP or RT-LAMP is the Bst polymerase. Exemplary heat-stable reverse transcriptases include but are not limited to the Moloney Murine Leukemia Virus (MMLV) reverse transcriptase and the avian myeloblastosis virus (AMV) reverse transcriptase.

Typical LAMP assays are performed using at least one set of primers comprising two inner and two outer primers that recognise six distinct regions of the target nucleic acid molecule (Fl , F2 and F3 and Bl , B2 and B3, respectively, see Fig. 10). The forward inner primer (FIP) consists of a F2 region and a complementary sequence of an Fl (Flc) region, while the backward inner primer (BIP) consists of the B2 region and a complementary sequence of the B l (B ic) region. The forward outer primer (F3) and backward outer primer (B3) have sequences that are complementary to the sequences of the F3c and B3c regions, respectively. These regions surround the desired amplified sequence. The primers used for LAMP assays can be optimized by a series of factors, such as the nucleotide base pair concentration and locations, the distance between the DNA regions, the thermodynamics of the primers, etc. Accessory primers, such as loop primers and swarm primers, may also be used to further increase the rate of amplification by utilising more sites in the target for amplification.

Methods herein may comprise a nucleic acid denaturation step prior to isothermal amplification. For example, the sample, optionally containing the reaction mix, may be heated to a temperature of about 80°C or above to denature the target nucleic acids prior to isothermal amplification.

The one or more target nucleic acid molecules may be linearly or exponentially amplified.

Argonautc (Ago) enzyme

Argonaute enzymes for use in the disclosed methods may be 'ild-type, mutant or engineered proteins. In one embodiment, the Argonaute enzyme is a thermostable enzyme. The thermostable Ago is preferably one that has cleavage activity at temperatures above 60°C.

Thermostable Ago enzymes can be e.g., from Pyrococcus furiosus, Thermus thermophilus, Thermococcus thioreducens (WP 055429304), Thermococcus onnurineus (WP 012572468), Thermococcus eurythermalis (WP_050002102J, Methanocaldococcus bathoardescens (WP_048201370), Methanocaldococcus sp. FS406-22 (WP_0I2979970), Methanocaldococcus fervens (WP 015791216), Methanocaldococcus jannaschii (WP_010870838), Methanotorris formiscicus (WP_052322764), Ferroglobus placidus (WP_012966655), Sulfolobus sp. (e.g., S. solfataricus), Methanopyrus kandleri, or Thermogladius cellulolyticus (WP_048163021).

In one embodiment, the Argonaute enzyme is a PfAgo enzyme derived from Pyrococcus furiosus, or a derivative thereof. In one embodiment, the Argonaute is TtAgo enzyme derived from Thermus thermophilus, or a derivative thereof. Ago derivatives include enzymes which have been engineered e.g., through targeted or random mutagenesis, for improved functionality, such as improved thermostability, improved rate of nucleic acid cleavage, expanded substrate specificity, etc.

In one embodiment, the Argonaute enzyme is present as a free enzyme in the reaction mix (i.e., not immobilized or conjugated to a surface). The inventors have found that the disclosed one-pot assays are sensitive enough to detect and quantify low concentrations of target nucleic acids without the need to immobilise or sequester the Ago enzyme during the amplification or detection steps.

The Argonaute enzyme may be provided at a concentration of <1 pM, <0.9 pM. <0.8 pM, <0.7 pM, <0.6 pM, <0.5 pM, <0.4 pM, <0.3 pM, <0.2 pM or <0.1 pM. In one embodiment, the Argonaute enzyme is provided at a concentration of about 0.18pM.

In some embodiments, Ago-mediated cleavage is performed at a temperature above 60°C. In some embodiments, Ago-mediated cleavage is performed at a temperature between 60°C to 90°C, such as at a temperature of about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, or about 90°C. In some embodiments, Ago- mediated cleavage is performed at a temperature above 90°C, such as at a temperature of about 95°C.

Guide nucleic acids

Ago enzymes utilise small DNA or RNA guides to cleave nucleic acid targets. A guide single stranded nucleic acid molecule herein is capable of hybridizing to a complementary sequence on a target nucleic acid molecule to form a structure recognized by the Argonaute enzyme. Binding of Ago to the structure generates an Ago complex. An Ago complex herein may comprise one or more guide single stranded nucleic acids, one or more Ago enzymes, and a target nucleic acid molecule. Full complementarity between a guide nucleic acid molecule and a target sequence is not required, provided there is sufficient complementarity to cause hybridization and promote formation of an Ago complex. Typically, in the context of an Ago system, formation of an Ago complex results in cleavage of the target nucleic acid molecule within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more base pairs from) the target sequence. Two or more guide nucleic acids may be used to induce cleavage at two or more positions in a target nucleic acid to generate single- stranded cleavage fragments. The reporter nucleic acid may be designed to have a complementary sequence to the single- stranded cleavage fragment, such that the cleavage fragment serves as a secondary' single stranded guide molecule, targeting Ago cleavage to the reporter nucleic acid molecule.

In one embodiment, the one or more guide single stranded nucleic acid molecules is a guide single- stranded DNA (ssDNA) or a guide single-stranded RNA (ssRNA). The single stranded guide molecules may be 5’-phosphorylatcd, 3 ’-phosphorylated, or phosphorylated at both the 5’ and 3’ ends. In some embodiments, the guide nucleic acid comprises a 2’ modification at one or both of the 3’ and 5’ termini. The 2’ modification may be, for example, a 2’-O-methyl (2’0Me), 2’-O-methoxyethyl (2’MOE) or 2’-fluoro (2’F) modification. Such chemical modifications can increase the binding affinity of the guide nucleic acid for Ago and thus enhance the efficiency of Ago cleavage. In some embodiments, methods herein comprise the use of two or more guide single stranded nucleic acid molecules.

Guide nucleic acids are not particularly limited. As shown in the working examples, one of ordinary skill in the ait can design sets of guide molecules that are specific for any target nucleic acid molecule sequence.

Pyrophosphatase (PPase)

Preferred pyrophosphatases (PPase) are ones that have enzyme activity at the temperature of the Ago-mediated nucleic acid cleavage reactions, so as to regenerate free Mg²⁺ while Ago is active.

In some embodiments, the pyrophosphatase is a thermostable pyrophosphatase. Exemplary thermostable pyrophosphatases include but are not limited to enzymes from Thermococcus litoralis, Pyrococcus horikoshii, and Sulfolobus acidocaldarius.

Reporter nucleic acids

Reporter nucleic acid molecules herein are capable of generating a detectable signal, such as a colorimetric or fluorescent signal, upon cleavage by the Ago enzyme. The reporter nucleic acid may have a sequence that is complementary to the sequence of a cleavage fragment from Ago cleavage of a target nucleic acid. Thus, sets of guide nucleic acids can be used to induce targeted Ago cleavage of the target nucleic acids to release single- stranded cleavage fragments, which act as secondary guides, targeting Ago cleavage to the reporter nucleic acids.

In one embodiment, the one or more reporter nucleic acid molecules comprises a fluorescent label. The reporter nucleic acid molecules may be detected based on FRET or change in fluorescence emission wavelength. Non-limiting examples of fluorescent labels include xanthene derivatives (such as fluorescein, rhodamine, Oregon green, eosin, and Texas red), cyanine derivatives (such as cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine), squaraine derivatives or ring-substituted squaraines (such as Seta and Square dyes), squaraine rotaxane derivatives, naphthalene derivatives (such as dansyl and prodan derivatives), coumarin derivatives, oxadiazolc derivatives (such as pyridyloxazolc, nitrobenzoxadiazole and benzoxadiazole), anthracene derivatives (such as anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange), pyrene derivatives (such as cascade blue), oxazine derivatives (such as Nile red, Nile blue, cresyl violet, and oxazine 170), acridine derivatives (such as proflavin, acridine orange and acridine yellow), arylmethine derivatives (such as auramine, crystal violet, malachite green), tetrapyrrole derivatives (such as porphin, phthalocyanine, bilirubin), or dipyrromethene derivatives (such as BODIPY, aza- BODIPY).

The reporter nucleic acid molecules may comprise a quencher label for quenching the detectable signal until Ago-mediated cleavage of the reporter nucleic acid.

As used herein, the term “quencher” refers to any moiety that can attenuate at least partly a signal (such as a colorimetric or fluorescent signal) emitted by the reporter nucleic acid, hi embodiments where the signal is a fluorescent signal from a fluorescent label on the reporter nucleic acid, excitation of the fluorophore in the presence of the quenching group may lead to an emission signal that is less intense than expected, of a different wavelength, or completely absent. Quenching typically occurs through energy transfer between the excited fluorophore and the quenching group. The quencher moiety may absorb energy from the fluorophore and then emit a signal (e.g., light at a different wavelength). Thus the quencher moiety may itself be a second fluorophore (e.g., a first fluorophore can be 6-carboxyfluorcsccin while the quencher or second fluorophore can be 6-carboxy-tetramethylrhodamine). The fluorophore-quencher pair can also be a FRET pair. Alternatively, the quencher may be a dark quencher. A dark quencher absorbs excitation energy and dissipates the energy in a different way (e.g., as heat). Thus, a dark quencher produces minimal to no fluorescence of its own (does not emit fluorescence). Examples of quenchers include, but are not limited to: a dark quencher, a Black Hole Quencher® (BHQ®) (e.g., BHQ-0, BHQ-1 , BHQ-2, BHQ-3), a Qxl quencher, an ATTO quencher (e.g., ATTO 540Q, ATTO 580Q, and ATTO 612Q), dimethylaminoazobenzenesulfonic acid (Dabsyl), Iowa Black RQ, Iowa Black FQ, IRDye QC-1, a QSY dye (e.g., QSY 7, QSY 9, QSY 21), AbsoluteQuencher, Eclipse, and metal clusters such as gold nanoparticlcs, and the like.

In some embodiments, each reporter nucleic acid molecule may have a spectrally distinct emission wavelength that allows it to be distinguished from other reporter nucleic acid molecules with a different emission wavelength in the same reaction tube (e.g. in multiplex reactions). In other embodiments, the reporter nucleic acid molecules are labelled with biotin or with a gold nanoparticle that allows detection on lateral flow.

In some embodiments, cleavage of a reporter nucleic acid is detected by measuring a colorimetric read-out. For example, the liberation of a fluorophore (e.g., liberation from a quencher/fluorophore pair) can result in a wavelength shift (and thus color shift) of a detectable signal. Thus, in some cases, cleavage of a reporter nucleic acid can be detected by a color shift. Such a shift can be expressed as a loss of an amount of signal of one color (wavelength), a gain in the amount of another color, a change in the ratio of one color to another, and the like.

In some embodiments, cleavage of the reporter nucleic acid is detected visually. Visual detection may be by direct observation (e.g., by eye or using a camera or a microscope) or via spectroscopic or spectrophotometric measurement.

In some embodiments, cleavage of the reporter nucleic acid is detected by measuring a change in a fluorescent signal produced by the reporter nucleic acid. The change in fluorescent signal may be an increase or a decrease in fluorescence.

Nucleic acid detection

In one embodiment, detecting amplified target nucleic acid is performed at a temperature that is equal or higher than the temperature(s) at which the amplification reaction is performed. In one embodiment, detecting amplified target nucleic acid is performed at a temperature that is above 60°C, above 65°C, above 70°C, above 75°C, above 80°C, above 85°C or above 90°C. In one embodiment, detecting amplified target nucleic acid is performed at a temperature that is above 90°C. In one embodiment, detecting amplified target nucleic acid is performed at about 95°C.

In some embodiments, signal detection is performed at the same temperature as Ago- mediated cleavage. In other embodiments, signal detection is performed at a different temperature from Ago-mediated cleavage.

In one embodiment, signal detection is performed at a lower temperature than Ago- mediated cleavage. The inventors have found that lowering the temperature after Ago cleavage can increase the signal-to-noise ratio and decrease the time taken for the signal to be generated and detected.

In some embodiments, Ago-mediated cleavage is performed at a temperature above 60°C, and signal detection is performed at a temperature below 60°C. In one embodiment, signal detection is performed at a temperature below 40°C.

Nucleic acid quantification

Methods herein may comprise quantifying the amount of the one or more target nucleic acid molecules in the sample.

In some embodiments, the methods are performed in a plurality of independent partitions. The method as defined herein may comprise partitioning the mixture of the sample and the reaction mix in step a) into a plurality of compartments. Alternatively, the sample may be partitioned into a plurality of compartments and the reaction mix added to each compartment. Each compartment may comprise on average not more than one target nucleic acid molecule.

The method may comprise determining the copy number of the one or more target nucleic acid molecules based on a Poisson distribution of the proportion of positive-to- negative compartments. A positive compartment is a compartment in which a signal is detected, and a negative compartment is a compartment in which a signal is not detected.

Methods herein may be performed in multiwell formats, microfluidic devices or chips.

The methods may use a digital sample partition system. The digital sample partition system may be a commercial digital PCR system or any sample partition device with a detector capable of detecting the signal generated by the reporter nucleic acid. The digital sample partition system may be a microfluidics-based, droplet-based, microcellbased, membrane-based or hydrogel-based partition system.

Disclosed herein is a method of detecting and/or quantifying one or more target nucleic acid molecules in a sample, the method comprising separating the sample into a plurality of compartments, performing the method as defined herein in each compartment to detect one or more target nucleic acid molecules in the sample. The method may comprise detecting two or more target nucleic acid molecules in the sample.

Kits

Disclosed herein is a reaction mix comprising: i) reagents for amplifying one or more target nucleic acids; ii) an Argonaute (Ago) enzyme, one or more guide single stranded nucleic acid molecules and one or more reporter nucleic acid molecules for detecting amplified target nucleic acids; and iii) a pyrophosphatase (PPase) for regenerating Mg²⁺ in the reaction mix. The reaction mix may comprise nucleotides (e.g. dNTPs) for amplifying the target nucleic acid. The reaction mix may comprise Mg²⁺. The reaction mix may further comprise a suitable buffer.

In some embodiments, the amplification reagents are reagents for LAMP or RT-LAMP amplification. Such reagents may comprise a DNA polymerase (such as a Bst polymerase or derivative thereof), LAMP primers (including inner and outer primers and optionally loop and swarm accessory primers), and optionally a reverse transcriptase. The DNA polymerase and reverse transcriptase are preferably thermostable enzymes. In some embodiments, the Ago enzyme and pyrophosphatase are thermostable enzymes.

In some embodiments, the Ago enzyme is present as a free enzyme in the reaction mix, i.e., the enzyme is not immobilized to a support or separately sequestered.

In some embodiments, the reaction mix comprises two or more guide single stranded nucleic acid molecules for each target nucleic acid.

Disclosed herein is a kit comprising a reaction mix as defined herein. The kit may comprise a microchip or compartmentalised reaction vessel for partitioning the sample and/or reaction volumes.

As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

EXAMPLES

Materials and methods

The LAMP primers, DNA guides and FQ reporters were synthesised by Integrated DNA Technologies. Bst 2.0 WarmStart polymerase, isothermal buffer, MgSO i, thermostable inorganic pyrophosphatase were from New England Biolabs. Human genomic DNA were from Roche.

Design and screening of primers and guide DNAs

(a) Consensus sequence analysis

For broader spectrum detection, such as targeting all commonly used retroviral vectors, the sequences of the targets arc first obtained from databases like NCBI. In the case of retroviral vectors, sequences can also be obtained from y-retroviral transfer plasmids from Addgene. Since only the region between the 5’ LTR and 3’ LTR on the transfer plasmid will be integrated into the genome, the 5’ LTR region is selected as the target region. The 5’ LTR sequences of different retroviral vectors are aligned to find the consensus sequence using UGENE software (v48.1) or other alignment software. The consensus sequence of the target is then used for primer design.

(b) LAMP primer design

For LAMP primer design, the specific sequence or consensus sequence is used as the target sequence, and PrimcrExplorcr V5 or the NEB LAMP Primer Design Tooi may be used to design primers. The distance between Flc and B 1c is set at a minimum of 32 bp to facilitate Argonaute-based detection. Typically 4-8 primer sets are designed and screened.

(c) LAMP primer screening

DNA targets containing the retroviral vector consensus sequence are mixed with 1.6 pM FIP/BIP, 0.2 pM F3/B3, and 0.4 pM LoopF/LoopB primers for each designed LAMP primer set, along with 2 mM dNTPs. Thermostable inorganic pyrophosphatase and Pf Argonaute are included in the reaction to mimic the MOTOPIA reaction mix, but are not required for the reaction. The mixture is heated to 95°C for 5-10 minutes and then immediately chilled on ice. Next, a LAMP master mix containing lx isothermal amplification buffer, 8 mM MgSCL, 1.2 U/pL Bst 2.0 WarmStart polymerase (New England Biolabs), 0.025 U/pL thermostable inorganic pyrophosphatase, 0.016 pg/pL Pf Argonaute, 0.2 pg/pL bovine scrum albumin or recombinant albumin (New England Biolabs), 50 mM taurine, 250 nM SYTO 9 green fluorescent nucleic acid stain (Invitrogen), and 250 nM Cyanine 680 succinimidyl ester (Biotium) is added to the 61 pL reaction.

For digital reactions, 40 pL of the reaction mixture is loaded into a QTAcuity digital nanoplate. These reactions axe automatically partitioned and incubated at 65°C for 25 minutes followed by endpoint fluorescence imaging, using FAM exposure duration (600 ms) and gain (6) for sample reading. In parallel, 20 pL reaction mixtures are divided into duplex and run on a LightCyclcr 96 qPCR machine (Roche) at 65°C for 30 minutes with fluorescence monitoring during the process. From the results, the primer set with the fastest speed and highest amplification efficiency (positive partition percentage) is selected to design guide DNA for the Argonaute reaction.

(d) Guide DNA design

Different sets of guide DNA arc designed using the sequence between the Flc and Bic regions of the chosen LAMP primer set based on the following principles.

(c) Guide DNA screening

The guide DNA sets are screened using the MOTOPIA-based quantification methods described below. The guide DNA set that provides the highest signal-to-noise ratio is chosen.

Different concentrations of serially diluted samples containing the target DNA (e.g., retroviral sequence) are quantified using the MOTOPIA-based quantification methods described below. The results are analyzed to ensure that the calculated copy number results have a linear relationship with the input retroviral vector DNA, indicating the successful design and development of the MOTOPIA assay for the target.

Argonaute protein preparation

77Ago was from New England Biolabs, while P/Ago was either from MCLAB or expressed and purified by Sangon Biotech. The open reading frame (ORF) of the Pyrococcus furiosus Argonaute (P/Ago) gene was optimized for codon usage bias of Escherichia coli. The ORF was synthesized by Sangon Biotech and cloned into a pET28a expression vector. This vector was then transformed into E. coli BL21 (DE3) and a 5 mL seed culture was grown at 37°C in LB medium containing 50 pg/mL kanamycin. This culture was transferred to 1 L of LB medium in a shake flask containing 50 pg/mL kanamycin and incubated at 37°C until an OD600 value of O.6-O.8 was reached. Protein expression was induced with the addition of IPTG to a final concentration of 1 mM, followed by 16 hr incubation at 15°C. Cells were harvested via centrifugation for 20 min at 6000 rpm and 4°C, and the cell pellet was collected for purification. Cell pellets were resuspended in lysis buffer (20 mM Tris/HCl, pH 8.0, 300 mM NaCl, 2 mM MnCh) and then disrupted using an Ultrasonic Homogenizer. After centrifuging at 12,000 rpm and 4°C for 30 min, the supernatant was collected, heated to 80°C for 30 min, and centrifuged again at 12,000 rpm and 4°C to remove denatured proteins. This supernatant was then applied to nickel-charged (Ni-NTA) beads for affinity purification. After washing the column twice with two column volumes of wash buffer (20 mM Tris/HCl pH8.0, 300 mM NaCl, 2 mM MnCh, 50 mM imidazole) and once with one column volume of elution buffer (20 mM Tris/HCl pH8.0, 300 mM NaCl, 2 mM MnCh, 500 mM imidazole), the target protein was recovered. The washing fractions containing the protein were then dialyzed into a storage buffer (20 mM Tris- HC1, pH 8.0, 300 mM NaCl, 0.5 mM MnCh, 10% (v/v) glycerol).

Argonaute guide DNA preparation

The selected DNA sequence (5 pM) was phosphorylated using T4 polynucleotide kinase (New England Biolabs), T4 PNK buffer (New England Biolabs) and ATP (New England Biolabs) at 37°C for 1 hour. The reaction was then heated to 65°C for 20 minutes to inactivate the T4 Polynucleotide Kinase. The resulting reaction containing the 5’ phosphorylated guide DNA can be used directly for Argonaute protein.

DNA target preparation

Synthetic DNA of lentiviral vector (RRE), human albumin gene (ALB), and VSVG gene were synthesized by IDT and inserted into a pUC-IDT-Amp vector. To obtain linearized DNA containing RRE or ALB, pUC-IDT-Amp containing the corresponding DNA sequence was amplified using pUC-Amp-F (tggtaagccctcccgtatcg) and pUC- Amp-R (tctggccccagtgctgcaatg) primers. Twenty cycles of PCR were performed using Platinum SuperFi PCR Master Mix at 98°C for 1 minute, followed by 20 cycles of 98°C for 10 seconds, 60°C for 10 seconds, and 72°C for 3 minutes; a final extension step was performed at 72°C for 5 minutes. The PCR product was purified using a QIAquick PCR Purification Kit (QIAGEN), and the DNA concentration was determined using a Nanodrop and dPCR. DNA of HSV and ADV viruses was obtained through extraction performed using a QTAamp DNA Mini Kit (QIAGEN), and CAR-T cell DNA was extracted using the DNeasy Blood & Tissue Kit (QIAGEN) starting from around 1 million CAR-T cells according to manufacturer’ s protocol.

Virus culture

Human adenovirus 1 (ATCC VR-1) was propagated in A549 human lung epithelial cells (ATCC CCL-185) grown in Ham's F-12K (Kaighn’s) Medium (Thermo Fisher Scientific). The virus was then purified using a chromatography-based Adeno-X Maxi Purification system (Takara Bio) following the manufacturer’s instructions. Herpes simplex virus 1 (ATCC VR-260) was propagated in Vero cells (ATCC CCL-81) grown in Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific). Concentration and purification were achieved through ultracentrifugation and iodixanol density gradient ultracentrifugation, respectively.

T cell isolation, activation, CAR transduction and CAR-T cell culture

Human CD3+ T cells were isolated from Human Peripheral Blood Mononuclea' Cells obtained from LONZA leukopak, using the EasyScpTM Human T Cell Isolation kit. The viable T cells were counted and activated using Dynabeads® Human T-Expander CD3/CD28 at a 1: 1 ratio. The activated T cells were cultured for one day in AIM-V medium supplemented with 2% human serum and interleukin-2 (IL-2) at 100 lU/mL. The following day, a viable cell count was obtained, and the cells were transduced with lentiviral vector encoding a second generation 4-lBB-CD3-zeta anti-CD19 CAR with GFP expression downstream of an IRES (1) produced from adherent HEK293T cells, using a Multiplicity of Infection (MOI) of 0.15, 0.5, 1.5, 5, 15, 50 and 150. The transduced T cells were left to grow for 12 days in a G-Rex 24 well plate at 37.0 °C, 5% CO2. Every two days after transduction 6 mL of AIM V spent medium was collected and replaced with 6 mL of fresh AIM V medium + IL-2 up till day 12. Around 1 million cells per condition were harvested on day six, eight and twelve and washed twice with PBS. Flow cytometry analysis for green fluorescent protein (GFP) was performed to identify the percentage of CAR positive T cells. The cell pellets were stored in -80°C for future experiments.

Digital PCR for Vector Copy Number Quantification

DNA extracted from around 1 million CAR-T cells (100 pL eluted DNA in total) was fragmented using 0.4 pL of 20,000 units/ml EcoRI-HF and 0.4x rCutSmart Buffer (New England Biolabs) in a 20 pL reaction. The fragmentation process involved incubation at 37°C for 30 minutes and 80°C for 2 minutes. This reaction was diluted with 140 pL of double distilled H2O, and 8.2 pL of the diluted solution was employed for digital PCR.

8.2 pL of fragmented DNA stated above was mixed with QIAcuity Probe PCR Master Mix, RRE forward and reverse primers at 1 pM, an RRE probe at 0.5 pM (FAM), Albumin forward and reverse primers at 0.6 pM, and an Albumin probe at 0.3 pM (HEX). Forty microliters of the reaction mixture were loaded onto the QIAcuity digital nanoplate and into the QIAcuity Digital PCR System. The reactions were automatically partitioned into 26,000 microwells, followed by 95°C incubation for 5 minutes and 40 cycles of 95°C for 30 seconds and 60°C for 1 minute, with end point fluorescence detection. To get the best signal to noise ratio, the exposure duration (200 ms to 800 ms) and gain (2 to 8) were adjusted for each experiment, usually at 600 ms/6 for sample reading. Positive partition percentages and nucleic acid concentration were calculated using the QIAcuity software.

Patient material

For viral copy number quantification, PBMCs were isolated from four anonymized lymphoma patients who were undergoing CD19-directed CAR T-cell therapy. The PBMCs were sourced from discarded leukaphcrcsis tubing sets (Spectra Optia Apheresis System, Terumo BCT). After isolation, the PBMCs were cryopreserved in CryoStor CS10 (STEMCELL Technologies) and later thawed for CAR T-cell production using either G-Rex or Breez bioreactors, following the protocol outlined in our previous study. For monitoring CAR-T cell expansion, samples from seven adult patients underwent BCMA-directed CAR-T cell therapy or Axi-cel treatment between 2023 and 2024 were analyzed. Blood samples were collected from patients at various time points post-treatment, and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque (STEMCELL Technologies, Cologne, Germany) through gradient centrifugation. The isolated PBMCs were then cryopreserved in CryoStor CS10 (STEMCELL Technologies) for subsequent flow cytometry and dMOTOPIA analysis. qPCR for virus detection

Extracted DNA from viruses spiked into CAR-T cells was used for qPCR as a comparison test to MOTOPIA-based virus detection. The DNA samples were mixed with TaqMan Fast Advanced Master Mix (Applied Biosystems), forward and reverse primers at 500 nM each, and probes at 250 nM each for RCL, ALB, hAdV, and HSV targets in a 20 pL reaction. All reactions were performed using the LightCycler qPCR instrument (Roche) with the following program: initial incubation at 95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds. Ct values were determined using Roche LightCycler 96 software.

Direct cell lysis

A cell pellet containing 0.01 million cells was mixed with 1% TritonX-100, 20 units/ml proteinase K, 0.25x rCutSmart Buffer (New England Biolabs), and nuclease-free water to a total volume of 20 pL. The mixture was then vortexed for 15 seconds and spun down, heated to 55°C for 20 minutes, vortexed and spun down again, and heated to 95°C for 10 minutes. After heating, the lysate was cooled on ice, vortexed, and spun down, and was then ready to be used for Argonautc-bascd detection or quantification.

Argonaute-based detection

Samples of extracted DNA or 15 pl cell lysate were mixed with 1.6 pM FIP/BIP, 0.2 pM F3/B3, 0.4 pM LoopF/LoopB, and 1.6 pM F1S/B1S primers for each detection target as well as 2.0 mM dNTPs. The mixture was heated to 95°C for 5-10 minutes, followed by an immediate chill on ice. Then, LAMP-Argonaute master mix containing lx isothermal amplification buffer, 8 mM MgSCL, 0.64 U/pL Bst 2.0 WarmStart polymerase, 0.025 U/uL thermostable inorganic pyrophosphatase, 0.016 pg/pL Pf Argonaute, 0.0002 mg/pL bovine serum albumin, 50 mM taurine, 0.05 pM corresponding guide DNAs and 1 pM probes for each target was added to the reaction. Finally, 20 pL of the mixture was run in a LightCyclcr 96 machine at 65°C for 30 minutes and 95°C for 10 minutes and 37°C for 60 seconds, with real-time fluorescent measurement every 20 seconds during the 95°C session and an end-point fluorescent measurement after the reaction.

Argonaute-based quantification

20 pL cell lysate from 0.01 million cells stated above were directly mixed with 0.4 pL 20,000 U/ml EcoRI-HF, followed by incubation at 37°C for 30 minutes and 80°C for 2 minute. The reaction was then diluted with 60 pL of double distilled H2O, and 12.3 pL of the diluted solution was subsequently used for Argonaute-based quantification. Additionally, DNA extracted from around 1 million CAR-T cells (100 pL eluted DNA in total) was fragmented using 0.4 pL of 20,000 U/ml EcoRI-HF and 0.4x rCutSmart Buffer (New England Biolabs) in a 20 pL reaction. The fragmentation process involved incubation at 37 °C for 30 minutes and 80°C for 2 minutes. This reaction was diluted with 140 pL of ddfEO. and 8.2 pL of the diluted solution was employed for Argonaute- based quantification.

Samples of extracted DNA or cell lysate were mixed with 1.6 pM FIP/BIP, 0.2 pM F3/B3 and 0.4 pM LoopF/LoopB for each detection target as well as 1.4 mM dNTPs. The mixture was heated to 95°C for 5-10 minutes, followed by an immediate chill on ice. Then, LAMP-Argonaute master mix containing lx isothermal amplification buffer, 8 mM MgSCL, 1.2 U/pL Bst 2.0 WarmStart polymerase, 0.025 U/pL thermostable inorganic pyrophosphatase, 0.016 pg/pL P/Ago. 0.0002 mg/pL bovine scrum albumin, 50 mM taurine, 0.05 pM corresponding guide DNAs, 250 nM Cyanine 680 succinimidyl ester (Biotium) and 0.5 pM probes for each target was added to the reaction. Finally, a total of 40 pL reaction mixtures were loaded into a QIAcuity digital nanoplate (~0.91 nL partition volume, -26,000 partitions per reaction). The reactions were then automatically partitioned and incubated at 65°C for 40 minutes, 95°C for 20 minutes and 35°C for 1 minute to cool the plate before end point fluorescence imaging. To maximize signal to noise ratio, exposure duration (200 ms to 800 ms) and gain (2 to 8) were adjusted for each experiment, and a 600 ms/6 was generally used for sample reading. The QIAcuity software calculated positive partition percentages and nucleic acid concentration based on the captured images.

MOTOPIA-based point-of-care testing

For MOTOPIA-based point-of-care testing, 60 pL of cell lysate (from 50 pL of cells) was mixed with 1.6 pM FIP/BIP, 0.2 pM F3/B3, 0.4 pM LoopF/LoopB, and 1.6 pM FIS/BIS primers for RCL and HSV targets, along with 2.0 mM dNTPs. The mixture was heated to 95°C for 5-10 minutes, followed by an immediate chill on ice. Next, the MOTOPIA master mix was prepared, containing lx isothermal amplification buffer, 8 mM MgSO4, 0.64 U/pL Bst 2.0 WarmStart polymerase (New England Biolabs), 0.025 U/pL thermostable inorganic pyrophosphatase, 0.016 pg/pL Pf Argonaute, 0.2 pg/pL bovine serum albumin or recombinant albumin (New England Biolabs), 50 mM taurine, 0.05 pM corresponding guide DNAs, and 2 pM probes for each target. This master mix was then added to the reaction, bringing the total volume to 200 pL. Subsequently, 100 pL of mineral oil was added on top of the reaction to prevent evaporation. The reaction was incubated in an Elite Dry Bath Incubator (Bio Lab) at 65°C for 40 minutes, followed by heating at 95°C for 30 minutes (for fluorometer detection). The endpoint fluorescence of FAM and Cy5 was detected at room temperature using a Qubit 4 Fluorometer (Thermo Fisher Scientific).

Table 1. Exemplary primer, probe and gDNA sequences

Example 1: Multiplex Omni-purpose Technology in One-Pot via Isothermal amplification and Argonaute (MOTOPIA)

To develop an Argonaute-based detection method, Argonaute proteins from Pyrococcus furiosus (pfAgo) and Thermits thermophilus (TtAgo) were tested in their ability to detect various concentrations of DNA targets (Fig. 3). pfAgo was chosen for subsequent experiments due to its higher sensitivity. It was found that Argonaute alone had a sensitivity of nanomolar level, which was insufficient for many detection requirements. Therefore, it was decided to combine an amplification step with the Argonaute-based detection. LAMP was selected as it could amplify target DNA ( 10⁹ copies in an hour) under isothermal conditions. Mn²⁺ was usually used as a cofactor for Argonaute activity. However, from previous studies, Mn²⁺ was found to inhibit LAMP reaction, thus slowing the reaction time and decreasing sensitivity of the one-pot reaction. Therefore, it was tested whether Argonaute could use Mg²⁺ as a cofactor for the reaction (Fig. 4). The results showed that around 5 mM Mg²⁺ could achieve similar effects as Mn²⁺ and enable Argonaute reaction.

In the proposed one-pot reaction with Mg²⁺ as a cofactor, the target is initially amplified by LAMP at 65°C, followed by increasing the temperature to 95°C to activate the cleavage of the target amplicons by Ago, guided by two or three tar get- specific gDNAs. This process generates a new gDNA from the amplicons that initiates secondary cleavage of reporters complementary to the target, releasing a fluorescence signal for end-point detection (Fig. la).

In an initial attempt to combine isothermal amplification with Argonaute-based detection in a one-pot reaction, it was observed that the LAMP worked well, while Argonaute-based detection failed (Fig. 2b). It was hypothesized that the LAMP was consuming Mg²⁺ by incorporating dNTPs into the DNA, leaving insufficient Mg²⁺ for the Argonaute reaction (Fig. 2a, left). To test this hypothesis, the inventors attempted to increase the Mg²⁺ or decrease the dNTPs in the one-pot reaction, but this approach doubled the LAMP reaction time and was not feasible (Fig. 2b-d). To solve this problem, a pyrophosphatase (PPasc) was introduced to recycle Mg²⁺ for the Argonaute reaction (Fig. 2a, right). It was found that, by using PPase, a one -pot reaction of LAMP and Argonaute was successfully achieved without compromising either enzyme’s activity (Fig. 2e). The PPase concentration was further optimized to minimize the delay in LAMP reaction (Fig. 5).

It was found that the concentration of the Ago protein significantly affects the sensitivity and kinetics of the LAMP reaction. High Ago concentrations delay the LAMP reaction and reduce sensitivity, whereas lower concentrations enhance sensitivity but slow down Ago-mediated cleavage kinetics (Fig. 2f and 6). Notably, at Ago levels of 0.016 mg/mL or below, fluorescent signals were not detected until 30 minutes after the start of the reaction (Fig. 6).

It was found that reducing the temperature from 95°C to 37°C after the one-pot LAMP- Ago reaction significantly decreases background fluorescence and increases target fluorescence across FAM, HEX, ROX, and Cy5 channels. This temperature adjustment enabled target detection within 10 minutes at 0.016 mg/mL Ago, shortening the reaction time by over ten-fold (Fig. 2g and 7). This effect was consistent for both LAMP- amplified loop DNA and PCR-amplified linear DNA (Fig. 2h-j and 8). Additionally, it was observed that there was decreased Ago cleavage activity when the DNA amplicon concentration exceeded 24 nM, possibly due to excess target DNA occupying Ago during the first cleavage round, leaving insufficient Ago for the second cleavage on the probe. Despite delayed second cleavage activity, lowering the temperature to 37°C still improved the signal-to-noisc ratio, allowing target detection in 10-20 minutes (Fig. 2g- h, 7 and 8).

Further analysis revealed that these temperature-dependent fluorescence changes are attributable to fluorescence quenching mechanisms. The increased fluorescence of the positive target at 37 °C might be due to reduced thermal quenching, as this effect was also observed on pure FAM or ROX dye. Conversely, the decreased fluorescence in negative controls at 37°C may be attributed to reduced flexibility of the fluorescent- quenched DNA probe at lower temperatures, promoting more efficient quenching by the attached quencher (Fig. 2k and 9). By utilizing end-point fluorescence measurements at below 37 °C, more sensitive and rapid detection of Ago-mediated cleavage can be achieved.

Based on the observations above, Multiplex Omni-purpose Technology in One-Pot via Isothermal amplification and Argonaute (MOTOPIA) was developed for single molecular level nucleic acids detection and quantification. In the one -pot reaction, the target is first amplified by LAMP at 65°C, followed by detection by pfAgo at 95°C. The entire reaction can be completed within one hour, allowing for simultaneous detection of multiple targets. Notably, a 95°C pre-treatment of the target was used to help open the nucleic acid strand and LAMP swarm primer was introduced for some applications to further improve the speed and increase sensitivity (Fig. 10).

Example 2: Multiplex nucleic acid quantification using digital MOTOPIA (dMOTOPIA)

To achieve absolute quantification using MOTOPIA, a digital sample partitioning strategy was developed. The reaction mix was divided into thousands of partitions, each on average containing a single target, and reactions were conducted in each partition. Positive partitions giving a signal were then quantified according to a Poisson distribution (Fig. le). Since most commercial digital chips partition reactions into volumes of approximately 1 nL, achieving a detection limit at least fM level (1 copy per 1 nL partition) is necessary for digital absolute quantification. Since PfAgo has superior sensitivity (0.25 nM) compared to TtAgo (Fig. 3), it was chosen for use in the digital MOTOPIA platform.

Digital MOTOPIA (dMOTOPIA) was used to quantify vector copy number (VCN), an essential parameter for assessing the clinical potency and safety of CAR-T cells. The clinical potency of CAR-T cells correlate with the number of transgene copies. However, higher copy numbers may increase the risk of cancer, thus safety testing is essential to maintain transgene copy numbers within a safe range. The US Food and Drug Administration (FDA) recommends that VCN be assessed for CAR-T cell products. To quantify VCN, a duplex assay targeting both the vector and human genome is necessary (Fig. lb).

Given that current CAR-T cell therapies primarily use lentiviral vectors for transgene integration, the VCN of lentivirus-transduced CAR-T cells was used as proof-of- concept. The sequences of 35 commonly used lentiviral vector systems were analysed, and a primer/gDNA/probe set (with the FAM dye/channel) targeting the 5’LTR-TVRRE (RRE) consensus region was designed. This region, located upstream of the CAR transgene, integrates into the genome along with the transgene. Assays targeting this region can be univers lly applied to detect second and third generation lentiviral vectors regardless of the CAR design. Additionally, a primer/gDNA/probe set (with EIEX or ROX dyes/channels) targeting the housekeeping gene albumin (ALB) was also designed.

To achieve absolute quantification, the MOTOPIA reaction was divided into approximately 26,000 individual partitions using a commercial digital chip, and the endpoint fluorescent signal was measured. The copy number of the target was quantified by the positive partition rate using Poisson distribution, eliminating the need for a standard curve (Fig. 12a). To test the quantification performance, the duplex digital MOTOPIA (dMOTOPIA) assay was mixed with different amounts of lentiviral vector and human DNA. It was found that dMOTOPIA could quantitatively detect both vector and human DNA down to the attomolar range (4.7 aM) (Fig. 12b). Moreover, the measurement of one target remains consistent regardless of the concentration of the other channels, indicating the robustness of the duplex assay (Fig. 12c). The copy number results of dMOTOPIA plotted against the input target concentrations showed a good linear relationship from around 40 aM to 4000 aM (R²>0.98), indicating the accurate quantification ability of dMOTOPIA (Fig. 12d). The dMOTOPIA VCN assay was also validated using the WHO lentiviral copy number standard quantified using digital PCR (dPCR). The results from both methods correlated well with the input WHO standard VCN (0.1 to 10 copies/genome, R²>0.99), while dMOTOPIA generated results faster (Fig. 12e). Beyond assays targeting lentiviral vectors, MOTOPIA also demonstrated flexibility and broad target applicability, as evidenced by the successful quantification of retroviral vector copy numbers using a primer/gDNA/probe set targeting the consensus sequence of retroviral vectors (Fig. 15). The design and validation process for retroviral vectors target can be completed in three days, demonstrating the broad utility of MOTOPIA.

Example 3: Quantification of CAR-T cell expansion and viral load in CAR-T manufacturing using dMOTOPIA

The use of dMOTOPIA for DNA quantification in CAR-T cell therapy was investigated. Normal qPCR and dPCR-based methods require DNA extraction for VCN quantification. To avoid sample loss during DNA extraction and accelerate the process, a sample-to-result test was developed that involved direct cell lysis followed by VCN quantification on lysate by dMOTOPIA. The direct lysis approach preserved more than 98% of the CAR-T cells and halved the time taken to obtain a result Human Tcells from healthy donors were infected with lentiviruses containing the CAR gene at different multiplicities of infection (MOI) and expanded for 12 days to mimic the CAR-T manufacturing process, producing CAR-T cells with varying VCNs (Fig. 13a). At different VCN levels, we observed a good linear relationship between the gold standard dPCR results and the dMOTOPIA results on both extracted DNA and cell lysate (R²>0.98), while dMOTOPIA is faster (Fig. 13b). As expected, higher MOIs yielded higher VCNs, and the VCN results from the three methods correlated with transduction efficiency (R²>0.96, Fig. 13c). Furthermore, it was demonstrated that the VCN remained constant across different days post-transduction, indicating stable insertion of the CAR into the T-cell genome and stable VCN during the CAR-T cell manufacturing process (Fig. 13d). The performance of dMOTOPIA on research-grade CAR-T cells generated from two different bioreactors (G-Rex and Breez) was also evaluated using PBMCs from four anonymized patients with lymphoma. Results from dMOTOPIA based on DNA and cell lysate samples correlated well with results from dPCR (R²>0.99 or 0.98), further confirming the performance of dMOTOPIA on complex patient samples (Fig. 13e and f). Monitoring CAR-T cell expansion

As CAR-T cells are living therapies, monitoring their expansion in vivo following infusion is essential for assessing treatment efficacy and identifying potential side effects. To evaluate and compare CAR-T cell numbers over time, precise quantification methods arc necessary. The application of dMOTOPIA for this purpose was investigated (Fig. 1c). The dMOTOPIA monitoring assay involves one quantification channel targeting the CAR transgene and another targeting human peripheral blood mononuclear cells (PBMCs), where an increase in CAR gene copies within PBMCs indicates greater CAR-T cell expansion. CAR-T cell expansion in patients with multiple myeloma (MM) was monitored following BCMA-directed CAR-T cell therapy (n=3, lentiviral vector), and in patients with diffuse large B-cell lymphoma (DLBCL) following axi-cel treatment (n=4, retroviral vector) (Fig. 14a). Peripheral blood samples were collected from patients at various days post-treatment, and PBMCs were analyzed using dMOTOPIA, with results compared to conventional flow cytometry (Fig. 14a-c). For all seven patients, dMOTOPIA accurately matched the cell expansion kinetics observed with flow cytometry, while requiring 98% less sample and 50% less reaction time (Fig. 14d). Even across 96 blood samples from 7 patients collected on different days, a positive correlation (R² = 0.915) is observed between dMOTOPIA assay results and flow cytometry measurements (Fig. 14c). These findings indicate that dMOTOPIA is highly effective for monitoring CAR-T cell expansion in peripheral blood.

The use dMOTOPIA for evaluating treatment prognosis was also assessed. For most patients, a peak in CAR-T cell expansion was followed by a slight decline, as detected by both methods (Fig. 14d). For MM patients receiving BCMA-directed CAR-T cell therapy (patients #1, #2, #3), CAR-T cell expansion peaked between days 10 and 15 post- infusion, with a peak around 1 to 1.2 transgcncs per PBMC. For DLBCL patients #5, #6 and #7 receiving axi-cel treatment, CAR-T cell expansion peaked slightly earlier, between days 5 and 10 post-transfusion, with a peak around 0.06 to 0.66 transgcncs per PBMC. Patient #5 with DLBCL had a lower copy number of the CAR transgene compared to other patients, ultimately resulting in disease relapse for this individual. For DLBCL patient #4, who received axi-cel treatment, the CAR transgene copy number was below 0.01, significantly lower than that of other patients receiving the same treatment, and this patient succumbed to disease progression. Despite the small patient cohort, these results indicate that responders exhibit higher CAR-T cell expansion than non -responders. The lack of expansion in non-responders underscores the importance of CAR-T cell monitoring to confirm functional CARs in vivo.

Monitoring adventitious viruses in CAR-T cell culture via MOTOPIA

Beyond in vivo monitoring, ensuring the safety of the CAR-T cell manufacturing process and final CAR-T product is important. dMOTOPlA was used to detect replication-competent lentivirus (RCL) and adventitious viruses in CAR-T cells during CAR-T (Fig. Id). The use of lentiviral vectors in CAR-T therapy poses a theoretical safety risk that could result in the introduction of RCL. Also, as a living cell therapy, CAR-T cell therapy is vulnerable to adventitious agent contamination, such as virus contamination from contaminated raw materials or during the manufacturing process. Thus, a MOTOPIA assay was developed to simultaneously detect RCL and adventitious viruses in CAR-T cell culture for both manufacture monitoring and release test (Fig. Ila). For RCL testing, a primer/guide/probe set in the FAM channel was developed targeting the vesicular stomatitis virus G glycoprotein (VSV-G) gene. This gene encodes an envelope protein crucial for replicating virus assembly and has been validated as a reliable target for RCL in previous studies. For adventitious viruses, human adenovirus (HAdV, ROX channel) and herpes simplex virus (HSV, Cy5 channel) were used as proof-of-concept targets as they are two reported viral contaminations in biologic manufacturing handling human or primate cell lines.

To identify conserved regions in HAdV and HSV, all strains accessible from the National Center for Biotechnology Information (NCBI) were aligned to identify target regions with sequence homology of more than 95% among all 128 human mastadenovirus C strains (covering human adenovirus 1, 2, 5 and 6) or all 41 human herpesvirus strains (covering herpes simplex virus 1 and 2). Degenerate sequences were used to ensure detection coverage. The human albumin gene was used as an internal positive reference target (HEX channel). The specificity of a 4-plex MOTOPIA assay was first assessed using different combinations of four target viral nucleic acids. The presence of each target was determined by a corresponding fluorescence readout from the MOTOPIA reaction, such as FAM fluorescence for RCL, HEX fluorescence for human DNA, ROX fluorescence for adenovirus and Cy5 fluorescence for human herpesvirus. It was found that the fluorescence only increased when there was the corresponding target. Also, the four detection channels did not interfere with each other, indicating the assay’s high specificity (Fig. 11b). Additionally, the LoD of 4-plex MOTOPIA to detect each individual virus using serially diluted DNA was assessed. The results showed that MOTOPIA could detect targets as low as 1 copy/pL for virus and human DNA and 5 copies/pL for RCL (Fig. l lc-f). The above results indicate the high specificity and sensitivity of MOTOPIA for multiplex detection.

After proving its sensitivity on extracted viral DNA, the assay was next tested on CAR- T cell cultures and compared with the conventional qPCR method (Fig. 16a). Various concentrations of viral particles or RCL plasmids were spiked into CAR-T cell cultures. From the results, both MOTOPIA and qPCR showed good sensitivity, detecting down to 0.04 pfu/pL HSV virus. At concentrations higher than 20 copies/pL RCL, 2 copies/pL HAdV, and 0.2 pfu/pL HSV, both methods have a 100% accuracy. At lower concentrations, MOTOPIA is slightly better at detecting HAdV while qPCR is slightly better at detecting HSV (Fig. 16b-d). Both methods showed an AUC above 0.90 (Fig. 16e-g). MOTOPIA is more suitable for virus monitoring in CAR-T cell therapy as it consumes fewer samples and is faster.

Towards point-of-care testing via MOTOPIA

Simplifying the assay readout in MOTOPIA could significantly broaden the potential uses of MOTOPIA, particularly in point-of-care (POC) settings. Rapid and cost- effective diagnostic capabilities provided by POC testing hold great promise, facilitating quicker treatment decisions and improving accessibility.

To optimize MOTOPIA for POC settings, rapid cell lysis was integrated with the MOTOPIA assay, with results measured using a portable fluorometer (Fig. 17a). This approach enabled a duplex assay to provide either a positive or negative test result. To prevent reaction evaporation and avoid cross-contamination in POC settings, mineral oil was added on top of the reaction. The entire process from sample to result was completed in under two hours. Challenging targets from previous experiments (RCL at the FAM channel and HSV at the Cy5 channel) were used for duplex MOTOPIA. Various concentrations of HSV virus particles and/or RCL plasmid were spiked into 1 million/mL CAR-T cell cultures, comparing the detection results from MOTOPIA to qPCR. MOTOPIA demonstrated the ability to detect as low as 2 copies/pL of RCL and 0.044 pfu/pL of HSV with 100% accuracy. In contrast, qPCR showed lower performance when both targets were present in the sample, only detecting 22 copies/pL of RCL when HSV was also present, whereas MOTOPIA was more sensitive and able to detect RCL at 2 copies/pL (Fig. 17d). Compared to qPCR, our portable MOTOPIA version saved 75% of the sample and 50% of the reaction time, showcasing superior sensitivity and accuracy for virus detection.

Example 4: Advantages of the MOTOPIA platform

Prokaryotic Argonaute proteins (pAgos) can be used for programmable DNA and RNA detection, and provide greater flexibility than CRISPR/Cas systems for target selection. Previous studies using Ago-based detection platforms have mostly focused on diagnostic applications, which do not usually require target quantification. Here, the inventors have developed a digital Ago-based detection platform (MOTOPIA) which can be used for rapid, sensitive, multiplex detection and quantification of target nucleic acids. In particular, the inventors show that MOTOPIA can be used to monitor various stages of CAR-T cell therapy, from cell manufacturing to post-administration patient monitoring.

A major advantage of MOTOPIA is its minimal cell requirement, made possible by the lysate tolerance of the LAMP and Argonaute reactions. Current methods for CAR-T cell assessment require millions of CAR-T cells, which is a significant fraction of the clinical dose (-1% of a traditional CAR-T cell therapy and -10% of a fast CAR-T cell therapy). In contrast, MOTOPIA reduces sample usage by up to 98%, preserving more therapeutic cells for treatment. Furthermore, Argonaute’ s multiplexing capability allows for simultaneous testing of multiple targets in a single reaction, further conserving precious samples.

Speed is another key advantage of M0T0P1A. Traditional methods such as culturebased techniques, flow cytometry, and qPCR/dPCR require lengthy procedures, including extensive incubation, wash steps, and DNA extraction. By utilizing cell lysates and a rapid LAMP- Ago reaction, MOTOPIA eliminates the extraction step and significantly reduces the overall processing and detection time. These advancements enable clinicians to make timely and informed decisions, which is crucial in CAR-T cell therapy due to the potential for severe side effects.

MOTOPIA also offers absolute quantification through digital sample partitioning, essential for various aspects of CAR-T cell therapy test, such as VCN quantification in cell therapy, viral titer quantification in gene therapy, and CAR-T cell transgene number monitoring in vivo. Previous Argonaute-based methods lacked the capability for a one- pot LAMP and Ago reaction with digital sample partitioning. By employing a Mg²⁺ recycling strategy, accurate VCN quantification and precise monitoring of CAR-T cell expansion was achieved. The ability to obtain precise and reliable quantification allows for accurate results despite patient-to-patient variations, facilitating personalized treatment adjustments and enhancing therapeutic efficacy.

Moreover, MOTOPIA is versatile and capable of identifying various clinically relevant nucleic acid targets, including pathogenic nucleic acids, nucleic acid vectors used in cell and gene therapy, and viral contaminants in biomanufacturing. Unlike CRISPR/Cas- based methods, MOTOPIA does not rely on a PAM sequence for target recognition, broadening the spectrum of detectable targets. MOTOPIA can be designed and developed for new targets within three days, making it valuable for rapid responses to epidemiological crises, such as epidemic outbreaks. Additionally, MOTOPIA is suitable for point-of-carc (POC) and in-ficld medical diagnostic testing, demonstrating compatibility with portable fluorometers. This POC adaptation maintains high sensitivity and accuracy while significantly reducing sample and time requirements compared to qPCR. The development of a portable device for visual output further enhances MOTOPIA’ s accessibility in diverse environments. CAR-T therapy has shown significant success in cancer treatment; however, clinical outcomes vary among patients. Efficient engraftment and expansion of CAR-T cells are critical for efficacy, but the impact of CAR-T cell expansion kinetics on clinical outcome is not fully understood. Accurate assessment of CAR-T cell expansion kinetics is essential for optimizing treatment strategics. The MOTOPIA platform can be used to measure and compare CAR-T cell expansion kinetics in different CAR-T cell therapies. Our study shows that MOTOPIA can accurately track CAR-T cell expansion kinetics with reduced sample and time requirements compared to conventional flow cytometry. The correlation between CAR-T cell expansion and clinical outcome in our study highlights MOTOPIA’ s potential for predicting therapeutic efficacy and disease prognosis. For example, patients who exhibited higher CAR-T cell expansion monitored by MOTOPIA tended to have better clinical responses, suggesting that MOTOPIA can serve as a valuable tool for early prediction of treatment success and facilitate personalized treatment adjustments based on individual patient profiles, enhancing therapeutic efficacy.

The potential applications of MOTOPIA extend beyond CAR-T cell therapy. Given its rapid, sensitive, and multiplex detection capabilities, MOTOPIA could be adapted for monitoring other therapies, such as CAR-NK cell therapy or gene therapy, where timely and precise quantification of transgene integration or disease marker is crucial for patient safety and therapeutic efficacy. Additionally, its minimal sample requirement and compatibility with direct cell lysates make it an attractive tool for infectious disease diagnostics. The ability to design assays quickly for new nucleic acid targets positions MOTOPIA as a flexible platform that can respond to evolving diagnostic needs in precision medicine.

Claims

1. A method of detecting and/or quantifying one or more target nucleic acid molecules in a sample, the method comprising: a) contacting the sample with a reaction mix comprising: i) reagents for amplifying the target nucleic acid molecules; ii) an Argonautc (Ago) enzyme, one or more guide single stranded nucleic acid molecules, and one or more reporter nucleic acid molecules for detecting amplified target nucleic acid; and iii) a pyrophosphatase (PPase) for regenerating Mg²⁺ in the reaction mix; b) partitioning the mixture of the sample and the reaction mix into a plurality of compartments; c) amplifying the one or more target nucleic acid molecules in each compartment under suitable conditions to obtain amplified target nucleic acid molecules; and d) detecting a signal in each compartment based on cleavage of the reporter nucleic acid molecules by the Argonaute (Ago) enzyme in the presence of the amplified target nucleic acid molecules and guide single stranded nucleic acid molecules, thereby allowing detection and/or quantification of the one or more target nucleic acid molecules in the sample.

2. The method of claim 1, wherein the sample is a cell lysate sample or a nucleic acid sample.

3. The method of claim 1 or 2, wherein the target nucleic acid molecule is a viral nucleic acid, and wherein the virus is selected from the group consisting of a lentivirus, a retrovirus, a human adenovirus, and a herpes simplex virus.

4. The method of any one of claims 1 to 3, wherein amplifying the one or more target nucleic acid molecules comprises performing an isothermal amplification reaction.

5. The method of claim 4, wherein the isothermal amplification reaction is loop- mediated isothermal amplification (LAMP).

6. The method of claim 5, wherein the isothermal amplification reaction is performed at a temperature between 60°C to 70°C.

7. The method of any one of claims 1 to 6, wherein the Ago enzyme is an enzyme from Pyrococcus furiosus (PfAgo) or a derivative thereof, or Thermus thermophiles (TtAgo) or a derivative thereof.

8. The method of any one of claims 1 to 7, wherein the Ago enzyme is present as a free enzyme in the reaction mix.

9. The method of any one of claims 1 to 8, wherein step d) is performed at a temperature that is above 60°C.

10. The method of any one of claims 1 to 8, wherein signal detection is performed at a lower temperature than Ago-mediated cleavage in step d).

11. The method of claim 10, wherein Ago-mediated cleavage is performed at a temperature above 60°C, and signal detection is performed at a temperature below 60°C.

12. The method of claim 11, wherein signal detection is performed at a temperature below 40°C.

13. The method of any one of claims 1 to 12, wherein the one or more guide single stranded nucleic acid molecules is a guide ssDNA or a guide ssRNA.

14. The method of any one of claims 1 to 13, wherein the one or more reporter nucleic acid molecules comprises a fluorescent label.

15. The method of any one of claims 1 to 14, wherein the one or more reporter nucleic acid molecules comprises a quencher label.

16. The method of any one of claims 1 to 15, wherein each compartment comprises on average not more than one target nucleic acid molecule.

17. The method of any one of claims 1 to 16, wherein the method comprises determining the copy number of the one or more tar get nucleic acid molecules based on a Poisson distribution of the proportion of positivc-to-ncgativc compartments.

18. A method of detecting and/or quantifying one or more target nucleic acid molecules in a sample, the method comprising: a) preparing a plurality of compartmentalised reaction volumes, each reaction volume comprising a volume of the sample and a reaction mix comprising: i) reagents for amplifying the target nucleic acid molecules; ii) an Argonaute (Ago) enzyme; iii) one or more guide single stranded nucleic acid molecules capable of hybridizing to the target nucleic acid molecules; iv) one or more reporter nucleic acid molecules capable of generating a detectable signal upon cleavage by the Ago enzyme; and v) a pyrophosphatase (PPase) for regenerating Mg²⁺ in the reaction mix; b) incubating the compartments under suitable conditions for i) amplifying the one or more target nucleic acid molecules to obtain amplified target nucleic acid molecules, and ii) cleavage of the one or more reporter nucleic acid molecules by the Ago enzyme in the presence of the amplified target nucleic acid molecules and guide single stranded nucleic acid molecules; and c) detecting a signal generated by cleavage of the reporter nucleic acid molecules in each compartment.

19. The method of claim 18, further comprising determining the copy number of the one or more target nucleic acid molecules based on a Poisson distribution of the proportion of positivc-to-ncgativc compartments.

20. A reaction mix comprising: i) reagents for amplifying one or more target nucleic acid molecules; ii) an Ago enzyme, one or more guide single stranded nucleic acid molecules and one or more reporter nucleic acid molecules for detecting amplified target nucleic acid; and iii) a pyrophosphatase (PPase) for regenerating Mg²⁺ in the reaction mix.

21. The reaction mix of claim 20, wherein the Ago is present as a free enzyme.

22. The reaction mix of claim 20 or 21, wherein the reaction mix comprises Mg²⁺.

23. A kit comprising a reaction mix of any one of claims 20 to 22.