Encoding and decoding of signals in multiplexed biochemical assays

Abstract

The application relates to the coding and decoding of signals in multiplexed biochemical assays. The disclosure provides methods, systems, compositions and test kits for the multiplexed detection of multiple analytes in samples. In some instances, the disclosure provides methods, systems, compositions and test kits, wherein, multiple analytes can be detected in a single sample volume by obtaining a cumulative measurement of at least one quantifiable component of a signal or multiple cumulative measurements. In some cases, the extra component of a signal or an extra signal (or its component) is also quantitatively measured. Each signal or each component of a signal can be used to construct a coding scheme, and then the coding scheme can be used to determine the presence or absence of any analyte.

Description

[0001] This application is a divisional application of an application with an application date of February 1, 2013, application number 201710674587.8, and invention name “Encoding and decoding of signals in multiplexed biochemical assays”.

[0002] The application with the application date of February 1, 2013, the application number of 201710674587.8, and the invention name of “Encoding and decoding of signals in multiplexed biochemical assays” is a divisional application of the application with the application date of February 1, 2013, the application number of 201380018760.9, and the invention name of “Encoding and decoding of signals in multiplexed biochemical assays”.

[0003] Cross-references

[0004] This application claims the benefit of U.S. Provisional Application No. 61 / 594,480, filed February 3, 2012, and U.S. Provisional Application No. 61 / 703,093, filed September 19, 2012, each of which is incorporated herein by reference in its entirety.

[0005] Sequence Listing

[0006] This instant application contains a sequence listing submitted in ASCII format via the EFS-webpage, and is incorporated herein by reference in its entirety. The ASCII copy, created on February 14, 2013, is named 38075-716.601_SL.txt and is 9,240 bytes in size. Technical Field

[0007] The present invention relates to encoding and decoding of signals in multiplexed biochemical assays. Background Art

[0008] Multiplexed reactions offer significant advantages over traditional single-channel reactions, including the ability to perform multiple reactions in parallel on the same sample, perform more reactions using the same chamber, and extract rich information from samples in a rapid and efficient manner. However, to achieve these advantages, multiplexed assays typically require complex reporting mechanisms, i.e., spectrally resolvable fluorescence or chemiluminescence (e.g., PCR, ELISA), spatially resolvable signals (e.g., microarrays, gel electrophoresis), temporally resolvable signals (e.g., capillary electrophoresis), or a combination thereof (e.g., Sanger sequencing). Summary of the invention

[0009] There is a need for multiplex reactions that can be performed in a single solution.

[0010] The present disclosure provides methods, compositions, systems and kits for multiplexed detection of analytes. In some cases, the present disclosure provides assays that can unambiguously detect the presence or absence of each of at least 7 analytes in a single sample volume in any combination of presence or absence without performing immobilization, separation, mass spectrometry, or melting curve analysis. In some instances, each analyte is encoded as the value of one (or at least one) component of a signal.

[0011] In some examples, the assays provided by the present disclosure are capable of specifically detecting the presence or absence of M analytes in any combination of presence or absence, where M = log2(F+1), and F is the maximum cumulative value of one component of the signal (e.g., intensity) when all analytes are present.

[0012] In some cases, each analyte is encoded as at least one first value in a first component of the signal (e.g., at least one intensity or range of intensities) and at least one second value in a second component of the signal (e.g., at least one wavelength or range of wavelengths). In some instances, each analyte is encoded as a first value in the first component of the signal for each second value in a plurality of second values ​​in the second component of the signal (e.g., a signal intensity or range of signal intensities at each wavelength or each wavelength range in a plurality of wavelengths or a plurality of wavelength ranges).

[0013] In some instances, the assays provided herein are capable of specifically detecting the presence or absence of M analytes in any combination of presence or absence, where M = C*log2(F+1), where C is the number of second values ​​used to encode the analytes and F is the maximum cumulative value of the first component of the signal for any second value when all analytes are present.

[0014] In some cases, the present disclosure provides assays capable of unambiguously detecting the presence or absence of M analytes in any combination of presence or absence, where M = (P*T) + 1, where P is the number of codes per layer in the coding scheme, and T is the number of layers. In some cases, the number of layers T = log4(F+1), and F is the maximum cumulative value of the first component of the signal for any second value when all analytes are present.

[0015] In some examples, the first value is an intensity or an intensity range. In some cases, the encoding scheme includes at least three intensities or intensity ranges.

[0016] In some cases, the second value is a wavelength or a range of wavelengths. In some examples, the encoding scheme includes at least five wavelengths or ranges of wavelengths.

[0017] In some embodiments, the signal is an electromagnetic signal. In some cases, the electromagnetic signal is a fluorescence emission signal. In some embodiments, the intensity of the fluorescence emission signal at at least four wavelengths or wavelength ranges is measured.

[0018] In some cases, the assays provided herein are performed using reagents that are lyophilized prior to use.

[0019] In some cases, the detection step is performed using a reagent comprising a hybridization probe. In some instances, the number of hybridization probes is greater than the number of analytes. In some cases, the hybridization probe group includes one or more hybridization probes specifically for different analytes, and includes the same fluorophore or a combination of fluorophores. In some instances, the sample is contacted with at least 18 of the hybridization probes. In some instances, the detection step is performed using a reagent comprising at least one pair of primers. In some cases, at least one pair of primers can amplify a region complementary to at least three of the hybridization probes.

[0020] In some instances, the measured signal is generated during a polymerase chain reaction. In some cases, the polymerase chain reaction is selected from the group consisting of an end-point polymerase chain reaction, a real-time polymerase chain reaction, a digital polymerase chain reaction, and combinations thereof.

[0021] In some examples, at least one cumulative measurement is performed on the solution.

[0022] In some examples, at least one analyte is encoded by at least one additional value (i.e., at least two values ​​together), wherein the at least one additional value is selected from the group consisting of: a value of at least one additional component from a signal, a value of at least one component from a different signal, and combinations thereof. For example, the at least one additional value may be selected from the group consisting of: fluorescence emission intensity, fluorescence emission wavelength, Resonance energy transfer (FRET) emission intensity, FRET emission wavelength, electrochemical signal, chemiluminescence wavelength, chemiluminescence intensity, fluorescence bleaching rate, chemiluminescence bleaching rate, and combinations thereof.

[0023] In some cases, a chromatogram is constructed. The chromatogram can be constructed by plotting all possible combinations of first values ​​and second values ​​for positive control samples for each analyte.

[0024] In some instances, at least one analyte comprises a polynucleotide. In some cases, the polynucleotide is from a source selected from the group consisting of: animals, plants, bacteria, fungi, parasites, and viruses. In some instances, the polynucleotide is from a source selected from the group consisting of: human immunodeficiency virus, herpes simplex virus, human papillomavirus, malaria parasite, mycobacterium, dengue virus, hepatitis virus, and influenza virus. In some cases, the polynucleotide is selected from the group consisting of: human immunodeficiency virus polyprotease, human immunodeficiency virus p17, human papillomavirus E6, and human papillomavirus E7.

[0025] In some cases, the sample is selected from the group consisting of: a clinical sample, a food sample, an environmental sample, a pharmaceutical sample, and a sample from a consumer product.

[0026] In some examples, information regarding the presence or absence of an analyte is transmitted via a computer network.

[0027] In some cases, information about the presence or absence of an analyte is provided to a physician. In some instances, clinical decisions are made based on such information.

[0028] In some instances, at least one step of the method provided herein is performed using instructions on a computer-readable medium. In some cases, the instructions are located on a remote server. In some instances, the instructions are located on a thermal cycler. In some cases, the instructions are located on a computer that communicates with a thermal cycler.

[0029] In some cases, at least one analyte is a positive control analyte.

[0030] In some instances, the coding scheme is non-degenerate. In some cases, the coding scheme is designed to be non-degenerate. In some instances, the coding scheme is made non-degenerate by enumerating every reasonable result that can be obtained from the coding scheme, identifying every reasonable result that is degenerate, and eliminating at least one potential analyte code from the coding scheme to eliminate degeneracy.

[0031] In some cases, the assays provided herein are endpoint assays. In some instances, the assays provided by the present disclosure end at a threshold number of cycles set by the detection limit of the instrument.

[0032] In some examples, the assays provided herein are liquid phase assays.

[0033] In some cases, the assays provided herein are quantitative.

[0034] In some cases, the present disclosure provides a method for detecting the presence or absence of each analyte in a plurality of analytes, the method comprising: (a) encoding each of the analytes as a first value of a signal, thereby generating a coding scheme, wherein each of the analytes is represented by the first value in the coding scheme, wherein the encoding is performed in a manner that eliminates degeneracy; (b) providing a sample that includes or may include at least one of the analytes; (c) contacting the sample with an analyte-specific reagent that produces the first value when each of the analytes is present as represented in the coding scheme; (d) cumulatively measuring the first value of the signal in the sample, thereby providing a cumulative measurement result; and (e) determining whether each of the analytes is present or absent based on the cumulative measurement result and the coding scheme.

[0035] In some instances, the method described in the previous paragraph is capable of unambiguously detecting the presence or absence of M analytes in any combination of presence or absence, where M=log2(F+1), and F is the maximum cumulative value of the first value when all analytes are present. In some cases, the first value is an intensity or intensity range. In some instances, the first value has a minimum value in the coding scheme, and the minimum value is selected from the group consisting of: at least 1, at least 2, at least 4, at least 8, at least 16, at least 32, and at least 64. In some cases, the first value is increased in the coding scheme by an amount equal to the cumulative maximum value plus one of the previous first value. In other cases, the first value is increased in the coding scheme by an amount greater than the cumulative maximum value plus one of the previous first value.

[0036] In some cases, the present disclosure provides a method for detecting the presence or absence of each analyte in a plurality of analytes, the method comprising: (a) encoding each of the analytes with at least one first value and at least one second value, wherein the first value is a value from a first component of a signal and the second value is a value from a second component of the signal, thereby generating a coding scheme, wherein each of the analytes is represented in the coding scheme by the at least one first value and the at least one second value, wherein the coding is performed in a manner that reduces or eliminates degeneracy; (b) providing a sample that includes or may include at least one of the analytes; (c) contacting the sample with an analyte-specific reagent represented in the coding scheme that produces the at least one first value and at least one second value when each of the analytes is present; (d) cumulatively measuring the first value and the second value in the sample, thereby providing a cumulative measurement result; and (e) determining whether each of the analytes is present or absent based on the cumulative measurement result and the coding scheme, wherein when degeneracy is eliminated, when each of the reagents produces only one second value, the method is capable of unambiguously detecting the presence or absence of each of at least six analytes in a single volume in any combination of presence or absence.

[0037] In some cases, each analyte in the method described in the previous paragraph is encoded as a first value in a first component of the signal for each second value in a plurality of second values ​​in a second component of the signal. In some cases, when degeneracy is eliminated, the method is capable of unambiguously detecting the presence or absence of each of at least 7 analytes in a single volume using 4 second values. In some instances, when degeneracy is eliminated, the encoding scheme includes T non-degenerate layers, where T=log4(F+1), and F is the maximum cumulative value of the first component of the signal for any second value when all analytes are present. In some cases, when degeneracy is eliminated, the method is capable of unambiguously detecting the presence or absence of M analytes in any combination of presence or absence, where M=(P*T)+1, and P is the number of codes per layer. In some instances, when degeneracy is eliminated, the method is able to unambiguously detect the presence or absence of M analytes in any combination of presence or absence, where M = C*log2(F+1), C is the number of second values ​​in the coding scheme, and F is the maximum cumulative value of the first component of the signal for any second value when all analytes are present.

[0038] The present disclosure also provides a non-degenerate encoding scheme that is capable of unambiguously encoding the presence or absence of each of at least 7 analytes in any combination of presence or absence in a single sample volume without performing immobilization, separation, mass spectrometry, or melting curve analysis.

[0039] In some instances, the non-degenerate coding scheme of the present disclosure is generated by a method comprising the following steps: (a) generating a code for each potential analyte, wherein each potential analyte is encoded by at least one value of at least one component of the signal; (b) enumerating each reasonable cumulative result for all possible combinations of each analyte present or absent; (c) identifying each reasonable degenerate result; and (d) eliminating at least one code to eliminate degeneracy. In some cases, the coding scheme can be expanded by adding additional codes, the additional codes being at least one unit larger than the sum of all previous codes in at least one component of the signal. In some cases, the coding scheme is generated by a mathematical iterative method that is structurally guaranteed to be non-degenerate.

[0040] In some cases, the present disclosure provides a system for detecting the presence or absence of each analyte in a plurality of analytes, the system comprising: (a) encoding each of the analytes as a first value of a signal, thereby generating a coding scheme, wherein each of the analytes is represented by the first value in the coding scheme, wherein the encoding is performed in a manner that eliminates degeneracy; (b) providing a sample that includes or may include at least one of the analytes; (c) contacting the sample with an analyte-specific reagent that produces the first value when each of the analytes is present, as represented in the coding scheme; (d) cumulatively measuring the first value of the signal in the sample, thereby providing a cumulative measurement result; and (e) determining whether each of the analytes is present or absent based on the cumulative measurement result and the coding scheme.

[0041] In some cases, the present disclosure provides a system for detecting the presence or absence of each analyte in a plurality of analytes, the system comprising: (a) encoding each of the analytes with at least one first value and at least one second value, wherein the first value is a value from a first component of a signal and the second value is a value from a second component of the signal, thereby generating a coding scheme, wherein each of the analytes is represented in the coding scheme by the at least one first value and the at least one second value, wherein the coding is performed in a manner that reduces or eliminates degeneracy; (b) providing a sample that includes or may include at least one of the analytes; (c) subjecting the sample to a sample containing at least one of the analytes; and (d) cumulatively measuring said first values ​​and said second values ​​in said sample to provide a cumulative measurement result; and (e) determining whether each of said analytes is present or absent based on said cumulative measurement result and said coding scheme, wherein, when degeneracy is eliminated, when each of said reagents produces only one second value, said method is capable of unambiguously detecting the presence or absence of each of at least 6 analytes in a single volume in any combination of presence or absence.

[0042] In some cases, the present disclosure provides a method for detecting the presence or absence of each analyte in a plurality of analytes for a third group, the method comprising: (a) obtaining an identity of each analyte from the group; (b) encoding each analyte as a first value of a signal, thereby generating a coding scheme, wherein each analyte is represented by the first value in the coding scheme, wherein the coding is performed in a manner that eliminates degeneracy; (c) providing to the group an analyte-specific reagent that produces the first value when each analyte is present as represented in the coding scheme, the group: (i) contacting a sample that includes or may include at least one analyte with the reagent; and (ii) cumulatively measuring the first value in the sample, thereby providing a cumulative measurement result; and (d) obtaining the cumulative measurement result from the group; (e) determining whether each analyte is present or absent based on the cumulative measurement result and the coding scheme; and (f) providing information about the presence or absence of each analyte to the group.

[0043] In some cases, the present disclosure provides a method for detecting the presence or absence of each analyte in a plurality of analytes for a third group, the method comprising: (a) obtaining an identity of each analyte from the group; (b) encoding each analyte into at least one first value and at least one second value, wherein the first value is a value from a first component of a signal and the second value is a value from a second component of the signal, thereby generating a coding scheme, wherein each analyte is represented by the at least one first value and the at least one second value in the coding scheme, wherein the coding is performed in a manner that reduces or eliminates degeneracy; (c) providing the analyte with a first value and a second value; and (d) providing the analyte with a second value. A group provides an analyte-specific reagent that generates at least one first value and at least one second value when each analyte is present as represented in the coding scheme, and the group: (i) contacts a sample that includes or may include at least one analyte with the reagent; and (ii) cumulatively measures the first value and the second value in the sample to provide a cumulative measurement result; and (d) obtains the cumulative measurement result from the group; (e) determines whether each analyte is present or absent based on the cumulative measurement result and the coding scheme; and (f) provides information about the presence or absence of each analyte to the group.

[0044] In some cases, the present disclosure provides a composition for detecting the presence or absence of each analyte in a plurality of analytes, the composition comprising analyte-specific reagents, each reagent producing a signal comprising a first value, wherein the reagents are capable of unambiguously detecting the presence or absence of each analyte of at least 7 analytes in any combination of presence or absence in a single sample volume without performing immobilization, separation, mass spectrometry, or melting curve analysis.

[0045] In some cases, the present disclosure provides a composition for detecting the presence or absence of each analyte in a plurality of analytes, the composition comprising analyte-specific reagents, each reagent producing a signal comprising at least one first value and at least one second value, the at least one first value being a value from a first component of the signal and the at least one second value being a value from a second component of the signal, wherein, when each of the reagents produces only one second value, the reagents are capable of detecting the presence or absence of each analyte of at least 6 analytes in a single volume in any combination of presence or absence.

[0046] In some cases, the present disclosure provides a kit for detecting the presence or absence of each analyte in a plurality of analytes, the kit comprising analyte-specific reagents, packaging, and instructions, each reagent producing a signal comprising a first value, wherein the kit is capable of unambiguously detecting the presence or absence of each analyte of at least 7 analytes in any combination of presence or absence in a single sample volume without performing immobilization, separation, mass spectrometry, or melting curve analysis.

[0047] In some examples, the present disclosure provides a kit for detecting the presence or absence of each analyte in a plurality of analytes, the kit comprising analyte-specific reagents, packaging, and instructions, each reagent producing a signal comprising at least one first value and at least one second value, the at least one first value being a value from a first component of the signal and the at least one second value being a value from a second component of the signal, wherein, when each of the reagents produces only one second value, the kit is capable of detecting the presence or absence of each of at least 6 analytes in any combination of presence or absence in a single volume.

[0048] In some cases, the present disclosure provides a kit for detecting the presence or absence of each analyte in a plurality of analytes, the kit comprising a box body 601, a snap-on slot arranged in the box body for placing a bottle, a bottle 602 comprising a probe for detecting the analyte, a bottle 603 comprising a primer for amplification, and a bottle 604 comprising a reagent for amplification, wherein the kit is capable of unambiguously detecting the presence or absence of each of at least 7 analytes in any combination of presence or absence in a single sample volume without performing immobilization, separation, mass spectrometry, or melting curve analysis.

[0049] In some examples, the present disclosure provides a kit for detecting the presence or absence of each analyte in a plurality of analytes, comprising: a box body 601, a snap-on slot arranged in the box body for placing a bottle, a bottle 602 comprising a probe for detecting the analyte, a bottle 603 comprising a primer for amplification, and a bottle 604 comprising a reagent for amplification, each probe generating a signal comprising at least one first value and at least one second value, the at least one first value being a value from a first component of the signal and the at least one second value being a value from a second component of the signal, wherein, when each of the probes generates only one second value, the kit is capable of detecting the presence or absence of each of at least 6 analytes in a single volume in any combination of presence or absence.

[0050] Incorporation by Reference

[0051] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description of illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings in which:

[0053] Figure 1 A comparison between a conventional encoding method for detecting four analytes in four colors and an encoding method of the present invention capable of detecting 16 or more sequences in four colors is shown. Colors are indicated by B, G, Y and R, which indicate blue, green, yellow and red, respectively.

[0054] Figure 2 Shown is a schematic diagram representing the detection of 6 analytes (including control) in four colors. Analytes are detected using hybridization probes connected to fluorophores (B, G, Y, R; blue, green, yellow and red, respectively) and quenchers (ovals marked with "X"). Control sequences and five other sequences (dengue = dengue virus; tuberculosis = Mycobacterium tuberculosis; P17 = HIV p17; malaria = Plasmodium falciparum; and herpes = herpes simplex virus 2) are all detected using probes marked with blue fluorophores. Each non-control analyte is detected using 1-3 additional probes, respectively. For example, the analyte of dengue virus is detected using three additional probes with green, yellow and red fluorophores; the analyte of herpes simplex virus 2 is detected using an additional probe with a red fluorophore; and so on.

[0055] Figure 3 Chromatograms showing the experimental results obtained as described in Example 2.

[0056] Figure 4 Exemplary embodiments of the invention are shown in which one or more steps of the methods provided herein are performed using a computer.

[0057] Figure 5 Schematic diagram showing two different methods of detecting the wavelength and intensity of an optical signal, such as a fluorescent emission signal.

[0058] Figure 6 A schematic diagram of an exemplary kit is shown.

[0059] Figure 7Chromatograms showing the experimental results obtained as described in Example 3.

[0060] Figure 8 Schematic diagram showing reasonable results (top) and unreasonable results (bottom). DETAILED DESCRIPTION

[0061] Fluorescence detection has been the preferred technique for multiplexed assays due to several desirable properties, including compatibility with biochemical assays, relatively small size of fluorescent labels, simple means of conjugating molecules of interest, affordability, low toxicity, stability, robustness, detectability using inexpensive optical systems, and the ability to be combined with spatial arrays. However, standard fluorescence has a wide range of emission spectra. Therefore, to avoid spectral overlap (i.e., to maintain spectral resolution) only a relatively small number of colors (e.g., 4 to 6) are typically used simultaneously in multiplexed fluorescence assays.

[0062] The traditional encoding method for multiplexed fluorescence assays has been to encode each analyte with a single color; that is, M=N, where M is the number of detectable analytes and N is the number of spectrally resolvable fluorescent probes. Whenever a higher multiplexing factor is required (i.e., M>N), fluorescence is often combined with other technical techniques, such as aliquoting, spatial arrays, or sequential processing. These additional processing steps are labor intensive and often require relatively expensive and complex optical and mechanical systems (e.g., spectrometers, mechanized microscope stages, microfluidics, droplet generators, scanners, and similar systems). Such systems are often impractical to configure in certain environments, particularly point-of-care and low-resource settings. Therefore, there is a significant need for multiplexed encoding and decoding methods that can provide inexpensive multiplexing while avoiding the use of expensive additional processing steps.

[0063] The present disclosure provides methods, systems, compositions and kits for the detection of multiple analytes in samples. Analytes are detected based on coding, analysis and decoding methods. In some instances, each analyte to be detected is encoded as a value of a signal (e.g., intensity), wherein the value is assigned so that the result of the determination clearly indicates the presence or absence of the analyte to be determined. In other instances, each analyte to be detected is encoded as a value in each of at least two components of the signal (e.g., intensity and wavelength). At least two components of the signal can be orthogonal. Similarly, as described more comprehensively elsewhere in the present disclosure, a plurality of orthogonal signals can be used, such as a combination of a fluorescent signal and an electrochemical signal. Analyte can be any suitable analyte, such as a polynucleotide, a protein, a small molecule, a lipid, a carbohydrate, or a mixture thereof. Signal can be any suitable signal, such as an electromagnetic signal, an optical signal, a fluorescent emission signal, an electrochemical signal, a chemiluminescent signal, and a combination thereof. At least two components of the signal can be any suitable two components, such as amplitude and frequency, or intensity and wavelength.

[0064] After encoding the analyte, a sample is provided, wherein the sample includes or may include at least one of the encoded analytes. The sample is contacted with one or more analyte-specific reagents that generate a specific signal when the analyte is present, as specified in the encoding scheme for each analyte. The reagent can be any suitable reagent that is capable of generating such a signal when its corresponding analyte is present, for example, an oligonucleotide probe (e.g., TAQMAN probe) connected to a fluorophore and a quencher. If the reagent is an oligonucleotide probe connected to a fluorophore and a quencher, a nucleic acid amplification that generates a signal can be performed.

[0065] After adding the reagent, the signal is quantified. In some cases, this quantification is performed by measuring a component of the signal (e.g., fluorescence intensity) and determining the presence and absence of certain analytes based on the value used to encode the presence of each analyte and the cumulative value of the signal.

[0066] In some cases, at least two components of the signal (e.g., intensity and wavelength) are cumulatively measured for the sample. For example, such measurements can be performed by measuring the intensity at a specific wavelength, or the intensity within a specific wavelength range. The presence or absence of the analyte can then be determined based on the value of each of the at least two components of the signal and the value used to encode the presence of the analyte (i.e., those values ​​in the encoding scheme).

[0067] Coding can be performed in a manner that reduces or eliminates the number of possible degenerate (e.g., ambiguous) results obtained by the method. As described elsewhere in this specification, the complete coding capabilities of a specific coding scheme can be enumerated, and some potential analyte codes can be eliminated from the coding scheme to reduce or eliminate degeneracy. Similarly, the coding scheme can be designed to be non-degenerate, so that the reduction or elimination of degeneracy is not necessary. A decoding matrix can be constructed to correspond to the constituent signals of the cumulative signal measurement results, and the cumulative signal measurement results (e.g., intensity or intensity at a specific wavelength) are interpreted as certain analytes are present or absent.

[0068] I. Definitions

[0069] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0070] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, with respect to the scope of the terms "including", "includes", "having", "has", "with", "such as", or variations thereof, when used in the specification and / or claims, these terms are not limiting and are intended to be included in a manner similar to the term "comprising".

[0071] As used herein, the term "about" generally refers to a range of 15% plus or minus 15% from a numerical value specified in the context of a particular usage. For example, "about 10" would include a range from 8.5 to 11.5.

[0072] As used herein, the terms "dimension" and "component", when referring to a signal, generally refer to aspects of the signal that can be quantified. For example, if the signal is generated by a fluorescent molecule, it can be quantified based on its wavelength (e.g., the first dimension or first component) and its intensity (e.g., the second dimension or second component).

[0073] The term "decoding," as used herein, generally refers to a method of determining which analytes are present based on a cumulative signal and an encoding scheme or decoding matrix capable of converting the cumulative signal into information regarding the presence or absence of one or more analytes.

[0074] The term "encoding", as used herein, generally refers to the process of representing an analyte using a code comprising a value of a signal (eg, intensity), or a value of each of at least two components of a signal or signals (eg, wavelength and intensity).

[0075] The terms "oligonucleotide probes connected to fluorophores and quenchers" and "TAQMAN probes" generally refer to hydrolysis probes used to detect the presence of analytes in polynucleotide amplification assays. These probes include oligonucleotide probes connected to fluorophores and quenchers. As long as the quencher and fluorophore are close, the quencher will quench the fluorescence emitted by the fluorophore excited by the light source. The sequence of the oligonucleotide probe is designed to be complementary to the polynucleotide sequence present in the analyte, and therefore, it is capable of hybridizing with the polynucleotide sequence present in the analyte. The hybridization of the oligonucleotide probe is performed in a nucleic acid amplification reaction (e.g., polymerase chain reaction) including primers. After the primer is extended by DNA polymerase, the 5' to 3' exonuclease activity of the polymerase degrades the probe, releasing the fluorophore and quencher into the medium. The proximity between the fluorophore and the quencher is destroyed, and the signal from the fluorophore is no longer quenched. Therefore, the amount of fluorescence detected is a function of the amount of analyte present. If no analyte is present, the probe will not hybridize with the analyte, and the fluorophore and quencher will remain in close proximity. Little or no signal will be generated.

[0076] The term "orthogonal" as used herein generally refers to at least two components of a signal that can be changed independently or approximately independently (e.g., wavelength and intensity), or at least two different signals (e.g., a fluorescence emission signal and an electrochemical signal). For example, when using fluorescent molecules, wavelength and intensity are considered to be orthogonal or approximately orthogonal. Among other factors, the wavelength of the fluorescence emission will depend on the composition of the fluorescent molecules, and the intensity of the fluorescence will depend on the amount of the molecules present. Although wavelength and intensity are examples of two components of a signal that can be changed approximately independently, the methods described herein are not limited to components that can be changed independently or approximately independently. Components of signals that are not changed independently, or multiple signals, can also be used, as long as the characteristics of the components or signals are determined well enough to be able to encode, measure and decode. For example, if a change in a component or signal affects a change in another component or signal, the two components or signals can still be used as long as the relationship between the changes is understood.

[0077] As used herein, the term "polynucleotide", "oligonucleotide", or "nucleic acid" is used herein to refer to a biological molecule comprising a plurality of nucleotides. Exemplary polynucleotides include deoxyribonucleic acid, ribonucleic acid, and synthetic analogs thereof, including peptide nucleic acids.

[0078] As used herein, the term "probe" generally refers to a reagent that can generate a signal in the presence of a specific analyte. The probe generally has at least two parts: a part that can specifically recognize the analyte, or a part thereof; and a part that can generate a signal in the presence of the analyte, or a part thereof. As described above and elsewhere in the present disclosure, the probe can be an oligonucleotide probe connected to a fluorophore and a quencher. The probe can also be any reagent that generates a signal in the presence of an analyte, such as an antibody that detects an analyte, having a fluorescent label that is emitted or quenched due to the binding of the antibody to the analyte. Any suitable probe can be used in the method proposed in the present disclosure, as long as the probe generates a quantifiable signal in the presence of an analyte. For example, the analyte-specific portion of the probe can be coupled to an enzyme that converts an uncharged substrate into a charged product in the presence of an analyte, thereby increasing the conductivity in the medium over time. In this case, different analytes can be encoded in different proportions by coupling the analyte-specific portion of the probe (such as a hybridization probe or antibody) to the enzyme. The rate of generation of increased conductivity in the medium will be accumulated for all analytes present in the medium. According to the methods provided herein, the encoded analyte can convert the conductivity measurement result into an unambiguous (i.e., non-degenerate) result, providing information about the presence or absence of a specific analyte. Similarly, the probe can include an enzyme that produces a chemiluminescent product from a substrate. The amount of chemiluminescence can then be used to encode the presence of a specific analyte.

[0079] II. Encoding and Decoding Methods

[0080] A. Traditional fluorescence encoding and decoding methods

[0081] A common method for determining the presence of analytes is to use four spectrally resolvable fluorescent molecules to indicate the presence or absence of four analytes. Figure 1 An example of this approach is shown on the left hand side of FIG. Figure 1 The left hand side of the diagram shows an encoding scheme where four analytes (sequence 1, sequence 2, sequence 3 and sequence 4) are each encoded by a single color (blue, green, yellow and red, respectively). The colors represent fluorophores attached to oligonucleotide probes that also include a quencher. Figure 1 In the system shown on the left hand side of FIG, there are four different oligonucleotide probes, each comprising a single fluorophore (blue, green, yellow, or red) and a quencher. The presence or absence of the analyte is determined based on the presence or absence of a signal in a particular color.

[0082] Figure 1The left-hand side graph shows the intensity versus color of a hypothetical sample containing two analytes: sequence 1 and sequence 3. The presence of these analytes is determined based on the measurement of the blue signal (corresponding to sequence 1) and the yellow signal (corresponding to sequence 3). The absence of sequence 2 and sequence 4 is indicated by the absence of green and red signals.

[0083] Table 1 shows an interpretation of the binary format of this encoding scheme. Each analyte is encoded as a value in each of the two components of the fluorescence signal: (1) color (also called wavelength; or wavelength range) and (2) intensity (indicated by the numbers in the table). For example, A (e.g., sequence 1) has a color of blue and an intensity of 1; B (e.g., sequence 2) has a color of green and an intensity of 1; and so on. The intensity of the signal within each color range is quantified as described herein, for example, by measuring the intensity of the signal within a specific wavelength range determined by a bandpass filter. A result of 1000 indicates that only analyte A is present; a result of 1100 indicates that analytes A and B are present; and so on.

[0084] Table 1. Traditional encoding of four analytes with four probes, each probe having a single color and one probe per analyte.

[0085]

[0086]

[0087] B. Coding methods using more than one color per analyte

[0088] The conventional methods described above suffer from the fact that they are limited by the number of spectrally resolvable fluorophores. More specifically, the number of detectable analytes is equal to the number of spectrally resolvable fluorophores. Therefore, the number of analytes can only be increased by increasing the number of spectrally resolvable fluorophores.

[0089] The present disclosure provides methods to overcome this limitation. More specifically, in some cases, by utilizing at least two components of the signal during the encoding process, the methods described herein can be used to detect more than one analyte per fluorophore. For example, using the methods provided herein, each fluorophore can detect 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 analytes. In some cases, the methods provided herein can be used to detect at least 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 analytes per fluorophore. In some cases, the methods provided herein can be used to detect 1.5-2, 2-4, 1.5-4, 4-6, 2-6, 6-10 analytes per fluorophore.

[0090] In some cases, the methods provided by the present disclosure may include the use of a control color. The control color may be connected to one or more probes that bind to the positive control analyte and each analyte to be detected in the sample. If the same sequence appears in the positive control analyte and each analyte to be detected, a single control probe may also be used. If the same sequence does not appear in the positive control analyte and each analyte to be detected, different probes may be used, but each probe may still be connected to the control color.

[0091] For example, based on the traditional method described above, a color (e.g., blue) can be used to encode the presence of a control analyte that is always present in the sample. The control analyte can be added to the sample, or it can be inherently present in the sample. The remaining colors (e.g., green, yellow, red) can be used to encode the presence of additional analytes. Table 2 shows an example of such a method.

[0092] Table 2. Four analytes (including controls) are coded in four colors with a maximum of two colors per analyte.

[0093] blue green yellow red Control (p) 1 0 0 0 A 1 0 0 1 B 1 0 1 0 C 1 1 0 0

[0094] In Table 2, the presence of the control group (p) is indicated by a result of 1000. The presence of the control and analyte A is indicated by a result of 2001. The presence of the control and all three other analytes is indicated by a result of 4111. The control color (blue) provides an indication of the normal operation of the assay. The intensity of the blue color reports the number of analytes present in the test sample. Of course, any color can be used for the control color. One of the skilled in the art will recognize that some practical considerations may make it preferred to use a color instead of another color as the control color. For example, if a color is better detected in a specific optical system (or fluorophore system), it may actually be preferred to use the color as the control color.

[0095] The coding scheme shown in Table 2 encodes each analyte using a control color and an additional color (up to 2 probes per analyte). However, as shown below, when up to 4 colors are used per analyte, the number of analytes that can be encoded increases. Table 3 shows an exemplary coding scheme in which each analyte is encoded by up to 4 colors. In the scheme shown in Table 3, the control color (blue) is produced in the presence of the control sequence and each analyte of the other seven analytes (AG). Each of the other seven analytes is encoded by the presence of one to three additional colors. Colors may be included by different probes (e.g., oligonucleotide probes connected to fluorophores and quenchers) or the same probe (which may have multiple fluorophores and multiple quenchers).

[0096] Table 3. Eight analytes (including controls) were coded in four colors with a maximum of four colors per analyte.

[0097]

[0098]

[0099] In Table 3, the presence of control and all other seven analytes is indicated by the result of 8444. Three analytes (A, B and C) are coded by two colors. Three analytes (D, E and F) are coded by three colors, and one analyte (G) is coded by four colors. The presence of control (p) is indicated by the result of 1000. The presence of control and analyte A is indicated by the result of 2001. The presence of control and analyte G is indicated by the result of 2111. These results express the cumulative intensity of signal in each color. For example, result 2111 has a signal intensity of 2X in the blue channel, and result 1000 only has a signal intensity of 1X in the blue channel.

[0100] Using Table 3, according to color (blue, green, yellow, red) and intensity (0-8), each possible cumulative determination result can be enumerated. Table 4 shows that by based on the coding method shown in Table 3, each possible cumulative determination result is enumerated, and the corresponding decoding result of each determination is provided according to the analyte present in the sample, and a "decoding matrix" is generated. By enumerating each possible cumulative determination result based on the coding scheme, and providing the corresponding decoding result of each determination according to the analyte present in the sample (or not present), a similar decoding matrix for any coding scheme described herein can be generated. In some cases, as described below, one or more analytes can be removed from the coding scheme to reduce or eliminate degeneracy.

[0101] Table 4. Decoding matrix for the encoding method shown in Table 3.

[0102]

[0103]

[0104]

[0105] The decoding matrix is ​​provided in Table 4, which was constructed using two assumptions. First, the decoding matrix assumes that the positive control (P) always produces a positive result. Second, the decoding matrix assumes that in each color, no matter which probe the signal may come from, the intensity is added and weighed in the same way as changing the probe concentration. This essentially means that the signal is additive and digital. The conditions on which these assumptions are based can be met by properly preparing the assay. As demonstrated in the examples provided herein, if a fluorescent signal is used, the intensity only needs to be approximately additive and digital.

[0106] Table 4 allows the cumulative measurement of intensity (i.e., signal intensity in each color range) in four ranges of fluorescence wavelength to be converted into the corresponding analyte present in the sample. For example, the result of 4321 indicates the presence of p, C, F and G, and the absence of other analytes. This result is called a "reasonable" result because it appears in the decoding matrix. In contrast, the result of 4000, although it may be measured, does not appear in the decoding matrix. More specifically, the result of 4000 cannot be achieved by adding any combination of the control code and the analyte code from Table 3. This result is called an "irrational" result. An unreasonable result may indicate a measurement failure. Therefore, the control (p) and the decoding matrix provide a means to verify whether the measurement is operating normally.

[0107] Table 4 exhaustively lists any combination of fluorophores that produce 4 resolvable emission spectra (e.g., colors). The term "rank" is used to describe the number of analytes detected including controls. In the example provided above, if the assay is operating normally, the rank is equal to the value of the blue signal. For example, rank 8 indicates that controls and all other seven analytes (AG) exist. Rank 2 indicates that controls and only one analyte exist. The lowest rank is rank 1, which indicates that only controls exist. The number of possibilities at each rank can be enumerated. For example, continuing to refer to the encoding and decoding methods described in Tables 3-4, there are 7 possibilities for rank 2. The number of possibilities at rank 3 can be calculated as a combination of 7 taking 2 or there are 7! / (5!*2!)=21 possibilities. More generally, the number of possibilities is a combination of N taking K, that is, N! / ((NK)!*K!). Similarly, at ranks 4, 5, 6, 7 and 8, the number of possibilities is 35, 35, 21, 7 and 1, respectively. Reference Table 4 shows that the table is consistent with theoretical predictions. Therefore, Table 4 is an exhaustive decoding matrix for the encoding methods provided in Table 3.

[0108] C. Reduce or eliminate degeneracy

[0109] As used herein, the terms "degenerate" and "degeneracy" generally describe situations where a reasonable result is ambiguous because it can indicate more than one possibility depending on the presence or absence of the analyte. For example, referring to Table 4, result 5233 is degenerate because it can be decoded as pADFG or pBDEG. Similarly, result 4222 is degenerate because it can be decoded as any of the following: pCDG, pAFG, pBEG, pDEF. In contrast, result 3110 can only indicate pBC and is therefore non-degenerate.

[0110] The present disclosure provides a method for reducing or eliminating degeneracy, thereby improving the confidence of the analyte detected by it. In one embodiment, the method for eliminating degeneracy includes: (i) encoding each potential analyte to be detected as a value of a signal, and optionally, encoding as a value in each component of at least two components of the signal; (ii) enumerating each reasonable result that can be obtained from the coding scheme; (iii) identifying each reasonable result that is degenerate; and (iv) eliminating at least one potential analyte (or potential analyte code) from the coding scheme, wherein eliminating the at least one potential analyte reduces or eliminates degeneracy. For example, with reference to the coding scheme described in Table 3, and the decoding matrix described in Table 4 (enumerating each reasonable result), eliminating any two of analytes D, E and F eliminates degeneracy. Eliminating any one of analytes D, E and F will not eliminate degeneracy, but will reduce it.

[0111] Continuing to refer to the coding scheme described in Table 3, any two of analytes D, E and F are eliminated from the coding scheme, resulting in a scheme that uses only 4 colors to analyze 6 analytes (including controls) without degeneracy. In contrast, multiplex conventional methods use 4 colors to allow reporting of only 3 analytes and 1 control. Therefore, the number of analytes that can be analyzed by using the method provided herein is nearly doubled.

[0112] Figure 2 An exemplary embodiment of the present invention is shown, wherein five analytes and one control are detected using four colors. The analytes are nucleic acids from dengue virus ("dengue"), mycobacterium tuberculosis ("tuberculosis"), human immunodeficiency virus (HIV) p17 (P17), Plasmodium ("malaria"), and herpes simplex ("herpes"). In this example, "color" is a fluorophore connected to an oligonucleotide probe that also includes a quencher. Oligonucleotide probes will usually be different for different analytes, and the color is the same. For example, all probes designated as probe 1 have a blue color, but will usually have different oligonucleotide sequences. Of course, if a complementary sequence is present in each analyte, the probe designated as probe 1 can also have the same oligonucleotide sequence. The mechanism of detecting with these probes is described elsewhere in this disclosure. Probe 1 (blue) hybridizes all 6 analytes including controls. Probe 2 (green) hybridizes analytes from dengue virus, mycobacteria and HIV p17. Probe 3 (yellow) hybridizes analytes from dengue virus and plasmodium. Probe 4 hybridization is from the analyte of dengue virus, mycobacterium and herpes simplex.The coding scheme defined by these probes is shown in Table 5.With reference to Table 3, potential analyte D (1011) and F (1110) have been eliminated from the coding scheme.Therefore, the coding scheme shown in Table 5 is non-degenerate, and each reasonable result from measuring corresponds to the presence or absence of the unique combination of analyte in the sample.

[0113] Table 5. Figure 2 Encoding schemes for the detection of dengue virus, mycobacteria, HIV, malaria parasites, and herpes simplex are illustrated.

[0114] blue green yellow red Control (p) 1 0 0 0 Herpes simplex 1 0 0 1 Plasmodium 1 0 1 0 HIV 1 1 0 0 Mycobacterium 1 1 0 1 Dengue virus 1 1 1 1

[0115] The method for encoding and decoding shown above, including the method for eliminating degeneracy, is equally applicable to the coding method using extra colors. For example, Table 3 can be expanded (further described below) by including extra colors and extra intensity. Then as mentioned above, enumeration can produce a decoding matrix for each reasonable result obtained from the coding scheme. A decoding matrix similar to the decoding matrix provided in Table 4 can be constructed for any coding scheme described in the present disclosure. Then identify the reasonable result of degeneration, and at least one potential analyte code is eliminated from the coding scheme to reduce or eliminate degeneracy. The method for eliminating degeneracy can be performed using software on a computer-readable medium or using a hardware configuration (e.g., microchip) for performing the method.

[0116] Although degeneracy can be reduced or eliminated by the method described above and elsewhere in the present disclosure, the present disclosure also provides a non-degenerate coding scheme by design. For example, after eliminating the degeneracy of the coding scheme described in Table 3, the coding scheme can be infinitely expanded in a non-degenerate manner, wherein the non-degeneracy is by design (see, for example, the coding scheme of example in Table 6, described more fully below). Similarly, the present disclosure provides a completely non-degenerate coding scheme by design, and therefore does not need any degeneracy to reduce or eliminate (see, for example, the coding scheme of example in Table 8, described more fully below). Therefore, the coding scheme provided by the present disclosure may have reduced or eliminated degeneracy by design, or is non-degenerate by design.

[0117] D. Encoding methods using more than one color and more than one intensity

[0118] The encoding methods described above (e.g., Tables 3-5) can be further expanded by allowing analytes to be encoded by intensities greater than 1. For example, each analyte encoded in the encoding scheme provided in Table 5 is encoded by a fluorescence intensity of 1 or 0. Allowing higher signal intensity values ​​in at least one color further increases the number of analytes that can be encoded by any method provided in the present disclosure. In some instances, in order to maintain the analyte counting capability of the control color, these higher intensity values ​​can be assigned to any color except the control color.

[0119] Table 6 shows the top three layers of an exemplary coding scheme utilizing four colors and multiple intensities. The first layer of Table 6 is a replication of Table 3, showing the coding of seven analytes and one control using four colors. As described above, any two of potential analytes D, E, and F can be eliminated from the coding scheme to produce a non-degenerate coding scheme. The first layer of Table 6 indicates that potential analytes D (1011) and F (1110) have been eliminated from the coding scheme to eliminate degeneracy. Therefore, as described above, the coding scheme shown in the first layer of Table 6 can determine the presence of five analytes and one control using four colors.

[0120] The coding scheme of layer 1 of Table 6 can be expanded to the second layer (layer 2) by allowing the intensity of any color to be increased. As described above, in order to maintain the sequence counting ability of the control, the intensity of the control color can be maintained at 1. Increasing the intensity of any of the remaining three colors will produce the codes 100Y, 10Y0, 1Y00, 10YY, 1Y0Y, 1YY0, and 1YYY, where Y>1. The minimum value of Y used to encode the new layer is equal to the cumulative maximum value from the existing layers plus 1. As described below, values ​​greater than 1 can also be used to maximize the difference between the intensities.

[0121] Therefore, in this context, the term "layer" is generally used to describe a code group that makes full use of a first value (e.g., intensity) and a second value (e.g., color) without degeneracy of a specific number to provide coding capabilities. For example, the first layer of Table 6 makes full use of the coding capabilities provided by four colors without degeneracy and up to 1 intensity per color. As shown in Table 6, the first layer, this will result in six coded analytes including controls. Introducing the second layer, as described above, the minimum value of Y can be increased to equal the cumulative maximum result plus 1 (or more than one) according to the existing layer. In the example provided in Table 6, the intensity of the blue (control) color is maintained at 1 to maintain the sequence counting ability of this color. Therefore, the second layer includes the possibility of five non-degenerate codes, which are obtained by increasing the intensity in the green, yellow and red channels to equal the cumulative maximum result plus one in each channel of these channels according to the first layer. All possibilities of these codes of layer 2 can then be enumerated, and the code that causes degeneracy can be eliminated, or the second layer can be made non-degenerate by design, using the information for eliminating the corresponding code from the first layer. Greater coding capacity can then be achieved by adding a third layer or more layers constructed according to a similar method. The coding scheme can have an unlimited number of layers, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 or more layers.

[0122] More specifically, referring to Table 6, analyte H in Layer 2 is encoded by 1004. The value of the control color remains at 1. The value of the red color is equal to the cumulative maximum result (3) based on the previous layer plus 1, or 4. Similarly, analytes I and J are encoded by 1030 and 1400, respectively. The combination of these codes is used to encode the remaining four analytes (KN), as completed for analyte DG in Layer 1. This completes Layer 2. In Layer 2, the content of potential analytes K (1034) and M (1430) leads to degeneracy. Therefore, these analytes have been eliminated from the encoding scheme to eliminate degeneracy.

[0123] Continuing to refer to Table 6, the third layer (layer 3) is constructed using the same principle described above. Analyte O is encoded with 1-0-0-16. The value of the control color remains 1. The value of the red color is equal to the cumulative maximum result (15) according to the previous layer plus 1, or 16. Similarly, analytes P and Q are encoded by 1-0-9-0 and 1-16-0-0, respectively. The combination of these codes is used to encode the remaining four analytes (RU), as completed for analyte DG in the first layer and for analyte KN in the second layer. In the third layer, the content of potential analytes R (1-0-9-16) and T (1-16-9-0) leads to degeneracy. Therefore, these analytes have been eliminated from the coding scheme to eliminate degeneracy.

[0124] The three-layer coding scheme shown in Table 6 shows the coding of 15 analytes and one control using four colors. This coding scheme can be expanded indefinitely by adding more intensities to create additional layers and / or adding more colors to create additional coding capabilities within a layer. As described in the present disclosure, methods of reducing or eliminating degeneracy can be used when adding each intensity and / or color to reduce or eliminate degenerate results.

[0125] More generally, the coding scheme depicted in Table 6 is a non-degenerate, infinite extension of the coding scheme depicted in Tables 3-5. The maximum intensity of the cumulative measurements at the first layer is 6. The maximum intensity of the cumulative measurements at the second layer is 15. The maximum intensity of the cumulative measurements at the third layer is 63, and so on. Given the maximum cumulative intensity value (F), the maximum number of layers (T) available in this coding scheme is T=log4(F+1). The coding scheme depicted in Table 6 provides five (P) non-degenerate codes per layer. Therefore, the maximum number of codes M=5*Log4(F+1), or M=P*T, where P is the number of non-degenerate codes per layer and T is the number of layers. For example, given F=63, for 5 non-degenerate codes per layer, the maximum number of codes (i.e., analytes) is 15. This formula does not include code 1000, which is reserved as a positive control in Table 6. To include the control in the total number of analytes, one can simply add one, providing the formula M=(P*T)+1.

[0126] The method provided in the present disclosure can be used to expand this coding scheme indefinitely. For example, by utilizing a combination of different intensities (i.e., first values) and colors (i.e., second values), anyone can encode any number (M) of analytes by changing the number (P) of non-degenerate codes of each layer and the number (T) of layers. For example, the number of non-degenerate codes of each layer is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 or more. 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, or more.

[0127] Table 6. Encoding of 15 analytes and 1 control using four colors and multiple intensities.

[0128]

[0129]

[0130] Figure 1 The right hand side of FIG. 1 shows an illustrative example of the method described above. More specifically, Figure 1The right hand side of the table 6 shows a coding method, wherein each of 9 or more than 9 analytes (sequence 1-sequence 9, etc.) is encoded by at least two colors and the intensity variation of each color. The color represents the fluorophore connected to the oligonucleotide probe that also includes a quencher. The system can be designed so that each probe is labeled with a single fluorophore, or each probe is labeled with more than one fluorophore. For example, the code for analyte H is 1004 in Table 6. The intensity of 4 in the red channel can be achieved by using an H-specific probe including 4 red fluorophores, or by using 4 H-specific probes each including a single red fluorophore. Of course, any combination of probes and fluorophores that produce a result of 4 in the red channel is also suitable, such as 2 probes each containing 2 red fluorophores, 1 probe containing 1 red fluorophore and 1 probe containing 3 red fluorophores, or just one probe containing one red fluorophore, but present in the reaction mixture in 4x quantity.

[0131] Figure 1 The encoding scheme depicted on the right hand side of may be as shown in Table 7. Figure 1 The analysis result shown on the right hand side of is 4112, or an intensity of 4 in the blue channel, 1 in the green channel, 1 in the yellow channel, and 2 in the red channel. Using the decoding matrix constructed as described herein, this result is decoded to indicate that sequence 1, sequence 3, sequence 4, and sequence 5 are present.

[0132] Table 7. Figure 1 The right-hand side of FIG. 1 illustrates the encoding scheme.

[0133]

[0134] E. Use one color and one intensity per analyte, but use different intensities within the analyte to encode

[0135] In some methods provided herein, each analyte is encoded by a single color and intensity combination. For example, in a four-color system, the first four analytes can be encoded by 1000, 2000, 4000, and 8000. The following four analytes can be encoded by 0100, 0200, 0400, and 0800. Analytes 9-12 and 13-16 will be similarly assigned, as shown in Table 8.

[0136] Like the encoding method described in Table 6, this encoding scheme is infinite, non-degenerate in theory by construction, and is limited only by the bandwidth of the instrument used by the measurement signal. However, this encoding scheme can quantify more analytes per unit bandwidth than the encoding method described in Table 6. The reason is that the available multipath of the signal is used with maximum efficiency because each intensity level is utilized in the encoding (i.e., there is no vacancy in the chromatogram; see the description of the chromatogram below). Table 8 shows an embodiment of the method, illustrating four layers of encoding based on four colors and intensities 1, 2, 4 and 8. The encoding scheme is non-degenerate, and if all 16 analytes are present, the result is 15-15-15-15. In terms of bandwidth utilization, this encoding method is more effective than the encoding method given above. However, this method does not have the proofreading ability of the first scheme, because all the results decoded as reasonable output results in the first scheme have vacancies in the chromatogram.

[0137] Table 8. Examples of encoding methods using one color and one intensity per analyte, but using different intensities among analytes.

[0138]

[0139] The methods shown above, and exemplified in Table 8, can be expanded by introducing additional colors and / or intensities. For example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, or more colors can be used. Additional intensity levels may also be used, such as 16, 32, 64, 128, 256, 512, 1024, 2048, 4096, 8192, 16384, 32768, 65536, and the like. The method is generalizable and can be used to encode an unlimited number of analytes. Each analyte is represented by a code in a single color, wherein the value of the code in the single color is equal to the sum of all previous values ​​plus 1. For example, if the first code in a particular color contains 1, the subsequent codes are 2, 4, 8, 16, 32, 64, 128, and the like. Other progressions are possible, but will not be considered effective in terms of bandwidth usage. However, one of ordinary skill in the art will recognize that when anyone wishes to maximize the intervals between values, less efficient bandwidth usage may be desired. Similarly, value 1 may be excluded from the encoding scheme, for example, to maximize the difference in intensity between the first coded value and the noise of the instrument. For example, in some cases, the coding scheme may start with a value of 2, which will provide a progression of 2, 4, 8, 16, 32, 64, 128, etc. for the analyte code, while the progression of the cumulative result will be 2, 4, 6, 8, 10, 12, 14, 16, etc. This approach produces uniform gaps in the possible cumulative results, allowing for a higher tolerance to noise than a progression of 1, 2, 4, 8, 16, 32, etc. analyte codes, and it corresponds to a progression of 1, 2, 3, 4, 5, 6, 7, 8, etc. cumulative results. In this example, the increased robustness in the measurement is at the expense of a reduced number of codes available in a fixed bandwidth of the multiplex. For example, if only 63 multiplex states can be reliably measured, then a coding scheme starting from 1 provides 7 codes per color, while a coding scheme starting from 2 provides 6 codes per color. If 4 colors are available, the former will provide 28 codes, while the latter will provide 24 codes. In summary, both the starting signal intensity and the magnitude (ie, the difference between intensity values) can be weighed to maximize the ability to resolve instrument noise and to maximize the difference between intensity values, thereby enhancing the ability to distinguish between different experimental results.

[0140] The coding scheme as described in Table 8 is non-degenerate by design. Although the coding scheme described in Table 8 uses both intensity and color to encode each of the 16 analytes, each analyte can also be encoded by simply utilizing intensity. For example, given the 16 analytes (AP) provided in Table 8, a single color coding scheme encodes all 16 analytes, and values ​​1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, 4096, 8192, and 16384 can be assigned to analytes A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, and P, respectively. This coding scheme is non-degenerate by design, and the cumulative intensity result can be clearly decoded to indicate the presence or absence of analyte AP. This coding scheme is capable of detecting the presence or absence of M analytes, where M=Log2(F+1), and F is the maximum cumulative value of the signal (e.g., signal intensity). By adding a second component to this signal (eg, color, as depicted in Table 8), the capacity of this coding scheme can be increased to M = C*Log2(F+1), where C is the number of colors used in the coding scheme.

[0141] As described throughout this specification, any suitable value can be used for C or F. For example, C can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, or more. F+1 can be at least about 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, 4096, 8192, 16384, 32768, 65536, 131072, 262144, 524288, 1048576, etc. As described elsewhere in this disclosure, the initial starting value of F and the step size in its progression can also be varied.

[0142] III. Analytes

[0143] Analyte can be any suitable analyte analyzed using the method and composition of the present invention, wherein the analyte can interact with a reagent to produce a measurable signal with at least two components. The analyte can be naturally occurring or synthetic. The analyte can be present in a sample obtained using any method known in the art. In some cases, the sample can be processed before it is used for the analysis of the analyte. The method and composition proposed in the present disclosure can be used in solution phase assays without the need for particles (such as beads) or solid supports.

[0144] In some cases, the analyte can be a polynucleotide, such as DNA, RNA, peptide nucleic acid, and any hybrid thereof, wherein the polynucleotide comprises any combination of deoxyribonucleotides and / or ribonucleotides. The polynucleotide can be single-stranded or double-stranded, or contain double-stranded or single-stranded sequence portions at the same time. The polynucleotide can comprise any combination of nucleotides or bases, including, for example, uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, and any nucleotide derivatives thereof. As used herein, the term "nucleotide" can include nucleotides and nucleosides, as well as nucleoside and nucleotide analogs, and modified nucleotides, including both synthetic species and naturally occurring species. The polynucleotide can be any suitable polynucleotide for which one or more agents (or probes) as described herein can be generated, including but not limited to cDNA, mitochondrial DNA (mtDNA), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), nuclear RNA (nRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small Cajal body-specific RNA (scaRNA), microRNA (miRNA), double stranded (dsRNA), ribozyme, riboswitch or viral RNA. The polynucleotide can be contained in any suitable vector, such as a plasmid, cosmid, fragment, chromosome, or genome.

[0145] Genomic DNA can be obtained from naturally occurring or genetically modified organisms, or from artificially or synthetically produced genomes. Genomic DNA alone can be obtained from any source, and any method known in the art can be used to obtain the analyte. For example, genomic DNA can be separated with or without amplification. Amplification can include PCR amplification, multiple displacement amplification (MDA), rolling circle amplification and other amplification methods. Genomic DNA can also be obtained by cloning or recombinant methods, such as those involving plasmids and artificial chromosomes or other conventional methods (see Sambrook and Russell, Molecular Cloning: A Laboratory Manual., supra). Polynucleotides can be separated using other methods known in the art, such as disclosed in Genome Analysis: A Laboratory Manual Series (Volumes I-IV) or Molecular Cloning: A Laboratory Manual. If the isolated polynucleotide is mRNA, it can be reverse transcribed into cDNA using conventional techniques, such as Sambrook and Russell, Molecular Cloning: A Laboratory Manual, supra, described in.

[0146] The analyte can be a protein, a polypeptide, a lipid, a carbohydrate, a sugar, a small molecule, or any other suitable molecule that can be detected with the methods and compositions provided herein. The analyte can be an enzyme or other protein. The analyte can be a drug or metabolite (e.g., an anticancer drug, a chemotherapeutic drug, an antiviral drug, an antibiotic drug, or a biologic). The analyte can be any molecule, such as a cofactor, a receptor, a receptor ligand, a hormone, a cytokine, a blood factor, an antigen, a steroid, or an antibody.

[0147] The analyte can be any molecule from any pathogen, such as a virus, bacteria, parasite, fungus, or prion (e.g., PrP Sc). Examples of viruses include those from the families Adenoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Picornaviridae, Polyomaviridae, Retroviridae, Rhabdoviridae, and Togaviridae. Specific examples of viruses include adenovirus, astrovirus, bocavirus, BK virus, coxsackievirus, cytomegalovirus, dengue virus, Ebola virus, enterovirus, Epstein-Barr virus, feline leukemia virus, hepatitis virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, herpes simplex virus (HSV), HSV type 1, HSV type 2, human immunodeficiency virus (HIV), HIV type 1, HIV type 2, human papillomavirus (HPV), HPV type 1, HPV type 2, HPV type 3, HPV type 4, HPV type 6, HPV type 10, HPV type 11, HPV type 16, HPV type 18, HPV type 26, HPV type 27, HPV type 28, HPV type 29, HPV type 30, HPV type 31, HPV type 33, HPV type 34, HPV type 35, HPV type 36, HPV type 37, HPV type 38, HPV type 39, HPV type 40, HPV type 41, HPV type 42, HPV type 43, HPV type 44, HPV type 45, HPV type 46, HPV type 47, HPV type 48, HPV type 49, HPV type 50, HPV type 51, HPV type 52, HPV type 53, HPV type 54, HPV type 55, HPV type 56, HPV type 57, HPV type 58, HPV type 59, HPV type 60, HPV type 61, HPV type 62, HPV type 63, HPV type 64, HPV type 65, HPV type 66, HPV type 67, HPV type 68, HPV type 69, HP HPV 34, HPV 35, HPV 39, HPV 40, HPV 41, HPV 42, HPV 43, HPV 44, HPV 45, HPV 49, HPV 51, HPV 52, HPV 54, HPV 55, HPV 56, HPV 57, HPV58, HPV 59, HPV 68, HPV 69, influenza virus, JC virus, Marburg virus, measles virus, mumps virus, Norwalk virus, parvovirus, polio virus, rabies virus, respiratory syncytial virus, retrovirus, rhinovirus, rotavirus, rubella virus, smallpox virus, vaccinia virus, West Nile virus and yellow fever virus.

[0148] Examples of bacteria include those from the genera Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, lla), Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, and Yersinia. Specific examples of bacteria include Bordetella parapertussis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatix, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, tetani), Corynebacterium diphtheriae, Enterococcus faecalisfaecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii), Salmonella choleraesuis, Salmonella dublin, Salmonella enteritidis, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, Yersinia pestis pestis and Yersinia enterocolitica.

[0149] Examples of parasites include those from the genera Acanthamoeba, Babesia, Balamuthia, Balantidium, Blastocystis, Cryptosporidium, Dientamoeba, Entamoeba, Giardia, Isospora, Leishmania, Naegleria, Pediculus, Plasmodium, Rhinosporidium, Sarcocystis, Schistosoma, Toxoplasma, Trichomonas, and Trypanosoma.Specific examples of parasites include Babesia divergens, Babesia bigemina, Babesia equi, Babesia microti, Babesia duncani, Balamuthia mandrillaris, Balantidium coli, Dientamoeba fragilis, Entamoeba histolytica, Giardialamblia, Isospora belli, Naegleria fowleri, Pediculus humanus, Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium ovale, Plasmodium vivax, Rhinosporidium seeberi, Sarcocystis bovihominis, Sarcocystis suihominis, Schistosoma mansoni, Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei, and Trypanosoma cruzi.

[0150] Examples of fungi include those from the genera Apophysomyces, Aspergillus, Blastomyces, Candida, Cladosporium, Coccidioides, Cryptococcus, Exserohilum, Fusarium, Histoplasma, Pichia, Pneumocystis, Saccharomyces, Sporothrix, Stachybotrys, and Trichophyton. Specific examples of fungi include Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Exserohilum rostratum, Fusarium verticillioides, Histoplasma capsulatum, Pneumocystis jirovecii, Sporothrix schenckii, Stachybotrys chartarum, and Trichophyton mentagrophytes.

[0151] In some cases, the method provided in the present disclosure can be used for detecting any one of the analytes described above or in other places of the specification sheets. In some cases, the method provided in the present disclosure can be used for detecting the combination (panel) of the analytes described above or in other places of the specification sheets. For example, the combination can include the analytes selected from the group consisting of any 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,60,70,80,90,100 or more analytes described above or in other places of the specification sheets.

[0152] Analyte can be obtained from any suitable position, including from organism, intact cell, cell preparation and from cell-free composition of any organism, tissue, cell or environment. Analyte can be obtained from environmental sample, biopsy, aspirate, formalin-fixed embedded tissue, air, agricultural sample, soil sample, petroleum sample, water sample or dust sample. In some cases, analyte can be obtained from body fluid, and it may include blood, urine, feces, serum, lymph, saliva, mucosal secretion, sweat, central nervous system fluid, vaginal fluid or semen. Analyte can also be obtained by the product of manufacture, such as cosmetics, food, personal care products, etc. Analyte can be the product of experimental operation, including recombinant cloning, polynucleotide amplification, polymerase chain reaction (PCR) amplification, purification method (such as genomic DNA or RNA purification) and synthesis reaction.

[0153] In each multiplex assay, more than one type of analyte may be detected. For example, polynucleotides, proteins, polypeptides, lipids, carbohydrates, sugars, small molecules, or any other suitable molecule may be detected simultaneously in the same multiplex assay with suitable reagents. Any combination of analytes may be detected at the same time.

[0154] The detection of analyte can be useful for any suitable application, and application includes scientific research, clinical, diagnosis, prognosis, forensic and monitoring applications. Typical applications include the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, criminal identification, system development, biological counter-terrorism, environmental monitoring, and DNA computing. For example, analyte can indicate disease or condition. Analyte can be used to make treatment decisions, or to assess the state of the disease. The presence of analyte can indicate infection with specific pathogens or any other disease such as cancer, autoimmune diseases, cardiopulmonary diseases, liver diseases, digestive tract diseases, etc. Therefore, the method provided herein can be used to make a diagnosis and make a clinical decision based on the diagnosis. For example, the result indicating the presence of bacterial polynucleotides in the sample collected from the subject may lead to treating the subject with antibiotics.

[0155] In some cases, the methods and compositions of the invention can be used to detect at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 , 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 analytes. In some cases, the methods and compositions of the invention can be used to detect 7-50, 8-40, 9-30, 10-20, 10-15, 8-12, or 7-12 analytes.

[0156] In some cases, the present disclosure provides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or 1000 analytes. In some cases, the present disclosure provides at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 analytes are assayed for the presence or absence of each analyte without immobilization, separation, mass spectrometry, or melting curve analysis.In some cases, the present disclosure provides less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 analytes are assayed for the presence or absence of an analyte without immobilization, separation, mass spectrometry, or melting curve analysis.

[0157] IV. Signals

[0158] The method proposed in the present disclosure can be used with any quantifiable signal. In some cases, the present disclosure provides a coding scheme and method for encoding an unlimited number of targets with non-degeneracy using a single component of the signal (e.g., intensity). For example, as described above and in Example 3, the coding scheme can rely on the multipath of the signal intensity without considering color. Although fluorescent probes have been used to illustrate this principle, as described elsewhere in the present disclosure, the coding scheme is equally suitable for any other method, which provides a quantifiable signal, including electrochemical signals and chemiluminescent signals.

[0159] The method proposed in the present disclosure can also utilize the signal measurement result in at least two dimensions, also referred to as the measurement result of at least two components of signal.For example, compared to the coding scheme described in the above paragraph, relying on signal intensity to distinguish analyte, utilizing at least two components of signal (for example, color and intensity) allows the unit of each signal intensity bandwidth to produce more different codes.When utilizing at least two components of signal, the cumulative measurement result of at least two components of single sample volume can be obtained.For example, in the coding scheme described in Table 5, the presence of each analyte results in a specific intensity (a component of signal) on each color of 4 colors (the second component of signal).The combination of these constituent signals results in the ability to measure cumulative signals by measuring the intensity in each wavelength or wavelength range.Then corresponding coding scheme or decoding matrix can be used to convert cumulative measurement result into determining whether analyte exists or not.

[0160] In some cases, the quantifiable signal includes a waveform having both a frequency (wavelength) and an amplitude (intensity). The signal can be an electromagnetic signal. The electromagnetic signal can be a sound, a radio signal, a microwave signal, an infrared signal, a visible light signal, an ultraviolet light signal, an x-ray signal, or a gamma ray signal. In some cases, the electromagnetic signal can be a fluorescent signal, such as a fluorescent emission spectrum that can be characterized in terms of wavelength and intensity.

[0161] In some parts of the present disclosure, signals are described and illustrated in terms of fluorescent signals. This is not meant to be restrictive, and one of ordinary skill in the art will readily recognize that the principles applicable to the measurement of fluorescent signals are also applicable to other signals. For example, similar to fluorescent signals, any electromagnetic signal described above can be characterized in terms of wavelength and intensity. The wavelength of the fluorescent signal can also be described in terms of color. The color can be determined based on measuring the intensity at a specific wavelength or wavelength range, for example, by determining the intensity distribution of fluorescence at different wavelengths, and / or by using a bandpass filter to determine the fluorescence intensity within a specific wavelength range. Such bandpass filters are commonly used in various laboratory instruments, including quantitative PCR devices. The intensity can be measured with a photodetector. Many wavelengths can be referred to as "channels."

[0162] In some cases, the methods provided in the present disclosure can be used for any signal, wherein the cumulative signal is measured with constituent signals of the same color, frequency, absorption band, etc. However, the cumulative signal does not need to be a digital signal or to scale linearly with the number and intensity of the constituent signals. For example, if the physical principle of the measurement is absorption, then due to the exponential nature of the Beer-Lambert law, the cumulative attenuation is the product of the constituent attenuations and the constituent concentrations are additive. Then the logarithm of the cumulative attenuation will be linearly adjusted with the constituent concentrations on each absorption band (equivalent to color if fluorescence detection is used). Therefore, the method of the present invention is applicable. The method of the present invention can also be used for chemiluminescent signals and electrochemical signals.

[0163] The number of signals, and the number of dimensions or components measured, can also be expanded to exceed the number shown in the exemplary embodiments of the present invention, resulting in the expansion of multi-path capabilities. The exemplary embodiments provided in the present disclosure utilize encoding schemes constructed using fluorescent signals with one or two components of measurement: wavelength and / or intensity. The number of analytes that can be encoded can be increased by increasing the number of wavelengths and / or intensities. The number of analytes that can be encoded can also be increased by increasing the number of signals, for example, by combining fluorescent signals with electrochemical signals or FRET signals (fluorescence resonance energy transfer).

[0164] In some cases, more than two components of a signal may be measured. For example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 20 or more components of a signal may be measured. At least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, or 20 components of a signal may be measured. At least 2, but less than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, or 20 components of a signal may be measured. In some cases, 2-3, 2-4, 2-5, 2-6, 3-5, 3-6, 3-8, or 5-10 components of a signal may be measured. These additional components may include kinetic components, such as the rate at which the signal decays and the rate of photobleaching.

[0165] If fluorescent signals are used, the number of analytes that can be encoded can be further expanded by utilizing additional fluorophores. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more fluorophores can be used. In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 fluorophores may be used. In some cases, less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 fluorophores may be used. In some cases, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 10-15, or 10-20 fluorophores may be used.

[0166] In general, the number of analytes that can be encoded can be further expanded by utilizing additional first values ​​(e.g., intensities) from the value or range of values ​​of the first component of the signal. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more first values ​​from the value or range of values ​​of the first component of the signal can be used. In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more first values ​​from a first component of a signal or a range of values ​​may be used. In some cases, less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 first values ​​from the values ​​or range of values ​​of the first component of the signal may be used. In some cases, 4-20, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 10-15, or 10-20 first values ​​from the values ​​or range of values ​​of the first component of the signal may be used.

[0167] In examples, where intensity is the component of the signal being quantified, such as when utilizing a fluorescent signal, the presence of an analyte can be encoded using multiple intensities or intensity ranges. For example, a coding scheme can utilize 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more intensities or intensity ranges. The encoding scheme may utilize at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more intensities or intensity ranges. The encoding scheme may utilize less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 intensities or intensity ranges. In some cases, the encoding scheme may utilize 2-20, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, or 5-10 intensities or intensity ranges.

[0168] The number of analytes that can be encoded can be further expanded by utilizing additional second values ​​(e.g., wavelengths) from the value or range of values ​​of the second component of the signal. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more second values ​​from the value or range of values ​​of the second component of the signal can be used. In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more second values ​​from a second component of the signal or a range of values ​​may be used. In some cases, less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more second values ​​from the values ​​or ranges of values ​​of the second component of the signal may be used. In some cases, 4-20, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 10-15, or 10-20 second values ​​from the values ​​or ranges of values ​​of the second component of the signal may be used.

[0169] In instances where wavelength is a component of the signal being quantified, such as when a fluorescent signal is utilized, the presence of an analyte can be encoded using a plurality of wavelengths or wavelength ranges. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more wavelengths or wavelength ranges can be used. 48, 49, 50, or more wavelengths or wavelength ranges may be used. In some cases, less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 wavelengths or wavelength ranges can be used. In some cases, 4-20, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 10-15, or 10-20 wavelengths or wavelength ranges can be used.

[0170] In some cases, when degeneracy is eliminated, the methods of the invention are capable of detecting the presence or absence of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more analytes in a single volume when each reagent used to produce a signal in the volume produces only one second value (e.g., each reagent emits only one wavelength of light).

[0171] In other cases, when degeneracy is eliminated, the methods of the invention are capable of detecting the presence or absence of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more analytes in a single volume using a total of four second values ​​in the volume (e.g., a total of four wavelengths or ranges of wavelengths, which may be implemented by using four spectrally resolvable fluorophores).

[0172] As described throughout the specification, the determination provided herein utilizes the cumulative measurement results of the sample. The cumulative measurement result can be, for example, a single measurement result of an intensity value, or a measurement result of an intensity value at one or more wavelengths or a range of wavelengths. Multiple cumulative measurements can be obtained. For example, intensity can be measured at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more wavelengths or wavelength ranges. 44, 45, 46, 47, 48, 49, 50, or more wavelengths or ranges of wavelengths. 44, 45, 46, 47, 48, 49, or 50 wavelengths or ranges of wavelengths.

[0173] More generally, cumulative measurements of any quantifiable component of a signal and any quantifiable component of a signal of another quantifiable component of the signal can be obtained. For example, at least one first component of a signal can be measured at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more second components of the signal. At least one first component of a signal may be measured at a second component of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more signals. At least one first component of a signal may be measured at less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 second components of the signal.

[0174] As will be apparent from the present disclosure, each analyte to be detected can be encoded as a suitable component utilizing any number of signals, or a code for any number of signals. For example, each analyte to be detected can be encoded in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more components or signals of the signal. In some cases, each analyte to be detected can be encoded in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more components or signals of the signal. In some cases, each analyte to be detected can be encoded in less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 components or signals of the signal. In some cases, each analyte can be encoded using 1-4, 4-20, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 10-15, or 10-20 components or signals of the signal. In the encoding scheme each analyte may be encoded by the same number of signal components, or signals, or a different number of signal components, or signals.

[0175] A. Probes, Primers, Fluorophores, and Quenchers

[0176] Some methods provided in the present disclosure utilize reagents that produce signals when analyte exists.Any suitable reagent can be used in the present invention.Generally, reagent will have analyte-specific components, and components that produce signals when analyte exists.In some cases, these reagents are referred to as probes.The probe can be a hybridization probe.The hybridization probe can be an oligonucleotide probe connected to a fluorophore and a quencher (e.g., TAQMAN probe).

[0177] The methods of the invention can use one or more reagents or probes to detect the presence or absence of each analyte. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more probes can be used to detect the presence or absence of each analyte. In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 probes can be used to detect the presence or absence of each analyte. In some cases, less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 probes can be used to detect the presence or absence of each analyte. In some cases, the number of probes used to detect the presence or absence of each analyte is 1-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 1-3, or 1-4.

[0178] In some cases, a sample is contacted with 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more probes to detect the presence or absence of all analytes. In some cases, a sample is contacted with at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more probes to detect the presence or absence of all analytes. In some cases, the sample is contacted with less than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 probes to detect the presence or absence of all analytes. In some cases, the number of probes contacted by the sample to detect the presence or absence of all analytes is 4-50, 4-40, 4-30, 4-20, 5-15, 5-10, or 3-10.

[0179] As described above, the oligonucleotide probe connected to the fluorophore and the quencher can be used to detect the presence of the analyte in the polynucleotide amplification assay. As long as the quencher and the fluorophore are close, the quencher quenches the fluorescence emitted by the light source excitation fluorophore. The sequence of the oligonucleotide probe is designed to be complementary to the polynucleotide sequence present in the analyte, and therefore, it is possible to hybridize with the polynucleotide sequence present in the analyte. The hybridization of the oligonucleotide probe can be performed in a nucleic acid amplification reaction (e.g., polymerase chain reaction) including primers. After the primer is extended by DNA polymerase, the 5' to 3' exonuclease activity of the polymerase degrades the probe, releasing the fluorophore and the quencher into the medium. The proximity between the fluorophore and the quencher is destroyed, and the signal from the fluorophore is no longer quenched. Therefore, the amount of fluorescence detected is a function of the amount of the analyte present. If there is no analyte, the probe will not hybridize with the analyte, and the fluorophore and the quencher will remain very close. Very little signal or no signal will be generated.

[0180] Each probe of the oligonucleotide probe can have one or more fluorophores and quenchers. For example, in some embodiments, the oligonucleotide probe can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more fluorophores. The oligonucleotide probe may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 fluorophores. 44, 45, 46, 47, 48, 49, or 50 fluorophores.

[0181] The oligonucleotide probe can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more quenchers. The oligonucleotide probe may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 quenchers. The oligonucleotide probe may include less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 quenchers.

[0182] The probe and the attachment of the quencher to the probe can be performed in the same reaction, or in a series of reactions.A series of reactions can be performed to label the probe with at least one fluorophore, and the reaction products can be mixed to produce a mixture of probes with different fluorophores.

[0183] If the oligonucleotide probe includes two or more fluorophores, these fluorophores can be arranged to allow (Fluorescence) resonance energy transfer (FRET) occurs between fluorophores. Simply put, FRET is the mechanism of energy transfer between fluorophores. Using a probe based on FRET allows an excitation source to generate two different fluorescence emission signals by the excitation of a single fluorophore. For example, the method described herein can use paired probes, wherein one probe in the pairing is connected to a fluorophore and a quencher, and the second probe is connected to two fluorophores (close enough for FRET to occur) and a quencher. Any combination of fluorophores and quenchers provided for FRET can be used. This method doubles the number of fluorescent probes that can be used with an excitation source, because a single excitation source can be used to generate two signals: one from a probe with a fluorophore, and one from a probe with two fluorophores close enough for FRET to occur. For example, using the encoding method described in Table 8, by pairing each probe in Table 8 with a corresponding FRET probe, the number of analytes that can be detected can be increased from 16 to 32.

[0184] Primer can be specific to a particular analyte, and can amplify the region complementary to the probe. In some cases, the number of the pairing of the primer used is equal to the number of probes. In other cases, the number of the probes used can exceed the number of the primer pairs used. In other cases, the number of the primer pairs used can exceed the number of the probes used. In some cases, the sample to be analyzed can be contacted with the pairing of 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 or more primers. In some cases, the sample to be analyzed can be contacted with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more primer pairs. In some cases, the sample to be analyzed can be contacted with less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 primer pairs. In some cases, the number of primer pairs is 2-10, 3-15, 4-20, 3-10, 4-10, 5-10, 6-8, or 6-10.

[0185] Primers can amplify regions of the polynucleotide to which different numbers of hybridization probes hybridize. For example, at least one pair of primers can amplify a region complementary to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 hybridization probes. In some cases, all of the pairs of primers can amplify regions that are complementary to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 hybridization probes.

[0186] In one example, the first probe can be labeled with a fluorophore 5'-fluorescein amide (5'-FAM) and a quencher black hole quencher 1 (BHQ-1). The second probe can be labeled with a 5'-FAM in close proximity (e.g., connected to) cyanine 5.5 (Cy5.5) and a quencher black hole quencher 3 (BHQ-3). Two fluorescence signals are generated from a single excitation wavelength (e.g., 470nm) according to the digestion of the probe by the nuclease activity of the polymerase. The fluorophore from the first probe emits fluorescence at about 520nm. The fluorophore on the second probe undergoes FRET. The donor fluorophore FAM is excited by an excitation light source (e.g., 470nm) and transfers its energy to the acceptor fluorophore Cy5.5. The acceptor fluorophore emits at about 705nm. These fluorophores and quenchers are exemplary only. Any fluorophore that can undergo FRET, and any quencher that can quench fluorescence are suitable for use in the present invention. Methods for producing FRET-based probes are described in Jothikumar et al., BioTechniques, 2009, 46(7): 519-524.

[0187] In certain embodiments, a single hybridization probe can be used for each analyte. In order to utilize multi-color coding (e.g., as shown in Table 3), each probe can be labeled with a plurality of fluorophores in a predetermined ratio. The ratio can be determined so that, in the same color, the positive control signal provides the same intensity as other positive control signals of the same intensity. This method reduces the number of probes that must be synthesized, and may be configured more cheaply than the method utilizing multiple probes. In addition, in the case of hybridization probes, it is easier for a hybridization probe to cooperate with the sequence of an analyte than multiple probes.

[0188] Although many aspects of the present invention are exemplified using nucleic acid-based probes, one of ordinary skill in the art will readily recognize that other forms of probes will work equally well with the present invention described in this disclosure. For example, binding molecules specific to the analyte can be used as probes. Non-limiting exemplary binding molecules include antibodies that recognize the analyte and generate a signal when the analyte is present.

[0189] In embodiments of the invention utilizing fluorescent labels, any suitable fluorescent label may be used. Exemplary fluorescent labels suitable for use in the invention include rhodamine, rhodol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodol, carboxyrhodol, chlororhodol, methylrhodol, sulfofluorescein, aminorhodamine, carboxyrhodamine ... chlororhodamine, methylrhodamine, sulforhodamine, thiorhodamine, cyanine, indolecarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7, oxadiazole derivatives derivatives), pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, pyrene derivatives, cascade blue, oxazine derivatives, Nile red, Nile blue, cresyl violet, oxazine 170, acridine derivatives, diaminoacridine (proflavin), acridine orange, acridine yellow, arylmethine derivatives, auramine, crystal violet, malachite green, tetrapyrrole derivatives, porphine, phthalocyanine pigments and bilirubin. Exemplary quenchers include black hole quenchers, such as BHQ-0, BHQ-1, BHQ-2, BHQ-3; ATTO quenchers, such as ATTO540Q, ATTO 580Q and ATTO 612Q; and the like.

[0190] Fluorophores that can be used with the present invention are not limited to any fluorophores described herein. For example, fluorophores with improved properties are constantly being developed, and these fluorophores can be easily used with the methods provided by the present disclosure. Such improved fluorophores include quantum dots, which can emit energy at different wavelengths after being excited at a single wavelength. The advantage of using such fluorophores is that only a single excitation source is required, but many different signals can be quantified, for example, based on color and intensity. In addition, fluorophores with narrow emission spectra will be particularly useful for the methods described herein because such fluorophores can be included in multiplexed assays with little or no overlap between their luminescence spectra, thereby providing more "colors" and significantly increasing the overall number of encoded analytes.

[0191] In some cases, one or more reagents are freeze-dried. Any suitable reagent can be freeze-dried. For example, probes, primers, enzymes, antibodies or any other reagents for detection can be freeze-dried. In some cases, samples comprising analytes can also be freeze-dried. Freeze-drying may be useful, for example, when distributing reagents and / or samples in developing regions, obtaining cold storage in these regions is expensive, difficult to use or can not be reliably used. In an example, freeze-dried reagents include any probe or analyte-specific reagents described in the present disclosure or otherwise suitable for use in the present invention. In another example, freeze-dried reagents include PCR primers. In another example, freeze-dried reagents include reagents suitable for performing PCR reactions.

[0192] V. Analytical Techniques and Instruments

[0193] The method provided herein is suitable for use with various detection methods. For example, the applicable method uses an analytical technique that measures the wavelength and intensity of the fluorescent signal. This can be achieved by measuring the intensity of the signal of the entire spectral wavelength, or by using a bandpass filter that limits the light of certain wavelengths, thereby only allowing the light of certain wavelengths to reach the photodetector. Many real-time PCR and quantitative PCR instruments include excitation light sources and bandpass filters that can detect fluorescent signals on four colors (e.g., blue, green, yellow and red). Therefore, the method of the present invention can be easily applied using instruments widely used in the art. Importantly, the method and composition provided herein can be used to detect multiple analytes by obtaining the cumulative measurement results of a single solution. There is no need for separation. The present invention does not need to use beads or solid phases. Of course, one of ordinary skill in the art should understand that the present invention can be used in conjunction with separation, beads, or solid phases if necessary.

[0194] Figure 5 Two examples of instrument configurations that can be used to obtain cumulative measurements useful for the methods of the present invention are shown. Figure 5, an instrument with a detector (e.g., a fluorometer) configured to detect both wavelength and intensity is shown at 501. Referring to 501, at least one excitation light source 502 is directed into a chamber 503 containing an analyte and a reagent that generates a signal when the analyte 504 is present. As described elsewhere, the analyte can be a nucleic acid, and the reagent that generates a signal can be a hybridization probe including a fluorophore and a quencher. If the analyte is present, an emission signal 505 is generated. In the configuration shown in 501, the wavelength and intensity of the emission signal are measured across the spectrum by a detector that is capable of generating a fluorescence emission spectrum 506.

[0195] Wavelength and intensity can also be determined using a combination of a photodetector and a bandpass filter. This configuration is used in several thermal cyclers known in the art. Figure 5 , an instrument with bandpass filters and a light spot detector is depicted in 507. Referring to 507, at least one excitation light source 508 is directed into a chamber 509 containing an analyte and a reagent that generates a signal when the analyte 510 is present. As described elsewhere, the analyte can be a nucleic acid, and the reagent that generates a signal can be a hybridization probe including a fluorophore and a quencher. If the analyte is present, an emission signal 511 is generated. In the configuration shown in 507, bandpass filters 512, 513, and 514 are used to limit the passage of light to emit light within a specific wavelength range. For example, bandpass filter 512 can limit the passage of light to emit light within wavelength range 1. Bandpass filter 513 can limit the passage of light to emit light within wavelength range 2. Bandpass filter 514 can limit the passage of light to emit light within wavelength range 3. Any number of bandpass filters (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more) can be used in the present invention. In addition, the position of the bandpass filters in 507 is not meant to be limiting. In some cases, the emitted light may pass through more than one bandpass filter at any time. In other cases, the bandpass filters can be configured so that all emitted light passes through only one bandpass filter at a time. After passing through the bandpass filters, the filtered light 515, 516, and 517 are detected by a photodetector. For example, the filtered light 515 may be light within wavelength range 1, the filtered light 516 may be light within wavelength range 2, and the filtered light 517 may be light within wavelength range 3. The intensity of each filtered light 515 , 516 , and 517 can then be quantified by photodetector 518 .

[0196] The method described in the present disclosure is compatible with various amplification methods, including quantitative PCR (qPCR) method, endpoint PCR method, reverse transcriptase PCR, and digital PCR method. Digital PCR method (e.g., droplet digital (BIORAD) and dynamic array (FLUIDIGM)) produces highly sensitive quantitative polynucleotide copy number. The method provided herein can be easily integrated into these systems, significantly expanding its working capacity by allowing multiplexing in droplets or dynamic arrays.

[0197] Similarly, the method described in the present disclosure can be applied in real-time PCR determination. For example, as the PCR operation is completed, the real-time data can be fully recorded. The endpoint value can then be decoded to indicate the presence or absence of the analyte. By detecting the maximum value of the second-order derivative of the real-time curve of the color, the value of each cycle threshold (Ct) can be analyzed for each color. Because specific analytes are amplified, fluorophores are released from their corresponding probes in the same ratio as the color diversity of the sequence encoding. Therefore, as the 1100 code will have the same Ct in blue and green, and the 1400 code will have a green Ct that is 2 cycles ahead of its blue Ct. This additional data can measure the identification and starting amount of each analyte.

[0198] The method described herein can also be used directly on a tissue sample. For example, a tissue sample can be obtained and a well can be directly constructed on the tissue sample using photolithography. The tissue sample can be fixed in advance to construct a well. The tissue sample can then be analyzed by distributing appropriate reagents into the wells in the tissue sample. The encoding and decoding methods described in the present disclosure can be used for multiplex detection of analytes etched into the wells in the tissue. Each well can correspond to a single cell or several cells, providing excellent spatial resolution when performing analyte analysis on different parts of the tissue. This method can be used to detect analytes in different regions of the same tissue.

[0199] In some cases, the instrument can be modified or constructed, for example, to provide additional excitation light sources at multiple wavelengths, and / or to provide additional bandpass filters or the ability to determine the entire spectrum. Including additional excitation sources will allow for the excitation of a greater number of various fluorophores. Including additional bandpass filters, or modifying or constructing the instrument to be able to determine the entire spectrum, allows for the detection of a greater variety of emissions from fluorophores. These techniques can be used to increase the number of fluorophores that can be used in the methods described in the present disclosure, and therefore can increase the number of analytes that can be detected simultaneously.

[0200] In some cases, the method described in the present disclosure utilizes the end-point PCR method. However, in some cases, the result can be obtained before the end point. For example, when the result is needed as soon as possible, this may be advantageous. For example, in the case of a calibration experiment, the different starting concentrations of the analyte are analyzed to determine that the signal reaches saturation of its cycle number, which is a function of the starting amount of the analyte. Using this data, a computer that monitors the PCR reaction in real time can be programmed to search for saturation of the maximum number of cycles according to the limit of detection (LOD) of a specific system. If the analyte is not amplified (for example, except for the positive control that may be present) at the maximum number of cycles, the result for the analyte is negative. If the analyte is amplified at the maximum number of cycles, the result for the analyte is positive, and the saturation intensity in each color can be used to decode the result using a coding scheme. In both cases, it is not necessary to run the PCR reaction to exceed the number of cycles set by the LOD for the instrument. Although the PCR method has been used to illustrate this quantitative method, a person of ordinary skill in the art will easily recognize that similar principles can be applied in any catalytic system, wherein the starting amount of the analyte limits the reaction rate, and the reaction rate can be measured and reported by the evolution of the signal intensity over a period of time.

[0201] VI. Compositions and Kits

[0202] The present disclosure also provides compositions and kits used with the methods described herein. The compositions may include any of the components, reaction mixtures and / or intermediates described herein, and any combination. For example, the present disclosure provides detection reagents used with the methods provided herein. Any suitable detection reagent may be provided, including hybridization probes labeled with fluorophores and quenchers and primers as described elsewhere in this specification.

[0203] In some cases, the composition comprises reagents for detecting at least seven analytes using four fluorophores. In some cases, the composition comprises reagents for detecting at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 analytes using four fluorophores. In some cases, the composition includes reagents for detecting at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 analytes using five fluorophores. In some cases, the composition includes reagents for detecting at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 analytes using six fluorophores. In some cases, the composition includes reagents for detecting at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 analytes using seven fluorophores.

[0204] In some cases, the composition includes primers. The composition can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 primer pairs.

[0205] The present invention also provides a test kit for implementing the method of the present invention. Therefore, various test kits are provided in suitable packaging. The test kit can be used for any one or more purposes described herein, and, accordingly, can contain instructions for detecting the presence or absence of each analyte in a plurality of analytes. The test kit can include a coding scheme, or a decoding matrix, to help users convert cumulative measurement results into results indicating the presence or absence of each analyte in a plurality of analytes. The test kit can be a diagnostic kit, for example, a diagnostic kit suitable for the detection of any analyte of the analyte listed herein. The test kit can include any composition provided in the present disclosure, including those listed above.

[0206] Figure 6 A schematic diagram of an exemplary kit of the invention is shown. Figure 6 , shows a test kit including a box body 601, which contains reagents 602 for detection of an analyte (e.g., a polynucleotide probe including a fluorophore and a quencher), and primers 603 for amplification of nucleic acids. The test kit may also contain any other reagents, such as reagents 604 suitable for performing an amplification reaction. In the embodiment depicted in 601, the reagents and primers for detection of the analyte are each contained in a single volume (such as a tube or bottle). However, these reagents may also be provided in separate volumes, as depicted by the box body shown in 605. Box body 605 comprises seven separate volumes. Three volumes 606, 607 and 608 contain reagents (or mixtures of such reagents) for detection of the analyte, and three volumes 609, 610 and 611 contain primers (or mixtures of primers). The test kit may also contain reagents 604 suitable for performing an amplification reaction.

[0207] supply Figure 6For illustrative purposes only. Any number of reagents for detection of an analyte (e.g., a probe), and any number of primers can be included in a single tube or bottle to suit the application. For example, in some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more reagents can be included in a single bottle for detection of an analyte. In other cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more primers can be included in a single bottle or tube for amplification. In some cases, primers and probes can be provided in the same bottle or tube. For example, each primer pair can provide 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more probes. 43, 44, 45, 46, 47, 48, 49, 50, or more primers can be provided per probe pair. The kit can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more bottles or tubes. Control analytes, and their associated detection reagents and primers, can also be included in the kit. These can be included in a separate bottle or tube, or, for example, in a bottle or tube 604 that provides reagents for an amplification reaction.As described elsewhere in this specification, any of the reagents in the kit may be lyophilized.

[0208] VII. Systems and Software

[0209] The method of the present disclosure can also be performed as part of a system. For example, one or more steps of the method described herein can be performed by software contained in a computer readable medium. The software can be used to encode analytes, check for degeneracy, reduce or eliminate degeneracy, and / or generate a decoding matrix. The software can also be used to convert the cumulative measurement results made on an instrument (e.g., a PCR machine) into information about the presence or absence of an analyte. For example, reference Figure 4 , the computer 401 can be connected to an input device 403, a display 402, and a printer 404. The computer can perform one or more steps of any of the methods described in the present disclosure, including, for example, the encoding method 410, the analysis method 420, and the decoding method 430. The computer can reduce or eliminate degeneracy, for example, as part of the encoding method 410. Similarly, the computer can generate a coding scheme that is non-degenerate by design, such as a coding scheme designed using mathematical iteration. The computer can also be connected to a network 405. The network can be a local area network and / or the Internet. The network can be used to perform the method of the present invention on multiple computers in different locations. For example, one computer can perform encoding, another computer can perform measurement, and another computer can perform decoding. A computer can perform any or all of these methods. The network can be used to transmit information obtained using the methods described in the present disclosure around the world, such as across national borders.

[0210] In one example, the software is embedded in the instrument that performs the measurement, such as a thermal cycler. In another example, the software is installed on a computer connected to the instrument that performs the measurement, such as a computer connected to a thermal cycler. In another example, the software is "cloud", i.e., on a computer that another computer can communicate with over a network.

[0211] Each element described above can be connected to a controller, and the controller communicates with each element, and coordinates their operation.For example, the controller can start a series of analytes to be encoded as the value of color and intensity.As described in the full text of the present disclosure, the controller can execute software to reduce or eliminate degeneracy by removing one or more potential analyte codes from the coding matrix.Then, the controller can automatically order the probe corresponding to this code from the manufacturer of the probe.Alternately, the controller can operate the oligonucleotide synthesizer that can synthesize the corresponding probe.After analyzing the sample (it can also be automatic) on the instrument, the controller can use the result of the decoding matrix processing analysis.Then, the controller can provide these results to the user.

[0212] VIII. Services

[0213] The method provided herein can also be performed as a service. For example, a service provider can obtain the identification of multiple analytes that a client wishes to analyze. The service provider can then encode each analyte to be detected by any method described herein, and provide the client with appropriate reagents for determination. The client can also perform determination and provide the service provider with results for decoding. The service provider can then provide the client with the decoded results. The client can also encode analytes, generate probes, and / or decode results by interacting with software installed locally (at the client's location) or remotely (for example, on a server accessible via a network). Exemplary clients include clinical laboratories, doctors, food and consumer product manufacturers, industrial manufacturers (for example, oil companies), etc. A client or group can be any suitable client or group that needs or wishes to use the method, system, composition, and kit of the present invention.

[0214] Example

[0215] Example 1: General Materials and Methods

[0216] 1. General reagents

[0217] TE buffer, pH 7 (LIFE TECHNOLOGIES, Carlsbad, CA); RNase-free ultrapure water (LIFETECHNOLOGIES, Carlsbad, CA); Taq 5x premix (FISHER SCIENTIFIC COMPANY, Tustin, CA).

[0218] 2. DNA sequences, primers and probes

[0219] Five nucleic acids from clinically relevant organisms were selected for exemplary studies: (1) human immunodeficiency virus 1 (HIV-1); (2) Plasmodium falciparum (malaria); (3) herpes simplex virus 2 (HSV-2); (4) Mycobacterium tuberculosis (TB); and (5) dengue virus type 3 (dengue fever).

[0220] Two diagnostically relevant regions from the HIV-1 genome, p17 and polyproteinase, were selected from the HIV-1 reference sequence at Los Alamos National Laboratory. The diagnostic sequence from the ChR 7 gene of Plasmodium falciparum was obtained from the UCSC Plasmodium falciparum genome browser. The herpes simplex virus 2 sequence was synthesized from a sequence obtained from the European Molecular Biology Library. Similarly, the diagnostic sequence of the rpoB gene in Mycobacterium tuberculosis was synthesized from a sequence obtained from the European Molecular Biology Library. The PCR diagnostic sequence of dengue virus type 3 was obtained from the National Institute of Health's Health Gene Sequence Database.

[0221] Oligonucleotides were synthesized by INTEGRATED DNA TECHNOLOGIES (IDT). Probe and primer pairs for each analyte were selected using an OLIGO analyzer from IDT. Sensing probes containing a fluorophore at the 5' end and a quencher at the 3' end were synthesized for all analytes. All oligonucleotides were lyophilized and reconstituted in TE buffer before use. Tables 9-14 show the sequence information, target sequence, primers, and probes for each of the six analytes.

[0222] Table 9. Sequence information of HIV-1 polyproteinase.

[0223]

[0224] Table 10. Sequence information of HIV p17.

[0225]

[0226] Table 11. Sequence information of Plasmodium ChR 7.

[0227]

[0228] Table 12. Sequence information of herpes simplex virus 2.

[0229]

[0230]

[0231] Table 13. Sequence information of Mycobacterium tuberculosis rpoB.

[0232]

[0233] Table 14. Sequence information of dengue virus type 3.

[0234]

[0235] 3. Polymerase chain reaction

[0236] All PCR reactions were performed on a ROCHE 480LIGHTCYCLER instrument. 45 cycles were performed with a hot start at 95°C for 60 seconds. The cycling conditions for denaturation, annealing, and extension were 45 seconds, 50 seconds, and 60 seconds at 95°C, 65°C, and 70°C, respectively. Each experiment was run in quintuplicate with a reaction volume of 15 μL. After each annealing step, fluorescence measurements were obtained in the following channels: 483nm to 533nm (FAM), 523nm to 568nm (Cy3), 558nm to 610nm (ROX), and 615nm to 670nm (Cy5). Fluorescence measurements can also be obtained after the hot start and 45 thermal cycles. The change in fluorescence intensity between these two measurements (after the hot start and at the end of 45 cycles) determines the endpoint fluorescence signal.

[0237] Example 2: Simultaneous detection of polynucleotide markers from pathogens using more than one color encoding method per analyte

[0238] This example describes the simultaneous detection of up to four polynucleotides selected from HIV (2 polynucleotides), HSV-2, Mycobacterium, Plasmodium, and Dengue virus. Using the reagents described in Table 15, 14 PCR reactions were assembled.

[0239] Table 15. Reagents for 14 PCR reactions.

[0240]

[0241]

[0242]

[0243]

[0244]

[0245] Referring to Table 15, reactions 1, 3, 5, 7, and 9 (containing analytes for HIV polyproteinase, Plasmodium, HSV, Mycobacterium, and Dengue, respectively) are positive controls that provide baseline fluorescence intensity for each polynucleotide analyte alone in the absence of the other analytes. As described more fully below, the change in fluorescence intensity in each color is used to provide a chromatogram ( Figure 3) is the expected cumulative intensity level for each color in the reaction. Reactions 2, 4, 6, 8 and 10 are negative controls, each used to determine the extent of nonspecific amplification. In these reactions, the primers provided are not specific for the analytes provided.

[0246] Experiments 11, 12, 13 and 14 were multiplex detection experiments. Encoding was performed as shown in Table 16. In each color, the fluorescence intensity measured for each assay is depicted as Figure 3 Black circle in the color spectrum.

[0247] Table 16. Coding scheme for pathogen detection.

[0248]

[0249]

[0250] To construct the chromatogram, in each color, the signal from the positive controls (reactions 1, 3, 5, 7, and 9) is used to assemble every possible combination of sequences present. The cumulative signal is plotted on a graph called a "chromatogram" ( Figure 3 ). The cumulative signals corresponding to all possible combinations of the same rank in the same color are organized into their own "band". Doing this for all four colors produces a level and band structure for which the experimental results for each combination can be evaluated (reactions 11, 12, 13, and 14). Figure 3 Three replicates of the chromatogram are shown, constructed using the positive control sample, and superimposed on the chromatogram with experimental results 4112, 3121 and 3102. The experimental results are indicated by black dots.

[0251] The intensity values ​​of each color in reactions 11-14 were measured in arbitrary fluorescence units (AFU), plotted in a color spectrum, and then assigned to bands based on two criteria. First, if the experimental result was within the band, then the result was assigned to a multiplexed band (e.g., Figure 3 Second, if the experimental result is between two bands, a two-step analysis is used. If the experimental result is between two plausible results, then the experimental result is assigned to the closest band (e.g., Figure 3 If the experimental result is between reasonable and unreasonable results, the result is assigned to the reasonable result (e.g., Figure 3 The value of FAM in the middle of the chromatogram is 3). Figure 8 Schematically shown are a plausible result of 4112 (upper panel), indicating the presence of HIV polyproteinase, Plasmodium, herpes simplex, and mycobacteria, and an illogical result of 4110 (lower panel), which could not be decoded and indicated a malfunction in the assay.

[0252] Using these criteria, the output of Experiment 12 is assigned as 4112, which is the correct answer. The output of Experiment 14 can be assigned as 2121 or 3121, but 2121 is unreasonable, so the result must be 3121, which is the correct answer. The output of Experiment 13 can be assigned as 3102 or 3101, both of which are reasonable, but the result is clearly closer to 2 than 1 in the Cy5 channel, so the correct answer, 3102, is assigned as the result. These results provide experimental validation of the methods presented in this article.

[0253] The results show a trend for the combined signal to be slightly lower than what would be expected from the simple sum of the positive control signals. This may be because the combination of the signals is not perfectly linear. This may be the result of additional effects of the chromatogram set-up that are not currently accounted for. These effects may be related to the absolute concentration of the quencher. The higher the absolute concentration of the quencher, the greater the percentage of the reaction volume it quenches, so that the same percentage of fluorophore released does not contribute as much to the cumulative signal as expected. One solution to this problem is to use low concentrations of probe so that the percentage loss in signal is negligible, regardless of how much quencher is released.

[0254] Figure 3 Each rank containing multiple combinations is shown to have a relatively wide band. The width of the band is ultimately the difference between the highest and lowest cumulative signals of the positive control samples. If all positive controls in a particular color produced exactly the same fluorescent signal, the width of each band would be zero. Because the signals instead differ, the resulting bands are relatively wide. However, there is a simple means to tighten the bands. As was done in the above experiment, instead of loading all probes at the same concentration, the concentration of each probe can be adjusted so that each resulting positive control signal is the same as the others in the same color. This approach will significantly tighten the bands, making it easier to determine the multiplexity of the signals of samples containing the analyte.

[0255] Example 3: Simultaneous detection of polynucleotide markers from pathogens using a color and different intensity encoding method based on each analyte

[0256] The present embodiment describes the simultaneous detection of up to three polynucleotides selected from dengue virus, HIV p17 and HIV polyproteinase. The coding scheme is as described in Table 11. As shown in Table 11, each analyte is encoded as a single value of fluorescence intensity in a single color. In this case, as described elsewhere in the present disclosure, the coding scheme is non-degenerate by design. This is achieved by assigning an intensity value to each analyte, and the intensity value assigned is equal to the cumulative intensity value of the previous analyte plus one. Of course, as described elsewhere in this manual, the initial assigned value can be greater than 1, for example, to better distinguish noise. Similarly, the value assigned to each analyte can be greater than the cumulative intensity value of the previous analyte plus one, for example, in order to increase the interval between the intensity, and to make the intensity assignment simpler.

[0257] Table 17. Coding scheme for detection of dengue virus, HIV polyproteinase and HIV p17.

[0258] FAM Dengue virus 1 HIV p17 2 HIV polyproteinase 4

[0259] The same reagents (e.g., template, primers, and probes) used in Example 2 are used here. The fluorescent signals produced by each probe in the positive control endpoint PCR reaction were measured and used to calculate the concentration of the probe that will produce a signal intensity of 1x, 2x, and 4x. The experiment is then set to detect seven non-empty combinations of analyte occurrence. Tables 18-24 provide the analyte PCR reaction components of each of these mixtures.

[0260] Table 18. Binary FAM assay 1 cocktail: HIV polyproteinase, HIV p17, and dengue virus.

[0261]

[0262] Table 19. Binary FAM assay 2 mix: Dengue virus and HIV p17.

[0263]

[0264]

[0265] Table 20. Binary FAM assay 3 mix: dengue virus and HIV polyproteinase.

[0266]

[0267] Table 21. Binary FAM Assay 4 Mix: HIV polyproteinase and HIV p17.

[0268]

[0269]

[0270] Table 22. Binary FAM Assay 5 Mix: Dengue Virus.

[0271]

[0272] Table 23. Binary FAM Assay 6 Mix: HIV p17.

[0273]

[0274]

[0275] Table 24. Binary FAM Assay 7 Mix: HIV Polyproteinase.

[0276]

[0277] The total fluorescence signal for each experiment was plotted on a chromatogram constructed as described above ( Figure 7 ). The width of the band around each positive control measurement is 1 x the uncertainty of the measurement spread. This uncertainty is equal to the standard deviation of the fluorescence signal of the last five PCR cycles of saturation.

[0278] Figure 7 The results of this experiment are shown in Table 11. The three results on the far right show the measured intensities of dengue virus alone (DEN), HIV p17 alone (P17), and HIV polyproteinase (TPP). The intensity assigned to each of these analytes is shown in Table 11, and Figure 7 It was confirmed that the three analytes, when present alone, produced the expected intensity results (see DEN, P17 and TPP). At the end of the assay, substantially all of the probes had been hydrolyzed, releasing substantially all of the fluorophores that emitted a signal. A fixed number of fluorophores produces a fixed and predictable amount of signal. Therefore, the target present should generally produce a fixed amount of signal, as determined by the number of probes added to the reaction volume.

[0279] As expected, the intensity value for the combination of analytes is equal to the sum of the assigned intensities. Because this encoding scheme is designed to be non-degenerate, each result can be unambiguously decoded into the presence or absence of a specific analyte. For example, from Figure 7 Starting from the left side of , the result of 7 indicates that all three analytes are present. The result of 3 indicates that the dengue virus and HIV p17 analytes are present, while the HIV polyproteinase is not present. The result of 5 indicates that the dengue virus and HIV polyproteinase are present, while the HIV p17 is not present. Finally, the result of 6 indicates that the HIV p17 and HIV polyproteinase are present, while the dengue virus is not present. As described elsewhere in this disclosure, this encoding scheme can be expanded to encode additional analytes. Sequence Listing <110> California Institute of Technology <120> Encoding and decoding of signals in multiplexed biochemical assays <130> 38075-716.601 <140> PCT / US2013 / 024509 <141> 2013-02-01 <150> 61 / 703,093 <151> 2012-09-19 <150> 61 / 594,480 <151> 2012-02-03 <160> 32 <170> PatentIn version 3.5 <210> 1 <211> 198 <212> DNA <213> Human immunodeficiency virus 1 <400> 1 ggaagctcta ttagatacag gagcagatga tacagtatta gaagaaatga gtttgccagg 60 aagatggaaa ccaaaaatga tagggggaat tggaggtttt atcaaagtaa gacagtatga 120 tcagatactc atagaaatct gtggacataa agctataggt acagtattag taggacctac 180 acctgtcaac ataattgg 198 <210> 2 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 2 ggaagctcta ttagatacag gagcag 26 <210> 3 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 3 ccaattatgt tgacaggtgt aggtcc 26 <210> 4 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <220> <223> 5'6-FAM <220> <223> 3'BHQ_1 <400> 4 tgagtttgcc aggaagatgg aaacca 26 <210> 5 <211> 199 <212> DNA <213> Human immunodeficiency virus 1 <400> 5 cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta 60 gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac 120 aagatagagg aagagcaaaa caaaagtaag aaaaaagcac agcaagcagc agctgacaca 180 ggacacagca atcaggtca 199 <210> 6 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 6 cagctacaac catcccttca gaca 24 <210> 7 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 7 tgacctgatt gctgtgtcct gtgt 24 <210> 8 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <220> <223> 5'6-FAM <220> <223> 3'BHQ_1 <400> 8 agcaaccctc tattgtgtgc atcaaagg 28 <210> 9 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <220> <223> 5'Cy3 <220> <223> 3'BHQ_2 <400> 9 aaagcacagc aagcagcagc tga 23 <210> 10 <211> 199 <212> DNA <213> Plasmodium falciparum <400> 10 gcctaacatg gctatgacgg gtaacgggga attagagttc gattccggag agggagcctg 60 agaaatagct accacatcta aggaaggcag caggcgcgta aattacccaa ttctaaagaa 120 gagaggtagt gacaagaaat aacaatgcaa ggccaattta aaaccttccc agagtaacaa 180 ttggagggca agtctggtg 199 <210> 11 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 11 gcctaacatg gctatgacgg gtaa 24 <210> 12 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 12 caccagactt gccctccaat tgtt 24 <210> 13 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <220> <223> 5'6-FAM <220> <223> 3'BHQ_1 <400> 13 attccggaga gggagcctga gaaata 26 <210> 14 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <220> <223> 5'6-ROXN <220> <223> 3'BHQ_2 <400> 14 aaggaaggca gcaggcgcgt aaatta 26 <210> 15 <211> 192 <212> DNA <213> Herpes simplex virus 2 <400> 15 tcagcccatc ctccttcggc agtatggagg gtgtcgcggc ggcgagccgc cgtccccaaa 60 gacgtgcggg tcgtacacgt acacgtacca gggcggcggg cctccgaccc ggtacgctct 120 cgtaaatgct tccctgctgg tgccgatctg ggaccgcgcc gcggagacat tcgagtacca 180 gatcgaactc gg 192 <210> 16 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 16 tcagcccatc ctccttcggc agtat 25 <210> 17 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 17 ccgagttcga tctggtactcgaatgt 26 <210> 18 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <220> <223> 5'6-FAM <220> <223> 3'BHQ_1 <400> 18 aaagacgtgc gggtcgtaca cgtaca 26 <210> 19 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <220> <223> 5'Cy5 <220> <223> 3'3IAbRQSp <400> 19 taaatgcttc cctgctggtg ccgat 25 <210> 20 <211> 200 <212> DNA <213> Mycobacterium tuberculosis <400> 20 gagtgcaaag acaaggacat gacgtacgcg gccccgctgt tcgtcacggc cgagttcatc 60 aacaacaaca ccggcgagat caagagccag acggtgttca tgggtgactt cccgatgatg 120 accgagaagg gcaccttcat catcaacggc accgagcgcg tcgtggtcag ccagctggtc 180 cgctcgcccg gtgtgtactt 200 <210> twenty one <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> twenty one gagtgcaaag acaaggacat gacg 24 <210> twenty two <211> 19 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> twenty two aagtacacac cgggcgagc 19 <210> twenty three <211> 25 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <220> <223> 5'6-FAM <220> <223> 3'BHQ_1 <400> twenty three cgctgttcgt cacggccgag ttcat 25 <210> twenty four <211> 26 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <220> <223> 5'Cy3 <220> <223> 3'BHQ_2 <400> twenty four agatcaagag ccagacggtg ttcatg 26 <210> 25 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <220> <223> 5'Cy5 <220> <223> 3'IAbRQSp <400> 25 aagggcacct tcatcatcaa cggca 25 <210> 26 <211> 200 <212> DNA <213> Dengue virus 3 <400> 26 atgccaactg tgattgagca cttagaaaga ctacaaagga aacatggagg aatgcttgtg 60 agaaatccac tctcacgaaa ctccacgcac gaaatgtatt ggatatccaa tggtacaggc 120 aacatcgtct cattcacaat gacacacagg agacccacca tagagaaaga tgtggattta 180 ggagcaggaa cccgacatgt 200 <210> 27 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 27 atgccaactg tgattgagca ct 22 <210> 28 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 28 acatgtcggg ttcctgctcc taaa 24 <210> 29 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <220> <223> 5'6-FAM <220> <223> 3'BHQ_1 <400> 29 acaaaggaaa catggaggaa tgcttgtga 29 <210> 30 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <220> <223> 5'Cy3 <220> <223> 3'BHQ_2 <400> 30 actctcacga aactccacgc acgaaa 26 <210> 31 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <220> <223> 5'6-ROXN <220> <223> 3'BHQ_2 <400> 31 acaatgacac acaggagacc caccat 26 <210> 32 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <220> <223> 5'Cy5 <220> <223> 3'IAbRQSp <400> 32 ggatatccaa tggtacaggc aacatcgt 28

Claims

1. A method for unambiguously detecting a plurality of M different amplicons in a single reaction mixture generated during a multiplex amplification reaction by: (1) encoding each amplicon into at least one fluorescence emission signal intensity (I), thereby generating a non-degenerate encoding scheme for the plurality of different amplicons; (2) Produce fluorescence emission intensity (i); (3) cumulatively measuring (i) corresponding to a plurality of different amplicons in the single reaction mixture, thereby providing a cumulative intensity measurement result; and (4) for each amplicon in the single reaction mixture, determining whether the amplicon is present or absent based on the cumulative intensity measurement result and (I) in the encoding scheme; wherein the M plurality of different amplicons are detected based on wavelengths emitted from sequence-specific probes or sequence-specific primers; wherein each sequence-specific probe or sequence-specific primer in a single reaction mixture produces no more than one fluorescent emission signal; and wherein the single reaction mixture undergoes a single set of program cycles, wherein the method is for non-diagnostic purposes and M is greater than the number of spectrally resolvable fluorescent probes.

2. The method according to claim 1, wherein M=log2(F+1), and F is the maximum cumulative value of (i) when all amplicons are present; or wherein (i) has a minimum value in a coding scheme selected from the group consisting of: at least 1, at least 2, at least 4, at least 8, at least 16, at least 32, and at least 64.

3. The method of claim 1, wherein the M plurality of different amplicons are detected based on wavelengths emitted from a fluorescent DNA binding dye.

4. The method of claim 1, wherein each (I) is increased in the encoding scheme by an amount equal to the cumulative maximum of all previous (I) plus one.

5. The method of claim 2, wherein each (I) is increased in the encoding scheme by an amount equal to the cumulative maximum of all previous (I) plus one.

6. The method of claim 3, wherein each (I) is increased in the encoding scheme by an amount equal to the cumulative maximum of all previous (I) plus one.

7. The method of any one of claims 1-6, wherein the amplicon is unambiguously detected without immobilization, separation, mass spectrometry, or melting curve analysis.

8. The method of any one of claims 1-6, wherein the method utilizes (I) and color of the fluorescent emission signal, wherein a cumulative measurement of the (I) and the color is obtained, and the cumulative measurement is converted to determine the presence or absence of each amplicon using a corresponding encoding scheme.

9. The method of claim 7, wherein the M plurality of different amplicons is 4 to 12.

10. The method of claim 8, wherein the M plurality of different amplicons is 4 to 12.

11. The method of claim 9 or 10, wherein the M plurality of different amplicons is 4.

12. The method of claim 9 or 10, wherein the M plurality of different amplicons is 5.

13. The method of claim 9 or 10, wherein the M plurality of different amplicons is 6.

14. The method of claim 9 or 10, wherein the M plurality of different amplicons is 7.

15. The method of claim 9 or 10, wherein the M plurality of different amplicons is 8.

16. The method of claim 9 or 10, wherein the M plurality of different amplicons is 9.

17. The method of claim 9 or 10, wherein the M plurality of different amplicons is 10 or 11.

18. The method of claim 9 or 10, wherein the M plurality of different amplicons is 12.

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