Method for detecting an analyte in a pathogen-containing sample

Abstract

The present disclosure relates to novel multiplexing methods and kits for detecting different analytes in a sample in parallel by sequential signal encoding of said analytes. In particular, the present disclosure relates to methods that result in inactivation of pathogens for in situ spatial transcriptomics (e.g., molecular mapping) without DNA or RNA preparation steps prior to analysis.

Description

[Technical field]

[0001] Disclosure Fields The technology provided herein relates to methods for detecting analytes in pathogen-containing samples, particularly by multiplex spatial transcriptomics methods, as well as detecting analytes in pathogen-inactivated samples by in vitro methods for the diagnosis of disease. [Background technology]

[0002] Methods for detecting specific molecular, cellular, and pathogenic targets (such as viruses, bacteria, or other pathogens) are fundamental tools for medical and veterinary diagnostics, environmental testing, and industrial quality control. Examples of methods for detecting specific targets in clinical medicine include over-the-counter rapid pregnancy tests, microbial culture tests for measuring the resistance of infectious pathogens to specific antibodies, and automated tests for cancer markers in blood samples. Detection of pathogen contaminants in food, high-throughput screening of candidate compounds for drug discovery, and quantification of active ingredients in pharmaceuticals are examples of industrial manufacturing applications that rely on methods for measuring the presence of specific targets. Environmental applications requiring testing for specific targets include water supply contamination, airborne biothreat pathogens, and household fungal contamination.

[0003] For example, the COVID-19 pandemic caused by SARS-CoV-2 highlights the clinical need to detect infections, track strain evolution, and identify biomarkers of disease progression. The analysis of gene expression relies on measurement equipment that must not be co-located in laboratories with higher safety levels used for pathogen detection. Analysis of these samples at higher safety levels is therefore not possible if the measurement equipment is located in the same laboratory. An alternative is to extract the biomolecules of interest and then reduce the safety level so that the analysis can be performed outside of the higher safety level laboratory. Typically, spatial transcriptomics analysis of biosafety samples is performed after RNA or DNA has been isolated. This is typical of scenarios using Visium technology (10x Genomics) or the GeoMx system (Nanostring; https: / / www.nature.com / articles / s41467-021-21361-7).

[0004] The analysis and detection of small amounts of analytes in biological and non-biological samples has become a routine task in clinical and analytical environments. Numerous analytical methods have been established for this purpose. Some of them use coding techniques that assign a specific readable code to a specific first analyte, which is different from the code assigned to a specific second analyte.

[0005] One of the prior arts in this field is the so-called "single molecule fluorescent in situ hybridization" (smFISH), which was developed substantially to detect mRNA molecules in a sample. In Lubeck et al. (2014), Single-cell in situ RNA profiling by sequential hybridization, Nat. Methods 11(4), p.360-361, the mRNA of interest is detected by a specific directly labeled probe set. After one round of hybridization and detection, a set of mRNA-specific probes is eluted from the mRNA, and the same probe set with another (or the same) fluorescent label is used in the next round of hybridization and imaging to generate a gene-specific color-coded scheme over several rounds. This technique requires several different tagged probe sets per transcript and requires denaturation of these probe sets after every detection round.

[0006] Further developments of this technology do not use directly labeled probe sets. Instead, the oligonucleotides of the probe set provide nucleic acid sequences that act as initiators for hybridization chain reaction (HCR), a technique that allows for signal amplification; see Shah et al. (2016), In situ transcription profiling of single cells reveals spatial organization of cells in the mouse hippocampus, Neuron 92(2), p.342-357.

[0007] Another technique called "multiplexed error-tolerant fluorescent in situ hybridization" (merFISH) has been reported by Chen et al. (2015), RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells, Science 348(6233):aaa6090. There, the mRNA of interest is detected by specific probe sets that provide additional sequence elements for the subsequent specific hybridization of fluorescently labeled oligonucleotides. Each probe set provides 4 different sequence elements out of a total of 16 sequence elements. After hybridization of the specific probe sets to the mRNA of interest, so-called readout hybridizations are performed. In each readout hybridization, one of 16 fluorescently labeled oligonucleotides that is complementary to one of the sequence elements is hybridized. All readout oligonucleotides use the same fluorescent color. After imaging, the fluorescent signal is destroyed by light irradiation and the next round of readout hybridization occurs without a denaturation step. As a result, a binary code is generated for each mRNA species. A unique signal signature of 16 rounds of 4 signals is generated using only one hybridization round for binding of a specific probe set to the mRNA of interest, followed by 16 rounds of hybridization of a readout oligonucleotide labeled with a single fluorescent color.

[0008] Further developments of this technology improve throughput by using two different fluorescent colors, eliminating the signal by disulfide cleavage between the readout oligonucleotide and the fluorescent label, and using alternative hybridization buffers; see Moffitt et al. (2016), High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization, Proc. Natl. Acad. Sci. US A. 113(39), p. 11046-11051.

[0009] A technique called "intron seqFISH" is reported in Shah et al. (2018), Dynamics and spatial genomics of the nascent transcriptome by intron seqFISH, Cell 117(2), p.363-376. There, the mRNA of interest is detected by a specific probe set that provides additional sequence elements for subsequent specific hybridization of fluorescently labeled oligonucleotides. Each probe set provides one of 12 possible sequence elements (representing the 12 "pseudocolors" used) per color-coding round. Each color-coding round consists of four successive hybridizations. In each of these successive hybridizations, three readout probes, each labeled with a different fluorophore, are hybridized to the corresponding elements of the mRNA-specific probe set. After imaging, the readout probes are removed with 55% formamide buffer, followed by the next hybridization. After five color-coding rounds, each containing four successive hybridizations, the color code is completed.

[0010] European Patent EP0611828 discloses the use of bridging elements to recruit signal generating elements to probes that specifically bind to analytes. More specifically, the description describes the detection of nucleic acids by specific probes that recruit bridging nucleic acid molecules. This bridging nucleic acid ultimately recruits signal generating nucleic acids. This patent document also describes the use of bridging elements with two or more binding sites for signal generating elements for signal amplification, such as branched DNA.

[0011] Player et al. (2001), Single-copy gene detection using branched DNA (bDNA) in situ hybridization, J. Histochem. Cytochem. 49(5), p. 603-611, describes a method in which a nucleic acid of interest is detected by a specific probe set that provides an additional sequence element. In a second step, a preamplifier oligonucleotide is hybridized to this sequence element. This preamplifier oligonucleotide contains multiple binding sites for amplifier oligonucleotides that are hybridized in a subsequent step. These amplifier oligonucleotides provide multiple sequence elements for label oligonucleotides. In this way, a branched oligonucleotide tree is constructed that results in an amplification of the signal.

[0012] A further development of this method, called RNAscope, was reported by Wang et al. (2012), RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues, J. Mol. Diagn. 14(1), p. 22-29, which uses an alternative design of mRNA-specific probes. Here, two mRNA-specific oligonucleotides must hybridize in close proximity to provide sequences that can recruit preamplifier oligonucleotides. Thus, the specificity of the method is increased by reducing the number of false positive signals.

[0013] Choi et al. (2010), Programmable in situ amplification for multiplexed imaging of mRNA expression, Nat. Biotechnol. 28(11), p.1208-1212, discloses a method known as "HCR-Hybridization Chain Reaction". The mRNA of interest is detected by a specific probe set that provides an additional sequence element. The additional sequence element is an initiator sequence to start the hybridization chain reaction. Essentially, the hybridization chain reaction is based on metastable oligonucleotide hairpins that self-assemble into a polymer after the first hairpin is released by the initiator sequence.

[0014] Further developments of this technology use so-called split-initiator probes, which, similar to the RNAscope technology, must hybridize in close proximity to form the initiator sequence for HCR, reducing the number of false positive signals; see Choi et al. (2018), Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145(12).

[0015] Mateo et al. (2019), Visualizing DNA folding and RNA in embryos at single-cell resolution, Nature Vol, 568, p. 49ff., disclose a method called "optical reconstruction of chromatin structures (ORCA)". This method aims to visualize chromosome folding.

[0016] European Patent No. EP2992115B1 describes a method of sequential single molecule hybridization, providing a technique for detecting and / or quantifying nucleic acids in cells, tissues, organs or organisms by sequential barcoding.

[0017] Against this background, it is an underlying objective of the present disclosure to provide a method for detecting an analyte in a pathogen-containing sample by means of which the drawbacks of prior art methods may be reduced or even avoided. Summary of the Invention

[0018] The present disclosure relates to a novel multiplexing method and kit for detecting different analytes in a sample in parallel by sequential signal coding of the analytes.In particular, the present disclosure relates to a method that provides pathogen inactivation for in situ spatial transcriptomics (e.g., molecular mapping) without a step of DNA or RNA preparation before analysis.One important advantage is that the analysis can be performed outside of a BSL3 or BSL4 laboratory. An advantageous embodiment of the present disclosure is a method for detecting an analyte in a pathogen-containing sample comprising the steps of: 1) collecting a sample containing a pathogen having a risk level >2; 2) inactivating pathogens in the sample without isolating RNA or DNA from the pathogens or sample; 3) Conducting spatial *omics analysis.

[0019] In a first aspect, embodiments of the present disclosure relate inter alia to a method for detecting an analyte in a pathogen-containing sample comprising: i) Inactivation of pathogens in a sample without isolation of RNA and / or DNA from the pathogen or sample; ii) Analyte detection by spatial transcriptomics.

[0020] In particular, spatial transcriptomics detection methods include multiplexing methods for detecting different analytes in a sample by sequential signal encoding of the analytes, comprising: (A) contacting the sample with at least twenty (20) different sets of analyte-specific probes for encoding at least twenty different analytes, each set of analyte-specific probes interacting with a different analyte, and when the analytes are nucleic acids, each set of analyte-specific probes includes at least five (5) analyte-specific probes that specifically interact with different substructures of the same analyte, each analyte-specific probe being: (aa) a binding element (S) that specifically interacts with one of the different analytes encoded therein; and (bb) an identifier element (T) comprising a nucleotide sequence unique to the analyte being encoded (the unique identifier sequence); wherein the analyte specific probes of a particular set of analyte specific probes are different from the analyte specific probes of another set of analyte specific probes in the nucleotide sequence of the identifier element (T); contacting, wherein the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and contain the same nucleotide sequence of an identifier element (T) unique to said analyte; and (B) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein each decoding oligonucleotide in each set of decoding oligonucleotides for a distinct analyte comprises: (aa) an identifier connector element (t) comprising a nucleotide sequence substantially complementary to at least a portion of a unique identifier sequence of an identifier element (T) of a corresponding analyte-specific probe set; and (bb) a translation element (c) comprising a nucleotide sequence allowing specific hybridization of a signal oligonucleotide; wherein a set of decoding oligonucleotides for a distinct analyte is different from another set of decoding oligonucleotides for a different analyte in the first connector element (t); and (C) contacting the sample with at least one set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translation connector element (C) comprising a nucleotide sequence substantially complementary to at least a portion of the nucleotide sequence of the translation element (c) contained in the decoding oligonucleotide; and (bb) Signal element contacting, including; (D) detecting a signal due to the signal element; (E) selectively removing the decoding oligonucleotides and the signal oligonucleotides from the sample, thereby substantially maintaining specific binding of the analyte-specific probe to the encoded analyte; (F) performing at least three further cycles comprising steps B) to E) to generate an encoding scheme comprising a code word for each analyte, in particular the last cycle may stop at step (D).

[0021] In a second aspect, an embodiment of the present disclosure relates to an in vitro method for the diagnosis of a disease resulting from a pathogen infection, such as a viral or bacterial infection, comprising the use of a method according to the present disclosure.

[0022] In a third aspect, embodiments of the present disclosure provide an in vitro method for the diagnosis of a disease in a plant caused by an infectious and / or parasitic origin, said method comprising the use of a method according to the present disclosure.

[0023] Before describing the present disclosure in detail, it should be understood that the present disclosure is not limited to the specific components of the steps of the described method. It should also be understood that the terms used herein are used for the purpose of describing specific embodiments only, and are not intended to be limiting. It should be noted that, as used in the specification and the appended claims, the singular forms "a", "an" and "the" include singular and / or plural referents unless the context clearly dictates otherwise. It should also be understood that when a range of a parameter bounded by numerical values ​​is given, the range is deemed to include these limits. [Brief description of the drawings]

[0024] [Figure 1] The embodiment in which the analyte is a nucleic acid and the probe set comprises oligonucleotides that specifically bind to the analyte, the probes comprising unique identifier sequences that allow hybridization of a decoding oligonucleotide. [Diagram 2] In an embodiment where the analyte is a protein and the probe set comprises a protein (here an antibody) that specifically binds to the analyte, the probe comprises a unique identifier sequence that allows hybridization of a decoding oligonucleotide. [Diagram 3] 1 is a flow chart of a method according to the present disclosure. [Figure 4] Another option for the application of decoding and signal oligonucleotides. [Diagram 5] Example of signal encoding of three different nucleic acid sequences with two different signal types and three detection rounds. In this example, the encoding scheme includes error detection. [Figure 6] The number of generated codewords versus the number of detection cycles (logarithmic scale). [Figure 7] Calculated total efficiency of the five-round encoding scheme based on single-step efficiencies. [Figure 8] Comparison of relative transcript abundance between different experiments. [Figure 9]Correlation of relative transcript abundance between different experiments. [Figure 10] Comparison of intercellular distribution of signals. [Figure 11] Comparison of the subcellular distribution of signals. [Figure 12] Distribution patterns of different cell cycle-dependent transcripts. [Figure 13] Detection of multiple targets using 8 rounds of code with two labels (A and B) and no label (-). Targets 1, 2, 3, 4, 5, 20, and n are shown. Rounds 1, 2, 3, and 8 of the coding scheme are shown. In this specification, blanks are part of the code. [Figure 14] Detection of multiple targets can be performed by a coding scheme using detectable markers. The termination scheme can also include "0" as a marker, meaning that no transcript is detected at a particular position. As a result, the coding scheme can be represented by the following constructs using only two gene-specific probes: 1) Contains detectable label F: detectable during imaging 2) Contains detectable label F and quencher Q: not detectable during imaging 3) Contains quencher Q: not detectable during imaging 4) Does not contain label F: not detectable during imaging 5) Does not contain signaling oligonucleotide: not detectable during imaging 6) Contains decoder oligonucleotide that cannot recruit signaling oligonucleotide 7) Does not contain decoder oligonucleotide: not detectable during imaging [Figure 15] Possible structures of the multi-decoder. The numbers show examples. (A) is a unique identifier sequence, (a) is the corresponding sequence of the decoding oligonucleotide or multi-decoder, and (c1)-(c3) are different sequence elements that specifically bind to different signal oligonucleotides. Examples 2-5 show different versions of the multi-decoder. The order of the different sequence elements as well as the number of signal oligonucleotide binding elements are not fixed. Example 1 shows a normal decoding oligonucleotide since there is only one signal oligonucleotide binding element (c1). [Figure 16]Example of signal encoding of three different nucleic acid sequences by using a multi-decoder and two different signal oligonucleotides that generate three different signal types and three detection rounds. In this example, the encoding scheme includes error detection and correction. [Figure 17] Number of generated codewords versus number of detection cycles (logarithmic scale). The number of codewords for merFISH does not grow exponentially with the number of detection cycles, but each additional round becomes less efficient. In contrast, the number of codewords for intronSeqFISH, the disclosed method without multiple decoders, and the method including multiple decoders grow exponentially. The slope of the curve for the method with multiple decoders is much larger than that of the prior invention, resulting in over 20,000,000 times more usable codewords after 20 rounds of detection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Disclosed herein are novel multiplexing methods and kits for detecting different analytes in a sample by sequential signal encoding of said analytes. This disclosure describes the use of a set of labeled and unlabeled nucleic acid sequences to perform specific quantitative and / or spatial detection of different analytes in parallel by specific hybridization. This technology allows the differentiation of more different analytes than the available different detection signals. Differentiation can be achieved by sequential signal encoding of the analytes, which is achieved by several cycles of specific hybridization, detection of the signal and selective elution of the hybridized nucleic acid sequences.

[0026] In contrast to other state-of-the-art methods, the oligonucleotide that provides detectable signal does not directly interact with sample-specific nucleic acid sequence, but is mediated by so-called "decoding oligonucleotide". This mechanism separates the dependency between analyte-specific oligonucleotide and signal oligonucleotide. The use of decoding oligonucleotide allows much higher flexibility while dramatically reducing the number of different signal oligonucleotides required, which in turn increases the coding capacity that can be achieved in a certain number of detection rounds.

[0027] The use of decoding oligonucleotides provides a sequential signal encoding technique that is more flexible, less expensive, simpler, faster, and / or more accurate than other methods.

[0028] A.Definition In the present disclosure, an "analyte" is an object that is specifically detected as present or absent in a sample and, if present, codes for it. It can be any type of substance, including a protein, polypeptide, protein or nucleic acid molecule (e.g., RNA, PNA or DNA) of interest. An analyte provides at least one site for specific binding with an analyte-specific probe. In the present specification, the term "analyte" may be replaced by "target". An "analyte" according to the present disclosure includes an object, such as a complex of at least two individual nucleic acid, protein or peptide molecules. In one embodiment of the present disclosure, an "analyte" excludes a chromosome. In another embodiment of the present disclosure, an "analyte" excludes a DNA.

[0029] In some embodiments, the analyte may be a "coding sequence," "encoding sequence," "structural nucleotide sequence," or "structural nucleic acid molecule," which refers to a nucleotide sequence that is translated into a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of a coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. Coding sequences may include, but are not limited to, genomic DNA, cDNA, ESTs, and recombinant nucleotide sequences.

[0030] A "sample" as referred to herein is a composition in liquid or solid form that is believed to contain the analyte to be encoded. In particular, the sample is a biological sample, preferably comprising biological tissue, more preferably comprising biological cells and / or extracts and / or cell parts. For example, the cells are prokaryotic or eukaryotic cells, particularly mammalian cells, especially human cells. In some embodiments, the biological tissue, biological cells, extracts and / or cell parts are fixed. In particular, the analytes in permeabilized samples, such as cell-containing samples, are fixed. In particular, the sample is a pathogen-containing sample, and may be any type of substance, tissue, biological material, organism, etc., that contains a pathogen. The sample may be any type of solid, fluid sample, smear, surface that contains a pathogenic substance. The sample may comprise eukaryotes, archaea, prokaryotes, or viruses. The sample may be from an organism, an environmental sample, excretion from an organism, or a surface.

[0031] "Pathogen" includes any kind of biological agent (eukaryotes, archaea, prokaryotes, viruses, viroids, proteins, pure RNA) that has a "risk level" higher than 2 and is defined as a risk for human, animal, plant and microbial populations (e.g. in the gut, in fermenters, in lakes, etc.). Risk levels are defined by local authorities and can vary regionally (e.g. from country to country). For example, in Germany risk levels are defined by BioStoffV. In other countries risk levels are defined by other laws and regulations (e.g. CDC in the USA). In all cases the risk level of a pathogen is defined by a scientific empirical procedure.

[0032] "Pathogen inactivation" specifically means that a pathogen risk level higher than 2 is reduced to at least a risk level of 2 or less. Ideally, the pathogen inactivation step does not prevent detection of the pathogen in the detection step. In some embodiments, the pathogen inactivation step does not reduce the detection efficiency in the detection step. Inactivation includes any kind of physical, chemical, biochemical, or biological treatment of the sample to reduce a risk level of more than 2 to a risk level of 2 or less. Inactivation also includes any kind of combination of physical, chemical, biochemical, or biological treatment. Thus, inactivation of pathogens includes preventing contamination of a sample of interest by pathogens in a given sample by making them non-infectious, for example, viral inactivation renders the virus incapable of infecting. Examples of methods for measuring pathogen inactivation are described in International Publication No. WO1993 / 015215A1.

[0033] Methods of pathogen inactivation may or may not achieve complete inactivation, and it is useful to consider a concrete example. A bacterial culture is said to be sterile if, when an aliquot of the culture is transferred to a fresh culture plate and allowed to grow, it is undetectable after a certain time. The time and growth conditions (e.g., temperature) define the "amplification factor." This amplification factor, together with the constraints of the detection method (e.g., visual inspection of the culture plate for the appearance of bacterial colonies), defines the sensitivity of the inactivation method. A minimum number of viable bacteria must be applied to the plate for a signal to be detectable. For an optimal detection method, this minimum number is one bacterial cell. For suboptimal detection methods, the minimum number of bacterial cells applied such that a signal is observed may be much higher than one. The detection method determines a "threshold" below which the method appears to be fully effective (and above which the method is, in fact, only partially effective).

[0034] Furthermore, viral inactivation can be demonstrated by biological assays such as enzyme assays (e.g., reduction in reverse transcriptase) or cell culture (e.g., virus-induced host cell death). A more sensitive method is reported by CV Hanson er.al. for quantifying HIV. See J. Clin. Microbiol. 23:2030 (1990). This method is a plaque assay employing HIV-susceptible cells in a monolayer. Fluorescent staining is used and detection is by visualization.

[0035] For example, spike tests are tests carried out to determine possible methods of inactivation of virus-like pathogens. The results of these tests are expressed as numbers, and based on these numbers, researchers can determine whether the process in which the tests are carried out is suitable for the viruses they are trying to extract and the manner in which they are trying to extract them. For example, spiking tests are tests carried out to determine whether the number of viruses (or the level of activity) in a sample is increased by 10% from the original amount. 4 or 10 5 It can be shown through experiments that a fold increase in the amount of virion in the sample will only change the viral inactivation rate by an order of magnitude. This finding indicates that the number of viruses (or activation levels) is 10 times that of the original sample. 4 or 10 5 A spike test was devised in which the activity is increased or "spiked" 100-fold. This new higher number or level of activity is then run through the process stream and purified. The number or level of activity is taken at the beginning and end of the process stream and used to calculate the reduction factor. The reduction factor (RF) for the various virus removal or inactivation steps is calculated using the following formula (please note Guidance on Virus Validation Studies: The Design, Contribution and Interpretation of Studies Validating the Inactivation and Removal of Viruses, EMEA CPMP BWP, 268 / 95 1996): RF step =log 10[(V1xT1) / (V2xT2)], where V1 = volume of feedstock spiked before the clearance step; T1 = virus concentration of the feedstock spiked before the clearance step; V2 = volume of material after the clearance step; and T2 = virus concentration of the material after the clearance step.

[0036] Furthermore, virucidal activity testing can be performed by using a quantitative suspension test involving a 30-second exposure time (as described in the World Health Organization, WHO Guidelines on Hand Hygiene in Healthcare: First World Patient Safety Challenge: Clean Care is Safe Care. Geneva: the Organization; 2009. [cited 2020 Apr 08]). One part virus suspension can be mixed with one part organic matter (0.3% bovine serum albumin as an interfering substance) and eight parts disinfectant solution of different concentrations. After 30 seconds of exposure, the samples are serially diluted and the 50% tissue culture infectious dose (TCID50) per milliliter is determined by crystal violet staining and subsequent scoring of the number of wells showing cytopathic effects. The TCID50 is calculated by the Spearman-Karber algorithm as described (George VG, Hierholzer JC, Ades EW. Cell culture. In: Virology methods manual. Mahy BWJ, Kangro HO, editors. Academic Press: London; 1996. p. 3-24). The cytotoxic effect of disinfectants can be monitored by using crystal violet staining and optical analysis of the altered density and morphology of cell monolayers in the absence of virus. We use the TCID 50 Cytotoxic effects were quantified, similar to viral infectivity, in 1000 cells / mL. Dose-response curves were plotted using normalized TCID 50 The reduction factor (RF) for each treatment condition can be calculated as the percent normalized viral inactivation versus the percent log disinfectant concentration by nonlinear regression using a robust fitting method for the data.

number

[0037] Physical inactivation treatments of pathogens according to the present disclosure may include, but are not limited to, any type of temperature change, electromagnetic waves, light (including visible and invisible light), incubation of the sample with radioactive materials, etc. that reduces the risk level of the pathogen from greater than 2 to at least a risk level of 2 or less. Combinations of these or other treatments are not excluded.

[0038] Some examples include, but are not limited to: Processing by drying temperature:>30℃,>40℃,>50℃,>60℃,>70℃,>80℃,>90℃,>100℃>110℃, etc. Treatment by temperature in solution:>30℃,>40℃,>50℃,>60℃,>70℃,>80℃,>90℃,>100℃>110℃ etc. Treatment with x-rays, radioactivity, UV light, blue light, red light, infrared light, etc.

[0039] Chemical inactivation treatments for pathogens according to the present disclosure may include any type of sample treatment by altering the pathogen environment, such as, but not limited to, pH, salt, the ratio of polar to non-polar solvents, oxidizing reagents, reducing reagents, reagents that bind covalently or non-covalently to the pathogen, or degrading reagents that cleave at least a portion of the pathogen. All reactions have in common that they reduce a pathogen risk level higher than 2 to at least a risk level of 2 or less. Combinations of these or other treatments are not excluded.

[0040] Some examples include, but are not limited to: Treatment with redox-reactive reagents such as, but not limited to, β-mercaptoethanol, dithiothreitol (DTT), or dithioerythritol, metal bases such as sodium borohydride (NaBH4) or lithium aluminum hydride (LiAlH4), peroxides (e.g., H2O2), ozone, potassium permanganate, organic peroxides (e.g., t-butyl hydroperoxide), or sodium perborate, hypochlorite, and other redox-reactive reagents that may be selected by the researcher from a potential series. Redox reagents are all reagents and conditions that can oxidize or reduce other chemical compounds. The redox reactivity of such reagents can be measured as an electrode potential, compared to a standard electrode potential. Redox-reactive agents can change their redox-reactive capabilities with certain reaction conditions that change temperature, pH, concentration, or other parameters (see texts on redox reactions). From this perspective, an experienced user can vary the agent and reaction parameters to find the appropriate redox-reactive substance. For example, a pathogen-containing sample is treated with a redox-reactive substance to cause the pathogen to adhere more tightly to the sample or surface, or the redox-reactive substance inactivates the pathogen's invasion mechanism or any other reaction required by the pathogen to grow in the organism. Typical pathogen inactivation reagents that cause cross-linking of pathogens are aldehydes, formaldehyde, glutaraldehyde, Boinsche Solution, etc. Typical pathogen inactivation reagents that cause a change in the lipophily of the solution are alcohols, e.g., methanol, ethanol, propanol, etc. Typical pathogen inactivation reagents that change the water tension are detergents, e.g., SDS, Triton, NP, etc.

[0041] For example, a composition for chemical inactivation treatment may consist of / include 80% (volume / volume) ethanol, 1.45% (volume / volume) glycerol, and 0.125% (volume / volume) hydrogen peroxide. A further composition may consist of / include 75% (volume / volume) 2-propanol, 1.45% (volume / volume) glycerol, and 0.125% (volume / volume) hydrogen peroxide. A further composition may consist of / include 80% (weight / weight) ethanol, 0.725% (volume / volume) glycerol, and 0.125% (volume / volume) hydrogen peroxide. A further composition may consist of / include 75% (weight / weight) 2-propanol, 0.725% (volume / volume) glycerol, and 0.125% (volume / volume) hydrogen peroxide.

[0042] Biochemical inactivation treatments for pathogens according to the present disclosure may include, but are not limited to, any type of enzymatic reaction, competitive reaction or reversible or irreversible reaction, addition of biomolecules (e.g., proteins, fatty acids, carbohydrates, nucleic acids, or mixtures thereof) that reduce the risk level of the pathogen from greater than 2 to at least a risk level of 2 or less. Combinations of these or other treatments are not excluded. Exemplary pathogen inactivation reagents include enzymes such as biomolecule degrading enzymes, proteolytic enzymes, enzymes that degrade lipophilic or amphipathic biomolecules, enzymes that degrade macromolecules, enzymes that degrade antibodies, and the like.

[0043] Biological inactivation treatments for pathogens according to the present disclosure may include, but are not limited to, treatment of any type of sample with a biological component that reduces the risk level of the pathogen from greater than 2 to a risk level of at least 2 or less. Biological inactivation treatments for pathogens according to the present disclosure may include, but are not limited to, treatment of any type of sample with a countervailing phage, virus, bacteria, or other organism or part of an organism that reduces the risk level of the pathogen from greater than 2 to a risk level of at least 2 or less. Combinations of these or other treatments are not excluded.

[0044] Spatial transcriptomics (or spatial*omics) according to the present disclosure refers to any type of analysis in which data from a sample is derived in a spatial manner from an in situ sample of a tissue or a whole organism. The in situ sample may be an organ or part of an organism. The in situ sample is not pre-processed or is pre-processed in a manner necessary to improve the results. Spatial*omics may include detection of small compounds, proteins, DNA, and / or RNA in tissues or cells. More advantageously, spatial*omics is limited to proteins, DNA, and / or RNA. More advantageously, spatial*omics is limited to DNA, and / or RNA. Even more advantageously, spatial*omics is limited to smFISH. Even more advantageously, spatial*omics is limited to any type of sequential smFISH.

[0045] In particular, spatial transcriptomics detection methods include multiplexing methods for detecting different analytes in a pathogen-containing sample by sequential signal encoding of the analytes, inactivation of the pathogen in the sample without isolation of RNA and / or DNA from the pathogen or sample, and (A) contacting the sample with at least twenty (20) different sets of analyte-specific probes for encoding at least twenty (20) different analytes, each set of analyte-specific probes interacting with a different analyte, and when the analyte is a nucleic acid, each set of analyte-specific probes includes at least five (5) analyte-specific probes that specifically interact with different substructures of the same analyte, each analyte-specific probe being: (aa) a binding element (S) that specifically interacts with one of the different analytes encoded therein; and (bb) an identifier element (T) comprising a nucleotide sequence unique to the analyte being encoded (the unique identifier sequence); wherein the analyte specific probes of a particular set of analyte specific probes are different from the analyte specific probes of another set of analyte specific probes in the nucleotide sequence of the identifier element (T); contacting, wherein the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and contain the same nucleotide sequence of an identifier element (T) unique to said analyte; and (B) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein each decoding oligonucleotide in each set of decoding oligonucleotides for a distinct analyte comprises: (aa) an identifier connector element (t) comprising a nucleotide sequence substantially complementary to at least a portion of a unique identifier sequence of an identifier element (T) of a corresponding analyte-specific probe set; and (bb) a translation element (c) comprising a nucleotide sequence allowing specific hybridization of a signal oligonucleotide; wherein a set of decoding oligonucleotides for a distinct analyte is different from another set of decoding oligonucleotides for a different analyte in the first connector element (t); and (C) contacting the sample with at least one set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translation connector element (C) comprising a nucleotide sequence substantially complementary to at least a portion of the nucleotide sequence of the translation element (c) contained in the decoding oligonucleotide; and (bb) signal element, contacting, including; (D) detecting a signal due to the signal element; (E) selectively removing the decoding oligonucleotides and the signal oligonucleotides from the sample, thereby substantially maintaining specific binding of the analyte-specific probe to the encoded analyte; (F) performing at least three further cycles comprising steps B) to E) to generate an encoding scheme comprising a code word for each analyte, in particular the last cycle may stop at step (D). Includes.

[0046] As used herein, "cells," "cell lines," and "cell cultures" are used interchangeably, and all such designations include progeny. Thus, the phrase "transformants" or "transformed cells" includes the primary subject cell and cultures derived therefrom, regardless of the number of passages. It will also be understood that all progeny may not be precisely identical in DNA content, due to deliberate or unintentional mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included.

[0047] One important advantage of the method according to the present disclosure is that the analysis can be performed outside of a BSL3 or BSL4 laboratory. Biosafety level (BSL), or pathogen / protection level, is a set of biocontainment precautions required to isolate hazardous biological agents in a closed laboratory. The levels of containment range from the lowest, Biosafety Level 1 (BSL-1), to the highest, Level 4 (BSL-4). In the United States, the Centers for Disease Control and Prevention (CDC) designated these levels. In the European Union, the same biosafety levels are defined in a directive. In Canada, the four levels are known as containment levels. Facilities with these designations are also sometimes designated as P1 to P4 (for pathogen or protection level), as in the term P3 laboratory. For example, Biosafety Level 3 (BSL-3) is appropriate for work involving pathogens that can cause severe and potentially fatal disease via the inhalation route. Biosafety Level 4 (BSL-4) is the highest level of biosafety precautions and is appropriate for working with pathogens that are readily aerosol transmitted in the laboratory and can cause severe to fatal disease in humans for which there is no available vaccine or treatment.

[0048] A "coding scheme" may characterize a set of code words associated with the analytes to be detected. Each code word refers to one type of analyte and is distinct from all other code words. A code word is thereby a sequence of codes imparted by the detection cycles of the method. A code within a code word is a detectable signal or the absence of a signal. A code word need not be composed of all the different signals used in the method. The number of codes in a code word is defined by the number of detection cycles.

[0049] As used herein, "oligonucleotide" refers to a short nucleic acid molecule, such as DNA, PNA, LNA, or RNA. The length of an oligonucleotide ranges from 4 to 200 nucleotides (nt), preferably 6 to 80 nt, more preferably 8 to 60 nt, more preferably 10 to 50 nt, more preferably 12 to 35 nt, depending on the number of consecutive sequence elements. A nucleic acid molecule can be fully or partially single-stranded. An oligonucleotide can be linear or can include a hairpin or loop structure. An oligonucleotide can include modifications, such as biotin, a labeling moiety, a blocking moiety, or other modifications.

[0050] An "analyte-specific probe" consists of at least two elements: a so-called binding element (S), which specifically interacts with one of the analytes, and a so-called identifier element (T), which contains a "unique identifier sequence". The binding element (S) can be a nucleic acid, such as a hybridization sequence or an aptamer, or a peptidic structure, such as an antibody.

[0051] A "probe" also consists of at least two elements, a so-called binding element (S), which specifically interacts with one of the analytes, and a so-called identifier element (T), which contains a "unique identifier sequence". The binding element (S) can be a nucleic acid, such as a hybridization sequence or an aptamer, or a peptidic structure, such as an antibody.

[0052] In particular, in some embodiments, the binding element (S) comprises an affinity portion derived from an affinity substance selected from the group consisting of an antibody, an antibody fragment, a receptor ligand, an enzyme substrate, a lectin, a cytokine, a lymphokine, an interleukin, an angiogenic or virulence factor, an allergen, a peptidic allergen, a recombinant allergen, an allergen-idiotypic antibody, an autoimmune-inducing structure, a tissue rejection-inducing structure, an immunoglobulin constant region, and derivatives, variants, or combinations thereof, or a moiety which is the affinity substance in its entirety. In further advantageous embodiments, the antibody fragment is a Fab, scFv; a single domain, or fragments thereof, a bis-scFv, Fab2, Fab3, a minibody, a maxibody, a diabody, a triabody, a tetrabody or a tandab, in particular a single chain variable fragment (scFv).

[0053] The "unique identifier sequence" contained by the analyte-specific probe is unique in its sequence compared to other unique identifiers. In this context, "unique" means that it specifically identifies only one analyte, e.g., cyclin A, cyclin D, cyclin E, etc., or that it specifically identifies only a group of analytes, regardless of whether the group of analytes includes a gene family. Thus, the analyte or group of analytes encoded by this unique identifier can be distinguished from all other analytes or groups of analytes encoded based on the unique identifier sequence of the identifier element (T). Or, in other words, there is only one "unique identifier sequence" for a particular analyte or group of analytes, not more than one, i.e. not even two. Due to the uniqueness of the unique identifier sequence, the identifier element (T) hybridizes to exactly one type of decoding oligonucleotide. The length of the unique identifier sequence is in the range of 8-60 nt, preferably 12-40 nt, more preferably 14-20 nt, depending on the number of analytes to be encoded in parallel and the required stability of the interaction. The unique identifier can be a sequence element of an analyte-specific probe, attached directly or by a linker, a covalent bond or a high affinity binding manner, e.g., antibody-antigen interaction, streptavidin-biotin interaction, etc. The term "analyte-specific probe" is understood to include multiple probes that may differ in their binding element (S), such that each probe binds to the same analyte but possibly to different parts thereof, e.g., to different (e.g., adjacent) or overlapping parts of a nucleotide sequence contained in the encoded nucleic acid molecule. However, each of the multiple probes contains the same identifier element (T).

[0054] A "dual-labeled probe" comprises a binding sequence capable of hybridizing with an analyte and a binding probe sequence capable of binding a detectable signal molecule, such as a fluorophore or a nucleic acid sequence containing a fluorophore.

[0055] A "decoding oligonucleotide" or "adapter" or "adapter segment" consists of at least two sequence elements: one sequence element capable of specifically binding to a unique identifier sequence, called the "identifier connector element" (t) or "first connector element" (t), and a second sequence element that specifically binds to a signal oligonucleotide, called the "translation element" (c). The length of the sequence elements ranges from 8 to 60 nt, preferably from 12 to 40 nt, more preferably from 14 to 20 nt, depending on the number of analytes to be coded in parallel, the stability of the interaction required and the number of different signal oligonucleotides used. The length of the two sequence elements may or may not be the same.

[0056] In some advantageous embodiments, the decoding oligonucleotide in the kit and / or method of the present disclosure can be a "multi-decoder". A "multi-decoder" is a decoding oligonucleotide consisting of at least three sequence elements. One sequence element (identifier connector element (t)) can specifically bind to a unique identifier sequence (identifier element (T)) and at least two other sequence elements (translation elements (c)) specifically bind a different signal oligonucleotide (each of these sequence elements specifically binds a signal oligonucleotide that is different from all other signal oligonucleotides recruited by other elements of the multi-decoder). The length of the sequence elements is in the range of 8-60 nt, preferably 12-40 nt, more preferably 14-20 nt, depending on the number of analytes to be detected in parallel, the required stability and the number of different signal oligonucleotides used. The length of the sequence elements may be the same or not.

[0057] Thus, in some advantageous embodiments, the decoding oligonucleotide comprises: - an identifier connector element (t) comprising a nucleotide sequence substantially complementary to at least a portion of the unique identifier sequence of an identifier element (T) of a corresponding analyte-specific probe set; and - at least two translation elements (c), each of which comprises a nucleotide sequence that allows specific hybridization of a different signal oligonucleotide; A multi-decoder including:

[0058] Thus, the first translation element binds a different signal oligonucleotide than the second translation element, in particular the signal oligonucleotides differ in the signal element contained in the signal oligonucleotide, for example in the type of fluorophore.

[0059] A "signal oligonucleotide" or "reporter" as used herein comprises at least two elements, the so-called "translation connector element" (C) or "second connector element" (C), having a nucleotide sequence capable of specifically hybridizing to at least a portion of the nucleotide sequence of the translation element (c) of the decoding oligonucleotide, and a "signal element" that provides a detectable signal. This element actively generates a detectable signal or provides such a signal through manipulation, e.g., excitation of fluorescence. Typical signal elements are, for example, enzymes, fluorophores, radioactive elements or dyes that catalyze a detectable reaction.

[0060] A "set" refers to a plurality of moieties or objects, such as analyte-specific probes or decoding oligonucleotides, whether the individual members of said plurality are identical or different from one another. In an analyte-specific probe set, the analyte-specific probes are identical in identifier element (T) for specifically interacting with the same analyte, but may contain different binding elements (S) for specifically interacting with different substructures of the same encoded analyte.

[0061] "Selective denaturation" can be the process of removing the bound decoding oligonucleotide and signal oligonucleotide with the highest efficiency, while at the same time, the target-specific probe must remain hybridized with the highest efficiency. The total efficiency of these two combined events can be at least 0.22 at 2 detection cycles, 0.37 at 3 detection cycles, 0.47 at 4 detection cycles, 0.55 at 5 detection cycles, 0.61 at 6 detection cycles, 0.65 at 7 detection cycles, 0.69 at 8 detection cycles, 0.72 at 9 detection cycles, 0.74 at 10 detection cycles, 0.76 at 11 detection cycles and 0.78 at 12 detection cycles.

[0062] In certain embodiments of the present disclosure, a single set refers to a plurality of oligonucleotides.

[0063] "Analyte-specific probe" refers to analyte-specific probes that bind to multiple moieties or targets, e.g., distinct from one another and to separate regions of an analyte. A single analyte-specific probe set is further characterized by the same unique identifier.

[0064] A "decoding oligonucleotide set" refers to a plurality of decoding oligonucleotides specific for a particular unique identifier required to recognize the encoding regardless of the length of the codeword. Each and every decoding oligonucleotide included in a "decoding oligonucleotide set" binds to the same unique identifier element (T) of an analyte-specific probe.

[0065] In certain embodiments, this pattern of binding or hybridization of the decoding oligonucleotides can be converted into a "code word". For example, the code word can also be "101" and "110" for an analyte, where a value of 1 represents binding and a value of 0 represents no binding. The code word can also have a longer length in other embodiments (see FIG. 13). A code word can be directly related to the specific unique identifier sequence of the analyte-specific probe. Thus, different analyte-specific probes will match a particular code word, which can then be used to identify different analytes of the analyte-specific probe based on the binding pattern of the decoding oligonucleotide. However, if no binding is evident, the code word would be "000" in this example.

[0066] The values ​​of each code word can also be assigned in different ways in some embodiments. For example, a value of 0 can represent binding, while a value of 1 represents no binding. Similarly, a value of 1 can represent binding of the secondary nucleic acid probe with one type of signal transduction agent, while a value of 0 can represent binding of the secondary nucleic acid probe with another type of distinguishable signal transduction agent. These signal transduction agents can be distinguished, for example, by different fluorescent colors. In some cases, the values ​​in the code word need not be limited to 0 and 1. The values ​​can also be derived from more alphabets, such as ternary (e.g., 0, 1, and 2) or quaternary (e.g., 0, 1, 2, and 3). Each different value can be represented, for example, by a different distinguishable signal transduction agent, including (in some cases) one value that can be represented by the absence of a signal.

[0067] The code words for each analyte may be assigned sequentially or randomly. For example, a first analyte may be assigned to 101, while a second nucleic acid target may be assigned to 110. Additionally, in some embodiments, the code words may be assigned using an error detection or correction system, such as a Hamming system, a Golay code, or an extended Hamming system (or a SECDED system, i.e., single error correction, double error detection). Generally speaking, such a system can be used to identify where an error occurred, and in some cases, such a system can also be used to correct the error and determine what the correct code word was. For example, a code word such as 001 may be detected as invalid and corrected to 101 using such a system, e.g., if 001 has not been previously assigned to a different target sequence. A variety of different error correction codes may be used, many of which have been previously developed for use within the computer industry; however, such error correction systems have not typically been used within biological systems. Additional examples of such error correction codes are discussed in more detail below.

[0068] "Substantially complementary" when referring to two nucleotide sequences means that both sequences can specifically hybridize with each other under stringent conditions, thereby forming a hybrid nucleic acid molecule with sense and antisense strands connected to each other via hydrogen bonds (Watson and Crick base pairing). "Substantially complementary" includes not only perfect base pairing along the complete strand, i.e., perfectly complementary sequences, but also imperfect complementary sequences that are still capable of hybridizing with each other under stringent conditions. It is widely accepted among experts that a "substantially complementary" sequence has at least 88% sequence identity to a fully or completely complementary sequence.

[0069] "Percent sequence identity" or "percent identity" in turn means that a sequence is compared to a described or claimed sequence ("reference sequence") after alignment of the sequence being compared ("comparison sequence") with the described or claimed sequence. The percent identity is then determined according to the following formula: percent identity = 100 [1 - (C / R)], where C is the number of differences between the reference and comparison sequences over the length of the alignment between the reference and comparison sequences; (i) each base or amino acid in the reference sequence that does not have a corresponding aligned base or amino acid in the comparison sequence; and (ii) each gap in the reference sequence, and (iii) each aligned base or amino acid in the reference sequence that constitutes a difference is different from the aligned base or amino acid in the comparison sequence, and (iiii) the alignment must begin at position 1 of the aligned sequence; and R is the number of bases or amino acids in the reference sequence over the length of the alignment with the comparison sequence, with any gaps made in the reference sequence also being counted as bases or amino acids.

[0070] If there is an alignment between a comparison sequence and a reference sequence for which the percent identity calculated above is about equal to or greater than the specified minimum percent identity, then the comparison sequence has a particular minimum percent identity to the reference sequence, even though there may be alignments for which the percent identity calculated herein above is less than the specified percent identity.

[0071] In an "incubation" step, as understood herein, the respective moieties or entities, such as probes or oligonucleotides, are brought into contact with each other under conditions well known to those skilled in the art that allow a specific binding or hybridization reaction, e.g., pH, temperature, salt conditions, etc. Such a step may therefore preferably be carried out in a liquid environment, such as a buffer system, well known in the art.

[0072] The "removal" step according to the present disclosure may include washing away the moiety or object to be removed, such as a probe or oligonucleotide, under specific conditions, such as pH, temperature, salt conditions, etc., as known in the art.

[0073] It will be appreciated that in certain embodiments of the method according to the present disclosure, multiple analytes may be coded in parallel. This requires the use of different analyte-specific probe sets in step (1). A particular set of analyte-specific probes is different from another set of analyte-specific probes. This means that the analyte-specific probes of set 1 bind to analyte 1, the analyte-specific probes of set 2 bind to analyte 2, the analyte-specific probes of set 3 bind to analyte 3, etc. In this embodiment, the method according to the present disclosure also requires the use of different sets of decoding oligonucleotides.

[0074] A particular set of decoding oligonucleotides is different from another set of decoding oligonucleotides, meaning that the decoding oligonucleotides of set 1 bind to the analyte specific probes of the analyte specific probes of set 1, the decoding oligonucleotides of set 2 bind to the analyte specific probes of the analyte specific probes of set 2, the decoding oligonucleotides of set 3 bind to the analyte specific probes of the analyte specific probes of set 3, etc.

[0075] In this embodiment where multiple analytes are coded in parallel, the different sets of analyte-specific probes may be provided as pre-mixtures of the different sets of analyte-specific probes and / or the different sets of decoding oligonucleotides may be provided as pre-mixtures of the different sets of decoding oligonucleotides. Each mixture may be contained in a single vial. Alternatively, the different sets of analyte-specific probes and / or the different sets of decoding oligonucleotides may be provided in multiple steps. It may also be provided alone.

[0076] A "kit" is a combination of individual elements useful for carrying out the uses and / or methods of the present disclosure, which elements are optimized for use together in the method. The kit also includes additional reagents, chemicals, buffers, reaction vials, etc., that may be useful for carrying out the methods according to the present disclosure. Such a kit integrates all the essential elements required to make the methods according to the present disclosure work. Thus, such a kit also allows unskilled laboratory staff to carry out the methods according to the present disclosure.

[0077] The term "quencher" or "quencher dye" or "quencher molecule" refers to a dye or equivalent molecule, such as the nucleoside guanosine (G) or 2'-deoxyguanosine (dG), which can reduce the fluorescence of a fluorescent reporter dye or a donor dye. The quencher dye may be a fluorescent dye or a non-fluorescent dye. When the quencher is a fluorescent dye, its fluorescence wavelength is usually substantially different from that of the reporter dye, and the quencher fluorescence is usually not monitored during the assay. Some embodiments of the present disclosure disclose signal oligonucleotides that include a quencher in combination with a quencher and / or a signal element (see FIG. 14), such that the signal oligonucleotide cannot be detected during imaging.

[0078] In some embodiments of the present disclosure, the sample is a biological sample, preferably comprising biological tissue, more preferably comprising biological cell.Biological sample can be derived from organ, organoid, cell culture, stem cell, cell suspension, primary cell; virus, bacteria or fungus infected sample, eukaryotic or prokaryotic sample, smear, disease sample, tissue section.

[0079] The methods are particularly suitable for encoding, detecting, counting or quantifying multiple analytes or single analyte molecules in a biological sample, i.e., a sample that contains nucleic acids or proteins as said analytes. It is understood that the biological sample may be in a form such as it is in its native environment (i.e., liquid, semi-liquid, solid, etc.) or that has been processed, such as a dried film on the surface of a device that can be re-liquidized before the method is performed.

[0080] In another embodiment of the present disclosure, prior to step (2), the biological tissue and / or biological cells are fixed. For example, in some embodiments, the cells and / or tissue are fixed prior to introducing the probe, for example to maintain the location of the analyte, such as nucleic acid, within the cell. Fixation of cells is known to those skilled in the art. As non-limiting examples, cells may be fixed using chemicals such as formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, methanol, acetone, acetic acid, and the like. In one embodiment, cells may be fixed using HEPES-glutamate buffer-mediated organic solvent (HOPE).

[0081] This method has the advantage that the analytes to be encoded, e.g., nuclei or proteins, are immobilized and cannot escape, thus preparing them for better detection or encoding by the methods according to the present disclosure.

[0082] In yet a further embodiment, within the set of analyte-specific probes, individual analyte-specific probes comprise binding elements (S1, S2, S3, S4, S5) that specifically interact with a different substructure of one of the encoded analytes.

[0083] This approach makes the method even more robust and reliable, because the signal intensity obtained at the end of the method or cycle, respectively, is increased. It is understood that the individual probes of a set, while binding to the same analyte, differ in their binding positions or binding sites at or on the analyte. The binding elements S1, S2, S3, S4, S5, etc. of the first, second, third, fourth, fifth, etc. analyte-specific probes therefore bind to different positions or positions that may or may not overlap.

[0084] A multiplexing method or assay allows for the simultaneous measurement of multiple analytes. In the present disclosure, it can be used to determine the presence or absence of multiple predetermined (known) analytes, such as nucleic acid target sequences, in a sample. The analytes can be "predetermined" in that their sequences are known in order to design probes that bind to the targets.

[0085] In some advantageous embodiments according to the present disclosure, at least 20, particularly at least 25, particularly at least 30 different analytes are detected and / or quantified in a sample in parallel. For example, there may be at least 5, at least 10, at least 20, at least 50, at least 75, at least 100, at least 300, at least 1,000, at least 3,000, at least 10,000, or at least 30,000 distinguishable analyte-specific probes applied to the sample simultaneously or sequentially.

[0086] In some advantageous embodiments, twenty (20) or more sets of analyte-specific probes for encoding at least twenty or more different analytes for multiplexing are required, particularly more than fifty, more than one hundred or more than two hundred sets. In particular, at least twenty different groups of analytes (e.g., mRNA molecules) or tags are targeted in the multiplexing methods of the present disclosure.

[0087] In some advantageous embodiments, at least four rounds are performed to gather information for the identification of the analyte, and multiple readouts increase the accuracy of the identification and avoid false positives. Unique tags can be identified by a variety of techniques, including, for example, direct or indirect hybridization with a labeled probe, or sequencing (by synthesis, ligation). In particular, the identity of the tag can be coded with one single signal (binary code), two or more signals, and the signals can be fluorescent labels (e.g., attached to oligonucleotides).

[0088] In some advantageous embodiments according to the present disclosure, the kit does not comprise a set of analyte-specific probes as defined in item A.

[0089] Preferably, when the analytes in the method according to the present disclosure are nucleic acids, each set of analyte-specific probes comprises at least 5 analyte-specific probes, in particular at least 15 analyte-specific probes, in particular at least 20 analyte-specific probes that specifically interact with different substructures of the same analyte. Nucleic acid analytes include specific DNA molecules, such as genomic DNA, nuclear DNA, mitochondrial DNA, viral DNA, bacterial DNA, extracellular or intracellular DNA, etc., and specific mRNA molecules, such as hnRNA, miRNA, viral RNA, bacterial RNA, extracellular or intracellular RNA, etc.

[0090] Preferably, when the analyte in the method according to the present disclosure is a peptide, polypeptide or protein, each set of analyte-specific probes comprises at least two (2) analyte-specific probes, in particular at least three (3) analyte-specific probes, in particular at least four (4) analyte-specific probes that specifically interact with different substructures of the same analyte.

[0091] In some advantageous embodiments according to the present disclosure, the method comprises at least two different sets of signal oligonucleotides, the signal oligonucleotides in each set comprising a different signal element and comprising a different connector element (C).

[0092] In particular, the method comprises at least two different sets of decoding oligonucleotides per analyte, the decoding oligonucleotides in the different sets comprising the same identifier connector element (t) comprising a nucleotide sequence substantially complementary to at least a portion of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and the decoding oligonucleotides of the different sets per analyte differ in a translation element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide.

[0093] In particular, the method comprises at least two different sets of decoding oligonucleotides per analyte, the decoding oligonucleotides in the different sets comprising the same identifier connector element (t) comprising a nucleotide sequence substantially complementary to at least a portion of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and the decoding oligonucleotides of the different sets for at least one analyte differ in a translation element (c) comprising a nucleotide sequence that allows specific hybridization of the signal oligonucleotide.

[0094] In some advantageous embodiments, the number of different sets of decoding oligonucleotides per analyte that contain different translation elements (c) corresponds to the number of different sets of signal oligonucleotides that contain different connector elements (C). However, the decoding oligonucleotides in a particular set of decoding oligonucleotides may interact with the same identifier element (T) that is unique to a particular analyte. In particular, all sets of decoding oligonucleotides for different analytes may contain the same type of translation element (c).

[0095] In particular, the disclosure relates to a method for detecting an analyte in a pathogen-containing sample, comprising the acts of exposing a pathogen-inactivated sample to a plurality of analyte-specific probes; for each of the analyte-specific probes, determining binding of the analyte-specific probe in the sample; generating a code word based on binding of the analyte-specific probe, the decoding oligonucleotide, and the signal oligonucleotide; and for at least some of the code words, matching the code word to a valid code word. In certain embodiments, this binding or hybridization pattern of the analyte-specific probe, the decoding oligonucleotide, and the signal oligonucleotide can be converted into a "code word." For example, the code word can be "100%" for a first analyte and a second analyte, respectively. 1 " and "1 10 ", where a value of 1 represents binding of the decoding oligonucleotide and a value of 0 represents no binding of the decoding oligonucleotide and / or binding of the signal oligonucleotide with or without a quenched signal element. The analyte during the detection round / cycle is therefore undetectable during imaging.

[0096] To generate such zeros (0) in the codeword for each individual analyte, the method includes: (D) at least one set of non-signal decoding oligonucleotides for binding to specific identifier elements (T) of the analyte-specific probe, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interact with the same different identifier elements (T); Each non-signal-decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence substantially complementary to at least a portion of the unique identifier sequence, and does not comprise a translation element (c) comprising a nucleotide sequence that permits specific hybridization of the signal oligonucleotide. This may include the use of

[0097] To generate such zeros (0) in the codeword for each individual analyte, the method includes: (D) at least one set of non-signal decoding oligonucleotides for binding to specific identifier elements (T) of the analyte-specific probe, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interact with the same different identifier elements (T); Each non-signal-decoding oligonucleotide comprises an identifier connector element (t) that comprises a nucleotide sequence that is substantially complementary to at least a portion of the unique identifier sequence, and comprises a translation element that does not interact / bind with the signal oligonucleotide because of an unstable binding sequence and / or because the translation element comprises a nucleotide sequence that allows specific hybridization of the signal oligonucleotide and is short (c). This may include the use of

[0098] In some advantageous embodiments, the method comprises: (D) at least two different non-signal decoding oligonucleotides for binding to at least two different identifier elements (T) of the analyte-specific probe, each set of non-signal decoding oligonucleotides interacting with a different identifier element (T); Each non-signal-decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence substantially complementary to at least a portion of the unique identifier sequence, and does not comprise a translation element (c) comprising a nucleotide sequence that allows specific hybridization of the signal oligonucleotide. This may include the use of

[0099] Moreover, in some advantageous embodiments, the method further comprises: (E) a set of non-signal oligonucleotides, each non-signal oligonucleotide being (aa) a translation connector element (C) comprising a nucleotide sequence substantially complementary to at least a portion of the nucleotide sequence of the translation element (c); and (bb) a set of non-signal oligonucleotides comprising a quencher (Q), a signal element and a quencher (Q), or no signal element. This may include the use of

[0100] In some advantageous embodiments, the method comprises: (E) at least two sets of non-signal oligonucleotides, each non-signal oligonucleotide (aa) a translation connector element (C) comprising a nucleotide sequence substantially complementary to at least a portion of the nucleotide sequence of the translation element (c); and (bb) at least two sets of non-signal oligonucleotides comprising a quencher (Q), a signal element and a quencher (Q), or no signal element. This may include the use of

[0101] In some advantageous embodiments, the different sets of non-signal oligonucleotides may be included in a pre-mixture of the different sets of non-signal oligonucleotides or may be present separately.

[0102] Furthermore, in some embodiments, the decoding oligonucleotides in a particular set of decoding oligonucleotides interact with the same identifier element (T) that is unique to a particular analyte.

[0103] In some advantageous embodiments, the different sets of decoding oligonucleotides may be included in a pre-mixture of different sets of decoding oligonucleotides or may be present separately. In some advantageous embodiments, the different sets of analyte-specific probes may be included in a pre-mixture of different analyte-specific probe sets or may be present separately. In some advantageous embodiments, the different sets of signal oligonucleotides may be included in a pre-mixture of different sets of signal oligonucleotides or may be present separately.

[0104] In some advantageous embodiments, a mixture of decoding oligonucleotides and / or multi-decoders is provided that specifically hybridize to the unique identifier sequence of the probe set. In some embodiments, the decoding oligonucleotide comprises at least two sequence elements, a first element that is complementary to the unique identifier sequence of the corresponding probe set and a second sequence element (translation element) that provides a sequence for specific hybridization of the signal oligonucleotide, the translation element defining the type of signal recruited to the decoding oligonucleotide. In some embodiments, a multi-decoder is used that comprises at least three sequence elements, a first element that is complementary to the unique identifier sequence of the corresponding probe set and at least an additional sequence element (translation element) that provides a sequence for specific hybridization of at least two different signal oligonucleotides. The translation element defines the type of signal recruited to the multi-decoder. Different possible structures of the multi-decoder can be seen in Figure 15. Since the multi-decoder recruits a complete signal oligonucleotide per translation element, the intensity of the signal in each channel is not lower than the intensity of the signal by the decoding oligonucleotide.

[0105] The use of multiple decoders further increases the efficiency of the encoding scheme. Figure 16 shows a possible encoding scheme using multiple decoders, based on the same conditions used in the example with a decoding oligonucleotide with two sequence elements. It can be clearly seen that the multiple decoder-based encoding scheme can generate a higher Hamming distance with the same number of rounds and the same number of different signal oligonucleotides used in the example of Figure 5.

[0106] As mentioned above, the analyte to be encoded may be a nucleic acid, preferably DNA, PNA or RNA, in particular mRNA, a peptide, a polypeptide, a protein and / or a mixture thereof.

[0107] In some advantageous embodiments, the binding element (S) comprises an amino acid sequence that allows specific binding to the encoded analyte. The binding element (S) may comprise an affinity portion derived from or a portion which is an affinity substance as a whole selected from the group consisting of antibodies, antibody fragments, anticalin proteins, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenic or virulence factors, allergens, peptidic allergens, recombinant allergens, allergen-idiotypic antibodies, autoimmune-inducing structures, tissue rejection-inducing structures, immunoglobulin constant regions, and combinations thereof.

[0108] In some advantageous embodiments, the binding element (S) may comprise or is an antibody or antibody fragment selected from the group consisting of Fab, scFv; single domains or fragments thereof, bis-scFv, F(ab)2, F(ab)3, minibody, diabody, triabody, tetrabody and tandab.

[0109] The present disclosure particularly relates to a method for detecting an analyte in a pathogen-containing sample, the method comprising: i) Inactivation of pathogens in a sample without isolation of RNA and / or DNA from the pathogen or sample; ii) Analyte detection by spatial transcriptomics Including, The spatial transcriptomics detection method may include or is a multiplex method for detecting different analytes in a sample by sequential signal encoding of the analytes, comprising the following steps: (A) contacting the sample with at least twenty (20) different sets of analyte-specific probes for encoding at least twenty (20) different analytes, each set of analyte-specific probes interacting with a different analyte, and when the analyte is a nucleic acid, each set of analyte-specific probes includes at least five (5) analyte-specific probes that specifically interact with different substructures of the same analyte, each analyte-specific probe being: (aa) a binding element (S) that specifically interacts with one of the different analytes encoded therein; and (bb) an identifier element (T) comprising a nucleotide sequence unique to the analyte being encoded (the unique identifier sequence); wherein the analyte specific probes of a particular set of analyte specific probes are different from the analyte specific probes of another set of analyte specific probes in the nucleotide sequence of the identifier element (T); the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and contain the same nucleotide sequence of an identifier element (T) unique to said analyte; and (B) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein each decoding oligonucleotide in each set of decoding oligonucleotides for a distinct analyte comprises: (aa) an identifier connector element (t) comprising a nucleotide sequence substantially complementary to at least a portion of a unique identifier sequence of an identifier element (T) of a corresponding analyte-specific probe set; and (bb) a translation element (c) comprising a nucleotide sequence allowing specific hybridization of a signal oligonucleotide; wherein the set of decoding oligonucleotides for an individual analyte is different from another set of decoding oligonucleotides for a different analyte in the first connector element (t); and (C) contacting the sample with at least one set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translation connector element (C) comprising a nucleotide sequence substantially complementary to at least a portion of the nucleotide sequence of the translation element (c) contained in the decoding oligonucleotide; and (bb) signal element, including, steps; (D) detecting a signal due to the signal element; (E) selectively removing the decoding oligonucleotides and the signal oligonucleotides from the sample, thereby substantially maintaining specific binding of the analyte-specific probe to the encoded analyte; (F) performing at least three further cycles comprising steps B) to E) to generate an encoding scheme comprising a code word for each analyte, in particular the last cycle may stop at step (D).

[0110] As mentioned above, the method according to the present disclosure includes selectively removing the decoding oligonucleotides and the signal oligonucleotides from the sample, thereby substantially maintaining the specific binding of the analyte-specific probes to the encoded analytes. In particular, all steps are performed sequentially. However, some steps, in particular the contacting steps A) to C), in particular steps B) and C), may be performed simultaneously.

[0111] This method achieves the requirement for another round / cycle of binding of additional decoding oligonucleotides to the same analyte-specific probe, thus ultimately resulting in a code or encoding scheme that includes two or more signals. This step is achieved by applying conditions and factors well known to those skilled in the art, such as pH, temperature, salt conditions, oligonucleotide concentration, polymers, etc.

[0112] In another embodiment of the present disclosure, the method may include repeating steps (B)-(E) at least three times to generate a coding scheme, whereby for four cycles / rounds performed by the user, four signal codes are determined, where "n" is an integer representing the number of rounds. The coding capacity of the method according to the present disclosure is increased depending on the nature of the analytes and the needs of the operator. In an embodiment of the present disclosure, the coding scheme is predetermined and assigned to the analyte to be coded.

[0113] However, this method allows for precise experimental configuration by providing for the proper order of the decoding and signal oligonucleotides used, and thus allows for precise allocation of specific analytes to the respective coding schemes. The decoding oligonucleotide used in the repeated steps (B)-(D2) may contain a translation element (c2) identical to the translation element (c1) of the decoding oligonucleotide used in the previous step (B)-(E). In another embodiment of the present disclosure, a decoding oligonucleotide is used in the repeated steps (B)-(E) that contains a translation element (c2) different from the translation element (c1) of the decoding oligonucleotide used in the previous step (B)-(E). It will be understood that the decoding element may or may not change from round to round, i.e., in the second round (B)-(E) contains the translation element c2, in the third round (B)-(E) contains the translation element c3, in the fourth round (B)-(E) contains the translation element c4 (and so on, where "n" is an integer representing the number of rounds.

[0114] The signal oligonucleotide used in the repeated steps (B)-(E) may contain a signal element identical to the signal element of the decoding oligonucleotide used in the previous steps (B)-(E). In further embodiments of the present disclosure, a signal oligonucleotide is used in the repeated steps (B)-(E) that contains a signal element different from the signal element of the decoding oligonucleotide used in the previous steps (B)-(E). In some embodiments, a non-signal oligonucleotide and / or a non-signal decoding oligonucleotide for a separate analyte is used, resulting in a code word value of 0 for this cycle / position. In some embodiments, no decoding oligonucleotide for a particular analyte in a repeated cycle is contacted with the sample, resulting in a code word value of 0 again for that cycle / position.

[0115] This method provides the same or a different signal in each round, resulting in an encoding scheme characterized by a signal sequence consisting of multiple different signals. This method allows for the generation of a unique code or code word that is distinct from the code words of all other encoding schemes. In another embodiment of the present disclosure, the binding element (S) of the analyte-specific probe comprises a nucleic acid that comprises a nucleotide sequence that allows specific binding to the encoded analyte, preferably specific hybridization to the encoded analyte.

[0116] In some advantageous embodiments, all steps are automated, particularly steps B) through F), particularly by using a robotic system and / or an optical multiplexing system according to the present disclosure. In some examples, these steps may be performed in a fluidic system.

[0117] As previously described, the method according to the present disclosure generates a coding scheme with a code word for each analyte. Thus, each analyte can be associated with a specific code word, said code word including multiple positions, each position corresponding to one cycle, resulting in multiple identifiable coding schemes with multiple code words. In particular, said coding schemes can be predetermined and assigned to the analyte being coded.

[0118] In some advantageous embodiments, the code words obtained for the individual analytes in the cycles carried out contain at least one element corresponding to the detected signal and additionally to no detected signal, such as 0, 1 or 0, 1, 2 (see also Fig. 13 and Fig. 14). In particular, when using a non-signal probe according to Fig. 14, No. 2-4, or a non-signal decoding oligonucleotide as shown in Fig. 14, No. 5, or when in one cycle the decoding oligonucleotide is not contacted with the corresponding identifier sequence contained in the analyte-specific probe that interacts with the corresponding analyte in the sample, no signal is detected for at least one analyte in at least one cycle. In this cycle, the position has the value zero (0).

[0119] In some advantageous embodiments, for at least one individual analyte, the position of the code word is zero (0). In particular, the code word zero (0) is generated without using a decoding oligonucleotide having an identifier connector element (t) that comprises a nucleotide sequence that is substantially complementary to at least a portion of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe for the individual analyte for the individual analyte. As mentioned above, in some embodiments, at least when the position of the code word is zero (0) in this cycle for one individual analyte, the corresponding decoding oligonucleotide having an identifier connector element (t) that comprises a nucleotide sequence that is substantially complementary to at least a portion of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe for the individual analyte for the individual analyte is not used.

[0120] Moreover, in some advantageous embodiments, the sample is contacted with at least two different sets of signal oligonucleotides, the signal oligonucleotides in each set comprising a different signal element and comprising a different connector element (C).

[0121] In a further particular embodiment, the sample is contacted with at least two different sets of decoding oligonucleotides for each analyte; The decoding oligonucleotides in these different sets contain the same identifier connector element (t) that comprises a nucleotide sequence that is substantially complementary to at least a portion of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set; and The different sets of decoding oligonucleotides for each analyte differ in a translation element (c) which comprises a nucleotide sequence allowing specific hybridization of the signal oligonucleotide. In a further particular embodiment, the sample is contacted with at least two different sets of decoding oligonucleotides for each analyte; The decoding oligonucleotides in these different sets contain the same identifier connector element (t) that comprises a nucleotide sequence that is substantially complementary to at least a portion of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set; and The different sets of decoding oligonucleotides for each analyte differ in a translation element (c) that comprises a nucleotide sequence that allows specific hybridization of the signal oligonucleotide; Only one set of decoding oligonucleotides per analyte is used per cycle, and / or different sets of decoding oligonucleotides are used in different cycles in combination with a corresponding set of signal oligonucleotides in the same cycle.

[0122] In some advantageous embodiments, the number of different sets of decoding oligonucleotides per analyte that contain different translation elements (c) corresponds to the number of different sets of signal oligonucleotides that contain different connector elements (C). All sets of decoding oligonucleotides for different analytes may contain the same type of translation element (c).

[0123] In some advantageous embodiments of the methods according to the present disclosure, the sample is contacted with at least one set of non-signal decoding oligonucleotides for binding to specific identifier elements (T) of the analyte-specific probe, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interact with the same different identifier elements (T), and each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence substantially complementary to at least a portion of the unique identifier sequence, and does not comprise a translation element (c) comprising a nucleotide sequence enabling specific hybridization of the signal oligonucleotide.

[0124] As described above, the sample can be contacted with at least two different sets of non-signal decoding oligonucleotides for binding to at least two different identifier elements (T) of the analyte-specific probes, each set of non-signal decoding oligonucleotides interacting with a different identifier element (T), each non-signal decoding oligonucleotide comprising an identifier connector element (t) comprising a nucleotide sequence substantially complementary to at least a portion of a unique identifier sequence, and not comprising a translation element (c) comprising a nucleotide sequence enabling specific hybridization of a signal oligonucleotide.

[0125] In some advantageous embodiments of the methods according to the present disclosure, the different sets of non-signal-decoding oligonucleotides may be included in a pre-mixture of the different sets of non-signal-decoding oligonucleotides or may be present separately.

[0126] Further, in some advantageous embodiments of the methods according to the present disclosure, the sample is contacted with a set of non-signal oligonucleotides, each non-signal oligonucleotide comprising (aa) a translation connector element (C) comprising a nucleotide sequence substantially complementary to at least a portion of the nucleotide sequence of the translation element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or no signal element.

[0127] In a further embodiment, the sample comprises: and contacting the nucleic acid with at least two sets of non-signal oligonucleotides, each non-signal oligonucleotide comprising: (aa) a translation connector element (C) comprising a nucleotide sequence substantially complementary to at least a portion of the nucleotide sequence of the translation element (c); and (bb) a quencher (Q), a signal element and a quencher (Q) or does not contain a signal element.

[0128] As discussed above, the different sets of non-signal oligonucleotides can be included in a pre-mixture of the different sets of non-signal oligonucleotides or can be present separately.

[0129] In a further embodiment, the decoding oligonucleotides in a particular set of decoding oligonucleotides interact with the same identifier element (T) that is unique to a particular analyte.

[0130] As mentioned above, the different sets of decoding oligonucleotides may be included in a pre-mixture of different sets of decoding oligonucleotides or may be present separately, and the different sets of analyte-specific probes may be included in a pre-mixture of different sets of analyte-specific probes or may be present separately, in addition, the different sets of signal oligonucleotides may be included in a pre-mixture of different sets of signal oligonucleotides or may be present separately.

[0131] In some advantageous embodiments of the methods according to the present disclosure, the binding element (S) comprises a nucleic acid comprising a nucleotide sequence that allows specific binding to the encoded analyte, preferably specific hybridization to the encoded analyte.

[0132] In some advantageous embodiments of the method according to the present disclosure, after step A) and before step B), unbound analyte-specific probes may be removed, in particular by washing, and further after step B) and before step C), unbound decoding oligonucleotides may be removed, in particular by washing, and further after step C) and before step D), unbound signal oligonucleotides may be removed, in particular by washing.

[0133] In some advantageous embodiments of the methods according to the present disclosure, the analyte-specific probes may be incubated with the sample, thereby allowing specific binding of the analyte-specific probes to the analytes to be encoded, and the decoding oligonucleotides may be incubated with the sample, thereby allowing specific hybridization of the decoding oligonucleotides to the identifier elements (T) of the respective analyte-specific probes, and the signal oligonucleotides may be incubated with the sample, thereby allowing specific hybridization of the signal oligonucleotides to the translation elements (T) of the respective decoding oligonucleotides.

[0134] As mentioned above, the encoded analyte may be a nucleic acid, preferably a DNA, a PNA, an RNA, in particular an mRNA, a peptide, a polypeptide, a protein, or a combination thereof. Thus, the binding element (S) may comprise an amino acid sequence that allows specific binding to the encoded analyte. Examples of binding elements (S) are affinity moieties derived from or as a whole affinity substance selected from the group consisting of antibodies, antibody fragments, anticalin proteins, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenesis or virulence factors, allergens, peptidic allergens, recombinant allergens, allergen-idiotypic antibodies, autoimmune-inducing structures, tissue rejection-inducing structures, immunoglobulin constant regions, and combinations thereof. In particular, the binding element (S) is an antibody or antibody fragment selected from the group consisting of Fab, scFv; single domains or fragments thereof, bis-scFv, Fab2, Fab3, minibodies, diabodies, triabodies, tetrabodies, and tandabs.

[0135] By this means, the method can be further developed to the extent that the coded analyte can be detected by any means adapted to visualize the signal element. Examples of detectable physical characteristics include, for example, light, chemical reaction, molecular weight, radioactivity, etc.

[0136] In some advantageous embodiments, the signal resulting from the signal element, and thus in particular the binding of the signal oligonucleotide to the decoding oligonucleotide, the interaction with the corresponding analyte probe, the binding to the respective analyte, is determined by: (a) imaging at least a portion of the sample; and / or (b) using optical imaging techniques; and / or (c) using fluorescent imaging techniques; and / or (d) multicolor fluorescence imaging techniques; and / or (e) Super-resolution fluorescence imaging technology It is measured by:

[0137] The methods according to the present disclosure may ideally be used for in vitro methods for the diagnosis of diseases selected from the group including cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases due to viral or bacterial infections, skin diseases, musculoskeletal diseases, dental diseases and prenatal diseases.

[0138] Furthermore, the method according to the present disclosure may also be used for in vitro methods for the diagnosis of diseases in plants, ideally selected from the group comprising diseases caused by biotic stress, preferably of infectious and / or parasitic origin, diseases caused by abiotic stress, preferably nutritional disorders, and / or unfavorable environment.

[0139] Additionally, the method according to the present disclosure would also ideally include: (a) contacting a test sample containing the sample with a substance and / or agent; (b) detecting various analytes in a sample by sequential signal encoding of said analytes by the method according to the present disclosure; The present invention may be used for in vitro methods for screening, identifying and / or testing substances and / or drugs, including:

[0140] In some advantageous embodiments, the spatial transcriptomics method described herein is used to specifically detect many different analytes in parallel. This technique allows the differentiation of a larger number of analytes than the different signals are available. The process involves at least four successive rounds of specific binding, signal detection and selective denaturation (if a next round is required), ultimately generating a signal code. To decouple the dependency between analyte-specific binding and the oligonucleotides that provide a detectable signal, so-called "decoding"-oligonucleotides are introduced. The decoding oligonucleotides transcribe the information of the analyte-specific probe set into the signal oligonucleotide.

[0141] In certain embodiments, the spatial transcriptomics method comprises the steps of: 1. providing one or more analyte-specific probe sets, each consisting of one or more probes that differ in a binding moiety that specifically interacts with an analyte, where all probes of a single probe set are tethered to a sequence element (unique identifier) ​​that is unique to the single probe set and that allows specific hybridization of a decoding oligonucleotide; 2. specific binding of the probe sets to the target binding sites of their analyte; 3. removing unbound probes (e.g., by a washing step); 4. providing a mixture of decoding oligonucleotides that specifically hybridize to the unique identifier sequences of the probe sets, where the decoding oligonucleotides have at least two sequence elements, a first element complementary to the unique identifier sequence of the corresponding probe set and a sequence for specific hybridization of a signal oligonucleotide. a second sequence element (translation element) providing a unique identifier sequence provided by the bound probe set, the translation element defining the type of signal to be recruited to the decoding oligonucleotide; 5. a step of specific hybridization of the decoding oligonucleotide to the unique identifier sequence provided by the bound probe set; 6. a step of removing (e.g. by a washing step) unbound decoding oligonucleotides; 7. a step of providing a mixture of signal oligonucleotides consisting of a mixture of nucleic acid sequences that specifically hybridize to a detectable signal and to the translation element of one of the decoding oligonucleotides used in the previous hybridization step; 8. a step of specific hybridization of the signal oligonucleotide; 9. a step of removing unbound signal oligonucleotides; 10. a step of detection of the signal; 11. a step of selective release of the decoding oligonucleotide and the signal oligonucleotide, during which the binding of the specific probe set to the analyte is almost or completely unaffected; 12.removing the released decoding and signal oligonucleotides (e.g., by a washing step) while the binding of the specific probe sets to the analytes is largely or completely unaffected, and repeating steps 4-12 at least three times until a sufficient number of signals are detected to generate a coding scheme for each different analyte of interest.

[0142] It is to be understood that the features mentioned above and those mentioned below can be used not only in the combinations indicated in each case, but also in other combinations or alone without departing from the scope of the present disclosure.

[0143] The present disclosure will now be further described by embodiments that provide the features, characteristics and advantages of the present disclosure. The embodiments are of purely exemplary nature and do not limit the scope or scope of the present disclosure. The features mentioned in certain embodiments are general features of the present disclosure and are not only applicable in certain embodiments, but also in an independent manner in any embodiment of the present disclosure.

[0144] The spatial transcriptomics method disclosed herein is used to specifically detect many different analytes in parallel. This technique allows the differentiation of a larger number of analytes than the available different signals. The process preferably includes at least two successive rounds of specific binding, signal detection and selective denaturation (if a next round is required), ultimately generating a signal code. To separate the dependency between the analyte-specific binding oligonucleotide and the oligonucleotide that provides a detectable signal, a so-called "decoding" oligonucleotide is introduced. The decoding oligonucleotide transcribes the information of the analyte-specific probe set into the signal oligonucleotide.

[0145] The disclosure further relates to a method for detecting an analyte containing a pathogen, the method comprising inactivating the pathogen in the sample without isolating RNA and / or DNA from the pathogen or sample, and - attaching a plurality of analyte-specific probes to the analyte, where the analyte-specific probes are independently attached to the analyte, and where the analyte-specific probes share a common identifier segment (T); - annealing a plurality of first decoding oligonucleotides to the analyte-specific probes, where the first decoding oligonucleotides share a first common region and a second common region that are reverse complementary to the common identifier segment; - annealing a first signal oligonucleotide to at least one of the plurality of first decoding oligonucleotides such that the oligo tethered to the first signal oligonucleotide is reverse complementary to a second common region; - detecting a first signal oligonucleotide; - removing a plurality of first decoding oligonucleotides; - annealing a plurality of second decoding oligonucleotides to the analyte-specific probes, where the second decoding oligonucleotides share a first common region that is reverse-complementary to the common identifier segment and a second common region of the second decoding oligonucleotides that is different from the second common region of the first decoding oligonucleotides; - annealing a second signal oligonucleotide to at least one of the plurality of second decoding oligonucleotides such that the oligonucleotide tethered to the signal oligonucleotide is reverse complementary to a second common region of the second decoding oligonucleotide; and Detecting the second signal oligonucleotide Includes.

[0146] In particular, in the above embodiment, a second aliquot of the plurality of first decoding oligonucleotides is annealed to the analyte-specific probe. Further, a first aliquot of the plurality of first decoding oligonucleotides is annealed to the analyte-specific probe.

[0147] In some embodiments, the second signal oligonucleotide is not annealed to at least one of the plurality of first decoding oligonucleotides, and in particular, the third signal oligonucleotide is not annealed to at least one of the plurality of first decoding oligonucleotides.

[0148] Methods and Examples In one application variant, the analyte or target is a nucleic acid, e.g., DNA or RNA, and the probe set comprises oligonucleotides that are partially or completely complementary to the entire sequence or a subsequence of the nucleic acid sequence to be detected (Figure 1). The nucleic acid sequence-specific oligonucleotide probe set comprises an analyte-specific probe (1) that comprises a binding element (S) that specifically hybridizes to the target nucleic acid sequence to be detected, and an identifier element (T) that comprises a nucleotide sequence (unique identifier sequence) that is unique to said set of analyte-specific probes.

[0149] In a further application variant, the analyte or target is a protein and the probe set comprises one or more proteins, such as antibodies (Figure 2). The protein-specific probe set comprises a binding element (T), such as a (hyper)variable region of an antibody, that specifically interacts with the target protein to be detected, and an analyte-specific probe (1) that comprises an identifier element (T).

[0150] In a further application variant, at least one analyte is a nucleic acid and at least a second analyte is a protein, and at least a first probe set binds to a nucleic acid sequence and at least a second probe set specifically binds to a protein analyte. Other combinations are also possible. One embodiment of the general method of the present disclosure may be: Step 1: Applying at least 20 analyte or target specific probe sets. The target nucleic acid sequence is incubated with a probe set consisting of oligonucleotides having sequences complementary to the target nucleic acid. In this example, a probe set of 5 different probes is shown, each containing a sequence element complementary to a distinct subsequence (S1-S5) of the target nucleic acid sequence. In this example, the regions do not overlap. Each of the oligonucleotides targeting the same nucleic acid sequence each contains an identifier element or unique identifier sequence (T). Step 2: Hybridization of the probe set. The probe set is hybridized to the target nucleic acid sequence under conditions that allow specific hybridization. After incubation, the probes hybridize to their corresponding target sequences and provide identifier elements (T) for the next step. Step 3: Removing Unbound Probe After hybridization, unbound oligonucleotides are removed, for example by a washing step. Step 4: Applying a decoding oligonucleotide. A decoding oligonucleotide is applied, consisting of at least two sequence elements (t) and (c). The sequence element (t) is complementary to the unique identifier sequence (T), while the sequence element (c) provides a region for subsequent hybridization of a signal oligonucleotide (translation element). Step 5: Hybridization of the Decoding Oligonucleotides. The decoding oligonucleotides are hybridized with the unique identifier sequence of the probe (T) via their complementary first sequence element (t). After incubation, the decoding oligonucleotides provide the translation sequence element (c) for the subsequent hybridization step. Step 6: Removing excess decoding oligonucleotides After hybridization, unbound decoding oligonucleotides are removed, for example by a washing step. Step 7: Applying a signal oligonucleotide. A signal oligonucleotide is applied. The signal oligonucleotide comprises at least one second connector element (C) substantially complementary to the translation sequence element (c) and at least one signal element that provides a detectable signal (F). Step 8: Hybridization of the signal oligonucleotide. The signal oligonucleotide is hybridized through a sequence connector element (C) that is complementary to the translation element (c) of the decoding oligonucleotide. After incubation, the signal oligonucleotides hybridize to their corresponding decoding oligonucleotides and provide a detectable signal (F). Step 9: Removing excess signal oligonucleotides After hybridization, unbound signal oligonucleotides are removed, for example, by a washing step. Step 10: Signal detection The signal provided by the signal oligonucleotide is detected. The next steps (steps 11 and 12) are unnecessary for the final detection round. Step 11: Selective denaturation. The hybridization between the unique identifier sequence (T) and the first sequence element (t) of the decoding oligonucleotide is eliminated. Destabilization can be achieved by different mechanisms well known to the trained person, for example: increasing the temperature, denaturing agents, etc. The target or analyte specific probes are not affected by this step. Step 12: Removing the modified decoding oligonucleotides. The modified decoding oligonucleotides and signal oligonucleotides are removed (e.g., by a washing step), leaving specific probe sets with free unique identifier sequences that can be reused in the next hybridization and detection round (steps 4-10). This detection cycle (steps 4-12) is repeated at least four times until the planned encoding scheme is completed.

[0151] Another embodiment of the general method of the present disclosure using multiple decoders may be the following (FIG. 16): Step 1: Target Nucleic Acids: In this example, three different target nucleic acids (A), (B) and (C) must be detected and differentiated by using only two different types of signal oligonucleotides. Before starting the experiment, a specific encoding scheme is set up. In this example, three different nucleic acid sequences are encoded with three rounds of detection by three different signal types (1), (2) and (1 / 2) and a resulting Hamming distance of 3 that allows for error detection. The planned code words are: Array A: (1)-(1)-(2) Array B: (2)-(2)-(1 / 2) Array C:(1 / 2)-(1 / 2)-(1) It is. Step 2: Hybridization of the probe sets: For each target nucleic acid, its own probe set is applied, which specifically hybridizes to the corresponding nucleic acid sequence of interest. Each probe set provides a unique identifier sequence (T1), (T2) or (T3). In this way, each different target nucleic acid is uniquely labeled. In this example, sequence (A) is labeled with (T1), sequence (B) with (T2) and sequence (C) with (T3). The description of FIG. 16 summarizes steps 1-3 of FIG. 3. Step 3: Hybridization of the decoding oligonucleotides and multi-decoders. For each unique identifier present, a specific decoding oligonucleotide or multi-decoder is applied, which hybridizes specifically to the corresponding unique identifier sequence by its first sequence element (here (t1) to (T1), (t2) to (T2) and (t3) to (T3)). Each of the decoding oligonucleotides or multi-decoders provides a translation element or two translation elements that define the signal generated after hybridization of the signal oligonucleotide. Here, the nucleic acid sequence (A) is labeled with (c1), (B) is labeled with (c2) and (C) is labeled with the translation elements (c1) and (c2), resulting in a signal (1 / 2). The illustration of FIG. 16 summarizes steps 4 to 6 of FIG. 3. Step 4: Hybridization of signal oligonucleotides: For each type of translation element, a signal oligonucleotide is applied that has a specific signal that can be distinguished from other signal oligonucleotides. This signal oligonucleotide can specifically hybridize to the corresponding translation element. The description of Figure 16 summarizes steps 7-9 of Figure 3. Step 5: Signal detection for the encoding scheme: Different signals are detected. Note that in this example, nucleic acids (A), (B) and (C) are already distinguishable after the first detection round. This is in contrast to step 5 in FIG. 5, which is illustrated by the additional signal type (1 / 2) that can be achieved by the multiple decoder. The nucleic acid sequences are already distinguishable, but the additional rounds contribute to the planned Hamming distance of 3. The illustration in FIG. 16 corresponds to step 10 in FIG. 3. Step 6: Selective Denaturation: The decoding (and signal) oligonucleotides and / or multidecoders of all nucleic acid sequences to be detected are selectively denatured and removed as described in steps 11 and 12 of Figure 3. The unique identifier sequences of the different probe sets can then be used in the next round of hybridization and detection. Step 7: Second round of detection: The next round of hybridization and detection is carried out as described in steps 3-5. Note that in this new round, the mixture of different decoding oligonucleotides and multi-decoders is changed. For example, the decoding oligonucleotides of the nucleic acid sequence (A) used in the first round consisted of sequence elements (t1) and (c1), while the new multi-decoder of round 2 consists of sequence elements (t1), (c1) and (c2). Note that now a Hamming distance of 2 is already given after two rounds, which is the final result of the example in Figure 3 after three rounds. Step 8: Third round of detection: Again, new combinations of decoding oligonucleotides and / or multiple decoders are used, resulting in new signal combinations. After signal detection, the resulting code words for the three different nucleic acid sequences are not only unique and therefore distinct, but also contain a Hamming distance of three to the other code words. Due to that Hamming distance, errors in the detection of the signal (signal exchange) should not result in a valid code word and therefore, in contrast to the encoding scheme of FIG. 3, can be detected and also corrected due to the Hamming distance of three. Thus, the three different nucleic acids can be distinguished in three detection rounds using two different signals, allowing error detection and correction.

[0152] It should be noted that the type of signal provided by a particular unique identifier in each round of detection is controlled by the use of a particular decoding oligonucleotide. As a result, the sequence of the decoding oligonucleotide applied during the detection cycle transcribes the binding specificity of the probe set into a unique signal sequence.

[0153] The decoding oligonucleotide hybridization steps (steps 4-6) and the signal oligonucleotide hybridization steps (steps 7-9) can be combined in two alternative ways as shown in FIG. Option 1: Simultaneous Hybridization. Instead of steps 4-9 of FIG. 3, specific hybridization of the decoding oligonucleotide and the signal oligonucleotide may be performed simultaneously, after removal of excess decoding and signal oligonucleotides, resulting in the same result as shown in step 9 of FIG. 3. Option 2: Pre-incubation. In addition to option 1 in Figure 3, the decoding and signal oligonucleotides can be pre-incubated in separate reactions with the specific probe sets already bound before being applied to the target nucleic acid.

[0154] 1. Example for signal encoding of three different nucleic acid sequences with two different signal types and three detection rounds Figure 3 shows a general concept of the generation and detection of specific signals mediated by decoding oligonucleotides. It does not show a general concept of the encoding that can be achieved by this method. To illustrate the use of the process shown in Figure 3 for the generation of encoding schemes, Figure 5 shows an example of multiple rounds using three different nucleic acid sequences. In this embodiment, the coding scheme includes error detection. Step 1: Target Nucleic Acids. In this example, three different target nucleic acids (A), (B) and (C) must be detected and differentiated by using only two different types of signals. Before starting the experiment, a specific encoding scheme is set up. In this example, three different nucleic acid sequences are encoded with three rounds of detection using two different signals (1) and (2) and a resulting Hamming distance of 2 that allows for error detection. The planned codewords are: Array A: (1)-(2)-(2); Array B: (1)-(1)-(1); Array C:(2)-(1)-(2) It is. Step 2: Hybridization of the probe sets. For each target nucleic acid, its own probe set is applied, which specifically hybridizes to the corresponding nucleic acid sequence of interest. Each probe set provides a unique identifier sequence (T1), (T2) or (T3). In this way, each different target nucleic acid is uniquely labeled. In this example, sequence (T) is labeled with (T1), sequence (B) with (T2) and sequence (C) with (T3). This description summarizes steps 1-3 of FIG. 3. Step 3: Hybridization of the Decoding Oligonucleotides. For each unique identifier present, a specific decoding oligonucleotide is applied, which hybridizes specifically to the corresponding unique identifier sequence by its first sequence element (here (t1) to (T1), (t2) to (T2), and (t3) to (T3)). Each of the decoding oligonucleotides provides a translation element that defines the signal that will be generated after hybridization of the signal oligonucleotide. Here, the nucleic acid sequences (A) and (B) are labeled with a translation element (c1) and the sequence (C) is labeled with (c2). This description summarizes steps 4 to 6 of FIG. 3. Step 4: Hybridization of the signal oligonucleotide. For each type of translation element, a signal oligonucleotide is applied that has a specific signal (2) that can be distinguished from other signal oligonucleotides. This signal oligonucleotide can specifically hybridize to the corresponding translation element. This description summarizes steps 7 to 9 of FIG. 3. Step 5: Signal detection for the encoding scheme. Different signals are detected. Note that in this example, nucleic acid sequence (C) can be distinguished from other sequences due to the unique signal (2) it provides, while sequences (A) and (B) provide the same type of signal (1) and are indistinguishable after the first cycle of detection. This is due to the fact that the number of different nucleic acid sequences detected exceeds the number of different signals available. This illustration corresponds to step 10 in FIG. 3. Step 6: Selective Denaturation. The decoding (and signal) oligonucleotides of all nucleic acid sequences to be detected are selectively denatured and removed as described in steps 11 and 12 of Figure 3. The unique identifier sequences of the different probe sets can then be used in the next round of hybridization and detection. Step 7: Second round of detection. The next round of hybridization and detection is carried out as described in steps 3-5. Note that in this new round, the mixture of different decoding oligonucleotides is changed. For example, the decoding oligonucleotides of the nucleic acid sequence (A) used in the first round consisted of sequence elements (t1) and (c1), while the new decoding oligonucleotides consist of sequence elements (t1) and (c2). Note that now all three sequences are clearly distinguishable due to the unique combination of the first and second round signals. Step 8: Third round of detection. Again, a new combination of decoding oligonucleotides is used, resulting in a new signal combination. After signal detection, the resulting code words for the three different nucleic acid sequences are not only unique and therefore distinct, but also contain a Hamming distance of 2 to the other code words. Due to that Hamming distance, an error in the detection of the signal (signal exchange) should not result in a valid code word and therefore can be detected. In this way, the three different nucleic acids can be distinguished in three detection rounds using two different signals, allowing error detection.

[0155] 2. Advantages over prior art Coding Strategy One particular advantage of the method according to the present disclosure over current methods is the use of decoding oligonucleotides that break the dependency between the target-specific probe and the signal oligonucleotide.

[0156] Without decoupling target-specific probes and signal generation, two different signals can only be generated for a specific target if two different molecular tags are used. Each of these molecular tags can only be used once. Multiple readings of the same molecular tag do not increase information about the target. To generate a coding scheme, a change of target-specific probe set after each round is required (SeqFISH), or multiple molecular tags must be present on the same probe set (like merFISH, intronSeqFISH).

[0157] According to the method of the present disclosure, different signals are achieved by using different decoding oligonucleotides that use the same unique identifier (molecular tag) and reusing a small number of different, generally costly, signal oligonucleotides, which, in contrast to other methods, offers several advantages. (1) The coding scheme is not defined by the target-specific probe set, as in all other methods of the prior art. Here, the coding scheme is transcribed by the decoding oligonucleotide. This allows much more flexibility in terms of the number of rounds for the codeword and the freedom of signal selection. Looking at the prior art methods (e.g., merFISH or intronSeqFISH), the coding scheme (number, type and sequence of detectable signals) for every target sequence is predefined by the presence of different tag sequences on the specific probe set (4 out of 16 per probe set for merFISH and 5 out of 60 per probe set for intronFISH). To generate a sufficient number of different tags per probe set, the method uses a rather complex oligonucleotide design with several tags on one target-specific oligonucleotide. To change the coding scheme for a particular target nucleic acid, the specific probe set needs to be replaced. The method according to the present disclosure describes the use of a single unique tag sequence (unique identifier) ​​per analyte, because it can be reused for each detection round to generate new information. The coding scheme is defined by the order of the decoding oligonucleotides used in the detection round. Therefore, the coding scheme is not predefined by specific probes (or unique tag sequences), but can be adjusted to different needs even during the experiment. This is achieved by simply changing the decoding oligonucleotides used in the detection round or in additional detection rounds. (2) The number of different signal oligonucleotides must match the number of different tag sequences in the prior art methods (16 for merFISH and 60 for intronSeqFISH). With the method according to the present disclosure, the number of different signal oligonucleotides matches the number of different signals used. For this reason, the number of signal oligonucleotides remains constant in the methods described herein and never exceeds the number of different signals, whereas in the prior art methods, it increases with the complexity of the coding scheme (the more detection rounds, the more different signal oligonucleotides are required). As a result, the methods described herein result in much lower complexity (unintended interaction of signal oligonucleotides with the environment or with each other) and dramatically reduce the cost of the assay, since most of the cost factor is signal oligonucleotides. (3) In the prior art methods, the number of different signals generated by a target-specific probe set is limited by the number of different tag sequences that the probe set can provide. Since each additional tag sequence increases the total size of the target-specific probe, there is a limit to the number of different tags that a single probe can provide. This limit is brought about by the size-dependent increase in several issues (unintended intra- and intermolecular interactions, cost, diffusion rate, stability, errors during synthesis, etc.). In addition, there is a limit to the total number of target-specific probes that can be applied to a particular analyte. In the case of nucleic acids, this limit is given by the length of the target sequence and the ratio of suitable binding sites. These factors result in a strict constraint on the number of different signals that a probe set can provide (4 signals for merFISH and 5 signals for intronSeqFISH). This limit substantially affects the number of different code words that can be generated in a given number of detection rounds. In the disclosed approach, only one tag is needed and can be freely reused for each detection round. This allows for low oligonucleotide complexity / length and, at the same time, the maximum possible coding efficiency (number of colors). ラウンド数) The large difference in the encoding capacity of our method compared to other methods is shown in Figures 1 and 5. With this approach of the present disclosure, a much smaller number of detection rounds is required to generate the same amount of information. A smaller number of detection rounds is associated with lower costs, less experimental time, lower complexity, higher stability and hit rate, less data volume to be collected and analyzed, and higher accuracy of the results.

[0158] Coding Capabilities All three methods compared in Table 1 below use specific probe sets that do not change between different rounds of detection. In the case of intronSeqFISH, there are four detection rounds required to generate the pseudocolor of one coding round, so data is only given for rounds 4, 8, 12, 16 and 20. The merFISH method uses a fixed number of four signals, so data starts with the minimum number of rounds possible. After eight detection rounds, our method exceeds the maximum coding capacity reached in 20 rounds of merFISH (indicated by one asterisk), and after 12 rounds of detection, the maximum coding capacity of intron FISH is exceeded (indicated by two asterisks). In the case of the method according to the present disclosure, the use of three different signals is assumed (as in intronSeqFISH). [Table 1]

[0159] Note that this maximum efficiency of encoding capacity is also achieved in the case of SeqFISH, where specific probes are denatured after every detection round, and a new set of probes hybridizes specifically to the target sequence in each detection round. However, this method has a major technical drawback of using only one specific hybridization for their encoding scheme (all other methods), as shown below: (1) For efficient denaturation of specific probes, rather harsh conditions must be used (high temperature, high concentration of denaturing agent, long incubation time), resulting in a much higher probability of loss or damage of the analyte. (2) For each detection round, a unique probe set must be used for every target nucleic acid sequence. Thus, the number of specific probes required for the experiment increases with the number of different signals required for the coding scheme. This dramatically increases the complexity and cost of the assay. (3) Because the hybridization efficiency of every target nucleic acid molecule is subject to several stochastic effects, the variation in signal intensity between different detection rounds is much higher than in methods using only one specific hybridization event, reducing the proportion of perfect codes. (4) The time required for specific hybridization is much longer than for hybridization of signal or decoding oligonucleotides (as seen in the methods of intronSeqFISH, merFISH and SeqFISH), which dramatically increases the time required to complete an experiment.

[0160] For these reasons, all other methods use a single specific hybridization event, accepting lower code complexity and therefore the need for more detection rounds, and higher oligonucleotide design complexity.

[0161] The method according to the present disclosure combines the advantages of SeqFISH (mainly the complete freedom regarding the encoding scheme) with all the advantages of methods using only one specific hybridization event, while eliminating the major drawbacks of such methods.

[0162] Note that the large number of code words generated after 20 rounds can also be used to introduce larger Hamming distances (differences) between different code words, allowing the detection of one, two, or even more errors and the correction of further errors. Thus, even much higher coding capabilities are still practically justified.

[0163] As mentioned above, the use of multiple decoders further increases the coding capacity of the coding scheme. Instead of being limited to having exactly the same number of different signal types as different signal oligonucleotides and corresponding translation elements, the use of multiple decoders increases the number of signal types that can be used to: (Nx(N+1)) / 2, where N is the number of different signal oligonucleotides used. For the code used in Table 1 with three different signal oligonucleotides, this means that seven different signal types can be used: (S1), (S2), (S3), (S1 / S2), (S1 / S3), (S2 / S3), (S1 / S2 / S3). The effect on coding efficiency can be seen in Table 1b and Figure 17. [Table 2]

[0164] 3. The selective denaturation, oligonucleotide assembly and reuse of unique identifiers is surprisingly efficient. The key factor of the method according to the present disclosure is the successive steps of decoding oligonucleotide binding, oligonucleotide binding, signal detection and selective denaturation. To generate the encoding scheme, this step needs to be repeated several times (depending on the length of the codeword). Since the same unique identifier is reused in every detection cycle, all events from the first to the last detection cycle are mutually dependent. Moreover, selective denaturation depends on two different events: the decoding oligonucleotide needs to be eluted from the unique identifier with the highest efficiency, while the specific probe must remain hybridized with the highest efficiency.

[0165] Therefore, the efficiency E of the entire encoding process can be described by the following equation:

number

[0166] Based on this formula, the efficiency of each single step can be estimated for a given total efficiency of the method. The calculation is thereby based on the assumption that each step has the same efficiency. The total efficiency describes the portion of the signal that can be successfully decoded of the total signal present.

[0167] The total efficiency of the method depends on the efficiency of each single step of different factors described by the formula. Under the assumption of equally distributed efficiency, the total efficiency can be plotted against the single step efficiency as shown in Figure 7. As can be seen, a practically reasonable total efficiency for an encoding scheme with 5 detection cycles can only be achieved with a single step efficiency clearly above 90%. For example, to achieve a total efficiency of 50%, an average efficiency within each single step of 97.8% is required. These calculations are further based on the assumption of 100% signal detection and analysis efficiency. Due to the broad DNA melting curves of oligonucleotides of various sequences, the inventors assumed prior to the experiment that selective denaturation may work with a lower efficiency for denaturation of decoding oligonucleotides and that the sequence-specific probe is not stable enough. In contrast to this assumption, we found a surprising efficiency of all steps and a high stability of the sequence-specific probe during selective denaturation.

[0168] Experimentally, we have achieved a total decoding efficiency of about 30% to 65% based on 5 detection cycles. Each single step (B sp , B de , B si , E de , S sp ) showed an average efficiency of about 94.4% to 98%. These high efficiencies are quite surprising and not easily predicted by someone well trained in this field.

[0169] 4. Experimental Data background The experiment shows the specific detection of 10-50 different mRNA species simultaneously with single molecule resolution. It is based on 5 detection cycles, 3 different fluorescent signals and a coding scheme with no signal gaps as well as a Hamming distance (error detection) of 2. The experiment proves the feasibility and functionality of the method according to the present disclosure.

[0170] Oligonucleotides and their sequences All oligonucleotide sequences (target-specific probes, decoding oligonucleotides, signal oligonucleotides) used in the experiments are listed in the sequence table in the supplementary material. The signal oligonucleotide R:ST05*O_Atto594 was ordered from biomers.net GmbH. All other oligonucleotides were ordered from Integrated DNA Technologies. Oligonucleotides were dissolved in water. Stock solutions (100 μM) were stored at −20° C.

[0171] Experimental Overview The 50 different target-specific probe sets are divided into 5 groups. The names of the transcripts detected and the names of the target-specific probe sets are the same (transcript variant names from www.ensemble.org). The term "novel" indicates the modified probe design. All oligonucleotide sequences of the probe sets can be found in the sequence table. The table lists the unique identifier names of the probe sets as well as the decoding oligonucleotide names used in the different detection cycles. The resulting codes indicate the sequence of the fluorescent signals generated during the 5 detection cycles ((G(reen)=Alexa Fluor488, O(range)=Atto594, Y(ellow)=Alexa Fluor546). [Table 3] TIFF2024528255000007.tif75159

[0172] Experimental Variations Several variations of the experiment were performed. Experiments 1-4 differ mainly in the number of transcripts detected in parallel. The groups listed as target-specific probe sets refer to Table 6. Experiments 5-8 are single round, single target controls for comparison with the decoded signal. [Table 4]

[0173] Experimental details A. Cell seeding and culture HeLa cells were grown to nearly 100% confluence in cell culture medium. HeLa cell culture medium contains DMEM (Thermo Fisher, Catalog No.: 31885) with 10% FCS (Biochrom, Catalog No.: S0415), 1% penicillin-streptomycin (Sigma-Aldrich, Catalog No.: P0781) and 1% MEM non-essential amino acid solution (Thermo Fisher, Catalog No.: 11140035). After aspiration of the cell culture medium, the cells were trypsinized by incubation with trypsin-EDTA solution (Sigma-Aldrich, Catalog No.: T3924) for 5 min at 37 °C after a washing step with PBS (1.424 g / l Na2HPO4·2H2O, 0.276 g / l NaH2PO4·2H2O, 8.19 g / l NaCl in water, pH 7.4). The cells were then seeded onto the wells of a μ-Slide 8 Well ibidiTreat (Ibidi, Cat. No.: 80826). The number of cells per well was adjusted to reach approximately 50% confluency after cell attachment. The cells were incubated overnight with 200 μl of HeLa cell medium per well. B. Fixation of cells After absorption of the cell culture medium and two washing steps with 200 μl of 37° C. warm PBS per well, the cells were fixed with 200 μl of pre-chilled methanol (−20° C., Roth, Cat. No.: 0082.1) for 10 min at −20° C. C. Counterstaining with Sudan Black Methanol was aspirated and 150 μl of 0.2% Sudan Black solution diluted in 70% ethanol was added to each well. The wells were incubated for 5 minutes in the dark at room temperature. After incubation, the cells were washed three times with 400 μl of 70% ethanol per well to remove excess Sudan Black solution. D. Analyte / Target-Specific Probe Hybridization Prior to hybridization, cells were equilibrated with 200 μl of sm-wash buffer, which contains 30 mM Na3 citrate, 300 mM NaCl, pH 7, 10% formamide (Roth, Catalog No. P040.1) and 5 mM ribonucleoside vanadyl complex (NEB, Catalog No. S1402S). For each target-specific probe set, 1 μl of a 100 μM oligonucleotide stock solution was added to the mixture. The oligonucleotide stock solution contains equimolar amounts of all target-specific oligonucleotides of the corresponding target-specific probe set. The total volume of the mixture was adjusted to 100 μl with water and mixed with 100 μl of 2x concentrated hybridization buffer. The 2x concentrated hybridization buffer contained 120 mM Na3 citrate, 1200 mM NaCl, pH 7, 20% formamide and 20 mM ribonucleoside vanadyl complex. 200 μl of the resulting hybridization mixture was added to the corresponding wells and incubated at 37° C. for 2 hours. The cells were then washed three times with 200 μl of target probe wash buffer per well for 10 minutes at 37° C. The target probe wash buffer contained 30 mM Na3 citrate, 300 mM NaCl, pH 7, 20% formamide and 5 mM ribonucleoside vanadyl complex. E. Hybridization of Decoding Oligonucleotides Prior to hybridization, cells were equilibrated with 200 μl of sm-wash buffer. For each decoding oligonucleotide, 1.5 μl of a 5 μM stock solution was added to the mixture. The total volume of the mixture was adjusted to 75 μl with water and mixed with 75 μl of 2x concentrated hybridization buffer. The resulting 150 μl of decoding oligonucleotide hybridization mixture was added to the corresponding wells and incubated at room temperature for 45 minutes. Cells were then washed three times with 200 μl of sm-wash buffer per well for 2 minutes at room temperature. F. Hybridization of Signal Oligonucleotides Prior to hybridization, cells were equilibrated with 200 μl of sm-wash buffer. The signal oligonucleotide hybridization mixture was the same for all rounds of experiments 1-4 and contained 0.3 μM of each signal oligonucleotide (see Table A3) in 1x concentrated hybridization buffer. In each round, 150 μl of this solution was added per well and incubated for 45 min at room temperature. The procedure was the same as for experiments 5-8, except that the final concentration of each signal oligonucleotide was 0.15 μM. Cells were then washed 3 times with 200 μl of sm-wash buffer per well for 2 min at room temperature. G. Fluorescence and White Light Imaging The cells were washed once with 200 μl imaging buffer per well at room temperature. For experiments without Trolox (see last column of Table 7), the imaging buffer contained 30 mM Na3 Citrate, 300 mM NaCl, pH 7, and 5 mM ribonucleoside vanadyl complex. For experiments with Trolox, the imaging buffer additionally contained 10% VectaCell Trolox Antifade Reagent (Vector laboratories, Catalog No.: CB-1000) to give a final Trolox concentration of 10 mM. A Zeiss Axiovert 200M microscope equipped with a 63x oil immersion objective (Zeiss, apochromat) with a numerical aperture of 1.4, a pco.edge4.2 CMOS camera (PCO AG) and an LED-light source (Zeiss, colibri 7) was used for imaging the fields. Filter sets and LED wavelengths were adjusted to the different optimum conditions of the fluorophores used. The exposure time per image was 1000 ms for Alexa Fluor546 and Atto594 and 400 ms for Alexa Fluor488. In each experiment, three regions were randomly selected for imaging. For each region, a z-stack of 32 images was detected with a z-step size of 350 nm. In addition, one white light image was acquired from the region. In experiments with more than one detection cycle, the regions from the first detection round were revisited and imaged in all subsequent rounds. H. Selective Denaturation For selective denaturation, all wells were incubated with 200 μl of sm-wash buffer for 6 min at 42° C. This procedure was repeated six times. Steps (E)-(H) were repeated five times in experiments 1-4. Step (H) was omitted in the fifth detection cycle. I.Analysis A semi-automated analysis of the raw data was performed based on a custom ImageJ plug-in to distinguish specific fluorescent signals from background. The resulting 3D point clouds of all three fluorescent channels were combined in a computer using a custom VBA script. The resulting combined 3D point clouds of five detection cycles were aligned to each other based on a VBA script. The resulting alignment strings represented the code word for each unique signal detected. The successfully decoded signals were used for quantitative and spatial analysis of the experiment based on a custom VBA script and plug-in.

[0174] result 1. Absolute number of decoded signals The absolute number of successfully decoded signals for all transcripts is listed for each region for each experiment in Table 4 below. In summary, the sum of correct codes indicates the total number of decoded signals that were assigned to transcripts detectable in the corresponding experiment, while the sum of incorrect codes indicates the total number of decoded signals that were not detectable in the corresponding experiment. The total number of signals includes successfully decoded signals as well as signals that failed to be decoded. [Table 5] TIFF2024528255000010.tif129159

[0175] conclusion The method according to the present disclosure generates a small amount of erroneously assigned code words and can therefore be considered specific. The percentage of successfully decodable signals is very high, furthermore it has a very large number of signals per region, and a very large number of transcripts are detected in parallel. The high percentage of assignable signals and the high specificity make the method practically useful.

[0176] Comparison of relative transcript abundance between different experiments The overlap of transcripts detected between experiments is used for analysis, as shown in Figure 8 for both comparisons (A and B). Each bar is the average abundance of all three experimental regions. The standard deviation between these regions is also shown.

[0177] Correlation of relative transcript abundance between different experiments As can be seen in Figure 9, the average relative abundance of transcripts from experiment 1 correlates with the abundance of overlapping transcripts from experiments 3, 4 and 2. The correlation coefficients as well as the equation for the linear regression are shown for each correlation.

[0178] Figure 8 shows low standard deviations, indicating low variation in relative abundance between different regions of one experiment. The differences in relative abundance between transcripts from different experiments are also very small. This is the case for the comparison of transcripts from group 1, detected in experiments 1, 2 and 3 (Figure 8A). It is also the case for the comparison of transcripts from groups 2, 3 and 4, overlapping in experiments 1, 2 and 4. The very high correlation of these abundances can also be seen in Figure 9. The abundance of transcripts from experiment 1 correlates very well with the abundances of the other multi-round experiments. The correlation coefficients are between 0.88 and 0.91, while the slopes of the linear regressions are between 0.97 and 1.05.

[0179] conclusion The relative abundance of transcripts correlates very well between different regions of one experiment, but also between different experiments. This can be clearly seen by comparing Figures 3 and 4. The main difference between experiments is the number of different targets, and therefore the total number of signals detected. Therefore, the number of transcripts detected, as well as the number and density of signals, do not hinder the method's ability to accurately quantify the number of transcripts. The very good correlation furthermore has a very high number of signals, confirming the specificity and stability of the method.

[0180] Comparison of intercellular distribution of signals In Figure 10, maximum projections of image stacks are shown: A: Region 1 of experiment 7 (single round, single transcript experiment detecting SPOCK1), B: 2D projection of all selected signals from experiment 1, region 1 assigned to SPOCK1, C: Region 1 of experiment 8 (single round, single transcript experiment detecting THRAP3), D: 2D projection of all selected signals from experiment 1, region 1 assigned to THRAP3.

[0181] Comparison of intracellular distribution of signals In Figure 11, maximum projections of image stacks are shown. Enlarged sub-regions of the corresponding regions are shown. A: Region 1 of experiment 8 (single round, single transcript experiment detecting THRAP3), B: 2D projections of selected signals from experiment 1, region 1 assigned to THRAP3, C: Region 1 of experiment 5 (single round, single transcript experiment detecting DDX5), D: 2D projections of all selected signals from experiment 1, region 1 assigned to DDX5.

[0182] Figure 10 shows the large differences in cell-to-cell distribution between the different transcripts. SPOCK1 appears highly abundant in some cells, but is nearly absent in others (Figure 10A). THRAP3 shows a more uniform distribution across all cells in the region (Figure 10C). These spatial distribution patterns are also clearly observable in the point clouds assigned to the corresponding transcripts from experiment 1 (Figures 10B and D).

[0183] Figure 11 shows the large differences in subcellular distribution between the different transcripts. THRAP3 is mainly observed in the periphery of the cell (cytoplasm) (Figure 11A), whereas DDX5 shows a higher abundance in the center of the cell (nucleus) (Figure 11C). These subcellular distributions can also be observed in the point clouds of experiment 1 assigned to THRAP3 and DDX5 (Figures 11B and D).

[0184] conclusion In addition to the reliability of the quantification, point clouds from multiple-round experiments also show the same intra- and intercellular distribution pattern of transcripts, as clearly demonstrated by direct comparison of assigned point clouds with signals from single-round experiments that detected only one distinctive mRNA species.

[0185] Distribution patterns of distinct cell cycle-dependent transcripts All images in Figure 12 show region 1 of experiment 1. In each image, a cloud of points is shown, which is assigned to a specific transcript: A: CCNA2, B: CENPE, C: CCNE1, D: all transcripts. Figure 12 shows the transcripts of three different cell cycle-dependent proteins. CENPE (Figure 12B), also known as centromere protein E, is deposited during G2 phase. It has been proposed to be involved in spindle elongation and chromosome movement. It is absent during interphase. CCNA2 (Figure 12A), also known as cyclin A2, regulates cell cycle progression by interacting with CDK1 during the G2 to M phase transition. Interestingly, there is a clear colocalization of both mRNA species. They are mainly present in the three central cells of region 1. CCNE1 (Figure 12C), also known as cyclin E1, interacts with CDK2 and is involved in the G1 to S phase transition. Figure 12 clearly shows that the transcript of this gene is absent in the three central cells, but is distributed quite evenly across the other cells. It therefore shows anti-localization with respect to the other two transcripts. The corresponding point cloud data is derived from a point cloud with a very high number of points and a very high point density (Figure 12D gives an impression).

[0186] conclusion The three decoded point clusters of cell cycle-dependent proteins shown in Figure 12 show distribution patterns that can be explained by their corresponding functions. These data strongly suggest that our method reliably generates biologically relevant data, with even fewer signals per cell (Figure 12C) and extremely high signal density (Figure 12D).

[0187] Sequence Listing SEQ ID NOs: 1-1247 in the attached sequence listing refer to the nucleotide sequences of exemplary target-specific oligonucleotides. The listed oligonucleotides consist of a target-specific binding site (at the 5' end), a spacer / linker sequence (gtaac or tagac), and a unique identifier sequence, which is the same for all oligonucleotides of a probe set.

[0188] SEQ ID NOs:1248-1397 in the attached sequence listing refer to the nucleotide sequences of exemplary decoding oligonucleotides.

[0189] SEQ ID NOs: 1398-1400 in the attached sequence listing refer to the nucleotide sequences of exemplary signal oligonucleotides. For each signal oligonucleotide, the corresponding fluorophore is present at two locations. One fluorophore is covalently attached to the 5' end and one fluorophore is covalently attached to the 3' end. SEQ ID NO: 1398 contains "5Alex488N" at its 5' end and "3AlexF488N" at its 3' end. SEQ ID NO: 1399 contains "5Alex546" at its 5' end and "3AlexF546N" at its 3' end. SEQ ID NO: 1400 contains "Atto594" at its 5' end and at its 3' end.

Claims

1. i) inactivation of pathogens within a sample without isolation of RNA and / or DNA from the pathogen or sample; ii) detecting analytes by spatial transcriptomics; 1. A method for detecting an analyte in a pathogen-containing sample, comprising: the pathogen-containing sample is selected from the group consisting of a solid, a fluid sample, a smear, and a surface containing a pathogenic material; the pathogen-containing sample is preferably a biological sample comprising biological tissue, more preferably comprising extracts and / or parts of biological cells and / or pathogen-containing cells; Detecting said spatial transcriptomics in step ii) comprises a multiplexing method for detecting different analytes in a pathogen-containing sample by sequential signal encoding of the analytes, comprising the following steps: (A) contacting the sample with at least twenty (20) different sets of analyte-specific probes for encoding at least twenty (20) different analytes, each set of analyte-specific probes interacting with a different analyte, and if the analytes are nucleic acids, each set of analyte-specific probes comprising at least five (5) analyte-specific probes that specifically interact with different substructures of the same analyte, each analyte-specific probe comprising: (aa) a binding element (S) that specifically interacts with one of the different encoded analytes; and (bb) an identifier element (T) comprising a nucleotide sequence unique to the encoded analyte (the unique identifier sequence); wherein the analyte-specific probes of a particular set of analyte-specific probes are different from the analyte-specific probes of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T); contacting, wherein the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and contain the same nucleotide sequence of an identifier element (T) unique to the analyte; and (B) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein each decoding oligonucleotide in each set of decoding oligonucleotides for a distinct analyte is (aa) an identifier connector element (t) comprising a nucleotide sequence substantially complementary to at least a portion of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set; and (bb) a translation element (c) comprising a nucleotide sequence allowing specific hybridization of a signal oligonucleotide; wherein the set of decoding oligonucleotides for a distinct analyte is different from another set of decoding oligonucleotides for a different analyte in the first connector element (t); and (C) contacting the sample with at least one set of signal oligonucleotides, each signal oligonucleotide comprising: (aa) a translation connector element (C) comprising a nucleotide sequence substantially complementary to at least a portion of the nucleotide sequence of the translation element (c) contained in the decoding oligonucleotide; and (bb) a signal element; contacting, including (D) detecting a signal resulting from the signal element; (E) selectively removing the decoding oligonucleotides and the signal oligonucleotides from the sample, thereby substantially maintaining specific binding of the analyte-specific probes to the encoded analytes; (F) performing at least three additional cycles comprising steps B) through E) to generate a coding scheme comprising a code word for each analyte, wherein the final cycle may stop at step (D). Including, A method wherein the code word for each analyte in the cycle performed additionally includes at least one element corresponding to no detected signal (code word zero (0)).

2. The method of claim 1 , wherein the pathogen-containing sample comprises eukaryotes, archaea, prokaryotes, and / or viruses.

3. 10. The method of claim 1, wherein the pathogen-containing sample comprises formalin-fixed, paraffin-embedded tissue containing the pathogen and / or the pathogen-containing sample is frozen or alcohol-stabilized.

4. 10. The method of claim 1, wherein the pathogen is inactivated by physical, chemical, biochemical and / or biological treatment.

5. the physical inactivation treatment is selected from the group consisting of temperature change, irradiation with electromagnetic waves and light, including visible light and invisible light such as UV; 5. The method of claim 4, wherein the temperature change treatment is selected from treatment at temperatures above 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 100°C or 110°C.

6. The method described in claim 4, wherein the treatment with electromagnetic waves is selected from treatment with X-rays, radioactivity, UV light, blue light, red light and / or infrared light, or a combination thereof.

7. 5. The method of claim 4, wherein the pathogen is inactivated by a chemical inactivation treatment selected from the group consisting of a pH change, a salt treatment, treatment with small amounts of polar or non-polar solvents, treatment with an oxidizing or reducing agent, treatment with an agent that binds covalently or non-covalently to the pathogen and treatment with a degrading agent that cleaves at least a portion of the pathogen, or a combination thereof.

8. The method according to claim 4, wherein the biochemical inactivation treatment is a treatment with an enzyme and / or a biomolecule.

9. 2. The method according to claim 1, wherein all steps are automated, in particular steps B) to F) are automated, in particular by using a robotic system.

10. 2. The method of claim 1, wherein the code words obtained for individual analytes in a performed cycle include at least one element corresponding to a detected signal and additionally to no detected signal.

11. 10. The method of claim 1, wherein no signal is detected for at least one analyte within at least one cycle.

12. 10. The method of claim 1, wherein the code word position for at least one distinct analyte is zero (0).

13. 2. The method of claim 1, wherein the code word zero (0) is generated without using a decoding oligonucleotide having an identifier connector element (t) comprising a nucleotide sequence substantially complementary to at least a portion of the unique identifier sequence of an identifier element (T) of a corresponding analyte-specific probe for an individual analyte.

14. 2. The method of claim 1, wherein if the code word position for at least one individual analyte is zero (0) in this cycle, then the corresponding decoding oligonucleotide having an identifier connector element (t) comprising a nucleotide sequence substantially complementary to at least a portion of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe for that individual analyte is not used.

15. the sample is contacted with at least two different sets of decoding oligonucleotides for each analyte; The decoding oligonucleotides included in these different sets contain the same identifier connector element (t) that contains a nucleotide sequence that is substantially complementary to at least a portion of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set; and The different sets of decoding oligonucleotides for each analyte differ in a translation element (c) comprising a nucleotide sequence that allows specific hybridization of the signal oligonucleotide; At least one set of non-signal decoding oligonucleotides is provided for binding to a particular identifier element (T) of the analyte-specific probe, and decoding oligonucleotides in the same non-signal decoding oligonucleotide set interact with the same different identifier element (T); each non-signal-decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence substantially complementary to at least a portion of the unique identifier sequence, and does not comprise a translation element (c) comprising a nucleotide sequence that allows specific hybridization of the signal oligonucleotide; The method of claim 1.

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