CN106701984A - Electrochemical luminescence nucleic acid detection method and kit based on branched DNA (Deoxyribonucleic Acid) amplification signal - Google Patents

Abstract

The present invention proposes an electrochemiluminescent nucleic acid detection method based on branched DNA amplified signals, amplifying the target gene by asymmetric PCR to obtain single-stranded target DNA, and adopting the principle of branched DNA amplified signal to design the electrochemiluminescence for the specific sequence of the target DNA Nucleic acid detection probe group, the single-stranded target DNA is hybridized with the electrochemiluminescence nucleic acid detection probe group, the hybridization product is detected by electrochemiluminescence, and the target DNA is qualitatively and quantitatively analyzed according to the presence or absence and intensity of the electrochemiluminescence signal, and finally To achieve the detection of target genes. The present invention also provides a kit for nucleic acid detection using the above method, which has the advantages of sensitive response, fast detection speed and accurate result.

Description

An electrochemiluminescent nucleic acid detection method and test based on branched DNA amplification signal Kit

technical field

The invention relates to the technical fields of molecular biology and nucleic acid detection, in particular to an electrochemiluminescence nucleic acid detection method based on branched DNA amplification signals, and also relates to an electrochemiluminescence nucleic acid detection kit based on branched DNA amplification signals.

Background technique

In recent years, nucleic acid testing (NAT) has been used in the prevention and control of infectious diseases, early diagnosis and precise treatment of malignant tumors, early screening of genetic diseases, prevention and control of animal and plant viruses, and detection of genetically modified genes. play an important role.

At present, the main methods for nucleic acid detection include gene chips, in situ hybridization, and PCR. Gene biosensors based on nucleic acid hybridization are one of the most rapidly developed nucleic acid detection methods in recent years. Among them, the electrochemiluminescence gene sensor combines the fast sensitivity of the electrochemiluminescence method with the high sequence specificity of nucleic acid molecule hybridization, and can realize the rapid, sensitive and accurate detection of nucleic acid.

The newly developed branched DNA (branched DNA, bDNA) signal amplification technology is an amplification technology for specific nucleotide detection in the form of microwells. Its principle is: use alkaline phosphatase-labeled oligodeoxynucleotide probes to sequentially hybridize with gene or pathogen nucleotides at the tissue or cell level, so that the hybridization signal cascades and amplifies, obtains chemiluminescent signals, and passes chemical The luminescence meter reads the luminescence value, and then calculates the copy number of the target DNA according to the standard curve drawn by the known concentration standard, so as to realize qualitative or quantitative detection. The bDNA technology has significant advantages: the release agent is used to directly release the target DNA, avoiding DNA extraction and saving time; no amplification is required, and false positives caused by improper operation or experimental environment pollution during the PCR reaction are avoided; equipment requirements It is not high, and the cost is lower; the operation is simple, the experimental steps are simple, and automatic detection can be realized.

Although the principle of bDNA technology has been proven to a certain extent, it has not been widely promoted in actual use. The reason is that there are many technical difficulties from theory to actual use, such as buffering in actual use. The construction of the liquid system and the design of the amplified probe set will directly affect the effect of use. In reality, although there are some related products, they are limited to only a few manufacturers. The composition of the buffer solution is not specified, and the design method and specific sequence of the amplification probe set involved are not disclosed, but a technical secret protection method is adopted. In addition, the specific experiment of the current electrochemiluminescence (ECL) technology The practice is not open, and the specific synthesis method of the luminescent labeling probe involved in it is rarely disclosed, resulting in many difficulties in the actual research and use of electrochemiluminescence that need to be faced and solved. At the same time, electrochemiluminescence also requires the use of special electrochemiluminescence Chemiluminescence instrument, and its development requires knowledge of physics, chemistry, biology and electronics, which also greatly limits the application of ECL technology. Furthermore, the combination of nucleic acid detection, bDNA technology and ECL technology requires that the required probe sequence be very clear to avoid problems such as non-specific hybridization and identification. For now, the sequences involved in these two technologies are rarely disclosed. , limiting the combination of these two technologies, especially in the field of nucleic acid detection.

Contents of the invention

The present invention combines the fast and sensitive advantages of the electrochemiluminescence detection method with the advantages of bDNA technology signal amplification, proposes an electrochemiluminescence nucleic acid detection method based on branched DNA amplification signals, and applies the method to pathogenic microorganisms, transgenes, etc. detection.

The technical solution of the present invention is realized in the following way: an electrochemiluminescence nucleic acid detection method based on branched DNA amplification signal, obtains single-stranded target DNA through asymmetric PCR amplification of the target gene, and adopts the principle of branched DNA amplification signal to design a specific method for the target DNA The electrochemiluminescence nucleic acid detection probe group of sex sequence, the single-stranded target DNA is hybridized with the electrochemiluminescence nucleic acid detection probe group, the hybridization product is detected by electrochemiluminescence, and the target DNA is detected according to the presence or absence and intensity of the electrochemiluminescence signal. Qualitative and quantitative analysis, and finally realize the detection of target genes.

Further, the steps are to extract target DNA, design and modify primers, design and synthesize the characteristic sequence of the capture probe set, design and synthesize the amplified probe set, design and synthesize the luminescent probe, and asymmetric PCR reaction, and then pass the probe set Hybridization, magnetic bead incubation and separation, electrochemiluminescence detection on the magnetic beads, qualitative and quantitative analysis of the target DNA according to the presence or absence and intensity of the electrochemiluminescence signal, and finally the detection of the target gene.

More specifically, the electrochemiluminescent nucleic acid detection method based on branched DNA amplified signal comprises the steps of:

(1) Extract the target DNA from the sample to be tested: the method of extracting the target DNA is different depending on the test object;

(2) Design primers according to the target gene sequence and modify the 5' end of the upstream primer with biotin: the modification of the primers is convenient for connecting with streptavidin-modified magnetic beads, and the design of the primers is mainly based on premier5 design and GenebankBLAST specificity analysis ;

(3) Design and synthesis of the detection probe set, including: design and synthesis of the characteristic sequence of the capture probe set, design and synthesis of the amplification probe set according to the principle of branched DNA, the amplification probe set includes the capture probe common Sequence, pre-amplification probe, amplification probe and luminescent labeling probe, luminescent substance labeling on the luminescent probe.

Design and synthesis of the characteristic sequence of the capture probe set: the design of the characteristic partial sequence is mainly based on the specificity analysis of Premier5 and Genebank BLAST;

Design and synthesis of amplified probe sets according to the principle of branched DNA: the design of amplified probe sets mainly uses Premier5 design and Genebank BLAST specificity analysis, and comprehensive probe length, annealing temperature, probe mismatch, dimer and specificity Sex and other related factors, after multiple screening, the specific sequence is finally determined;

Design and synthesis of luminescent probes: follow the principle of complementary base pairing, consider mismatches with other probes, and self-specific analysis, and modify active groups such as amino, carboxyl, and sulfhydryl groups at the end of the probes to facilitate attachment of luminescent labels thing.

(4) Use asymmetric PCR to amplify the target gene to obtain single-stranded target DNA labeled with biotin at the 5' end: due to different detection target DNA, the required asymmetric ratio is not necessarily the same. Generally speaking, the upstream and downstream primers Perform asymmetric PCR according to the ratio of 1:1 to 200:1, and then screen out the single-stranded DNA fragment amplified by the optimal upstream primer;

(5) The single-stranded target DNA labeled with biotin at the 5' end is hybridized with the detection probe set: according to capture probe-pre-amplifier probe hybridization, pre-amplifier probe-amplifier probe hybridization, target DNA-capture probe hybridization and Amplifying probe-luminescent labeling probe hybridization is carried out in sequence, and the conditions can be 50-60°C, 30-50min;

(6) Incubate the hybridization product with streptavidin-modified magnetic beads, remove the supernatant, collect the magnetic beads and disperse them in the buffer, the incubation conditions can be 37°C, 15-30min;

(7) Electrochemiluminescence detection of magnetic beads: luminescence detection includes: electrochemiluminescence, chemiluminescence, fluorescence, etc.;

(8) According to whether the electrochemiluminescence signal value is higher than the threshold value, it is judged whether the target DNA component is contained in the sample;

(9) Develop a standard curve, and perform quantitative analysis on the target DNA components in the positive samples according to the standard curve.

Further, in step (1), the DNA can be DNA of tumor markers, DNA of pathogenic microorganisms and DNA of genetically modified organisms, and is also suitable for viral nucleic acids and the like.

Further, in step (3), the capture probe includes a characteristic sequence and a universal sequence, the universal sequence is shared by all detection targets, and the characteristic sequence is a specific sequence designed for different target DNAs; the capture probes included in the amplification probe group are Universal sequence for capture probes.

Further, in step (3), the luminescent probes are not limited to ruthenium probes, and other electrochemiluminescent probes such as luminol, acridines, peroxalic acid esters, fused-ring aromatic hydrocarbons and quantum dots can also be used, And CdTe, CdSe, CdS, ZnS, C ₃ N ₄ , terbium, graphene oxide and other quantum dot fluorescent probes, chemiluminescence probes, etc.; in order to facilitate the connection of nucleic acid and the above markers, amino and carboxyl groups can be modified at the end of the probe , mercapto and other active groups.

Further, in step (6), the hybridization product is connected by biotin- and streptavidin-modified magnetic beads modified on the single-stranded target DNA.

Another object of the present invention is to propose a nucleic acid detection kit obtained by the electrochemiluminescent nucleic acid detection method based on branched DNA amplification signals, which is simple, fast and low in cost, and includes target DNA sequence primers, amplified probe sets , DNA polymerase, MgSO ₄ , dNTP, 10×PCR Buffer, Marker, positive control and negative control.

Further, in the electrochemiluminescent nucleic acid detection kit based on branched DNA amplification signal, the sequence of the amplification probe set is as follows:

The sequence of the pre-amplification probe is SEQ ID NO: 1; the sequence of the general part of the capture probe sequence is SEQ ID NO: 2; the sequence of the amplification probe is SEQ ID NO: 3; the sequence of the labeling probe is SEQ ID NO: 4.

Further, the positive control is the target DNA plasmid, and the negative control is ddH ₂ O.

Compared with the existing nucleic acid detection methods, the electrochemiluminescent nucleic acid detection method based on branched DNA amplification signal of the present invention has the following prominent features:

1. The primers for pathogenic bacteria were designed based on the conserved regions of the 16S rDNA genes of 9 types of foodborne pathogenic bacteria.

2. The transgene primers are designed according to the CaMV35S promoter and NOS terminator. This method is also suitable for primers of cancer genes, etc.

2. Design of capture probes: DNA sequencing to obtain related sequences, combined with related sequences in GenBank, to determine the tag sequence of each target bacteria in the primer amplification area, and design probes in the tag sequence area to achieve specific detection.

3. The design of the amplified probe set. According to the principle of complementary base pairing and non-homologous hybridization, a general amplified probe set was designed through BLAST functional screening in GenBank.

4. Design and synthesis of luminescence-labeled probes. Based on complementary base pairing, the characteristic sequences were screened by BLAST and used as luminescence-labeled probe sequences, synthesized and labeled with ruthenium at the 3' end.

5. The upstream and downstream primers are used for asymmetric PCR amplification according to the ratio of 1:1 to 200:1, and then the single-stranded target DNA product is obtained.

6. Biotin-streptavidin modification of primer-magnetic beads, the primer is modified with biotin at the 5' end, and the magnetic beads are coated with streptavidin to facilitate the enrichment of target DNA. The purpose of the magnetic beads is to adsorb and further amplify the signal, which is convenient for separation and observation.

7. Advantages of branched DNA technology, there are no problems such as DNA or RNA purification and PCR false positives, and the required equipment is simple, easy to operate, and convenient for automatic detection of DNA targets.

8. The universal kit can be used for the detection of pathogenic microorganisms, genetically modified and cancer genes, etc.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those skilled in the art, other drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 is the detection flowchart provided by the embodiment of the present invention;

Fig. 2 is the asymmetric electrophoresis diagram of Staphylococcus aureus provided by the embodiment of the present invention, wherein, M: Maeker, 1: negative control, 2, 3, 4 are normal experimental group 300bp is single-stranded DNA, 400bp is double-stranded DNA, 5: positive control;

Fig. 3 is the ECL diagram of the detection of Staphylococcus aureus provided by the embodiment of the present invention (the sample to be tested is tested normally in the electrochemiluminescence instrument, and a trigger voltage of 1.25V is applied in 15-30s).

detailed description

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

The present invention proposes an electrochemiluminescent nucleic acid detection method based on branched DNA amplification signals, using branched DNA signal amplification technology, and simultaneously adopting a mode in which a combination of electrochemiluminescent signals corresponds to a sequence, and amplifying target DNA through asymmetric PCR , to detect the specific sequence of the PCR product, so as to realize the detection of the target gene.

The electrochemiluminescent nucleic acid detection method based on branched DNA amplifying signal comprises the following steps:

(1) Extract the target DNA from the sample to be tested: the method of extracting the target DNA is different depending on the test object; DNA can be DNA of tumor markers, DNA of pathogenic microorganisms and DNA of genetically modified organisms, and is also suitable for viral nucleic acids, etc. .

(2) Design primers according to the target gene sequence and modify the 5' end of the upstream primer with biotin: the modification of the primers is convenient for connecting with streptavidin-modified magnetic beads, and the design of the primers is mainly based on premier5 design and GenebankBLAST specificity analysis ; Use the primer design software Primer5 to design primers, select three pairs of optimal primers for experimental screening according to the parameters, and then modify the 5' end of the upstream primer with biotin for subsequent binding with streptavidin-modified magnetic beads.

(3) Design and synthesis of the characteristic partial sequence of the capture probe set: the design of the capture probe is mainly based on Premier5 and Genebank BLAST specificity analysis; finally, the characteristic sequence of each target bacteria in the primer amplification area is determined by using the primer design software Primer5, and then Screen out the optimal capture probe sequence.

(4) Design and synthesize the amplification probe set according to the principle of branched DNA: the design of the amplification probe set (including the general part of the capture probe, the pre-amplification probe, the amplification probe and the luminescence labeling probe) is mainly based on premier5 and GenebankBLAST Specificity analysis, based on complementary base pairing and non-homologous hybridization principles, comprehensive probe length, annealing temperature, probe mismatch, dimer and other related factors, multiple screening, and finally determine the universal amplification probe set.

The sequence of the pre-amplification probe is SEQ ID NO: 1; the sequence of the general part of the capture probe sequence is SEQ ID NO: 2; the sequence of the amplification probe is SEQ ID NO: 3; the sequence of the labeling probe is SEQ ID NO: 4.

(5) Luminescent probe design and synthesis: Luminescent probe modification, depending on the luminescent label, the modification at the end of the probe is also different. Luminescent probes are not limited to ruthenium probes, and can also be used such as luminol, acridine Classes, peroxyoxalates, fused-ring aromatic hydrocarbons and other electrochemiluminescence probes of quantum dots, as well as quantum dot fluorescent probes such as CdTe, CdSe, CdS, ZnS, C ₃ N ₄ , terbium, graphene oxide, etc., chemical Luminescent probes, etc. The sequence of the luminescent probe follows the principle of complementary base pairing, and considers mismatches with other probes, as well as self-specific analysis, and modifies active groups such as amino, carboxyl, and sulfhydryl groups at the end of the probe to facilitate attachment of luminescent labels.

(6) Use asymmetric PCR to amplify the target DNA to obtain single-stranded target DNA labeled with biotin at the 5' end: due to different detection target DNA, the required asymmetric ratio is not necessarily the same. Generally speaking, the upstream and downstream primers Perform asymmetric PCR according to the ratio of 1:1 to 200:1, and then screen out the single-stranded DNA fragment amplified by the optimal upstream primer.

(7) The single-stranded target DNA labeled with biotin at the 5' end is hybridized with the probe set: according to capture probe-pre-amplification probe hybridization, pre-amplification probe-amplification probe hybridization, target DNA-capture probe hybridization and amplification Probe-luminescent labeled probe hybridization is carried out in sequence, the conditions can be 50-60°C, 30-50min;

Take a certain amount of Tris–HCl (pH 7.1, 20mM Tris–HCl, 140mM NaCl, 5mM KCl, 1mM CaCl ₂ , 1mM MgCl ₂ ) hybridization solution, add a certain proportion of capture probes, pre-amplification probes and amplification probes, 50- Shake and incubate at 60°C for 30min, then add a certain proportion of target DNA, shake and incubate at 50-60°C for 30min, finally add a certain proportion of luminescent labeled probes, shake and incubate at 50-60°C for 30min, and then obtain the target DNA of the amplified probe set branched molecules.

(8) Incubation and separation of hybridization products and streptavidin-modified magnetic beads: the incubation conditions can be 37°C, 15-30min;

Take a certain amount of the above-mentioned hybrid single-stranded target DNA, add streptavidin-modified magnetic beads (7.2 mg/mL) and incubate at 37°C for 30 min with slight shaking, then place in a magnetic separator, remove the supernatant by magnetic separation, and use Tris– Wash with HCl for 3 times, and finally disperse into Tris–HCl solution to obtain functionalized magnetic beads with single-stranded target DNA attached.

(9) Electrochemiluminescence detection of magnetic beads, including: electrochemiluminescence, chemiluminescence, fluorescence, etc.;

Take a certain amount of magnetic beads and tripropylamine (TPA) and add them to the sample cell together. The magnetic sheet placed at the bottom of the working electrode will adsorb the magnetic beads to the surface of the working electrode, and the voltage between the working electrode and the reference electrode is given to perform electrochemiluminescence detection. . Judging whether the sample contains the target DNA component according to whether the light signal value is higher than the threshold; formulate a standard curve, and perform quantitative analysis on the target DNA component in the positive sample according to the standard curve.

A nucleic acid detection kit based on an electrochemiluminescent nucleic acid detection method based on branched DNA amplification signals, including target DNA sequence primers, amplification probe sets, DNA polymerase, MgSO ₄ , dNTP, 10×PCR Buffer, Marker, positive Control and negative control, the positive control is the target DNA plasmid, and the negative control is ddH ₂ O.

Further, in the electrochemiluminescent nucleic acid detection kit based on branched DNA amplification signal, the sequence of the amplification probe set is as follows:

The sequence of the pre-amplification probe is SEQ ID NO: 1; the sequence of the general part of the capture probe sequence is SEQ ID NO: 2; the sequence of the amplification probe is SEQ ID NO: 3; the sequence of the labeling probe is SEQ ID NO: 4.

In order to facilitate the understanding of the technical solutions of the present invention, examples are used for specific description.

Example 1

Foodborne Staphylococcus aureus detection

At present, harmful pathogenic microorganisms not only affect people's health and endanger people's lives, but also cause panic in the whole society and affect normal social operation. The traditional identification of pathogenic microorganisms mainly relies on methods such as bacterial culture, serology, biochemistry, and colony morphology for classification and identification. While reliable, these methods often take days or even weeks to obtain results; and colony morphology can also be affected by the environment. Nucleic acid detection of microorganisms can quickly and accurately identify pathogenic microorganisms, thereby minimizing the damage of pathogens to the human body.

1) Strain culture

Staphylococcus aureus is cultured in 7.5% NaCl broth, Vibrio cholerae and Vibrio parahaemolyticus are cultured in alkaline peptone water, Streptococcus pyogenes is cultured in glucose meat infusion broth, and other bacteria are generally cultured in nutrient broth or nutrient agar nourish.

2) Target DNA extraction

The DNA of the strain samples was extracted by boiling lysis method. The details are as follows: take 1ml of overnight cultured bacterial solution and centrifuge to obtain bacterial cells, or obtain a bacterial lawn from a nutrient agar plate; suspend the bacterial cells with 100ul sterile water, heat at 100°C for 10min, cool to room temperature, centrifuge at 14000rpm for 8min, and take the supernatant as a template for PCR amplification.

3) Asymmetric PCR amplification

According to the design, the upstream primer sequence of S. aureus (Staphylococcus aureus) is located at 679-696 of 16S RNA, the sequence is 5'-CGCACATCAGCGTCAGTT-3', and the downstream primer is located at 1032-1053 of 16S rDNA, the sequence is 5 '-ATACGTAGGTGGCAAGCGTTAT-3'; F':R'100:1 for asymmetric PCR amplification, and the F' primer was labeled with biotin to obtain biotin-labeled single-stranded target DNA.

A 25ul reaction system was used for PCR, containing 10×buffer (50mM KCl, 10mM Tris-HCl, pH 8.6), 1.5mM MgCl ₂ , 0.4uM each universal primer, 0.2mM dNTP, 0.5ul Taq enzyme, and 0.5ul DNA template. After the PCR products were separated by 2.0% agarose gel electrophoresis, the gel was tapped and recovered, and finally the DNA content was estimated by electrophoresis (compared with the brightness of DNAmarker of known concentration).

4) The single-stranded target DNA labeled with biotin at the 5' end is hybridized with the probe set

Take a certain amount of 70ul Tris–HCl (pH 7.1, 20mM Tris–HCl, 140mM NaCl, 5mM KCl, 1mMCaCl ₂ , 1mM MgCl ₂ ) hybridization solution, add 2ul capture probes, 2ul pre-amplification probes and 6ul amplification probes, 50 Shake and incubate at -60°C for 30min, then add 2ul target DNA, shake and incubate at 50-60°C for 30min, finally add 18ul luminescence-labeled probes, shake and incubate at 50-60°C for 30min, and obtain the target DNA branch of the amplified probe set shape molecule.

5) Incubation and separation of hybridization products with streptavidin-modified magnetic beads

Take 50ul of the above-mentioned hybridized single-stranded target DNA, add streptavidin-modified magnetic beads (7.2mg/mL) and incubate with slight shaking at 37°C for 30min, then place in a magnetic separator, remove the supernatant by magnetic separation, and wash with Tris–HCl Wash 3 times, and finally disperse into Tris–HCl solution, and finally obtain functionalized magnetic beads with single-stranded target DNA attached.

6) Electrochemiluminescence detection of functionalized magnetic beads

Take 50ul of incubation magnetic beads and 2ul of tripropylamine (TPA) into the sample pool together, and set a voltage of 1.25V between the working electrode and the reference electrode for electrochemiluminescence detection. According to whether the light signal value is higher than the threshold value, it can be judged whether the target DNA component is contained in the sample; a standard curve is established, and the target DNA component in the positive sample is quantitatively analyzed according to the standard curve.

Example 2

CaMV35S Promoter Screening Kit

The safety of genetically modified products is controversial. Most transgenic organisms contain CaMV35S promoter and NOS terminator, and these two genes are commonly used regulatory elements for exogenous gene expression in plants, which can be used as markers of transgenic organisms. In this example, the CaMV35S promoter was selected as the target gene to be tested for the transgenic product.

The kit includes the following components: Taq DNA polymerase, MgSO ₄ , dNTP, 10×PCR Buffer, Marker, positive control and negative control, the positive control is a recombinant plasmid containing the CaMV35S promoter gene sequence, and the negative control The reference substance is ddH ₂ O.

Use of the kit: the kit selects the detection object of the CaMV35S promoter, and combines the detection method of branched DNA electrochemiluminescence for the detection of transgenic products.

Preservation of the kit: the contents in the kit should be stored at -20°C, protected from light, and the validity period is 1 year. Store at -4°C after the first use, valid within 3 months.

The CaMV35S promoter-specific detection primer sequence of the kit is as follows:

Upstream primer sequence: AACAGAACTCGCCGTAAAGACT;

Downstream primer sequence: CAGATAGCTGGGCAATGGAA;

The sequence of the specific detection amplification probe set is as follows:

Pre-amplified probes:

TATAGTGAGACGTCCATTTTATAGTGAGACGTCCATTTTATAGTGAGACGTCCATTTTGATAGATAGGTTCTCA

Capture probe:

CATACGCGCGACGATACGGCTTTTGAGAACCTATCTATCA

Zoom probe:

TGGACGTCTCACTATATTTAGCTGAGTCAAAGCATTTTAGCTGAGTCAAAGCATTTTAGCTGAGTCAAAGCAT

Labeled probe: ATGCTTTGACTCAGCT

The method for using the kit includes: extracting transgenic product DNA; asymmetric PCR with upstream and downstream primers; hybridizing the amplified probe group with the target DNA; hybridizing the luminescent label probe with the hybridization product;

The electrochemiluminescent branched DNA method involved in the present invention is also applicable to the fluorescent branched DNA method, and is also applicable to the chemiluminescent branched DNA method, etc.; such methods can be applied to the detection of genetically modified food and the detection of food-borne pathogenic bacteria and cancer genetic testing.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

SEQ ID NO: 1

Preamplifier Probe 74

TATAGTGAGACGTCCATTTTATAGTGAGACGTCCATTTTATAGTGAGACGTCCATTTTGATAGATAGGTTCTCA

SEQ ID NO: 2

capture probe 23

CATACGCGCGACGATACGGCTTT

SEQ ID NO: 3

Zoom Probe 73

TGGACGTCTCACTATATTTAGCTGAGTCAAAGCATTTTAGCTGAGTCAAAGCATTTTAGCTGAGTCAAAGCAT

SEQ ID NO: 4

Labeled probes 16

ATGCTTTGACTCAGCT

Claims (9)

1. it is a kind of based on branched DNA amplify signal electrochemical luminescence nucleic acid detection method, it is characterised in that：By asymmetric PCR amplification target genes obtain single-stranded target DNA, and signal principle design is amplified for target dna specificity sequence using branched DNA The electrochemical luminescence detection of nucleic acids probe groups of row, single-stranded target DNA and electrochemical luminescence detection of nucleic acids probe set hybridisation, to hybridization Product carries out electrochemical luminescence detection, and qualitative and quantitative is carried out to target dna according to the presence or absence of electrochemical luminescence signals and intensity Analysis, realizes the detection of target gene.

2. the electrochemical luminescence nucleic acid detection method of signal is amplified based on branched DNA as described in the appended claim 1, it is characterised in that Comprise the following steps：

(1) target gene DNA is extracted from testing sample；

(2) primer is designed according to target gene sequence and biotin modification is carried out to the end of sense primer 5 '；

(3) design of detection probe group and synthesis, including：The design of capture probe group characteristic sequence and synthesis, according to branched DNA Principle amplifying probe group is designed with synthesis, the amplifying probe group include capture probe universal sequence, preceding amplifying probe, Amplifying probe and luminescent marking probe, carry out shiner mark on luminescence probe.

(4) target gene is expanded using asymmetric PCR, obtains the single-stranded target DNA of 5 ' end mark biotins；

The single-stranded target DNA of (5) 5 ' end mark biotins hybridizes with detection probe group；

(6) hybrid product is incubated with Streptavidin modification magnetic bead, removes supernatant, collects magnetic bead and is scattered in buffer solution In；

(7) electrochemical luminescence detection is carried out to magnetic bead；

(8) whether whether target dna composition higher than in threshold decision sample is contained according to electrochemical luminescence signals value；

(9) standard curve is formulated, quantitative analysis is carried out to the target dna composition in positive according to standard curve.

3. the electrochemical luminescence nucleic acid detection method of signal is amplified based on branched DNA as described in the appended claim 1, and its feature exists In：In step (1), the target dna includes：Tumor markers DNA, pathogenic microorganism DNA, genetically modified organism DNA, viral core Acid.

4. the electrochemical luminescence nucleic acid detection method of signal is amplified based on branched DNA as described in the appended claim 1, and its feature exists In：In step (3), capture probe includes characteristic sequence and universal sequence, and universal sequence is shared for all detection targets, feature sequence It is classified as the specific sequence designed for different target DNA；The capture probe that amplifying probe group includes is logical for capture probe Use sequence.

5. the electrochemical luminescence nucleic acid detection method of signal is amplified based on branched DNA as described in the appended claim 1, and its feature exists In：In step (3), luminescence probe includes ruthenium probe, C₃N₄Probe, terbium probe, electrochemical luminescence probe, quantum dot fluorescence probe, Chemiluminescence probe.

6. the electrochemical luminescence nucleic acid detection method of signal is amplified based on branched DNA as claimed in claim 5, and its feature exists In：In step (3), the active base group modification of probe end, including amino, carboxyl, sulfhydryl reactive group.

7. the electrochemical luminescence nucleic acid detection method of signal is amplified based on branched DNA as described in the appended claim 1, and its feature exists In：In step (6), hybrid product is connected by the magnetic bead that the biotin and Streptavidin modified on single-stranded target DNA are modified.

8. the electrochemical luminescence nucleic acid detection method system of signal is amplified according to any in claim 1-7 based on branched DNA Standby kit for detecting nucleic acid, it is characterised in that：Including target dna sequence primer, capture probe group, general amplifying probe group, Archaeal dna polymerase, MgSO₄, dNTP, 10 × PCR Buffer, Marker, positive control and negative control.The positive control is Target dna plasmid, the negative controls are ddH₂O.The target dna plasmid is mutually strained according to the difference of detection target Change, equally, capture probe characteristic sequence is the specific sequence designed for different target DNA.

9. the electrochemical luminescence kit for detecting nucleic acid of signal is amplified based on branched DNA as recited in claim 7, and its feature exists In：Preceding amplifying probe sequence is SEQ ID NO：1；Capture probe universal sequence is SEQ ID NO：2；Amplifying probe sequence is SEQ ID NO：3；Luminescent marking probe sequence is SEQ ID NO：4.