Digital parallel detection method for joint detection of nucleic acid and protein and application

Abstract

The invention discloses a digital parallel detection method for joint detection of nucleic acid and protein. The method comprises the following steps: S1, preparing a PCR reaction system for specific detection of a nucleic acid target; s2, preparing an immune PCR reaction system for specific recognition of a protein target; in the step S2, a DNA template in positive correlation with the protein concentration is generated through enzyme-linked immunosorbent assay and biotin-avidin specific recognition reaction; s3, preparing a digital PCR reaction system for specific detection of nucleic acid and protein; s4, preparing a digital PCR single molecule reaction system; s5, performing a digital PCR amplification reaction for parallel detection of nucleic acid and protein; and S6, carrying out data analysis on digital PCR amplification results of nucleic acid and protein parallel detection. According to the invention, synchronous detection and quantitative analysis of any one or more nucleic acid and protein molecules can be realized, and accurate quantification of nucleic acid copy number and protein concentration in a sample can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid and protein detection, and more specifically to a digital parallel detection method for nucleic acid and protein joint detection and its application. Background Art

[0002] Nucleic acid and protein markers play an important role in the diagnosis, treatment and prevention of diseases. Among them, nucleic acid markers usually refer to DNA or RNA molecules that can reflect the physiological or pathological state of the body, such as circulating tumor DNA (ctDNA), microRNA (miRNA), etc. Protein markers refer to protein molecules whose expression levels or structures change abnormally under disease conditions, such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), etc. Studies have shown that quantitative detection of the expression levels and changes of nucleic acid and protein markers in circulating body fluids can provide important basis for early diagnosis, efficacy monitoring and prognosis judgment of diseases (see Crosby D, Science, 2022, 375(6586):eaay9040; Von-Felden J, Gut, 2020, 69(11):2025-2034). In addition, combining different types and numbers of molecular markers for disease screening and diagnosis can help further improve the sensitivity of clinical screening. Therefore, it is of great significance to achieve parallel detection and precise quantification of nucleic acid and protein molecules.

[0003] For a long time, real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) have been classical methods for nucleic acid and protein molecule detection, respectively. However, both qPCR and ELISA are detection methods under macroscopic reaction systems, and the detection sensitivity is greatly affected by the concentration of the target molecule, which is prone to missed detection and cannot guarantee the accurate quantitative detection of trace molecular markers in circulating body fluids. In addition, both qPCR and ELISA are relative quantitative methods, which not only require calibration with the help of internal reference genes or standard products, but also require quantification with the help of standard curves, and the quantitative accuracy is difficult to guarantee (see Bustin SA, Clinical Chemistry, 2009, 55 (4): 611-622).

[0004] In recent years, digital detection, an advanced quantitative detection technology, has developed rapidly and has gradually become a new tool for molecular quantitative detection research and application. The digital detection scheme does not require the use of internal reference genes or standards throughout the process, does not rely on standard curves, and can achieve absolute quantification of molecules. However, there are significant differences between digital PCR for digital detection of nucleic acids and digital ELISA for digital detection of proteins in terms of reaction conditions, reagent systems, detection equipment, etc., and these two technical schemes are performed separately on different devices. This will not only affect the correlation analysis of nucleic acid and protein detection results, but also restrict the output timeliness of the test report. Up to now, there is no solution that can realize digital parallel detection of nucleic acids and proteins. Therefore, it is of great significance to develop a new method for two-dimensional digital parallel detection of nucleic acids and proteins. Summary of the invention

[0005] The present invention aims to solve the problem of joint detection and accurate quantification of two different types of molecules, nucleic acids and proteins, so as to completely overcome the technical barriers to parallel detection of nucleic acid and protein molecular markers and provide a digital parallel detection method for joint detection of nucleic acids and proteins.

[0006] The technical solution adopted by the present invention is: a digital parallel detection method for nucleic acid and protein joint detection, the method comprising the following steps: S1, preparing a PCR reaction system for nucleic acid target specific detection; S2, preparing an immune PCR reaction system for protein target specific recognition; in step S2, a DNA template positively correlated with protein concentration is generated through enzyme-linked immunosorbent reaction and biotin-avidin specific recognition reaction; S3, preparing a digital PCR reaction system for nucleic acid and protein specific detection; S4, preparing a digital PCR single molecule reaction system; S5, performing a digital PCR amplification reaction for nucleic acid and protein parallel detection; S6, performing data analysis on the digital PCR amplification results for nucleic acid and protein parallel detection.

[0007] As a further improvement of the present invention, in step S3, the preparation of the digital PCR reaction system for nucleic acid and protein specific detection includes: it is composed of two parallel and independent digital PCR reaction systems dPCR-Ia and dPCR-Ib to realize the dual detection function of different reaction systems; PCR-Ia and dPCR-Ib respectively contain primers, probes, templates, and reaction raw materials required for direct detection of nucleic acids and indirect detection of proteins; except for the differences in primer and probe sequences and DNA templates between dPCR-Ia and dPCR-Ib, the final concentrations of primers and probes in the system, reaction raw materials and dosage are the same; the reaction raw materials include enzymes, dNTPs and buffers. The constructed dPCR-Ia and dPCR-Ib systems are used to realize digital PCR detection of nucleic acid and protein targets, respectively; using different reaction systems for detection can provide favorable conditions for further expansion of detection throughput.

[0008] As a further improvement of the present invention, the reporter groups of the two probes of dPCR-Ia and dPCR-Ib are any one or two of Atto425, FAM, VIC / HEX, ROX, Cy5, Cy5.5, and Cy7 fluorescent groups, and the two probes can choose to use the same reporter group or different reporter groups.

[0009] As a further improvement of the present invention, in step S3, the preparation of the digital PCR reaction system for nucleic acid and protein specific detection includes: being composed of a digital PCR reaction system dPCR-II to realize the dual detection function of the same reaction system; the dPCR-II simultaneously contains primers, probes, templates, and reaction raw materials for parallel detection of nucleic acids and proteins; the two probes in the dPCR-II system have and can only choose to use two different reporter groups; the reaction raw materials are enzymes, dNTPs, and buffers. The dual detection using the same reaction system can effectively reduce reagent consumption and reduce costs; at the same time, it can effectively improve the accuracy and stability of quantitative detection.

[0010] As a further improvement of the present invention, the reporter groups of the two probes are any two of Atto425, FAM, VIC / HEX, ROX, Cy5, Cy5.5, and Cy7 fluorescent groups. Since a multiple PCR reaction system is used to construct the dPCR-II system, different reporter groups should be used for control detection.

[0011] As a further improvement of the present invention, in step S4, the single-molecule reaction system is prepared by any one of the schemes of microdroplet type, microarray type, micropit or microwell type, and nanopore type.

[0012] As a further improvement of the present invention, in step S5, the amplification reaction includes the dPCR-Ia and dPCR-Ib single molecule reaction systems simultaneously performing parallel independent ramping thermal cycling reactions or the dPCR-II single molecule reaction system performing ramping thermal cycling reactions, and the ramping thermal cycling reactions include 1 cycle A and 35-50 cycles B; cycle A: heating at 93-98°C for 30s-10min; cycle B: heating at 93-98°C for 5s-30s, and heating at 50°C-62°C for 20s-60s.

[0013] As a further improvement of the present invention, for the detection of protein targets, further deduction must be made to obtain the results. Specifically, in step S6, the data analysis of the digital PCR amplification results includes the following two parts: Q1, for nucleic acid targets, the copy number obtained by data analysis is the absolute concentration of nucleic acid molecules in the sample to be tested; Q2, for protein targets, the value obtained by data analysis needs to be substituted into the formula for converting nucleic acid copy number to protein concentration, and then the absolute concentration of protein molecules is calculated; the establishment of the conversion formula for nucleic acid copy number to protein concentration is jointly affected by the enzyme-linked immunosorbent assay in step S2 and the efficiency of the biotin-avidin specific recognition reaction; the conversion formula for nucleic acid copy number to protein concentration is shown in formula (I):

[0014] C 皮克 =k N 拷贝数 +b(I)

[0015] in:

[0016] C is the protein concentration (picogram, pg);

[0017] N is the number of nucleic acid copies;

[0018] k is the slope;

[0019] b is the intercept.

[0020] Digital PCR technology can indirectly detect the protein concentration in the sample through this formula, thus making an important contribution to the realization of the entire technology.

[0021] As a further improvement of the present invention, the nucleic acid target includes one or more of DNA and RNA molecular markers; the protein target includes one or more of antigen, antibody, cytokine, enzyme, and hormone protein markers.

[0022] As a further improvement of the present invention, the detection method is applied to the detection of blood markers of diseases.

[0023] The present invention has the beneficial effects: the digital parallel detection method for nucleic acid and protein joint detection provided by the present invention can not only realize the synchronous detection and quantitative analysis of any one or more nucleic acid and protein molecules, but also realize the accurate quantification of nucleic acid copy number and protein concentration in the sample. In the future, it will provide new detection technology solutions for the research and application related to the joint use of nucleic acid and protein molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A schematic diagram of the technical principle of the digital parallel detection method for nucleic acid and protein joint detection provided by the present invention;

[0025] Figure 2 The results of scanning and data analysis of Example 1 of the present invention;

[0026] Figure 3 This is the result of scanning and data analysis of Example 2 of the present invention. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with the accompanying drawings.

[0028] Example 1, a digital parallel detection method for the joint detection of microRNA 21 (miR-21) and alpha-fetoprotein (AFP), the method comprising the following steps:

[0029] S1. preparing a PCR reaction system for specific detection of miR-21;

[0030] Prepare PCR reaction system A for miR-21 specific detection directly in tube A;

[0031] S2. preparing an immuno-PCR reaction system for AFP specific identification;

[0032] First, an immune PCR reaction system for AFP specific recognition is prepared in tube B, and a DNA template (SEQ ID NO: 7) positively correlated with the AFP concentration is generated through enzyme-linked immunosorbent assay and biotin-avidin specific recognition reaction, and then the template is used to prepare PCR reaction system B;

[0033] Since steps S1-S2 are prior art, they will not be described here.

[0034] S3, preparing a digital PCR reaction system for specific detection of miR-21 and AFP;

[0035] In this embodiment, PCR reaction systems A and B prepared by S1 and S2 are used to prepare digital PCR reaction systems (dPCR-Ia and dPCR-Ib), respectively, to achieve dual detection functions of different reaction systems;

[0036] dPCR-Ia contains the primer set required for direct detection of miR-21 (0.5 μM F1 and 0.5 μM R1) (SEQ ID NO: 1 and SEQ ID NO: 2), 0.4 μM probe P1-HEX (SEQ ID NO: 3), and reaction materials (0.2 U / μL DNA polymerase, 0.125 mM dNTPs, 1× Buffer);

[0037] dPCR-Ib contains the primer set required for indirect detection of AFP (0.5 μM F2 and 0.5 μM R2) (SEQ ID NO: 4 and SEQ ID NO: 5), 0.4 μM probe P2-CY5 (SEQ ID NO: 6), and reaction materials (0.2 U / μL DNA polymerase, 0.125 mM dNTPs, 1× Buffer);

[0038] S4, preparing a digital PCR single-molecule reaction system;

[0039] 20 μL dPCR-Ia and 20 μL dPCR-Ib were added to the reagent loading pools 1 and 2 on the biochip, respectively, and 100 μL fluorinated oil was added to the fluorinated oil loading pools 1 and 2. More than 20,000 droplet digital PCR single-molecule reaction systems dPCR-Ia and dPCR-Ib were generated through the "oil-in-water" process and spread flat in the biochip reaction chamber.

[0040] S5, performing digital PCR amplification reaction for parallel detection of miR-21 and AFP;

[0041] The biochip is placed in a PCR amplification instrument to carry out digital PCR amplification reaction.

[0042] S6. Data analysis of the digital PCR amplification results for parallel detection of miR-21 and AFP;

[0043] After the reaction is completed, the amplified biochip is placed in a biochip reader for biochip scanning and data analysis. Figure 2 shown.

[0044] The data obtained from the digital PCR amplification results include the following two parts:

[0045] Q1. Figure 2 a The amplification results of the droplet digital PCR single-molecule reaction system dPCR-Ia for quantitative detection of miR-21. The concentration of miR-21 is about 98 copies;

[0046] Q2, Figure 2b is the amplification result of the droplet digital PCR single molecule reaction system dPCR-Ib for AFP quantitative detection, and the result is about 15872 copies. Substitute this result into the formula for converting nucleic acid copy number to protein concentration

[0047] C 皮克 =0.13N 拷贝数 +26.92

[0048] The calculated absolute concentration of AFP is about 2.09╳10 3 Pique.

[0049] Example 2, a digital parallel detection method for the joint detection of microRNA 21 (miR-21) and alpha-fetoprotein (AFP), the method comprising the following steps:

[0050] S1. preparing a PCR reaction system for specific detection of miR-21;

[0051] Prepare PCR reaction system A for miR-21 specific detection directly in tube A;

[0052] S2. preparing an immuno-PCR reaction system for AFP specific identification;

[0053] First, an immune PCR reaction system for AFP specific recognition is prepared in tube B, and a DNA template positively correlated with the AFP concentration is generated through enzyme-linked immunosorbent reaction and biotin-avidin specific recognition reaction, and then the template is used to prepare PCR reaction system B;

[0054] S3, preparing a digital PCR reaction system for specific detection of miR-21 and AFP;

[0055] In this embodiment, the PCR reaction systems A and B prepared by S1 and S2 are used to prepare a digital PCR reaction system dPCR-II to achieve dual detection functions of the same reaction system;

[0056] dPCR-II contains not only the primer set (0.5 μM F1 and 0.5 μM R1) (SEQ ID NO: 1 and SEQ ID NO: 2) and 0.4 μM probe P1-HEX (SEQ ID NO: 3) required for direct detection of miR-21, but also the primer set (0.5 μM F2 and 0.5 μM R2) (SEQ ID NO: 4 and SEQ ID NO: 5) and 0.4 μM probe P2-CY5 (SEQ ID NO: 6) required for indirect detection of AFP, as well as reaction materials (0.2 U / μL DNA polymerase, 0.125 mM dNTPs, 1× Buffer);

[0057] S4, preparing a digital PCR single-molecule reaction system;

[0058] 20 μL of dPCR-II was added to the reagent loading pool 1 on the biochip, and 100 μL of fluorinated oil was added to the fluorinated oil loading pool 1. More than 20,000 droplet digital PCR single molecule reaction systems dPCR-II were generated through the "oil-in-water" process and spread flat in the biochip reaction chamber.

[0059] S5, performing digital PCR amplification reaction for parallel detection of miR-21 and AFP;

[0060] The biochip is placed in a PCR amplification instrument to carry out digital PCR amplification reaction.

[0061] S6. Data analysis of the digital PCR amplification results for parallel detection of miR-21 and AFP;

[0062] After the reaction is completed, the amplified biochip is placed in a biochip reader for biochip scanning and data analysis. Figure 3 shown.

[0063] The data obtained from the digital PCR amplification results include the following two parts:

[0064] Q1. Figure 3 a is the result of quantitative detection of miR-21 by droplet digital PCR single-molecule reaction system dPCR-II, with a concentration of about 116 copies;

[0065] Q2, Figure 3 b is the result of quantitative detection of miR-21 by droplet digital PCR single molecule reaction system dPCR-II, the concentration is about 9136 copies. Substitute this result into the formula for converting nucleic acid copy number to protein concentration C 皮克 =0.13N 拷贝数 +26.92

[0066] The calculated absolute concentration of AFP is about 1.21╳10 3 Pique.

[0067] The digital parallel detection method for nucleic acid and protein joint detection provided by the present invention can not only realize the synchronous detection and quantitative analysis of any one or more nucleic acid and protein molecules, but also realize the accurate quantification of nucleic acid copy number and protein concentration in the sample. In the future, it will provide a new detection technology solution for the research and application related to the joint use of nucleic acid and protein molecules.

[0068] Those skilled in the art should be aware that the protection scheme of the present invention is not limited to the above-mentioned embodiments, and various arrangements, combinations and transformations can be performed on the basis of the above-mentioned embodiments. Without violating the spirit of the present invention, various transformations of the present invention fall within the protection scope of the present invention.

Claims

1. A digital parallel detection method for nucleic acid and protein joint detection, the method comprising the following steps: S1. preparing a PCR reaction system for nucleic acid target specific detection; S2. preparing an immune PCR reaction system for specific identification of protein targets; In step S2, a DNA template positively correlated with the protein concentration is generated through an enzyme-linked immunosorbent assay and a biotin-avidin specific recognition reaction; S3, preparing a digital PCR reaction system for nucleic acid and protein specific detection; S4, preparing a digital PCR single-molecule reaction system; S5, performing digital PCR amplification reaction for parallel detection of nucleic acid and protein; S6. Perform data analysis on the digital PCR amplification results for parallel detection of nucleic acids and proteins.

2. A digital parallel detection method for nucleic acid and protein joint detection according to claim 1, characterized in that In the step S3, the preparation of the digital PCR reaction system for specific detection of nucleic acids and proteins includes: it is composed of two parallel and independent digital PCR reaction systems dPCR-Ia and dPCR-Ib to realize the dual detection function of different reaction systems; PCR-Ia and dPCR-Ib respectively contain primers, probes, templates, and reaction raw materials required for direct detection of nucleic acids and indirect detection of proteins; except for the differences in primer and probe sequences and DNA templates, the final concentrations of primers and probes, reaction raw materials and usage in the system are the same between dPCR-Ia and dPCR-Ib; the reaction raw materials include enzymes, dNTPs and buffers.

3. A digital parallel detection method for nucleic acid and protein joint detection according to claim 2, characterized in that The reporter groups of the two probes of dPCR-Ia and dPCR-Ib are any one or two of Atto425, FAM, VIC / HEX, ROX, Cy5, Cy5.5, and Cy7 fluorescent groups. The two probes can use the same reporter group or different reporter groups.

4. A digital parallel detection method for nucleic acid and protein joint detection according to claim 1, characterized in that In step S3, the preparation of the digital PCR reaction system for nucleic acid and protein specific detection includes: being composed of a digital PCR reaction system dPCR-II to realize the dual detection function of the same reaction system; the dPCR-II simultaneously contains primers, probes, templates, and reaction raw materials for parallel detection of nucleic acids and proteins; the two probes in the dPCR-II system have and can only choose to use two different reporter groups; the reaction raw materials are enzymes, dNTPs and buffers.

5. A digital parallel detection method for nucleic acid and protein joint detection according to claim 4, characterized in that The reporter groups of the two probes are any two of Atto425, FAM, VIC / HEX, ROX, Cy5, Cy5.5, and Cy7 fluorescent groups.

6. A digital parallel detection method for nucleic acid and protein joint detection according to claim 1, characterized in that In the step S4, the single molecule reaction system is prepared by any one of the schemes of microdroplet type, microarray type, micropit or microwell type, and nanopore type.

7. A digital parallel detection method for nucleic acid and protein joint detection according to claim 1, characterized in that In step S5, the amplification reaction includes the dPCR-Ia and dPCR-Ib single molecule reaction systems simultaneously performing parallel independent ramping thermal cycling reactions or the dPCR-II single molecule reaction system performing ramping thermal cycling reactions, and the ramping thermal cycling reactions include 1 cycle A and 35-50 cycles B; cycle A: heating at 93-98°C for 30s-10min; cycle B: heating at 93-98°C for 5s-30s, and heating at 50°C-62°C for 20s-60s.

8. A digital parallel detection method for nucleic acid and protein joint detection according to claim 1, characterized in that In step S6, the data analysis of the digital PCR amplification results includes the following two parts: Q1, for nucleic acid targets, the copy number obtained by data analysis is the absolute concentration of nucleic acid molecules in the sample to be tested; Q2, for protein targets, the value obtained by data analysis needs to be substituted into the formula for converting nucleic acid copy number to protein concentration, and then the absolute concentration of protein molecules is calculated; the establishment of the conversion formula for nucleic acid copy number to protein concentration is jointly affected by the enzyme-linked immunosorbent assay in step S2 and the efficiency of the biotin-avidin specific recognition reaction; the conversion formula for nucleic acid copy number to protein concentration is shown in formula (I): C 皮克 =k*N 拷贝数 +b(I) in: C is the protein concentration (picogram, pg); N is the number of nucleic acid copies; k is the slope; b is the intercept.

9. A digital parallel detection method for nucleic acid and protein joint detection according to claim 8, characterized in that the nucleic acid target includes one or more of DNA and RNA molecular markers; the protein target includes one or more of antigen, antibody, cytokine, enzyme, and hormone protein markers.

10. An application of a digital parallel detection method for nucleic acid and protein joint detection according to any one of claims 1 to 9, characterized in that: This detection method is applied to the detection of blood markers of diseases.

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