WO2024199149A1 - Multi-target detection kit based on droplet coding, and application thereof - Google Patents

Abstract

A multi-target detection kit based on droplet color scale coding, and an application thereof, belonging to the technical field of biological detection. Provided is a multi-target detection kit, which comprises a detection reagent, a color scale coding reagent, and a droplet generation reagent; the detection reagent is used for detecting a specific target in a sample to be detected; the color scale coding reagent is used for differentiating target types in the sample to be detected; each group of detection reagents comprises a fluorescent detection system or a self-luminous detection system capable of recognizing with specificity one type of target; the fluorescent detection system comprises a fluorescent marker; the self-luminous detection system comprises a combination of a light-emitting marker and an initiator thereof, or a self-luminous marker; the color scale coding reagent comprises a fluorescent marker; the emission wavelengths of different fluorescent markers in the color scale coding reagent do not interfere with one another; and the emission wavelengths between the fluorescent marker in the detection reagent and the fluorescent marker in the color scale coding reagent do not interfere with one another. The kit can achieve high-throughput multi-target detection.

Description

A multi-target detection kit based on droplet encoding and its application

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to a Chinese patent application filed with the Chinese Patent Office on March 24, 2023, with application number 202310300964.7 and invention name “A multi-target detection kit based on droplet color coding and its application”, the entire contents of which are incorporated herein by reference.

Technical Field

The present application relates to a multi-target detection kit based on droplet color-scale coding and its application, belonging to the field of biological detection technology.

Background Art

Precision medicine, which accurately diagnoses each patient and selects the most appropriate treatment method to minimize iatrogenic damage, minimize medical costs, and maximize patient benefits, is the continuous pursuit of clinical diagnosis and treatment. Accurate diagnosis is the basic prerequisite for achieving precision treatment. With the continuous development of biological research and clinical medicine, there are more and more scenarios for clinical diagnosis based on multiple different indicators, such as tumor typing and companion diagnosis, virus typing and variants, flora analysis, microbial testing, etc. With the continuous deepening of research, the sensitivity and specificity of detection technology continue to improve, but most detection technologies can only detect a single target in samples, which limits further promotion and application.

For example, SARS-CoV-2 is the seventh known coronavirus that can infect humans, and the disease caused by the virus is named COVID-19. Due to the high number of self-replications of SARS-CoV-2 and the high probability of mutation, a variety of mutant strains have been produced, and cases of reinfection continue to emerge (see the literature: R.L.Tillett, J.R.Sevinsky, P.D.Hartley, et al. Genomic evidence for reinfection with SARS-CoV-2: a case study. Lancet Infect. Dis. 2020; 21: 1.). According to incomplete statistics, SARS-CoV-2 mutant strains have developed to about 4,000, and there are significant differences in the transmission rate and mortality rate between different mutant strains. Accurate detection of multiple SARS-CoV-2 mutant strains is a necessary means to support its epidemiological investigation. Clarifying the subtypes of SARS-CoV-2 mutant strains is a necessary prerequisite for exploring its clinical consequences (morbidity, mortality) and treatment methods.

For example, cervical cancer, also known as cervical cancer, is a malignant tumor that occurs in the cervix of the uterus. More than 90% of cervical cancers are caused by HPV infection (see the literature: J.X.Wen, N.Sheng, R.Y.Wang, et al. Visualized Closed-Tube PCR with Hamming Distance 2 Code for 15 HPV Subtype Typing. Anal. Chem. 2021; 93: 5529-5536.). HPV is a circular double-stranded closed DNA virus, and about 40 subtypes have been identified as being associated with human reproductive system lesions. Based on the possibility of HPV infection leading to cancer development, these subtypes can be further divided into high-risk (HR-HPV) and low-risk (LR-HPV) subtypes. Due to the significant differences in the pathogenic mechanism, carcinogenic risk and prognosis of different subtypes of HPV, the realization of multiple HPV typing detection is of great significance for the clinical diagnosis and treatment of patients.

For example, colon cancer is a malignant tumor originating from the colon mucosal epithelium. Through the detection of 71 mutations in the EGFR pathway of 747 patients with colon cancer, it was found that the remission rate of patients with wild-type mutations in K-ras, BRAF, NRAS and PIK3CA after cetuximab treatment increased by nearly 70% (see the literature: W. De Roock, B. Claes, D. Bernasconi, et al. Lianidou. Development and Validation of Multiplex Liquid Bead Array Assay for the Simultaneous Expression of 14 Genes in Circulating Tumor Cells. Anal. Chem. 2019; 91, 5: 3443-3451.). By detecting multiple members of the entire EGFR pathway and conducting a comprehensive analysis of colon cancer, we can provide personalized diagnosis and treatment for colon cancer patients, design better drug combinations, and provide more guidance for clinicians' diagnosis, prognosis, and treatment (see literature: C. Parisi, A. Markou, A. Strati, et al. Development and Validation of Multiplex Liquid Bead Array Assay for the Simultaneous Expression of 14 Genes in Circulating Tumor Cells. Anal. Chem. 2019; 91, 5: 3443-3451.).

It can be seen that with the continuous variation of the target to be tested and the multi-factor signal transduction of the signal pathway, single target detection can no longer meet the clinical diagnosis needs. Therefore, it is urgent to develop a multi-target detection technology to provide the necessary technical support for variant sample detection and multi-factor detection.

Currently, genome sequencing (Next-generation sequence, NGS) can detect variant subtypes, but its high cost, The long detection time and reliance on professional bioinformatics analysis have limited further promotion and application. Nucleic acid amplification based on reverse transcription polymerase chain reaction (RT-PCR) can achieve specific and sensitive detection of subtypes. However, due to the limitation of the detection channel, the number of target genes that can be detected in a single reaction of RT-PCR is often limited to 4 to 6, which greatly limits the application and promotion of this technology under complex multi-target conditions. Based on the CRISPR/Cas system, it is expected to provide a new method for the highly sensitive detection of new coronavirus nucleic acid by utilizing its high specificity and efficient cutting characteristics (see literature: XKCheng, Y.Li, J.Kou, et al. Novel non-nucleic acid targets detection strategies based on CRISPR/Cas toolboxes: A review. Biosens. Bioelectron. 2022; 215: 114559.). However, due to the non-specific characteristics of the enzyme cleavage activity of the CRISPR/Cas system after activation, the development and application of this technology in the direction of multi-target/multiple detection is hindered.

Digital PCR (dPCR) technology is to divide the sample into many independent PCR microreactors, each reaction contains a small amount or no target sequence, and after PCR amplification, the concentration of the target sequence is quantified with statistically defined accuracy using the Poisson statistic and the amplification positive segmentation score. Digital PCR overcomes the weakness of RT-qPCR precise quantification and can absolutely quantify the target nucleic acid present in the sample. Claudia Alteri's team used dPCR to detect 55 throat swab samples from patients with negative RT-qPCR test results for the new coronavirus, screened out 19 new coronavirus positive samples, and was confirmed in clinical practice (see literature: C. Alteri, V. Cento, M. Antonello, et al. Detection and quantification of SARS-CoV-2 by droplet digital PCR in real-time PCR negative nasopharyngeal swabs from suspected COVID-19 patients. PLoS One. 2020; 15: e0236311.). In recent years, many teams have been working hard to apply dPCR technology to the field of multiplex detection. Miguel Alcaide et al. used dPCR technology for clinical KRAS gene variant subtype analysis, which can identify 13 variant subtypes of KRAS gene in clinical samples (see literature: M. Alcaide, M. Cheung, K. Bushell, et al. A Novel Multiplex Droplet Digital PCR Assay to Identify and Quantify KRAS Mutations in Clinical Specimens. J Mol Diagn. 2014). 019; 21:214-27.), Miguel Alcaide et al. used dPCR to identify suboptimal samples and abnormal cfDNA size distribution, and selected genes associated with high levels of circulating tumor DNA (ctDNA) (see literature: M. Alcaide, M. Cheung, J. Hillman, et al. Evaluating the quantity, quality and size distribution of cell-free DNA by multiplex droplet digital PCR. Sci Rep. 2020; 10:12564.).

Liquid chip technology is a high-throughput luminescence detection technology based on coded microspheres and flow technology. In essence, it achieves color coding by mixing fluorescent dyes of different concentrations in polystyrene microspheres. Different color levels correspond to different targets to achieve multiple detection. Yong Yan's team used Luminex xMAP liquid chip analyzer to detect six common respiratory viruses such as influenza virus (see literature: Y. Yan, J. Y. Luo, Y. Chen, et al. A multiplex liquid-chip assay based on Luminex xMAP technology for simultaneous detection of six common respiratory viruses. Oncotarget. 2017 Jun 17; 8(57): 96913-96923.). Wang Changchun's team proposed a new method for sensitive detection of specific single-stranded DNA (single Stranded DNA) sequences based on surface enhanced Raman spectroscopy (SERS) liquid chip, which achieved the detection of three types of ssDNA (see literature: J.M.Li, W.F.Ma, L.J.You, et al. Highly sensitive detection of target ssDNA based on SERS liquid chip using suspended magnetic nanospheres as capturing substrates. Langmuir. 2013; 29: 6147-55.).

Digital PCR technology and liquid chip technology have their own unique advantages in multiple target analysis, but they also have some limitations. Limited by the selection of fluorescent probes and the limitations of detection methodology, the throughput of digital PCR multiple detection is not enough to meet the needs of virus variant detection; the inevitable heating and cooling process in the PCR reaction can easily cause the droplets to break or merge, and the relevant results have to be discarded. The synthesis of microspheres of different color levels is the basic premise of liquid chip technology, which has high requirements for synthesis technology and workload; at the same time, most of the related reactions are completed on the surface of the microspheres, and the reaction efficiency of the microsphere interface is 1 to 3 orders of magnitude lower than that of the pure liquid phase reaction, which has a great impact on the detection sensitivity (see literature: X. Zhai, J. Li, Y. Cao, et al. A nanoflow cytometric strategy for sensitive ctDNA detection via magnetic separation and DNA self-assembly. Anal Bioanal Chem. 2019; 411: 6039-47.). If the advantages of digital PCR technology and liquid phase chip technology can be combined, it is expected to achieve multiplex detection with high specificity and high sensitivity, targeting multiple SARS-CoV-2 variants at the same time, but there is still no relevant research.

Summary of the invention

To solve the above problems, the present application provides a multi-target detection kit based on droplet color-scale coding, the multi-target detection kit comprising at least one group of detection reagents, at least one group of color-scale coding reagents and at least one group of droplet generation reagents; the detection reagents are used to perform qualitative or quantitative detection of specific targets in a sample to be tested; the color-scale coding reagents are used to distinguish the types of targets in the sample to be tested; The droplet generation reagent is used to generate droplets of an aqueous phase obtained by mixing a detection reagent, a color scale coding reagent and a sample to be tested; each group of detection reagents comprises at least one fluorescence detection system or a self-luminescent detection system that can specifically identify a target; the fluorescence detection system comprises at least one fluorescent marker; the self-luminescent detection system comprises at least one combination of a luminescent marker and its initiator or at least one self-luminescent marker; the color scale coding reagent comprises at least one fluorescent marker; the emission wavelengths of different fluorescent markers in the color scale coding reagent do not cross-talk with each other; the emission wavelengths of the fluorescent marker in the detection reagent and the fluorescent marker in the color scale coding reagent do not cross-talk with each other.

In the present application, the non-crosstalk means that it can be effectively distinguished by an instrument or software algorithm.

In one embodiment of the present application, the fluorescence detection system comprises a one-pot fluorescence detection system. The one-pot fluorescence detection system refers to a fluorescence detection system that completes multiple steps of reactions that require separate reactions to synthesize a target molecule in the same reaction vessel without separation and purification steps of intermediate products.

The one-pot fluorescence detection system includes at least one of a PCR fluorescence detection system, a constant temperature amplification fluorescence detection system, a fluorescence immunoassay system, a chemiluminescence detection system or a CRISPR/Cas-based fluorescence detection system.

In one embodiment of the present application, the PCR fluorescence detection system includes an upstream primer, a downstream primer and a probe; the probe is modified with a fluorescent marker.

In one embodiment of the present application, the fluorescent marker includes at least one of a fluorescent group, a fluorescent nanomaterial or a fluorescent protein.

In one embodiment of the present application, the fluorescent group includes at least one of FAM, CY5, HEX, VIC, APC, Cy5.5, APC-Cy7, PC5.5, PC7, APC-A700, APC-H7, V450, PC5, DRAQ7, Cy3, Cy7, PBXL-1, PBXL-3, ROX, rare earth chelates or fluorescent probe chelates; the fluorescent nanomaterial includes at least one of fluorescent quantum dots, fluorescent polystyrene microspheres, fluorescent silica, fluorescent polyphosphazene nanomaterials, upconversion fluorescent particles, rare earth-doped fluorescent nanomaterials, fluorescent labeled PEG nanomaterials or fluorescent nanoclusters.

In one embodiment of the present application, the fluorescent group includes at least one of aggregation-induced emission probes and their derivatives, derivatives of the above-mentioned fluorescent groups, doped or labeled materials of fluorescent groups and their derivatives, temperature-controlled luminescent materials, magnetically controlled luminescent materials, and field-controlled luminescent materials.

In one embodiment of the present application, the emission wavelength of FAM is 518 nm; the emission wavelength of CY5 is 664 nm; the emission wavelength of HEX is 556 nm; the emission wavelength of VIC is 554 nm; the emission wavelength of APC is 660 nm; the emission wavelength of Cy5.5 is 712 nm; the emission wavelength of APC-Cy7 is 780 nm; the emission wavelength of PC5.5 is 685 nm; the emission wavelength of PC7 is 780 nm; the emission wavelength of APC- The emission wavelength of A700 is 712nm; the emission wavelength of APC-H7 is 780nm; the emission wavelength of V450 is 440nm; the emission wavelength of PC5 is 685nm; the emission wavelength of DRAQ7 is 780nm; the emission wavelength of Cy3 is 562nm; the emission wavelength of Cy7 is 773nm; the emission wavelength of PBXL-1 is 666nm; the emission wavelength of PBXL-3 is 662nm; and the emission wavelength of ROX is 602nm.

In one embodiment of the present application, the target is at least one of nucleic acid, protein, biological small molecule or cell.

The present application also provides a multi-target detection method based on droplet color coding, which is not intended for diagnosis and treatment of diseases. The multi-target detection method uses the multi-target detection kit to detect samples to be tested.

In one embodiment of the present application, the method comprises the following steps:

Step 1: respectively mixing the sample to be tested and the detection reagent with the color scale coding reagent of different fluorescence concentrations and/or different emission wavelengths to obtain a mixed system; using a droplet generation reagent to generate droplets of the mixed system to obtain droplets, so as to simultaneously establish the color scale coding system and the detection system in the same droplet;

Step 2: mixing the droplets and incubating them to obtain an incubation solution; performing fluorescence detection on the incubation solution to obtain a fluorescence detection result;

Step three: Distinguish the target type in the sample to be tested according to the fluorescence detection result of the fluorescent marker in the coding reagent, and perform qualitative or quantitative detection of the specific target in the sample to be tested according to the fluorescence detection result of the fluorescent marker in the detection reagent; during the detection process, one target corresponds to one group of detection reagents.

In one embodiment of the present application, in step 2, fluorescence detection is performed using a flow cytometer, a digital PCR instrument, or a fluorescence imaging device.

In one embodiment of the present application, the target is at least one of nucleic acid, protein, biological small molecule or cell.

The present application also provides a droplet color scale coding kit, which includes at least one group of color scale coding reagents and at least one group of droplet generation reagents; the droplet generation reagents are used to generate droplets from an aqueous phase containing color scale coding reagents; the color scale coding reagents include at least one fluorescent marker; the emission wavelengths of different fluorescent markers in the color scale coding reagents do not cross-talk with each other; the color scale coding reagents perform color scale coding on the generated droplets through the two-dimensional information of the fluorescent markers contained in the droplets on the fluorescence concentration and emission wavelength.

In one embodiment of the present application, the coding capacity of the color-scale coding reagent = color-scale number 1 × color-scale number 2… × color-scale number n, wherein n = the number of probe types, which is an arbitrary positive integer; the color-scale number i=1~n of the color-scale coding reagent refers to the number of corresponding probe concentrations that can be effectively distinguished by the detection instrument, and each color-scale number is an arbitrary positive integer.

In one embodiment of the present application, the fluorescent marker includes at least one of a fluorescent group, a fluorescent nanomaterial or a fluorescent protein.

In one embodiment of the present application, the fluorescent group includes at least one of FAM, CY5, HEX, VIC, APC, Cy5.5, APC-Cy7, PC5.5, PC7, APC-A700, APC-H7, V450, PC5, DRAQ7, Cy3, Cy7, PBXL-1, PBXL-3, ROX, rare earth chelates or fluorescent probe chelates; the fluorescent nanomaterial includes at least one of fluorescent quantum dots, fluorescent polystyrene microspheres, fluorescent silica, fluorescent polyphosphazene nanomaterials, upconversion fluorescent particles, rare earth-doped fluorescent nanomaterials, fluorescent labeled PEG nanomaterials or fluorescent nanoclusters.

In one embodiment of the present application, the emission wavelength of FAM is 518 nm; the emission wavelength of CY5 is 664 nm; the emission wavelength of HEX is 556 nm; the emission wavelength of VIC is 554 nm; the emission wavelength of APC is 660 nm; the emission wavelength of Cy5.5 is 712 nm; the emission wavelength of APC-Cy7 is 780 nm; the emission wavelength of PC5.5 is 685 nm; the emission wavelength of PC7 is 780 nm; the emission wavelength of APC- The emission wavelength of A700 is 712nm; the emission wavelength of APC-H7 is 780nm; the emission wavelength of V450 is 440nm; the emission wavelength of PC5 is 685nm; the emission wavelength of DRAQ7 is 780nm; the emission wavelength of Cy3 is 562nm; the emission wavelength of Cy7 is 773nm; the emission wavelength of PBXL-1 is 666nm; the emission wavelength of PBXL-3 is 662nm; and the emission wavelength of ROX is 602nm.

The present application also provides a droplet color scale coding method, which uses the above-mentioned droplet color scale coding kit to perform droplet generation and droplet color scale coding on a sample to be coded.

In one embodiment of the present application, the method comprises the following steps:

Step 1: Mix the sample to be encoded with color-scale coding reagents of different fluorescence concentrations and/or different emission wavelengths to obtain a mixed system; use a droplet generation reagent to generate droplets of the mixed system to obtain droplets, so as to establish a color-scale coding system in the droplets;

Step 2: mixing the droplets and incubating them to obtain an incubation solution; performing fluorescence detection on the incubation solution to obtain a fluorescence detection result;

Step 3: Color code the sample to be coded according to the fluorescence detection result of the fluorescent marker in the coding reagent.

The present application also provides the use of the above-mentioned multi-target detection kit or the above-mentioned multi-target detection method or the above-mentioned droplet color scale coding kit or the above-mentioned droplet color scale coding method in target detection, and the application is for non-disease diagnosis and treatment purposes.

In one embodiment of the present application, the target is at least one of nucleic acid, protein, biological small molecule or cell.

The technical solution of this application has the following advantages:

The present application provides a multi-target detection kit based on droplet color scale coding, the multi-target detection kit comprising at least one group of detection reagents, at least one group of color scale coding reagents and at least one group of droplet generation reagents; the detection reagents are used to perform qualitative or quantitative detection of specific targets in the sample to be tested; the color scale coding reagents are used to distinguish the types of targets in the sample to be tested; each group of detection reagents comprises at least one fluorescence detection system or self-luminescence detection system that can specifically identify a target; the fluorescence detection system comprises at least one fluorescent marker; the self-luminescence detection system comprises at least one self-luminescence marker; the color scale coding reagent comprises at least one fluorescent marker; the emission wavelengths of different fluorescent markers in the color scale coding reagent do not cross-talk with each other; the emission wavelengths of the fluorescent marker in the detection reagent and the fluorescent marker in the color scale coding reagent do not cross-talk with each other. The multi-target detection kit comprehensively utilizes the advantages of solution phase reaction in droplet coding technology and the characteristics of color-scale coding in liquid chip technology, and uses two-dimensional information of fluorescence intensity and emission wavelength for coding, and proposes a multiple target detection strategy of droplet color-scale coding. On the one hand, by utilizing the characteristics and advantages of detection technologies such as flow analysis technology, digital PCR technology, or fluorescence imaging technology in terms of fluorescence intensity, fluorescence channels, etc., it is expected to achieve more than 100 double-color multiplexing and more than 1,000 triple-color multiplexing. High-throughput multi-target detection (the coding capacity of droplet color-scale coding using the multi-target detection kit = color-scale number 1 × color-scale number 2… × color-scale number n, where n = number of probe types); on the other hand, by selecting different fluorescent channels, and by selecting fluorescent markers with non-overlapping emission wavelengths, a color-scale coding system and a detection system are simultaneously established in the same droplet, so that the two do not interfere with each other, and the two systems are mutually independent, and then multi-target, multi-channel, and multi-excitation are performed to achieve effective distinction of fluorescence signals and construct a controllable droplet color-scale coding system (for example, FAM fluorescent groups are selected to perform qualitative or quantitative detection of three targets respectively, and at the same time, different concentrations of Cy5 fluorescent groups are used to encode and distinguish the three targets at different color levels to achieve the purpose of multi-target detection); in addition, the multi-target detection kit is designed with a droplet coding system with a high coding capacity, and is relatively independent of the detection system, making it easier to expand. In summary, the multi-target detection kit has the following advantages:

High encoding capacity: It uses information from two dimensions, probe type and fluorescence intensity, to encode, breaking through the bottleneck of traditional multiplex fluorescence intensity detection;

Strong compatibility: This coding system is a homogeneous system and a liquid phase coding system. It does not need to be associated with the detection system related elements. It is compatible with homogeneous high-sensitivity detection systems and avoids the limitations of various complex processes such as the synthesis, surface modification and solid phase reaction of liquid phase chip microspheres.

Great scalability: The coding probe used in this coding system is independent of the detection system. As long as the emission wavelength of the detection system and the coding probe do not crosstalk with each other, it can be combined with this coding system to achieve multiple detections.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Schematic diagram of the principle of the color-coded multi-target nucleic acid detection kit.

Figure 2: Schematic diagram of the principle of color-coded multi-target detection.

Figure 3: Schematic diagram of the structure of the droplet generation chip.

Figure 4: Droplet generation information data sheet.

Figure 5: Microscope image of generated droplets.

Figure 6: Microscopic results of generated droplets after storage at 4°C.

Figure 7: Fluorescence emission spectra of FAM encoded with different Cy5 concentrations.

Figure 8: FAM channel digital PCR instrument results under the condition of Cy5 encoding concentration of 0nM.

Figure 9: FAM channel digital PCR instrument results under the condition of Cy5 encoding concentration of 20nM.

Figure 10: FAM channel digital PCR instrument results under the condition of Cy5 encoding concentration of 100nM.

Figure 11: FAM channel digital PCR instrument results under the condition of Cy5 encoding concentration of 250nM.

Figure 12: Digital PCR instrument results with color coding for different FAM concentrations.

Figure 13: Digital PCR instrument results with color coding for different Cy5 concentrations.

FIG. 14 : FAM channel result diagram of target detection of PCR in a droplet system.

Figure 15: FAM and Cy5 dual-channel results of multi-target encoding detection in the droplet system.

DETAILED DESCRIPTION

The following examples are provided for a better understanding of the present application, but are not limited to the best implementation mode described, and do not limit the content and protection scope of the present application. Any product identical or similar to the present application obtained by anyone under the inspiration of the present application or by combining the features of the present application with other prior arts shall fall within the protection scope of the present application.

If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

Example 1: A multi-target nucleic acid detection kit based on color coding

The present embodiment provides a multi-target nucleic acid detection kit based on color-scale coding, wherein the multi-target nucleic acid detection kit is composed of at least one group of detection reagents and one group of color-scale coding reagents; the detection reagents are composed of at least one PCR fluorescence detection system that can specifically identify a nucleic acid target; the PCR fluorescence detection system is composed of an upstream primer, a downstream primer and a probe; the probe is modified with a fluorescent group FAM (emission wavelength 518nm); the color-scale coding reagent is composed of a fluorescent dye CY5 (emission wavelength 664nm) (the detection principle is shown in Figure 1).

Example 2: A multi-target nucleic acid detection method based on color coding

This embodiment provides a multi-target nucleic acid detection method based on color scale coding, the method using the multi-target nucleic acid detection kit of embodiment 1, comprising the following steps:

Step 1: The sample to be tested and the detection reagent are respectively mixed with the color-scale coding reagent of the fluorescence concentration gradient to obtain a mixed system; the mixed system is subjected to droplet generation using a droplet generation reagent to obtain droplets, so as to simultaneously establish the color-scale coding system and the detection system in the same droplet (see Figure 2 for the detection principle);

Step 2: mixing the droplets and incubating them to obtain an incubation solution; using a flow cytometer, a digital PCR instrument, or a fluorescence imaging device to perform fluorescence detection on the incubation solution to obtain a fluorescence detection result;

Step three: Distinguish the target type in the sample to be tested according to the fluorescence detection result of the fluorescent marker in the coding reagent, and perform qualitative or quantitative detection of the specific target in the sample to be tested according to the fluorescence detection result of the fluorescent marker in the detection reagent; during the detection process, one target corresponds to one group of detection reagents.

Experimental Example 1: Feasibility Verification of Droplet Generation

The generation of droplets is the basis of the encoding system. In order to verify the feasibility of the multi-target nucleic acid detection kit described in Example 1 and the multi-target nucleic acid detection method described in Example 2 in terms of droplet generation, this experimental example verifies and characterizes the droplets generated. The experimental process is as follows:

The FAM fluorescent dye (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was diluted with dimethyl sulfoxide (DMSO) to a concentration of 500 nM to obtain a FAM fluorescent dye dilution solution; the FAM fluorescent dye dilution solution and the premixed oil (purchased from Bio-Rad, USA) were mixed at a volume ratio of 1:1 to obtain a mixed solution; 30 μL of the mixed solution was first added to the small hole of the droplet generation chip (purchased from Bio-Rad, USA, the structure is shown in Figure 3), and then 80 μL of the droplet generation oil (purchased from Bio-Rad, USA) was added to the small hole of the droplet generation chip. o-Rad Company) was added into the middle hole of the droplet generation chip, and then the droplet generation chip was placed in a droplet generator (purchased from Suzhou Zhongke Xilai Technology Co., Ltd.) to react at room temperature (25°C) for 2 minutes, so that the mixed solution and droplet generation oil generated droplets and converged in the large holes of the droplet generation chip; 50 μL of the droplets generated in the large holes of the droplet generation chip were taken and placed in an eight-tube strip, analyzed with a digital PCR instrument (purchased from Suzhou Zhongke Xilai Technology Co., Ltd.), and observed with a microscope. The analysis and observation results are shown in Figures 4 to 5.

As shown in FIG4 , the 50 μL system generates nearly 20,000 droplets.

As shown in Figure 5, the generated droplets have uniform particle size and intact morphology, and the particle size is measured to be about 110 μm.

Experimental Example 2: Verification of the stability of droplet generation

The stability of droplet generation is the basis of the encoding system. In order to verify the droplet generation stability of the multi-target nucleic acid detection kit described in Example 1 and the multi-target nucleic acid detection method described in Example 2, this experimental example explored the stability of the generated droplets. The experimental process is as follows:

The droplets obtained in Experimental Example 1 were stored at 4° C. for one month. During the storage period, they were observed under an optical microscope every other week. The observation results are shown in FIG6 .

As can be seen from Figure 6, the droplet morphology has not changed significantly, indicating that the droplet has good stability and can meet the subsequent experimental requirements.

Experimental Example 3: Feasibility Verification of Dual-Fluorophore Color-Scale Coding

The droplet coding system requires the participation of multiple fluorescent groups. In order to verify the feasibility of the multi-target nucleic acid detection kit described in Example 1 and the multi-target nucleic acid detection method described in Example 2 in encoding multiple fluorescent groups, this experimental example explored whether multiple fluorescent groups would cause crosstalk. The experimental process is as follows:

CY5 fluorescent dye (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was diluted with dimethyl sulfoxide (DMSO) to a concentration of 500nM, 200nM, 40nM, and 0nM, respectively, to obtain CY5 fluorescent dye dilution solutions of different concentrations; FAM fluorescent dye (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was diluted with dimethyl sulfoxide (DMSO) to a concentration of 400nM, to obtain FAM fluorescent dye dilution solutions; CY5 fluorescent dye dilution solutions of different concentrations were taken and mixed with FAM fluorescent dye dilution solutions at a volume ratio of 1:1 to obtain to a mixed fluorescent dye solution containing different concentrations of CY5 fluorescent dye; first, 30 μL of the mixed fluorescent dye solution containing different concentrations of CY5 fluorescent dye was added to the small hole of the droplet generation chip (purchased from Bio-Rad, USA, the structure is shown in Figure 3), and then 80 μL of droplet generation oil (purchased from Bio-Rad, USA) was added to the middle hole of the droplet generation chip, and then the droplet generation chip was placed in a droplet generator (purchased from Suzhou Zhongke Xilai Technology Co., Ltd.) to react at room temperature (25°C) for 2 minutes, so that the mixed fluorescent dye solution and droplet generation oil were generated. The droplets converge in the large holes of the droplet generation chip; 50 μL of the droplets generated in the large holes of the droplet generation chip are placed in an eight-tube strip and analyzed using a fluorescence spectrophotometer and a digital PCR instrument (purchased from Suzhou Zhongke Xilai Technology Co., Ltd.). The analysis results are shown in Figures 7 to 11 (Figure 7 shows that the CY5 concentration is the final concentration).

As shown in Figure 7, the addition of different concentrations of CY5 did not significantly affect the fluorescence emission spectrum of FAM. As shown in Figures 8 to 11, the addition of CY5 also did not significantly affect the FAM fluorescence intensity of the droplets.

Experimental Example 4: Feasibility verification of droplet encoding with different fluorescent group concentration gradients

The droplet encoding is performed by fluorescent groups of different concentrations. Whether the droplet groups can be effectively distinguished is the key to the droplet encoding system. In order to verify the effectiveness of the multi-target nucleic acid detection kit described in Example 1 and the multi-target nucleic acid detection method described in Example 2 in distinguishing droplet groups, the feasibility of fluorescent group concentration gradient droplet encoding was explored in this experimental example. The experimental process is as follows:

Experiment 1: FAM fluorescent dye (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was diluted with dimethyl sulfoxide (DMSO) to concentrations of 200nM, 400nM, 650nM, 900nM, and 1300nM, respectively, to obtain FAM fluorescent dye dilutions of different concentrations; the FAM fluorescent dye dilutions of different concentrations were mixed with premixed oil (purchased from Bio-Rad, USA) at a volume ratio of 1:1 to obtain mixed solutions; 30μL of the mixed solution was first added to the small droplet generation chip (purchased from Bio-Rad, USA, structure shown in Figure 3) The droplet generation chip was placed in a droplet generator (purchased from Suzhou Zhongke Xilai Technology Co., Ltd.) for reaction at room temperature (25°C) for 2 minutes, so that the mixed solution and the droplet generation oil generated droplets and converged in the large hole of the droplet generation chip; 50 μL of the droplets generated in the large hole of the droplet generation chip were placed in an eight-tube strip, analyzed with a digital PCR instrument (purchased from Suzhou Zhongke Xilai Technology Co., Ltd.), and observed with a microscope. The analysis and observation results are shown in Figure 12.

Experiment 2: CY5 fluorescent dye (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was diluted with dimethyl sulfoxide (DMSO) to concentrations of 20nM, 30nM, 40nM, 50nM, 65nM, 100nM, 150nM, and 200nM, respectively, to obtain CY5 fluorescent dye dilutions of different concentrations; CY5 fluorescent dye dilutions of different concentrations were mixed with premixed oil (purchased from Bio-Rad, USA) at a volume ratio of 1:1 to obtain mixed solutions; 30μL of the mixed solution was first added to the droplet generation chip (purchased from Bio-Rad, USA, structure 3), then 80 μL of droplet generation oil (purchased from Bio-Rad, USA) was added to the middle hole of the droplet generation chip, and then the droplet generation chip was placed in a droplet generator (purchased from Suzhou Zhongke Xilai Technology Co., Ltd.) to react at room temperature (25°C) for 2 minutes, so that the mixed solution and droplet generation oil generated droplets and converged in the large hole of the droplet generation chip; 50 μL of the droplets generated in the large hole of the droplet generation chip were placed in an eight-tube strip, analyzed with a digital PCR instrument (purchased from Suzhou Zhongke Xilai Technology Co., Ltd.), and observed with a microscope. The analysis and observation results are shown in Figure 13.

As shown in Figure 12, five different concentrations of FAM can achieve effective distinction and have the ability of color coding.

As shown in Figure 13, changing FAM to CY5 did not significantly interfere with the experimental results, and adding 8 different concentrations of CY5 can still achieve effective distinction and has the ability of color coding.

Experimental Example 5: Feasibility verification of droplet encoding under PCR system

In this experiment, BCR-ABL (the nucleotide sequence of BCR-ABL is shown in SEQ ID NO.1) was used as the target nucleic acid, and the droplet encoding feasibility of the multi-target nucleic acid detection kit described in Example 1 and the multi-target nucleic acid detection method described in Example 2 under the PCR system was verified (the nucleotide sequence of the upstream primer F1 targeting BCR-ABL used in the PCR system is shown in SEQ ID NO.2, the nucleotide sequence of the downstream primer R1 is shown in SEQ ID NO.3, and the nucleotide sequence of the probe Probe1 is shown in SEQ ID NO.4. The 5' end of the probe Probe1 is connected with FAM and the 3' end is connected with TAMRA. The sequences used in the PCR system are synthesized by Shanghai Shenggong Biological Co., Ltd.). The verification process is as follows:

10 μM upstream primer F1, 10 μM downstream primer R1, 10 μM probe Probe1 and 100 nM target nucleic acid BCR-ABL were mixed and incubated with ddH ₂ O to make its volume 15 μL, to obtain a PCR system; add 15 μL of premixed solution (purchased from Bio-Rad, USA) in the middle of the PCR system, and mix it thoroughly to obtain a mixed solution; solution; first take 30 μL of the mixed solution and add it to the small hole of the droplet generation chip (purchased from Bio-Rad, USA, the structure is shown in Figure 3), then add 80 μL of droplet generation oil (purchased from Bio-Rad, USA) to the middle hole of the droplet generation chip, and then place the droplet generation chip in a droplet generator (purchased from Suzhou Zhongke Xilai Technology Co., Ltd.) to react at room temperature (25°C) for 2 minutes, so that the mixed solution and the droplet generation oil generate droplets and converge in the large hole of the droplet generation chip; take the droplets generated in the large hole of the droplet generation chip and place them in a PCR instrument (purchased from Xi'an Tianlong Technology Co., Ltd.) for PCR reaction to obtain a reaction solution; take 50 μL of the reaction solution and place it in an eight-tube strip, and analyze it with a digital PCR instrument (purchased from Suzhou Zhongke Xilai Technology Co., Ltd.), The analysis results are shown in FIG14 ; wherein the conditions for the PCR reaction are: ① 95° C. for 10 min; ② 94° C. for 30 s, 60° C. for 1 min (39 cycles); ③ 98° C. for 10 min.

As shown in Figure 14, when the target nucleic acid BCR-ABL is present, the droplet fluorescence value is significantly enhanced, indicating that the system is feasible.

Experimental Example 6: Feasibility verification of multiple droplet encoding under PCR system

In this experiment, BCR-ABL (the nucleotide sequence of BCR-ABL is shown in SEQ ID NO.1) and ABL (the nucleotide sequence of ABL is shown in SEQ ID NO.5) were used as target nucleic acids to verify the feasibility of droplet encoding in the PCR system of the multi-target nucleic acid detection kit described in Example 1 and the multi-target nucleic acid detection method described in Example 2 (the nucleotide sequence of the upstream primer F1 targeting BCR-ABL used in the PCR system is shown in SEQ ID NO.2, the nucleotide sequence of the downstream primer R1 is shown in SEQ ID NO.3, and the probe Pro The nucleotide sequence of be1 is shown in SEQ ID NO.4, the 5' end of probe Probe1 is connected with FAM, and the 3' end is connected with TAMRA, the nucleotide sequence of the upstream primer F2 targeting ABL used in the PCR system is shown in SEQ ID NO.6, the nucleotide sequence of the downstream primer R2 is shown in SEQ ID NO.7, the nucleotide sequence of the probe Probe2 is shown in SEQ ID NO.8, the 5' end of probe Probe2 is connected with FAM, and the 3' end is connected with TAMRA, the sequences used in the PCR system are synthesized by Shanghai Shenggong Biological Co., Ltd., and the verification process is as follows:

10 μM upstream primer F1, 10 μM downstream primer R1, 10 μM probe Probe1 and 0 nM target nucleic acid BCR-ABL were mixed with 30 nM and 50 nM CY5 fluorescent dyes (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), respectively, and the volume was supplemented to 15 μL with ddH ₂ O to obtain the first PCR system; 10 μM upstream primer F1, 10 μM downstream primer R1, 10 μM probe Probe1 and 100 nM target nucleic acid BCR-ABL were mixed with 30 nM and 50 nM CY5 fluorescent dyes (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), respectively, and the volume was supplemented to 15 μL with ddH ₂ O to obtain the second PCR system; 10 μM upstream primer F1, 10 μM downstream primer R1, 10 μM probe Probe1 and 200 nM target nucleic acid BCR-ABL were mixed with 30 nM and 50 nM CY5 fluorescent dyes (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), respectively, and the volume was supplemented to 15 μL with ddH 2 O. CY5 fluorescent dye (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was mixed, and the volume was supplemented to 15 μL with ddH ₂ O to obtain the third group of PCR systems; 15 μL of premixed solution (purchased from Bio-Rad, USA) was added to the three groups of PCR systems respectively, and they were fully mixed to obtain three groups of mixed solutions; first, 30 μL of the mixed solution was added to the small hole of the droplet generation chip (purchased from Bio-Rad, USA, the structure is shown in Figure 3), and then 80 μL of droplet generation oil (purchased from Bio-Rad, USA) was added to the middle hole of the droplet generation chip, and then the droplet generation chip was placed in a droplet generator (purchased from Suzhou Zhongke Xilai Technology Co., Ltd.) at room temperature (25°C) for 2 minutes, so that the three groups of mixed solutions and the droplet generation oil respectively generated three groups of droplets and gathered in the large hole of the droplet generation chip; the three groups of droplets generated in the large hole of the droplet generation chip were placed in a PCR instrument (purchased from Xi'an Tianlong Technology Co., Ltd.) for PCR reaction to obtain reaction solutions A1~A3;

10 μM upstream primer F2, 10 μM downstream primer R2, 10 μM probe Probe2 and 0 nM target nucleic acid ABL were mixed with 150 nM and 200 nM CY5 fluorescent dyes (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), respectively, and the volume was made up to 15 μL with ddH ₂ O to obtain the fourth PCR system; 10 μM upstream primer F2, 10 μM downstream primer R2, 10 μM probe Probe2 and 100 nM target nucleic acid ABL were mixed with 150 nM and 200 nM CY5 fluorescent dyes (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), respectively, and the volume was made up to 15 μL with ddH ₂ O to obtain the fifth PCR system; 10 μM upstream primer F2, 10 μM downstream primer R2, 10 μM probe Probe2 and 200 nM target nucleic acid ABL were mixed with 150 nM and 200 nM CY5 fluorescent dyes (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), respectively, and the volume was made up to 15 μL with ddH 2 O to obtain the fifth PCR system. CY5 fluorescent dye (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), and _ddH2O was used to make up its volume to 15 μL to obtain the sixth group of PCR systems; 15 μL of premixed solution (purchased from Bio-Rad, USA) was added to the three groups of PCR systems respectively, and they were fully mixed to obtain three groups of mixed solutions; first, 30 μL of the mixed solution was added to the small hole of the droplet generation chip (purchased from Bio-Rad, USA, the structure is shown in Figure 3), and then 80 μL of droplet generation oil (purchased from Bio-Rad, USA) was added to the middle hole of the droplet generation chip, and then the droplet generation chip was placed in a droplet generator (purchased from Suzhou Zhongke Xilai Technology Co., Ltd.) for reaction at room temperature (25°C) for 2 minutes, so that the three groups of mixed solutions and the droplet generation oil respectively generated three groups of droplets and gathered in the large hole of the droplet generation chip; the three groups of droplets generated in the large hole of the droplet generation chip were placed in a PCR instrument (purchased from Xi'an Tianlong Technology Co., Ltd.) for PCR reaction to obtain reaction solutions B1 to B3;

Reaction solutions A1-A3 and reaction solutions B1-B3 were mixed to obtain a mixed solution; 50 μL of the mixed solution was placed in an eight-tube strip, and analyzed using a digital PCR instrument (purchased from Suzhou Zhongke Xilai Technology Co., Ltd.). The analysis results are shown in Figure 15; wherein, the PCR reaction conditions were: ① 95°C for 10 min; ② 94°C for 30 s, 60°C for 1 min (39 cycles); ③ 98°C for 10 min.

As shown in Figure 15, four concentrations of CY5 are used as the horizontal axis for encoding to distinguish target sequences of different concentrations or types, while the intensity of FAM fluorescence on the vertical axis can detect the presence or absence of target sequences, and the detection results are almost non-overlapping, which can be effectively distinguished. It shows that the multiple droplet encoding system is feasible.

Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims (16)

A multi-target detection kit based on droplet color-scale coding, characterized in that the multi-target detection kit comprises at least one group of detection reagents, at least one group of color-scale coding reagents and at least one group of droplet generation reagents; the detection reagents are used to perform qualitative or quantitative detection of specific targets in the sample to be tested; the color-scale coding reagents are used to distinguish the types of targets in the sample to be tested; the droplet generation reagents are used to generate droplets of an aqueous phase obtained by mixing the detection reagents, the color-scale coding reagents and the sample to be tested; each group of detection reagents comprises at least one fluorescence detection system or self-luminescence detection system that can specifically identify a target; the fluorescence detection system comprises at least one fluorescent marker; the self-luminescence detection system comprises at least one combination of a luminescent marker and its initiator or at least one self-luminescence marker; the color-scale coding reagent comprises at least one fluorescent marker; the emission wavelengths of different fluorescent markers in the color-scale coding reagents do not cross-talk with each other; the emission wavelengths of the fluorescent markers in the detection reagents and the fluorescent markers in the color-scale coding reagents do not cross-talk with each other. The multi-target detection kit according to claim 1, characterized in that the fluorescence detection system comprises a one-pot fluorescence detection system. The multi-target detection kit according to claim 2, characterized in that the one-pot fluorescence detection system includes at least one of a PCR fluorescence detection system, a constant temperature amplification fluorescence detection system, a fluorescence immunoassay system, a chemiluminescence detection system, or a CRISPR/Cas-based fluorescence detection system. The multi-target detection kit according to claim 3, characterized in that the PCR fluorescence detection system comprises an upstream primer, a downstream primer and a probe; and the probe is modified with a fluorescent marker. The multi-target detection kit according to any one of claims 1 to 4, characterized in that the fluorescent marker comprises at least one of a fluorescent group, a fluorescent nanomaterial or a fluorescent protein. The multi-target detection kit according to any one of claims 1 to 5, characterized in that the fluorescent group includes at least one of FAM, CY5, HEX, VIC, APC, Cy5.5, APC-Cy7, PC5.5, PC7, APC-A700, APC-H7, V450, PC5, DRAQ7, Cy3, Cy7, PBXL-1, PBXL-3, ROX, rare earth chelates or fluorescent probe chelates; the fluorescent nanomaterial includes at least one of fluorescent quantum dots, fluorescent polystyrene microspheres, fluorescent silica, fluorescent polyphosphazene nanoparticles, upconversion fluorescent particles, rare earth-doped fluorescent nanomaterials, fluorescent labeled PEG nanomaterials or fluorescent nanoclusters. The multi-target detection kit according to any one of claims 1 to 6, characterized in that the target is at least one of a nucleic acid, a protein, a small biological molecule or a cell. A multi-target detection method based on droplet color coding, the method is not for the purpose of disease diagnosis and treatment, characterized in that the multi-target detection method uses the multi-target detection kit described in any one of claims 1 to 7 to detect the sample to be tested. The multi-target detection method according to claim 8, characterized in that the method comprises the following steps: Step 1: respectively mixing the sample to be tested and the detection reagent with the color scale coding reagent of different fluorescence concentrations and/or different emission wavelengths to obtain a mixed system; using a droplet generation reagent to generate droplets of the mixed system to obtain droplets, so as to simultaneously establish the color scale coding system and the detection system in the same droplet; Step 2: mixing the droplets and incubating them to obtain an incubation solution; performing fluorescence detection on the incubation solution to obtain a fluorescence detection result; Step three: Distinguish the target type in the sample to be tested according to the fluorescence detection result of the fluorescent marker in the coding reagent, and perform qualitative or quantitative detection of the specific target in the sample to be tested according to the fluorescence detection result of the fluorescent marker in the detection reagent; during the detection process, one target corresponds to one group of detection reagents. A droplet color scale coding kit, characterized in that the droplet color scale coding kit comprises at least one group of color scale coding reagents and at least one group of droplet generation reagents; the droplet generation reagents are used to generate droplets from an aqueous phase containing the color scale coding reagents; the color scale coding reagents comprise at least one fluorescent marker; the emission wavelengths of different fluorescent markers in the color scale coding reagents do not cross-talk with each other; the color scale coding reagents perform color scale coding on the generated droplets through two-dimensional information of the fluorescent markers contained in the droplets on fluorescence concentration and emission wavelength. The droplet color scale coding kit as described in claim 10 is characterized in that the coding capacity of the color scale coding reagent = color scale number 1 × color scale number 2... × color scale number n, wherein n = the number of probe types, which is an arbitrary positive integer; the color scale number i = 1~n of the color scale coding reagent refers to the number of corresponding probe concentrations that can be effectively distinguished by the detection instrument, and each color scale number is an arbitrary positive integer. A droplet color scale coding method, characterized in that the droplet color scale coding method uses the droplet color scale coding kit described in claim 10 or 11 to perform droplet generation and droplet color scale coding on a sample to be coded. The droplet color scale coding method according to claim 12, characterized in that the method comprises the following steps: Step 1: Mix the sample to be encoded with color-scale coding reagents of different fluorescence concentrations and/or different emission wavelengths to obtain a mixed system; use a droplet generation reagent to generate droplets of the mixed system to obtain droplets, so as to establish a color-scale coding system in the droplets; Step 2: mixing the droplets and incubating them to obtain an incubation solution; performing fluorescence detection on the incubation solution to obtain a fluorescence detection result; Step 3: Color code the sample to be coded according to the fluorescence detection result of the fluorescent marker in the coding reagent. The use of the multi-target detection kit according to any one of claims 1 to 7 or the multi-target detection method according to claim 8 or 9 or the droplet color scale coding kit according to claim 10 or 11 or the droplet color scale coding method according to claim 12 or 13 in target detection is characterized in that the application is for non-disease diagnosis and treatment purposes. The multi-target detection kit as described in claim 1 is characterized in that the non-crosstalk means that it can be effectively distinguished by an instrument or software algorithm. The multi-target detection kit according to any one of claims 1 to 6, characterized in that the fluorescent group includes at least one of aggregation-induced emission probes and derivatives thereof, derivatives of the fluorescent group according to claim 6, doped or labeled materials of the fluorescent group and its derivatives, temperature-controlled luminescent materials, magnetically controlled luminescent materials, and field-controlled luminescent materials.