CN111139285A

CN111139285A - Method for detecting contents of different target proteins in multiple samples to be detected in high-throughput manner and special kit thereof

Info

Publication number: CN111139285A
Application number: CN201811299647.3A
Authority: CN
Inventors: 赵芳; 李舟; 章文蔚; 刘二凯; 张长领; 陈奥
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd; Shenzhen BGI Life Science Research Institute
Priority date: 2018-11-02
Filing date: 2018-11-02
Publication date: 2020-05-12

Abstract

The invention discloses a method for detecting contents of different target proteins in a plurality of samples to be detected in a high-throughput manner and a special kit thereof. The invention combines PEA and DNA sequencing technology to realize the simultaneous detection of the multi-protein content of multiple samples, and the specific method comprises the following steps: the ss-DNA (protein bar code) with different sequences is marked aiming at the antibodies of different types, and the protein types are distinguished according to the difference of the ss-DNA sequences; converting protein information into DNA information by means of PEA technology; adding a section of different Barcode tag sequences (sample barcodes) for different samples during library construction for distinguishing different samples; the sequencer respectively detects the sample barcode sequence, the protein barcode sequence and the number of the protein barcode sequences, so that the simultaneous detection of the contents of various proteins in a plurality of samples is realized. The invention has important application value.

Description

Method for detecting contents of different target proteins in multiple samples to be detected in high-throughput manner and special kit thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for detecting the content of different target proteins in a plurality of samples to be detected in a high-throughput manner and a special kit thereof.

Background

The methods for detecting the trace amount of the polyprotein mainly comprise a Luminex (Luminex) technology, a biorad bio-plex (Bio-plex) technology and a protein chip technology.

The Luminex technology integrates microspheres and a flow cytometer, utilizes microspheres with color codes to covalently cross-link monoclonal antibodies, combines the monoclonal antibodies with target molecules to be detected, adds a fluorescein-labeled detection antibody, and identifies the single microspheres and detects fluorescence intensity by laser scanning fluorescence codes to determine the concentration of the molecules to be detected. Since different color-coded microspheres can be used to detect different proteins, analysis of multiple target molecules can be accomplished simultaneously in one experiment. However, the detection instrument is expensive, the detection reagent needs to use the biological modified fluorescent microspheres, the detection sample amount is 10-20 mu L, the consumption of the reaction antibody is high, and the final detection cost is overhigh.

The biorad bio-plex technology integrates the microsphere and suspension chip technologies: in the liquid phase chip, microspheres with different colors for different detection objects (the microspheres are provided with proteins for different detection objects) are mixed, then the detection objects are added, the microspheres and the detection objects are specifically combined in suspension, and fluorescent labels are added, so that a plurality of indexes of the same sample can be detected simultaneously in the same reaction system. The method can detect a plurality of proteins in the same sample, but can not simultaneously detect a plurality of samples containing a plurality of indexes. In addition, the reagent cost is high due to the reference of Luminex microsphere technology.

The protein chip technology is a high-density protein microarray which is prepared by simultaneously fixing a plurality of known protein molecules on different solid phase carriers. Usually, the protein molecule to be detected and the chip are incubated for a period of time, and then the labeled protein molecule is added to react with the chip-protein molecule complex. Since the position and sequence of each molecule are known, the distribution of various protein molecules can be known by detecting the concentration or presence of the protein by a biochip scanner. The method has the advantages of being a quantitative detection method and ensuring that the higher-order structure of the protein can be detected; the disadvantage is that it is sometimes difficult to find a pair of protein molecules that can be attached to the same molecule and matched, and furthermore, it is difficult to prepare a high density microarray using this method, which makes high throughput detection very challenging.

The Proximity Extension Analysis (PEA) is a technique in which a pair of antibodies are respectively modified with single-stranded DNA (ss-DNA), and when a protein molecule to be detected reacts specifically with the antibodies, the distance between the antibodies and the ss-DNA is reduced, and an amplifiable detection signal is generated, so that the detection of the protein is converted into the detection of the DNA, and the analysis of trace proteins is realized. The method for simultaneously quantifying DNA of various sequences generally adopts Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR, QPCR).

Disclosure of Invention

The invention aims to simultaneously detect the contents of different target proteins in a plurality of samples to be detected.

The invention firstly protects a kit for detecting the contents of different target proteins in a sample to be detected, which can comprise a plurality of reagents for detecting different target proteins, a library-building PCR upstream primer, a library-building PCR downstream primer, a sequencing primer A and a sequencing primer B;

each reagent for detecting the target protein can comprise a single-chain DNA molecule A, a target protein antibody A, a single-chain DNA molecule B and a target protein antibody B;

the single-stranded DNA molecule A sequentially comprises a DNA fragment 1, a DNA fragment 2 and a DNA fragment 3 from the 5 'end to the 3' end; the single-stranded DNA molecule B can comprise a DNA segment 4, a DNA segment 5 and a DNA segment 6 from the 5 'end to the 3' end in sequence; the DNA segment 3 and the DNA segment 6 are reverse complementary; in each reagent for detecting the target protein, the nucleotide sequences of the DNA segment 2 and the DNA segment 5 are different, and the reagents are used for detecting different target proteins;

the library-building PCR upstream primer can comprise a DNA fragment a and a DNA fragment b from the 5 'end to the 3' end in sequence; the DNA fragment b and the DNA fragment 1 are completely identical;

the library-building PCR downstream primer can comprise a DNA fragment c and a DNA fragment d from the 5 'end to the 3' end in sequence; the 5' ends of the DNA fragment d and the DNA fragment 4 are completely consistent;

the sequencing primer A sequentially comprises a DNA fragment X and a DNA fragment Y from the 5 'end to the 3' end; the DNA fragment X is reverse complementary to the DNA fragment c, and the DNA fragment Y is completely identical to the DNA fragment a;

and the sequencing primer B is reversely complementary with the sequence of the DNA fragment 4.

In the kit, the single-stranded DNA molecule A can be composed of a DNA fragment 1, a DNA fragment 2 and a DNA fragment 3 from the 5 'end to the 3' end. The single-stranded DNA molecule B can be composed of a DNA segment 4, a DNA segment 5 and a DNA segment 6 from the 5 'end to the 3' end.

In the kit, the pool-building PCR upstream primer can be specifically composed of the DNA fragment a (sequencing adaptor sequence A) and the DNA fragment b from the 5 'end to the 3' end. In embodiments of the invention, sequencing is performed using BGI500 platform sequencing. Accordingly, the nucleotide sequence of sequencing adapter sequence a (from 5 'to 3') may be GAACGACATGGCTACGATCCGACTT. The purpose of adding a sequencing joint sequence A into the library-building PCR upstream primer is to facilitate sequencing, and the sequence A is used for detecting extension products of a single-stranded DNA molecule A and a single-stranded DNA molecule B and obtaining information of protein barcodes (aiming at distinguishing different proteins). According to the difference of sequencing platform, the sequencing linker sequence composed of other nucleotide sequence can be replaced.

In the kit, the library-building PCR downstream primer may specifically consist of the DNA fragment c (sequencing adaptor sequence B) and the DNA fragment d from the 5 'end to the 3' end. In embodiments of the invention, sequencing is performed using BGI500 platform sequencing. Accordingly, the nucleotide sequence of sequencing linker sequence b (from 5 'to 3') can be TGTGAGCCAAGGAGTTG. According to the difference of sequencing platform, the sequencing linker sequence composed of other nucleotide sequence can be replaced.

In the above kit, the sequencing primer A may specifically consist of the DNA fragment X and the DNA fragment Y from the 5 'end to the 3' end.

In the above kit, the target protein antibody a and the target protein antibody b may be one target protein capture antibody and one target protein detection antibody.

In the kit, the reagents for detecting different target proteins have no cross reaction.

Any one of the above kits can specifically comprise a plurality of reagents for detecting different target proteins, a library construction PCR upstream primer, a library construction PCR downstream primer, a sequencing primer A and a sequencing primer B. Each reagent for detecting a target protein may specifically be composed of the single-stranded DNA molecule A, the target protein antibody A, the single-stranded DNA molecule B, and the target protein antibody B.

The DNA segment 1 and the DNA segment 4 can be single-stranded DNA molecules consisting of 20-35 (such as 20-23, 23-32, 32-35, 20, 23, 32 or 35) nucleotides. The DNA segment 2 and the DNA segment 5 can be single-stranded DNA molecules consisting of 5-9 (such as 5-7, 7-9, 5, 7 or 9) nucleotides. The DNA segment 3 and the DNA segment 6 can be single-stranded DNA molecules consisting of 5-11 (such as 5-9, 9-11, 5, 9 or 11) nucleotides. The DNA fragment a can be a single-stranded DNA molecule consisting of 15-30 (such as 15-25, 25-30, 15, 25 or 30) nucleotides. The DNA fragment c can be a single-stranded DNA molecule consisting of 15-30 (such as 15-17, 17-30, 15, 17 or 30) nucleotides.

In the above, the 5 ' end of the DNA segment d and the 5 ' end of the DNA segment 4 are identical, specifically, 20 to 35 (e.g., 20 to 23, 23 to 32, 32 to 35, 20, 23, 32, or 35) nucleotides of the 5 ' end of the DNA segment d and the DNA segment 4 are identical, and specifically, 21 nucleotides are identical.

The single-stranded DNA molecule A can be a single-stranded DNA molecule consisting of 30-50 (such as 30-39, 39-50, 30 or 50) nucleotides. The single-stranded DNA molecule B can be a single-stranded DNA molecule consisting of 30-50 (such as 30-39, 39-50, 30 or 50) nucleotides. The pool-building PCR upstream primer can be a single-stranded DNA molecule consisting of 45-50 (such as 45-48, 48-50, 45, 48 or 50) nucleotides. The library PCR downstream primer can be a single-stranded DNA molecule consisting of 45-50 (such as 45-48, 48-50, 45, 48 or 50) nucleotides. The sequencing primer A can be a single-stranded DNA molecule consisting of 40-50 (such as 40-42, 42-50, 40, 42 or 50) nucleotides.

The 5' ends of the single-stranded DNA molecule A and the single-stranded DNA molecule B can be modified by azide or amino.

The library-building PCR downstream primer also can comprise a Barcode marking sequence; the Barcode marker sequence is located downstream of the DNA fragment c and upstream of the DNA fragment d.

The purpose of adding the Barcode marker sequence in the library-building PCR downstream primer is to detect information of barcodes of a plurality of samples to be detected (distinguish different samples to be detected) during sequencing so as to detect (high-throughput detection) the content of different target proteins in the plurality of samples to be detected.

The library PCR downstream primer can comprise the DNA fragment c, the Barcode marker sequence and the DNA fragment d from the 5 'end to the 3' end in sequence. The library PCR downstream primer can be specifically composed of the DNA fragment c, the Barcode marker sequence and the DNA fragment d from the 5 'end to the 3' end.

Any one of the above kits may further comprise reagents for detecting a DNA standard; the reagent for detecting the DNA standard substance can comprise the DNA standard substance, a library-building PCR upstream primer-standard substance and a library-building PCR downstream primer-standard substance. The library-building PCR upstream primer-standard substance and the library-building PCR downstream primer-standard substance can be used for detecting DNA standard substances. The reagent for detecting the DNA standard can be used as a positive control.

The DNA standard may contain specific DNA molecules (double strands). The specific DNA molecule can comprise a DNA segment m, a random DNA segment and a DNA segment n from the 5 'end to the 3' end in sequence. The library PCR upstream primer-standard can comprise the DNA fragment a and the DNA fragment M from the 5 'end to the 3' end in sequence. The DNA fragment M and the DNA fragment M are completely identical. The library PCR downstream primer-standard can comprise the DNA fragment c and the DNA fragment N from the 5 'end to the 3' end in sequence. The 3' ends of the DNA fragment N and the DNA fragment N are reversely complementary.

The reagent for detecting the DNA standard substance specifically comprises the DNA standard substance, the library building PCR upstream primer-standard substance and the library building PCR downstream primer-standard substance.

The library-building PCR downstream primer-standard can also comprise a Barcode marker sequence; the Barcode marker sequence is located downstream of the DNA fragment c and upstream of the DNA fragment N.

The DNA standard may be a specific DNA molecule (double strand) solution. The solvent of the DNA standard product can be water. The specific DNA molecule may specifically consist of the DNA fragment m, the random DNA fragment and the DNA fragment n from the 5 'end to the 3' end.

Each strand of the specific DNA molecule (double strand) may consist of 60-100 (e.g., 60-80, 80-100, 60, 80, or 100) nucleotides. The DNA fragment m can be a single-stranded DNA molecule consisting of 15-30 (such as 15-19, 19-30, 15, 19 or 30) nucleotides. The DNA fragment n may be a single-stranded DNA molecule consisting of 25 to 50 (e.g., 25 to 32, 32 to 50, 25, 32 or 50) nucleotides. The random DNA fragment may be a single-stranded DNA molecule consisting of 25-50 (e.g., 25-29, 29-50, 25, 29, or 50) nucleotides. The pool-building PCR upstream primer-standard can be a single-stranded DNA molecule consisting of 40-50 (such as 40-44, 44-50, 40, 44 or 50) nucleotides. The library PCR downstream primer-standard can be a single-stranded DNA molecule consisting of 45-50 (such as 45-48, 48-50, 45, 48 or 50) nucleotides.

In the above, the 3 'terminal reverse complementarity of the DNA fragment N and the DNA fragment N may specifically be 20-35 (e.g., 20-23, 23-32, 32-35, 20, 23, 32, or 35) nucleotides reverse complementarity of the 3' terminal of the DNA fragment N and the DNA fragment N, and specifically may be 21 nucleotides reverse complementarity.

The library PCR upstream primer-standard may specifically consist of the DNA fragment a (sequencing adaptor sequence A) and the DNA fragment M from the 5 'end to the 3' end. A sequencing adaptor sequence A is added to the library-building PCR upstream primer-standard so as to facilitate sequencing and be used for detecting DNA standards with different concentrations. According to the difference of sequencing platform, the sequencing linker sequence composed of other nucleotide sequence can be replaced.

The library PCR downstream primer-standard may specifically consist of the DNA fragment c and the DNA fragment N from the 5 'end to the 3' end. The library-building PCR downstream primer-standard can comprise the DNA fragment c (sequencing linker sequence B), the Barcode marker sequence and the DNA fragment N from the 5 'end to the 3' end in sequence. The library PCR downstream primer-standard specifically comprises the DNA fragment c, the Barcode marker sequence and the DNA fragment N from the 5 'end to the 3' end.

The sequencing linker sequence B and the Barcode marker sequence are added into the library-building PCR downstream primer, so that sequencing is facilitated, and the library-building PCR downstream primer is used for detecting DNA standard substances with different concentrations.

The nucleotide sequence of any of the Barcode marker sequences described above may be any of F1) -F8): F1) ACTGGTAAGA, respectively; F2) AAGCTCCTGA, respectively; F3) CTGGGGCTAT, respectively; F4) CCCAGTCAGG, respectively; F5) GGATTTGGTT, respectively; F6) TACTAATGGC, respectively; F7) TTTTCATTTT, respectively; F8) CTGAGCTCCT are provided.

Any one of the above kits may specifically comprise a plurality of reagents for detecting different target proteins, a library-creating PCR upstream primer, a library-creating PCR downstream primer, a sequencing primer A, a sequencing primer B, and the reagents for detecting DNA standards.

The application of any one of the above-mentioned kits in detecting the contents of different target proteins in a sample to be detected also belongs to the protection scope of the invention.

The invention also provides a method for detecting the content of different target proteins in a sample to be detected, which comprises the following steps (a), (b), (c) and (d):

the step (a) may include the steps of:

(a-1) connecting the target protein antibody A with the single-chain DNA molecule A to obtain a probe 1; connecting any target protein antibody B with the single-chain DNA molecule B to obtain a probe 2;

(a-2) after the step (a-1) is completed, mixing the probe 1 and the probe 2 to obtain a mixed probe;

(a-3) preparing different mixed probes according to the method of the steps (a-1) and (a-2), wherein the different mixed probes form a mixed probe set; each mixed probe in the mixed probe group is used for detecting one target protein;

the step (b) may include the steps of:

(b-1) adding a sample to be detected into each mixed probe in the mixed probe group, uniformly mixing and extending to obtain an extended product;

(b-2) respectively taking each extension product obtained in the step (b-1) as a template, and carrying out PCR amplification on any one of the library-building PCR upstream primer and the library-building PCR downstream primer to obtain a PCR amplification product;

(b-3) respectively taking the PCR amplification products obtained in the step (b-2), and sequencing by using any one of the sequencing primer A and the sequencing primer B to obtain the DNA copy numbers corresponding to different target proteins in a sample to be detected;

the step (c) may include the steps of:

(c-1) adding each target protein standard solution into each mixed probe in the mixed probe group, uniformly mixing and extending to obtain an extended product;

(c-2) respectively taking each extension product obtained in the step (c-1) as a template, and carrying out PCR amplification on any one of the library-building PCR upstream primer and the library-building PCR downstream primer to obtain a PCR amplification product;

(c-3) respectively taking the PCR amplification products obtained in the step (c-2), and sequencing by using any one of the sequencing primer A and the sequencing primer B to obtain the DNA copy number corresponding to different target proteins in each target protein standard solution;

the step (d): and (c) drawing a standard curve according to the concentration of the target protein in each target protein standard solution and the corresponding copy number, and substituting the copy number obtained in the step (b-3) into the standard curve to obtain the content of different target proteins in each sample to be detected.

In the step (a-1), the linkage may be performed by a coupling agent; the coupling agent may provide a chemical group that reacts with the protein antibody of interest and the single-stranded DNA molecule.

In the step (a-1), the step of obtaining a probe by linking the target protein antibody and the single-chain DNA molecule (e.g., linking the target protein antibody a and the single-chain DNA molecule a, or linking the target protein antibody b and the single-chain DNA molecule b) may comprise the steps of:

(a-1-1) mixing a coupling agent (excessive coupling agent can be added, and all target protein antibodies can react) with the target protein antibodies to obtain a mixed system 1; taking the mixed system 1, and reacting to obtain a compound of the target protein antibody and the coupling agent;

(a-1-3) adding single-stranded DNA molecules (excessive single-stranded DNA molecules can be added, and all 'complexes of the target protein antibody and the coupling agent' can react) into the system which is subjected to the step (a-1-1) to obtain a mixed system 2; and (3) taking the mixed system 2 for reaction to obtain a target protein antibody, a compound of the single-chain DNA molecule and the coupling agent (the target protein antibody, the compound of the single-chain DNA molecule and the coupling agent are probes).

In the step (a-1-1), the mixed system 1 may further contain a PBS buffer. The concentration of the coupling agent in the mixed system 1 may be 6 to 10nmol (e.g., 6 to 8nmol, 8 to 10nmol, 6nmol, 8nmol, or 10 nmol). The concentration of the target protein antibody may be 0.8 to 1.2. mu.g/. mu.L (e.g., 0.8 to 1.0. mu.g/. mu.L, 1.0 to 1.2. mu.g/. mu.L, 0.8. mu.g/. mu.L, 1.0. mu.g/. mu.L, or 1.2. mu.g/. mu.L). The reaction condition can be 35-39 deg.C (such as 35-37 deg.C, 37-39 deg.C, 35 deg.C, 37 deg.C or 39 deg.C) standing for 20-40min (such as 20-30min, 30-40min, 20min, 30min or 40 min).

In the step (a-1-3), the concentration of the single-stranded DNA molecule A in the mixed system 2 may be 0.3 to 0.8nM (e.g., 0.3 to 0.6nM, 0.5 to 0.8nM, 0.3nM, 0.5nM, 0.6nM or 0.8 nM). The reaction conditions may be 35-39 deg.C (such as 35-37 deg.C, 37-39 deg.C, 35 deg.C, 37 deg.C or 39 deg.C) and standing for 3-7h (such as 3-5h, 5-7h, 3h, 5h or 7 h).

In the step (a-1), before the step (a-1-3) is performed after the step (a-1-1) is completed, the method may further include the step (a-1-2): taking the system which completes the step (a-1-1), and removing the excessive coupling agent. The "removal of excess coupling agent" may be carried out by subjecting the system subjected to the step (a-1-1) to ultrafiltration centrifugation 1 to 3 times (e.g., 1 time, 2 times or 3 times).

The step (a-1) may further include the step (a-1-4): taking the system after completion of step (a-1-3), and removing the excess single-stranded DNA molecules (in order to ensure high purity of the probe). The "removal of excess single-stranded DNA molecules" can be carried out by subjecting the system subjected to the step (a-1-3) to ultrafiltration centrifugation 1 to 3 times (e.g., 1 time, 2 times or 3 times).

The step (a-1) may further include the step (a-1-5): and (c) taking the system which finishes the step (a-1-4), and adding ultrapure water for dilution. This step is performed to control the concentration of the probe prepared each time to be constant, thereby facilitating the subsequent QC. In the step (a-1-5), the concentration of the probe may be 20 to 40. mu.g/mL (e.g., 20 to 30. mu.g/mL, 30 to 40. mu.g/mL, 20. mu.g/mL, 30. mu.g/mL, or 40. mu.g/mL)

In the step (a-2), "mixing probe 1 and probe 2" may specifically be mixing 1 part by volume of

probe

1 and 1 part by volume of probe 2.

The step (b-1) may include the steps of:

(b-1-1) mixing the sample to be tested with each mixed probe in the mixed probe set, and incubating;

(b-1-2) adding DNA polymerase (such as Bst DNA polymerase) and dNTP to the system after the step (b-1-1) is completed to obtain a reaction system f;

(b-1-3) taking the reaction system f, and extending to obtain an extension product.

In the step (b-1-1), the incubation may be performed at 35-39 deg.C (e.g., 35-37 deg.C, 37-39 deg.C, 35 deg.C, 37 deg.C or 39 deg.C) for 20-40min (e.g., 20-30min, 30-40min, 20min, 30min or 40 min).

In the step (b-1-1), "mixing the sample to be tested and each mixed probe in the mixed probe set" may specifically be mixing the sample to be tested, each mixed probe in the mixed probe set and PBS buffer.

In the step (b-1-3), the extension refers to reverse complementary binding of the DNA fragment 3 on the probe and the DNA fragment 6 on the probe, and the extension is performed on the basis of the reverse complementary binding to obtain an extension product. One end of the extension product may be the end of the DNA fragment 1 and the other end may be the end of the DNA fragment 4.

The step (c-1) may comprise the steps of:

(c-1-1) mixing each target protein standard solution with each mixed probe in the mixed probe set, and incubating;

(c-1-2) adding DNA polymerase (such as Bst DNA polymerase) and dNTP to the system after the step (c-1-1) is completed to obtain a reaction system m;

(c-1-3) taking the reaction system m, and extending to obtain an extension product.

In the step (c-1-1), the incubation may be performed at 35-39 deg.C (e.g., 35-37 deg.C, 37-39 deg.C, 35 deg.C, 37 deg.C or 39 deg.C) for 20-40min (e.g., 20-30min, 30-40min, 20min, 30min or 40 min).

In the step (c-1-1), "mixing each target protein standard solution and each mixed probe in the mixed probe set" may specifically be mixing each target protein standard solution, each mixed probe in the mixed probe set, and PBS buffer.

In the step (c-1-3), the extension refers to reverse complementary binding of the DNA fragment 3 on the probe and the DNA fragment 6 on the probe, and the extension is performed on the basis of the reverse complementary binding to obtain an extension product. One end of the extension product may be the end of the DNA fragment 1 and the other end may be the end of the DNA fragment 4.

Any of the above extension conditions may specifically be: 10min at 37 ℃ and 10min at 85 ℃.

Any one of the above PBS buffers may specifically be 0.01mM PBS buffer at pH 7.2.

Any of the above coupling agents may specifically be DBCO-NHS.

According to the above method, in one embodiment of the present invention, a kit for detecting the amount of alpha-fetoprotein in a sample to be tested is prepared. The kit comprises a single-chain DNA molecule A (hereinafter named ss-DNA1), an alpha fetoprotein antibody A (specifically an AFP capture antibody), a single-chain DNA molecule B (hereinafter named ss-DNA2), an alpha fetoprotein antibody B (specifically an AFP detection antibody), a library construction PCR upstream primer, a library construction PCR downstream primer (a library construction PCR downstream primer 1, a library construction PCR downstream primer 2, a library construction PCR downstream primer 3 or a library construction PCR downstream primer 4), a DNA standard (a double-chain specific DNA molecule aqueous solution), a library construction PCR upstream primer-standard, a library construction PCR downstream primer-standard (a library construction PCR downstream primer 1-standard, a library construction PCR downstream primer 2-standard, a library construction PCR downstream primer 3-standard or a library construction PCR downstream primer 4-standard), a sequencing primer A and a sequencing primer B. The library PCR upstream primer and the library PCR downstream primer are used for detecting the extension products of ss-DNA1 and ss-DNA 2. The library-building PCR upstream primer-standard substance and the library-building PCR downstream primer-standard substance are used for detecting the DNA standard substance. The specific nucleotide sequences and descriptions of the primers, DNA standards and single-stranded DNA molecules are detailed in Table 1. Experiments prove that the kit can detect the alpha fetoprotein content and has higher accuracy.

TABLE 1

The invention combines PEA and DNA sequencing technology to realize the simultaneous detection of the multi-protein content of multiple samples, and specifically comprises the following steps: 1. the ss-DNA (protein bar code) with different sequences is marked aiming at the antibodies of different types, and the protein types are distinguished according to the difference of the ss-DNA sequences; 2. converting protein information into DNA information by means of PEA technology; 3. adding a section of different Barcode tag sequences (sample barcodes) for different samples during library construction for distinguishing different samples; 4. the sequencer respectively detects the sample barcode sequence, the protein barcode sequence and the number of the protein barcode sequences, so that the simultaneous detection of the contents of various proteins in a plurality of samples is realized. The invention has important application value.

Drawings

FIG. 1 is a schematic diagram of high throughput sequencing for detecting the amount of multiple proteins.

FIG. 2 is a plot of linear regression with AFP concentration on the X-axis and copy number on the Y-axis.

FIG. 3 is a graph showing linear regression with the input amount of DNA standard on the X-axis and the copy number on the Y-axis.

FIG. 4 is a plot of linear regression with the logarithm of AFP concentration on the X-axis and the CT mean on the Y-axis.

FIG. 5 is a correlation analysis of QPCR quantitative detection and sequencer quantitative detection of AFP.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The AFP capture antibody and the AFP detection antibody are both products of Shenzhen Shenpengpen bio-corporation, and the product numbers are MAB-AFP-7 and MAB-AFP-28 respectively. Bst DNA polymerase is a product of NEB, Inc., and has a product number of M0275S. dNTP (10mM) was a product of ENZYMATICS, cat # N205L-1000.

Example 1 preparation of a kit for high throughput sequencing of multiple protein content in multiple samples

The invention discloses a kit for detecting the multi-protein content of a multi-sample by high-throughput sequencing, which is prepared by the inventor based on a PEA technology and a DNA sequencing technology. The kit can be used for simultaneously detecting a plurality of proteins in a plurality of samples to be detected (namely, a plurality of samples), and the detection cost is low. The principle of the kit for detecting the content of multiple proteins in multiple samples by high-throughput sequencing is shown in figure 1: in FIG. 1, A is the preparation process of the hybrid probe. In FIG. 1, B is the conversion of protein information into DNA information by reactions such as immunization, extension, and PCR amplification. In FIG. 1, C is the principle of high throughput sequencing for detecting multiple proteins in multiple samples.

The kit for detecting the multi-protein content of the multi-sample by high-throughput sequencing consists of a plurality of reagents for detecting different target proteins, a library-building PCR upstream primer, a library-building PCR downstream primer, a sequencing primer A and a sequencing primer B. There is no cross-reaction between reagents used to detect different proteins of interest. Each reagent for detecting the target protein comprises a single-chain DNA molecule A, a target protein antibody A, a single-chain DNA molecule B and a target protein antibody B.

The single-stranded DNA molecule A (from 5 'to 3') is composed of a DNA fragment 1, a DNA fragment 2 and a DNA fragment 3.

The single-stranded DNA molecule A is a single-stranded DNA molecule consisting of 30-50 (such as 39) nucleotides.

The single-stranded DNA molecule B (from 5 'to 3') is composed of a DNA fragment 4, a DNA fragment 5 and a DNA fragment 6.

The single-stranded DNA molecule B is a single-stranded DNA molecule consisting of 30-50 (such as 50) nucleotides.

The 5' ends of the single-stranded DNA molecule A and the single-stranded DNA molecule B are modified by azide or amino.

The DNA segment 1 and the DNA segment 4 are single-stranded DNA molecules consisting of 20-35 (such as 23, 32) nucleotides.

The DNA fragment 2 and the DNA fragment 5 are single-stranded DNA molecules consisting of 5-9 (such as 7 and 9) nucleotides.

DNA fragment 3 and DNA fragment 6 are both single-stranded DNA molecules consisting of 5-11 (e.g., 9) nucleotides.

DNA fragment 3 and DNA fragment 6 are complementary in reverse orientation.

In each reagent for detecting a target protein, the nucleotide sequences of the DNA fragment 2 and the DNA fragment 5 are different, so that different target proteins can be detected;

the pool-building PCR upstream primer (from 5 'to 3') consists of a DNA fragment a (sequencing adaptor sequence A) and a DNA fragment b. DNA fragment b is identical to DNA fragment 1. And carrying out phosphorylation modification on the 5' end of the upstream primer of the library-building PCR.

The library PCR downstream primer (from 5 'to 3') consists of a DNA fragment c (sequencing adaptor sequence B), a Barcode tag sequence and a DNA fragment d. The 5' ends of DNA fragment d and DNA fragment 4 were identical.

The DNA fragment a is a single-stranded DNA molecule consisting of 15-30 (such as 25) nucleotides.

The DNA fragment c is a single-stranded DNA molecule consisting of 15 to 30 (for example, 17) nucleotides.

The pool-building PCR upstream primer is a single-stranded DNA molecule consisting of 45-50 (such as 48) nucleotides.

The downstream primer of the library-building PCR is a single-stranded DNA molecule consisting of 45-50 (such as 48) nucleotides.

The purpose of adding a sequencing adaptor sequence A into the PCR upstream primer of the library is to facilitate sequencing and to detect ss-DNA, namely information of protein barcodes (distinguishing different proteins). According to the difference of sequencing platform, the sequencing linker sequence composed of other nucleotide sequence can be replaced. In embodiments of the invention, sequencing is performed using BGI500 platform sequencing. Accordingly, the nucleotide sequence of the sequencing adapter sequence A (from 5 'to 3') is GAACGACATGGCTACGATCCGACTT.

The purpose of adding sequencing linker sequences B and Barcode marker sequences into the library PCR downstream primer is to facilitate sequencing and to detect information of sample barcodes (distinguishing different samples). According to the difference of sequencing platform, the sequencing linker sequence composed of other nucleotide sequence can be replaced. In embodiments of the invention, sequencing is performed using BGI500 platform sequencing. Accordingly, the nucleotide sequence of sequencing adapter sequence B (from 5 'to 3') was TGTGAGCCAAGGAGTTG.

The sequencing primer A (from 5 'to 3') consists of a DNA fragment X and a DNA fragment Y. The DNA fragment X and the DNA fragment c (sequencing linker sequence B) are reversely complementary, and the DNA fragment Y and the DNA fragment a (sequencing linker sequence A) are completely consistent. Because the BGI500 platform sequencing is used in the invention, the library is circular, and is circled at the positions of the DNA segment a and the DNA segment c, the sequencing primer A consists of the reverse complementary sequence of the DNA segment c and the DNA segment a. The nucleotide sequence of sequencing primer A (from 5 'to 3') was CAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT.

The sequencing primer A is a single-stranded DNA molecule consisting of 40-50 (such as 42) nucleotides.

And the sequencing primer B is reversely complementary with the sequence of the DNA fragment 4. Since the DNB generated by DNB sequencing is identical to the original sequence (i.e., DNA fragment 4), the sequencing primer B is reverse complementary to the original sequence (i.e., DNA fragment 4). The nucleotide sequence of sequencing primer B (from 5 'to 3') was AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAA.

The kit for detecting the multi-sample multi-protein content by high-throughput sequencing can also contain a DNA standard, and a library-building PCR upstream primer and a library-building PCR downstream primer for detecting the DNA standard, which are respectively named as a library-building PCR upstream primer-standard and a library-building PCR downstream primer-standard.

The DNA standard is double-strand specific DNA molecule solution. The solvent may be water.

The double-stranded specific DNA molecule (from 5 'to 3') consists of a DNA fragment m, a random DNA fragment and a DNA fragment n.

Each strand of a double-stranded specific DNA molecule consists of 60-100 (e.g., 80) nucleotides.

The DNA fragment m consists of 15 to 30 (e.g., 19) nucleotides.

The DNA fragment n consists of 25 to 50 (e.g., 32) nucleotides.

Random DNA fragments consist of 25-50 (e.g., 29) nucleotides.

The pool-building PCR upstream primer-standard (from 5 'to 3') consists of a DNA fragment a (sequencing adaptor sequence A) and a DNA fragment M. The DNA fragment M and the DNA fragment M are identical.

The library PCR downstream primer-standard (from 5 'to 3') consists of DNA fragment c (sequencing linker sequence B), Barcode tag sequence and DNA fragment N. The 3' ends of the DNA fragment N and the DNA fragment N are reversely complementary.

The library PCR upstream primer-standard is a single-stranded DNA molecule consisting of 45-50 (such as 44) nucleotides.

The library PCR downstream primer-standard is a single-stranded DNA molecule consisting of 45-50 (such as 48) nucleotides.

Example 2 preparation of kit for high throughput sequencing detection of alpha-fetoprotein content

The invention discloses a kit for detecting alpha-fetoprotein content by high-throughput sequencing. The kit comprises a single-chain DNA molecule A (hereinafter named ss-DNA1), an alpha fetoprotein antibody A (specifically an AFP capture antibody), a single-chain DNA molecule B (hereinafter named ss-DNA2), an alpha fetoprotein antibody B (specifically an AFP detection antibody), a library construction PCR upstream primer, a library construction PCR downstream primer (a library construction PCR downstream primer 1, a library construction PCR downstream primer 2, a library construction PCR downstream primer 3 or a library construction PCR downstream primer 4), a DNA standard (a double-chain specific DNA molecule aqueous solution), a library construction PCR upstream primer-standard, a library construction PCR downstream primer-standard (a library construction PCR downstream primer 1-standard, a library construction PCR downstream primer 2-standard, a library construction PCR downstream primer 3-standard or a library construction PCR downstream primer 4-standard), a sequencing primer A and a sequencing primer B. The library PCR upstream primer and the library PCR downstream primer are used for detecting the extension products of ss-DNA1 and ss-DNA 2. The library-building PCR upstream primer-standard substance and the library-building PCR downstream primer-standard substance are used for detecting the DNA standard substance. Specific nucleotide sequences of the primers, DNA standards and single-stranded DNA molecules are shown in Table 1.

TABLE 1

Example 3 establishment of method for detecting alpha-fetoprotein content Using the kit prepared in example 2

Preparation of first and second mixing probes

1. Adding DBCO-NHS and 30 mu g of AFP capture antibody into 30 mu L of PBS buffer solution with pH7.2 and 0.01mM to obtain a reaction system; in the reaction system, the concentration of DBCO-NHS was 8 nmol.

2. Taking the reaction system obtained in the step 1, standing for 30min at 37 ℃, and then carrying out ultrafiltration and centrifugation for 2 times.

3. After the step 2 is completed, ss-DNA1 is added to obtain a reaction system; in this reaction system, the concentration of ss-DNA1 was 0.5 nM.

4. Taking the reaction system obtained in the step 3, standing for 5 hours at 37 ℃, and then carrying out ultrafiltration and centrifugation for 2 times.

5. After completion of step 4, dilution with ultrapure water was added to obtain an AFP capture antibody-ss-DNA 1 probe with a DNA concentration of 30. mu.g/mL.

6. According to the steps 1-5, replacing the AFP capture antibody with an AFP detection antibody, replacing the ss-DNA1 with ss-DNA2, and obtaining the AFP detection antibody-ss-DNA 2 probe with the DNA concentration of 30 mug/mL.

7. Mixing 1 volume part of AFP capture antibody-ss-DNA 1 probe and 1 volume part of AFP detection antibody-ss-DNA 2 probe to obtain a mixed probe.

Second, extension reaction

1. mu.L of a sample to be tested (AFP aqueous solution 1 at a concentration of 2ng/mL, AFP aqueous solution 2 at a concentration of 8ng/mL, AFP aqueous solution 3 at a concentration of 32ng/mL or AFP aqueous solution 4 at a concentration of 128 ng/mL), 2. mu.L of the mixed probe and 17. mu.L of PBS buffer solution of pH7.2 and 0.01mM were mixed, and then allowed to stand at 37 ℃ for 30 min.

2. To the system which completed

step

1, 1. mu.L of LBst DNA polymerase, 2. mu.L of dNTP (10mM) and 17. mu.L of PBS buffer solution of pH7.2 and 0.01mM were added to obtain a reaction system.

3. After the step 2 is completed, the reaction system is taken and extended (the extension conditions are: 37 ℃ for 10min and 85 ℃ for 10min), and an extension product is obtained.

Third, PCR amplification

1. Preparing a reaction system A and a reaction system B

The reaction system A is 20 μ L, and comprises 4 μ L of 5 XTaq DNA polymerase buffer solution, 1 μ L of dNTP (10mM), 0.2 μ L of DNA polymerase (concentration is 1U/. mu.L), 2 μ L of aqueous solution of the upstream primer of the library-building PCR, 2 μ L of aqueous solution of the downstream primer of the library-building PCR (different downstream primers of the library-building PCR corresponding to different samples to be tested), 4 μ L of extension product and 6.8 μ L H₂And (C) O. In the reaction system A, the concentrations of the library-establishing PCR upstream primer and the library-establishing PCR downstream primer are both 10 mu M.

The reaction system B was 20. mu.L, and consisted of 4. mu.L of 5 XTaq DNA polymerase buffer, 1. mu.L of dNTP (10mM), 0.2. mu.L of DNA polymerase (1U/. mu.L in concentration), 2. mu.L of an aqueous solution of the forward primer-standard of the library-building PCR, 2. mu.L of an aqueous solution of the reverse primer-standard of the library-building PCR (different amounts of the DNA standard added correspond to different amounts of the reverse primer-standard of the library-building PCR), 4. mu.L of the LDNA standard (12.5 fmol, 50fmol, 200fmol or 800fmol added), and 6.8. mu. L H2O. In the reaction system B, the concentrations of the library-establishing PCR upstream primer-standard substance and the library-establishing PCR downstream primer-standard substance are both 10 mu M.

2. PCR amplification

And (3) respectively taking the reaction system A or the reaction system B prepared in the step (1) to carry out PCR amplification to obtain a PCR amplification product.

Fourthly, quantitative detection of sequencer

And (4) respectively taking the PCR amplification products obtained in the third step, and sequencing by adopting a BGISEQ500 platform.

Based on the sequencing results, the DNA copy number of each sample was obtained.

The results are shown in Table 2.

TABLE 2

Sample(s)	DNA copy number (strip)
		AFP aqueous solution 1 at a concentration of 2ng/mL	85562
AFP aqueous solution 2 at a concentration of 8ng/mL	152631
		AFP aqueous solution 3 at a concentration of 32ng/mL	271119
AFP aqueous solution 4 at a concentration of 128ng/mL	657590
		DNA standard (input amount is 12.5fmol)	227266
DNA standard (input amount is 50fmol)	675476
		DNA standard (input 200fmol)	4918080
DNA standard (input amount is 800fmol)	24661499

Linear regression plots were performed with the concentration of antigen (i.e., AFP) in reaction A as the X-axis and the copy number as the Y-axis. The results are shown in FIG. 2. The results show that AFP concentration is linear with copy number, the correlation coefficient (R)²) The value can reach 0.99.

And (3) performing linear regression plotting by taking the input amount of the DNA standard substance in the reaction system B as an X axis and the copy number as a Y axis. The results are shown in FIG. 3. The results show that the input amount of the DNA standard substance is linear with the copy number, and the correlation coefficient (R)²) The value can reach 0.99.

Fifth, establishment of method for detecting alpha-fetoprotein content in sample to be detected by using kit prepared in example 2

The method for detecting the alpha fetoprotein content in the sample to be detected by adopting the kit prepared in the embodiment 2 comprises the following specific steps:

1. preparing solutions of alpha-fetoprotein with different concentrations, sequentially performing extension reaction, PCR amplification and sequencing according to the steps from one step to four steps, and then drawing an alpha-fetoprotein standard curve by taking the concentrations of the alpha-fetoprotein with different concentrations as abscissa and the corresponding DNA copy numbers as ordinate.

2. Taking a sample to be detected, and sequentially carrying out extension reaction, PCR amplification and sequencing according to the first step to the fourth step to obtain the corresponding DNA copy number of the sample to be detected; and then obtaining the content of the alpha fetoprotein in the sample to be detected according to the standard curve of the alpha fetoprotein.

Example 4 quantitative determination of alpha-fetoprotein content by QPCR

The experiment was repeated twice and the average was taken. The steps for each repetition are as follows:

preparation of first and second mixing probes

A mixed probe was prepared in the same manner as in the first step of example 3.

Second, extension reaction

2. To the system which completed

step

3. After the completion of the step 2, the reaction system was taken out and elongated (elongation conditions: 20min at 37 ℃) to obtain an elongated product.

Quantitative detection by QPCR

1. Preparing a reaction system

The reaction system is 10 muL, and consists of 4 muL of extension product, 0.25 muL of upstream primer (nucleotide sequence: 5 ' -TGTCATAAGGTTACCTAAGGGACTCACTGAATAAGGC) aqueous solution, 0.25 muL of downstream primer (nucleotide sequence: 5'-TTGTCTTCCTAAGACCGCTTGGCCTCC-3') aqueous solution, 5 muL of Universal SYBR qPCR Master Mix and 0.5 muLH 2O. In the reaction system, the concentration of the forward primer and the concentration of the reverse primer are both 10. mu.M.

The Universal SYBR qPCR Master Mix is a product of Biotechnology Inc. of Nanjing Novowed, Inc., having a product number of Q711-03. The Universal SYBR qPCR Master Mix contains Taq enzyme and dNTP mixture.

2. QPCR detection

And (3) carrying out QPCR detection on the reaction system prepared in the step (1) to obtain the CT value of each sample to be detected.

Reaction conditions are as follows: 95 ℃ for 3s, 60 ℃ for 30s, 40 cycles.

The results are shown in Table 3.

TABLE 3

With the logarithm of the antigen (i.e., AFP) concentration as XAxis, CT mean Y axis, linear regression plots were performed. The results are shown in FIG. 4. The results show that AFP concentration is linear with CT value, and the correlation coefficient (R)²) The value can reach 0.99.

Correlation between quantitative detection of QPCR and quantitative detection of sequencer

The results of QPCR quantitative detection and sequencer quantitative detection of the antigen (i.e., AFP) were analyzed for correlation.

The analytical results are shown in FIG. 5. The result shows that the quantitative detection of QPCR and the quantitative detection of the antigen by a sequencer have higher consistency. Therefore, the kit prepared in the embodiment 2 can be used for detecting the alpha-fetoprotein content, and has high accuracy.

Claims

1. A kit for detecting the content of different target proteins in a sample to be detected comprises a plurality of reagents for detecting different target proteins, a library-building PCR upstream primer, a library-building PCR downstream primer, a sequencing primer A and a sequencing primer B;

each reagent for detecting the target protein comprises a single-chain DNA molecule A, a target protein antibody A, a single-chain DNA molecule B and a target protein antibody B;

the single-stranded DNA molecule A sequentially comprises a DNA fragment 1, a DNA fragment 2 and a DNA fragment 3 from the 5 'end to the 3' end;

the single-stranded DNA molecule B sequentially comprises a DNA fragment 4, a DNA fragment 5 and a DNA fragment 6 from the 5 'end to the 3' end;

the DNA segment 3 and the DNA segment 6 are reverse complementary;

in each reagent for detecting the target protein, the nucleotide sequences of the DNA segment 2 and the DNA segment 5 are different, and the reagents are used for detecting different target proteins;

the library-building PCR upstream primer sequentially comprises a DNA fragment a and a DNA fragment b from the 5 'end to the 3' end; the DNA fragment b and the DNA fragment 1 are completely identical;

the library-building PCR downstream primer sequentially comprises a DNA fragment c and a DNA fragment d from the 5 'end to the 3' end; the 5' ends of the DNA fragment d and the DNA fragment 4 are completely consistent;

2. The kit of claim 1, wherein:

the DNA segment 1 and the DNA segment 4 are single-stranded DNA molecules consisting of 20-35 nucleotides;

the DNA segment 2 and the DNA segment 5 are single-stranded DNA molecules consisting of 5-9 nucleotides;

the DNA segment 3 and the DNA segment 6 are single-stranded DNA molecules consisting of 5-11 nucleotides;

the DNA fragment a is a single-stranded DNA molecule consisting of 15-30 nucleotides;

the DNA fragment c is a single-stranded DNA molecule consisting of 15-30 nucleotides.

3. The kit of claim 1, wherein:

the single-stranded DNA molecule A is a single-stranded DNA molecule consisting of 30-50 nucleotides;

the single-stranded DNA molecule B is a single-stranded DNA molecule consisting of 30-50 nucleotides;

the library-building PCR upstream primer is a single-stranded DNA molecule consisting of 45-50 nucleotides;

the library-building PCR downstream primer is a single-stranded DNA molecule consisting of 45-50 nucleotides;

the sequencing primer A is a single-stranded DNA molecule consisting of 40-50 nucleotides.

4. The kit of any one of claims 1 to 3, wherein: the library-building PCR downstream primer also comprises a Barcode marker sequence; the Barcode marker sequence is located downstream of the DNA fragment c and upstream of the DNA fragment d.

5. The kit of any one of claims 1 to 4, wherein: the kit also comprises a reagent for detecting the DNA standard; the reagent for detecting the DNA standard comprises the DNA standard, a library-building PCR upstream primer-standard and a library-building PCR downstream primer-standard;

the DNA standard contains specific DNA molecules; the specific DNA molecule sequentially comprises a DNA fragment m, a random DNA fragment and a DNA fragment n from the 5 'end to the 3' end;

the library-building PCR upstream primer-standard comprises the DNA fragment a and the DNA fragment M from the 5 'end to the 3' end in sequence; the DNA fragment M and the DNA fragment M are completely identical;

the library-building PCR downstream primer-standard comprises the DNA fragment c and the DNA fragment N from the 5 'end to the 3' end in sequence; the 3' ends of the DNA fragment N and the DNA fragment N are reversely complementary.

6. The kit of claim 5, wherein: the library-building PCR downstream primer-standard also comprises a Barcode marker sequence; the Barcode marker sequence is located downstream of the DNA fragment c and upstream of the DNA fragment N.

7. Use of the kit of any one of claims 1 to 6 for detecting the amount of different proteins of interest in a test sample.

8. A method for detecting the content of different target proteins in a sample to be detected comprises the following steps (a), (b), (c) and (d):

the step (a) includes the steps of:

(a-1) connecting the target protein antibody A of any one of claims 1 to 4 with the single-chain DNA molecule A to obtain a probe 1; connecting the target protein antibody B and the single-chain DNA molecule B in any one of claims 1 to 4 to obtain a probe 2;

the step (b) comprises the steps of:

(b-2) respectively using each extension product obtained in the step (b-1) as a template, and carrying out PCR amplification by using the library-building PCR upstream primer and the library-building PCR downstream primer in any one of claims 1 to 4 to obtain PCR amplification products;

(b-3) sequencing the PCR amplification products obtained in the step (b-2) by using the sequencing primer A and the sequencing primer B according to any one of claims 1 to 4 to obtain the DNA copy numbers corresponding to different target proteins in a sample to be detected;

the step (c) comprises the steps of:

(c-2) respectively using each extension product obtained in the step (c-1) as a template, and carrying out PCR amplification by using the library-building PCR upstream primer and the library-building PCR downstream primer in any one of claims 1 to 4 to obtain PCR amplification products;

(c-3) sequencing the PCR amplification products obtained in the step (c-2) by using the sequencing primer A and the sequencing primer B according to any one of claims 1 to 4 to obtain the DNA copy number corresponding to different target proteins in the target protein standard solution;

9. The method of claim 8, wherein:

in the step (a-1), the linking is performed by a coupling agent; the coupling agent may provide a chemical group that reacts with the protein antibody of interest and the single-stranded DNA molecule.

10. The method of claim 8, wherein:

the step (b-1) comprises the steps of:

(b-1-2) adding DNA polymerase and dNTP to the system after the step (b-1-1) is completed to obtain a reaction system f;

(b-1-3) taking the reaction system f, and extending to obtain an extension product;

the step (c-1) comprises the steps of:

(c-1-2) adding DNA polymerase and dNTP to the system after the step (c-1-1) is completed to obtain a reaction system m;