CN102703442A - Bio-barcode for detecting canine parvovirus and application thereof - Google Patents

Abstract

The invention provides a bio-barcode for detecting canine parvovirus and a complentary NP (nanoparticle) probe chain used with the bio-barcode. The complentary NP probe chain has nucleotide sequences respectively shown as SEQ ID No.1 and SEQ ID No.2. The invention further provides a method and an assay kit for fluorescent quantitative detection of the bio-barcode for canine parvovirus. The method has the advantages of accurate detection, high sensibility, high specificity, simplicity, convenience and quickness, and has excellent specimen detectability.

Description

A biological barcode for detecting canine parvovirus and its application

technical field

The invention relates to a barcode detection method, in particular to the assembly of primers, probes and fluorescent quantitative PCR detection kits used in the biological barcode detection technology for detecting canine parvovirus.

Background technique

Bio-bar codes assay (BCA) is a new type of marker immunoassay technology first reported by Professor Mirkin of Northwestern University in 2003. Its outstanding feature is that it has extremely high sensitivity, which can reach 106 of the conventional ELISA method. times. The basic principle of BCA technology is to use the magnetic microsphere probe (magnetic microparticles, MMPs) labeled with the monoclonal antibody of the object and the gold nanoparticle probe (nanoparticle, NP) labeled with the polyclonal antibody of the object and specific DNA double strands. After forming the MMP-analyte-NP sandwich complex, dehybridization is used to release the specific DNA strands marked on the NP probe, and these released DNA strands are identified by conventional PCR methods or chip detection methods (gold standard silver staining method) The presence of the detected object can be confirmed.

NP probes are gold nanoparticles coated with double-stranded DNA and polyclonal antibodies against the object to be tested. One of the double-stranded DNA is connected to the colloidal gold particle through the Au-S bond, and the other is complementary to the former to indicate The barcode DNA of the object to be tested; the MMP probe is a magnetic microsphere probe with a diameter of about 1 μm corresponding to the sorting magnetic field, and its surface is coated with a monoclonal antibody against the object to be tested.

It can be seen from the detection procedure of BCA technology that its basic principle is similar to the double antibody sandwich ELISA antigen detection method. The indirect detection of the detected object is realized by detecting the barcode DNA, and the detection sensitivity is greatly improved due to the amplification effect of the PCR reaction and chip detection.

The biological barcode detection process can be divided into two aspects: the acquisition of barcode DNA and the detection of barcode DNA. The acquisition process of barcode DNA includes three steps. The first step is the interaction of antigen and antibody. First, the MMP probe functionalized with monoclonal antibody against the object to be detected specifically binds to the possible object in the sample, and then the MMP probe is immobilized by a magnetic field. The needle is washed repeatedly with PBS solution to remove unbound analytes and irrelevant impurities; the second step: elution and dehybridization, adding NP probes modified by double-stranded DNA and polyclonal antibodies against analytes, Sandwich the object to be tested to form a sandwich structure, then apply a magnetic field, fix the MMP probe with a magnet, and wash repeatedly to remove unconnected NP probes; the third step: DNA separation, this step is a step to remove In the hybridization step, the sandwich structure is fixed by a magnetic field. Under high temperature and low salt conditions, hundreds of barcode DNA bound to the NP probe is released into the solution, and then the barcode DNA chain is quantified to indirectly detect the analyte. There are currently two ways to detect the barcode DNA chain: one is to perform ordinary PCR amplification on the obtained barcode DNA chain, and then detect it by gel electrophoresis; the other is to detect it through a chip. The chip detection system requires three kinds of nucleic acids with different functions: 1. ) a capture probe (slide that captures DNA modification) attached to a solid support (slide, etc.); 2) a detection probe that generates a signal (DNA-modified colloidal gold); 3) a target nucleic acid (barcoded DNA), In this system the target nucleic acid acts as a link between the capture probe and the signal detection probe. If the above three components exist in the system, the target nucleic acid can hybridize with the above two nucleotides, and a clear detection signal can be obtained after elution to remove impurities. If the target nucleic acid does not hybridize with the above two probes, the signal probe It cannot be connected to the solid phase, and there is no signal response on the solid phase after elution, and the obtained signal can be analyzed with a common flatbed scanner.

BCA technology has made good progress in the detection of some pathogenic markers. Nam et al. first used BCA technology to detect prostate specific antigen (PSA). The detection limit of PSA using BCA technology can reach 3 a mol/L, which is 6 orders of magnitude lower than the existing traditional ELISA method; Georganopoulou et al. used BCA technology to detect amyloid β-derived diffusible ligands (ADDL), and its detection limit could detect 50 ADDL molecules in 15 μL of cerebrospinal fluid; Stoeva et al. BCA technology was used to jointly detect three tumor markers PSA, human chorionic gonadotropin (HCG) and α-fetoprotein (AFP) in serum, and the detection results showed that the limit of detection was acceptable. Up to 170fmol/L.

In summary, compared with ELISA technology, BCA technology has very significant advantages, which are embodied in the following aspects: 1) BCA technology shows a sensitivity of six orders of magnitude (1,000,000 times) higher than conventional ELISA method 2) Design barcode DNA with different lengths and sequences for different tested objects, which can analyze multiple substances in complex samples; 3) High specificity, which depends on the monoclonal antibody used in the detection system The specificity of the detection system can be guaranteed as long as the monoclonal antibody with high specificity against the target object is used; 4) The detection range is wide, as long as the object has monoclonal antibody and polyantibody, it can be detected , which makes it a great advantage in detecting some trace substances that are not suitable for detection by PCR technology.

However, as can be seen from the detection process and display system of BCA technology, this technology has obvious defects, which are mainly reflected in the detection of barcode DNA, specifically: 1) traditional PCR detection is to use terminal PCR to quantify samples The amount of template in the medium is usually separated by gel electrophoresis and fluorescent staining is used to detect the final amplification product of the PCR reaction. However, in the PCR reaction, due to factors such as the aggregation of templates, reagents, and pyrophosphate molecules, the polymerase reaction is affected, and eventually the PCR reaction no longer proceeds exponentially and enters a "plateau stage", and the final products of some reactions are slower than others Therefore, the quantification of the end-point PCR reaction method is inaccurate; secondly, the end-point PCR is prone to cross-contamination and produces false positives; Time and environmental factors vary a lot; 3) Neither the PCR detection system nor the chip detection system can effectively quantify the detected object, which is compared with the development requirements and development direction of the current immune detection technology: not only qualitative, but also quantitative Outdated, so it is very important to improve the conventional BCA technology so that it can overcome the above shortcomings and can accurately quantify the analyte.

Combining BCA technology with real-time fluorescent quantitative PCR (Real-time fluorescent quantitative PCR, FQ-PCR) technology, based on the analysis and optimization of key data, the establishment of fluorescent quantitative biological barcode detection technology can overcome the above defects and deficiencies.

FQ-PCR technology was founded in 1996 and launched by Applied Biosystems in the United States. It is a nucleic acid quantitative technology developed on the basis of PCR qualitative technology. FQ-PCR technology is to add a fluorescent group to the PCR reaction system, and use the change of the fluorescent signal to detect the change of the amount of the amplification product in each cycle of the PCR amplification reaction in real time. Perform quantitative analysis. FQ-PCR technology integrates the advantages of PCR technology and DNA probe hybridization technology, and directly detects the change of fluorescent signal during PCR, so that the PCR amplification and analysis process are completed under the same closed system, and realized with the support of computer analysis software The dynamic monitoring and automatic quantification of PCR amplification products have the following characteristics: 1) The quantitative principle is unique, and the amount of amplified products is displayed by the amount of fluorescent signal generated. Dynamic real-time continuous fluorescence monitoring has excellent 2) Fluorescent signals are obtained by embedding fluorescent dyes into double-stranded DNA, or double-labeled sequence-specific fluorescent probes or energy signal transfer probes, which greatly improve the sensitivity, specificity and accuracy of detection ;3) It is only necessary to open the lid once when adding samples, and the subsequent process is completely closed-tube operation, which avoids the contamination of samples and products, and there is no complicated subsequent processing of products, which is efficient and fast; 4) The reproducibility of results Well, the quantitative dynamic range is up to five orders of magnitude. As the development of PCR technology, FQ-PCR has shown its superiority in many fields of scientific research and clinical diagnosis since 1996. Now it has been widely used in biology, basic medicine, disease diagnosis, food, water quality environmental protection, medicine It is considered the gold standard for nucleic acid quantification.

Canine parvovirus (Canine Parvovirus, CPV) can cause canine parvovirus disease, which is a highly contagious infectious disease of dogs. The clinical symptoms are mainly characterized by hemorrhagic enteritis or non-suppurative myocarditis. one of the most serious diseases in the industry. CPV belongs to the family Parvoviridae, the genus Parvovirus, a linear negative-sense single-stranded DNA virus with a genome length of 5323bp, encoding structural proteins VP1, VP2 and VP3 and non-structural proteins NS1 and NS2. VP2 is the main capsid protein of CPV, which encodes the main antigenic determinant of CPV, can induce the body to produce neutralizing antibodies, and is also considered as a receptor binding protein. VP2 plays an extremely important role in the process of virus infection, and the host range of CPV is determined by its ability to bind to receptors on the cell surface or other cellular ligands. VP2 is the main exposed protein on the virus core particle, suggesting that VP2 can be used as an ideal target for antibody detection of canine parvovirus.

Contents of the invention

The object of the present invention is to provide a biological barcode for detecting canine parvovirus and its application.

Another object of the present invention is to provide a fluorescent quantitative biological barcode detection technology.

The invention provides a biological barcode for detecting canine parvovirus (Canine Parvovirus, CPV), which has the nucleotide sequence shown in SEQ ID No.1 or a specific fragment thereof.

The present invention provides specific primers for amplifying the above-mentioned biological barcodes, the nucleotide sequence of which is:

Primer 1: 5'-CTTACACTGTTGCTGCTGCC-3' (SEQ ID No.3),

Primer 2: 5'-TCCTCTACTTCGGGTGGGAA-3' (SEQ ID No.4).

The present invention provides a complementary probe NP chain used in conjunction with the above-mentioned biological barcode, its nucleotide sequence is:

5'-TCCTCTACTTCGGGTGGGAAGTCGGGGTTCA ₁₀ -(CH ₂ ) ₆ -SH-3' (SEQ ID No. 2).

The invention provides a biological barcode detection method for canine parvovirus, comprising the following steps,

1) Preparation of NP probe: Gold nanoparticles, anti-VP2 protein polyclonal antibody, nucleotide sequence are barcode DNA chain of SEQ ID No.1 and nucleotide sequence is SEQ ID No.2 by conventional method Complementary probe NP strands prepare NP probes;

2) Preparation of MMP probes: MMP probes were prepared with magnetic microspheres and anti-VP2 protein monoclonal antibodies;

3) Biological barcode detection of canine parvovirus: use NP probe, MMP probe and canine parvovirus to prepare NP-VP2-MMP sandwich complex through the action of antigen and antibody, and then release the barcode DNA chain under high temperature and low salt conditions, Finally, the fluorescent quantitative PCR method was used to detect the obtained barcode DNA chain.

Wherein, the diameter of the gold nanoparticles described in step 1) is 30nm.

Wherein, the diameter of the magnetic microspheres described in step 2) is 1 μm.

The primers used in the fluorescent quantitative PCR method are as described in SEQ ID No.3 and SEQ ID No.4 respectively.

The invention also provides a biological barcode detection kit for canine parvovirus, which includes the MMP probe coated with the monoclonal antibody against VP2 protein and the NP probe coated with the polyclonal antibody against VP2 protein.

Further, the barcode DNA chain connected by the NP probe of the above kit has the nucleotide sequence shown in SEQ ID No.1, and the NP chain of the complementary probe has the nucleotide sequence shown in SEQ ID No.2.

Further, the above kit also includes a pair of primers, the nucleotide sequences of which are shown in SEQ ID No.3 and SEQ ID No.4 respectively.

The invention uses fluorescent quantitative PCR technology to replace ordinary PCR detection and chip detection, thereby realizing accurate quantification of the detected object. Compared with the conventional biological barcode detection method, the fluorescent quantitative biological barcode detection technology established by the present invention can accurately quantify the barcode DNA chain, thereby accurately quantifying the canine parvovirus antigen, overcoming the negative and positive limits of the conventional biological barcode detection method Unclear and other issues. The detection method and the detection kit of the present invention have the advantages of accurate detection, high sensitivity, strong specificity, simplicity and speed, and good specimen detection ability.

Description of drawings

Figure 1 is the sensitivity diagram of the detection of CPV VP2 protein by fluorescent quantitative biological barcoding technology. The concentration of VP2 protein from left to right is as follows: 1: 1pg/mL; 2: 100fg/mL; 3: 10fg/mL; 4: 1fg/mL ; 5: 100ag/mL.

Figure 2 shows the sensitivity detection results of fluorescent quantitative biological barcoding technology (canine parvovirus). The titers of canine parvovirus from left to right are: 1:10 ^-1 TCID ₅₀ /0.1mL; 2:10 ^-2 TCID ₅₀ /0.1 mL; 3: 10 ^-3 TCID ₅₀ /0.1 mL; 4: ^{10 -4} TCID ₅₀ /0.1 mL; ^{5: 10 -5} TCID ₅₀ /0.1 mL.

Detailed ways

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents used in the examples are commercially available.

Example 1 Design and Synthesis of Biological Barcodes for Detecting Canine Parvovirus

The oligonucleotide chain used in the detection method of the present invention is as follows:

Barcoded DNA strands:

5′-ACACTGTTGCTGCTGCCTCTACGTTCAATGGCTTCCAGAGGCCGACGATCTACTATGTGATGTCAGGGCCTGCGTGGCAACTCATGCAGCAATTCCAGAACCCCGACTTCCCACCCGAAGTAGAGGA-3′ (SEQ ID No. 1);

Complementary probe NP strand:

5'-TCCTCTACTTCGGGTGGGAAGTCGGGGTTCA ₁₀ -(CH ₂ ) ₆ -SH-3' (SEQ ID No. 2).

The above barcode DNA was synthesized by Beijing Sanbo Polygala Biological Company, and the complementary probe NP chain was synthesized by Dalian Bao Biological Engineering Co., Ltd.

The establishment of the biological barcode detection method of embodiment 2 canine parvovirus

1. Preparation of gold nanoparticles

In this embodiment, gold nanoparticles with a diameter of 30 nm are selected. Gold nanoparticles were prepared by sodium citrate reduction method.

Take 500mL of 0.01% HAuCl4 solution, heat it to boiling while stirring, then quickly and accurately add 7.5mL of 1% trisodium citrate aqueous solution, continue heating and boiling for 15min, and stir with a magnetic stirrer throughout the whole process; when the color of the solution gradually deepens , and finally dark purple, stop heating; filter the prepared gold nanoparticles with a 0.45 μm nylon membrane, cool to room temperature, and store at 4°C in the dark.

2. Preparation of polyclonal antibody against canine parvovirus

Using purified canine parvovirus, the strain CPV-2a used was isolated and identified by our laboratory, and New Zealand white rabbits (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were immunized to prepare polyclonal antibodies against canine parvovirus.

Before each male New Zealand white rabbit was immunized, 2 mL of blood was collected from the ear vein, left to stand to separate the serum, and stored at -20°C as a negative control. For the first immunization, each rabbit was injected with 2mL of emulsified antigen, subcutaneously injected at multiple points around the paws and cubital fossa lymph nodes, on both sides of the back, under the neck, behind the ears, etc., and the control rabbit was injected with the same amount of normal saline. Two weeks later, booster immunization with the same dose (containing equal volume of Freund's incomplete adjuvant), multi-point injection on the back, and booster immunization again 2 weeks later. The third booster immunization was carried out 10 days later, and the last immunization was carried out about 10 days later, and 2 mL of emulsified antigen was directly injected intravenously. 7 to 10 days after each booster immunization, 2 to 3 mL of blood was collected from the vein of the rabbit’s ear, and the antibody titer was detected by indirect ELISA. 7 days after the last immunization, blood was collected by carotid artery bleeding, left standing at 4°C overnight, and 4000 rpm the next day. Centrifuge for 10 min, collect the supernatant, and store at -20°C after aliquoting.

3. Preparation of gold nanoparticles-canine parvovirus polyclonal antibody complex

Take 1 mL of gold nanoparticles and an appropriate amount of anti-canine parvovirus polyclonal antibody, and adjust the pH to 9.0 with 0.1 mol/L K ₂ CO ₃ . Mix the gold nanoparticles and the antibody under stirring, and then add a certain amount of stabilizer 5% BSA after 10 minutes to make the final concentration 1%; or add 1% polyethylene glycol (PEG, molecular weight 20kD) to the marker 1/10 of the total amount to prevent aggregation and precipitation of antibody proteins and gold nanoparticles.

4. Preparation of NP probes

The complementary probe NP chain (final concentration is 2 μmol/L) of embodiment 1 is added in the gold nanoparticle (concentration is 4.5nmol/L) that is modified by anti-canine parvovirus polyclonal antibody, room temperature is placed 16 hours; Adjust the pH of the buffer to 7.0, increase the ion concentration to 0.1mol/LNaCl, place it at room temperature for 40 hours, and centrifuge at 10,000g for 30 minutes to obtain gold nanoparticles modified with complementary probe NP chains; (pH7.0) solution was washed 3 times to remove unlabeled gold nanoparticles; the barcode DNA chain obtained in Example 1 (final concentration of 2 μmol/L) was added to the above-mentioned gold nanoparticles marked with complementary probe NP chain modification , hybridized at room temperature for 4 hours, and centrifuged at 14,000 g for 30 minutes to obtain NP probes.

5. Preparation of anti-canine parvovirus monoclonal antibody

Using purified CPV for primary immunization, re-immunization, and booster immunization of Balb/c mice, the spleen was taken and fused with SP20 cells to prepare hybridoma cells, and the recombinantly expressed VP2 protein was used to coat the microtiter plate, and the positive cell lines were cloned and screened by limiting dilution method . The identified positive hybridoma cells were immunized to BALB/c mice to prepare anti-canine parvovirus monoclonal antibody.

6-8 weeks old female Balb/c mice were used, and 0.5 mL of sterilized liquid paraffin was injected intraperitoneally 7 days before the injection of hybridoma cells; each mouse was intraperitoneally injected with ^0.5 mL of culture medium; when the mice produce ascites (1-3 weeks), release the ascites (1-5mL) by abdominal puncture, and puncture the next day until the mice die; centrifuge at 3000rpm for 15min to remove the cells in the ascites; store the supernatant aseptically or Add 0.01% sodium azide and store in a -70°C refrigerator for later use.

6. Preparation of MMP probes

Dissolve 100 μg of monoclonal antibody in 20 mL of PBS, add it to activated magnetic beads (Dynabeads M-280 magnetic beads are purchased from Invitrogen), shake vigorously, and then place the flask on a mixer for 16 to 24 hours of reaction; Separation, discard supernatant; resuspend magnetic beads in 40mL PBS; add 20mL quencher (glycine), shake vigorously, put it on the mixer for 30min reaction; magnetic separation, discard supernatant; wash with PBS for 3 Second, magnetic separation; resuspend the magnetic beads with 50mL washing solution, and store at 4°C.

7. Preparation of detection probes

Add the probe NP chain (the double-stranded hybridization combination of the complementary probe chain and the barcode DNA chain) (final concentration: 2 μmol/L) into gold nanoparticles (concentration: 4.5 nmol/L), and place it at room temperature for 16 hours; Adjust the pH to 7.0 with salt, increase the ion concentration to 0.1mol/L NaCl, place at room temperature for 40 hours, and centrifuge at 10,000g for 30min to obtain the detection probe; suspend with 0.3mol/L NaCl, 10mmol/L PBS with pH7.0, and wash by centrifugation for two times, and finally dissolved with a certain amount of 0.3mol/L PBS, which is the detection probe.

8. Establishment of a fluorescent quantitative biological barcode detection method for canine parvovirus

Add 50 μL of anti-VP2 antigen monoclonal antibody functionalized magnetic microspheres (5 mg/mL) to 10 μL of known concentration of VP2 protein and known titer of canine parvovirus, shake vigorously at 37°C for 1 hour; add magnetic field fixation Wash MMP probes repeatedly with PBS solution to remove unbound VP2 antigen and irrelevant proteins; add 50 μL of 0.1 nmol/L gold nanoparticle probe modified by double-stranded DNA and polyantibody against VP2 protein, and shake vigorously for 30 Minutes, a sandwich structure is formed; add a magnetic field, while immobilizing MMPs with a magnet, wash with 500 μL of PBS buffer repeatedly 4 times to remove unattached NP probes.

After the sandwich structure was formed, a magnetic field was added, and while the MMPs were fixed with a magnet, the 500 μL PBS buffer was repeatedly washed 4 times to remove unconnected NP probes; 50 μL ultrapure water was added to the sandwich structure and heated at 60 °C. Shake for 30 minutes to dehybridize the barcoded DNA strands. The sandwich complex was magnetically separated, and the supernatant was collected, containing barcoded DNA strands, for qualitative and quantitative detection by fluorescent quantitative PCR.

9. Fluorescent quantitative PCR detection of barcoded DNA strands

Fluorescent PCR detection was performed on the obtained barcoded DNA strands. The SYBRGreen I dye method was used for detection, and the reagents used were purchased from Dalian Bao Biological Engineering Co., Ltd.

The reaction system is 25 μL:

The reaction conditions are as follows:

Primer 1: 5'-CTTACACTGTTGCTGCTGCC-3',

Primer 2: 5'-TCCTCTACTTCGGGTGGGAA-3'.

10. Data Analysis

When analyzing the results, the baseline (baseline) and threshold (threshold) can take the default value of the instrument. Generally, the fluorescence signal of the first 6 to 15 cycles is taken as the baseline. When performing data analysis, it is generally necessary to perform consignment settings and change the vertical axis of the data to linear (Figure 1, Figure 2). Through the analysis of Figure 1 and Figure 2, it shows that the established method can detect 100ag/mL VP2 protein and 10-5TCID ₅₀ /0.1mL CPV virus titer, and presents a good linear relationship. It shows that the established method can be used for the detection of trace CPV virus and protein.

Example 3 Biological barcode detection method specificity verification of canine parvovirus

Use the established detection method to simultaneously detect rabies virus (the CTN strain is preserved for this laboratory), and canine distemper virus (the strain for making seedlings is preserved for this laboratory), and the detection Ct value is greater than 40, the results are all negative, and the CPV detection is positive , indicating that this method does not react with other canine infectious viruses and has good specificity.

Preparation of the biological barcode fluorescence quantitative detection kit of embodiment 4 canine parvovirus

The NP probe prepared in step 4 of Example 2, the MMP probe prepared in step 6, and the primer pair in step 9 were packaged together to obtain a biological barcode fluorescence quantitative detection kit for canine parvovirus.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, some improvements and modifications can also be made. These improvements and modifications It should also be regarded as the protection scope of the present invention.

Claims (10)

1. A biological barcode for detecting canine parvovirus (Canine Parvovirus), which has a nucleotide sequence shown in SEQ ID No.1 or a specific fragment thereof. 2. be used to amplify the specificity primer of biological barcode described in claim 1, its nucleotide sequence is: Primer 1: 5'-CTTACACTGTTGCTGCTGCC-3', Primer 2: 5'-TCCTCTACTTCGGGTGGGAA-3'. 3. The complementary probe NP chain used in conjunction with the biological barcode described in claim 1, its nucleotide sequence is: 5'-TCCTCTACTTCGGGTGGGAAGTCGGGGTTCA ₁₀ -(CH ₂ ) ₆ -SH-3'. 4. A biological barcode detection method for canine parvovirus, comprising the following steps, 1) Preparation of NP probes: prepare NP probes with gold nanoparticles, polyclonal antibodies against VP2 protein, the barcode DNA chains described in claim 1 and the complementary probe NP chains described in claim 3 by conventional methods; 2) Preparation of MMP probes: MMP probes were prepared with magnetic microspheres and anti-VP2 protein monoclonal antibodies; 3) Biological barcode detection of canine parvovirus: use NP probe, MMP probe and canine parvovirus to prepare NP-VP2-MMP sandwich complex through the action of antigen and antibody, and then release the barcode DNA chain under high temperature and low salt conditions, Finally, the fluorescent quantitative PCR method was used to detect the obtained barcode DNA chain. 5. The biological barcode detection method according to claim 4, characterized in that the gold nanoparticles in step 1) have a diameter of 30nm. 6. The biological barcode detection method according to claim 4, characterized in that the diameter of the magnetic microspheres in step 2) is 1 μm. 7. The detection method according to any one of claims 4-6, wherein the primers used in the fluorescent quantitative PCR method are as described in claim 2. 8. A biological barcode detection kit for canine parvovirus, comprising an MMP probe coated with a monoclonal antibody against VP2 protein and an NP probe coated with a polyclonal antibody against VP2 protein. 9. test kit as claimed in claim 8, is characterized in that, the barcode DNA chain that described NP probe connects has the nucleotide sequence shown in SEQ ID No.1, and complementary probe NP chain has SEQ ID No. The nucleotide sequence shown in 2. 10. The kit of claim 9, further comprising the primers of claim 2.

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