CN116515840B - Kit and detection method for detecting bovine viral diarrhea virus type 3 - Google Patents
Kit and detection method for detecting bovine viral diarrhea virus type 3 Download PDFInfo
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Abstract
The invention relates to a kit and a detection method for detecting bovine viral diarrhea virus type 3, belonging to the technical field of biological detection. The kit comprises crRNA, wherein the crRNA is a combination of crRNA-1 and crRNA-2; the sequence of crRNA-1 is shown as SEQ ID NO. 1; the crRNA-2 sequence is shown as SEQ ID NO. 2. The kit for detecting bovine viral diarrhea virus type 3 provided by the invention effectively solves the problem of identifying and detecting bovine viral diarrhea virus type 3 strains in the prior art, and provides clear guidance for the subsequent treatment of bovine viral diarrhea. Compared with the traditional detection method, the detection method provided by the invention has the advantages of strong relative bit variability, low detection limit, high sensitivity, low price, simplicity and rapidness in operation and convenience in carrying equipment and instruments.
Description
Technical Field
The invention relates to a kit and a detection method for detecting bovine viral diarrhea virus type 3, belonging to the technical field of biological detection.
Background
Bovine viral diarrhea, also known as bovine viral diarrhea-mucosal disease (BVD-MD), is a stronger infectious disease caused by bovine viral diarrhea virus (bovine viral diarrhea virus, BVDV). The virus can infect pigs, deer, sheep, camels, rabbits and other wild ruminants in addition to cattle, and has been reported to infect more than 40 animals, and the host is quite extensive. Three genotypes of BVDV are reported to be BVDV-1, BVDV-2 and BVDV-3, respectively.
Wherein BVDV-3 is a new-appearing pestivirus infecting cattle, the clinical manifestation of the pestivirus is similar to the clinical symptoms caused by BVDV-1 and BVDV-2, and the amino acid sequence of BVDV-3 has higher homology with BVDV-1 and BVDV-2, but the antigenicity is obviously different. Therefore, genotyping of the virus is particularly important.
The current detection methods for BVDV viruses mainly comprise etiology detection, serology detection and molecular biology detection. The etiology detection comprises electron microscopy and indirect immunofluorescence staining (IFA), but is only suitable for laboratory examination because of high requirements on equipment by electron microscopy; the IFA technology is not suitable for large-scale screening of a large-scale cattle farm due to long detection period and complicated operation; the most widely used serological assay is the enzyme-linked immunosorbent assay (ELISA). Commercial antigen capture ELISA kits can be used to confirm BVDV infection, but cannot distinguish which type of infection BVDV-1, BVDV-2 and BVDV-3 is. Molecular biological assays include reverse transcription-polymerase chain reaction (RT-PCR) and gene chips, but gene chips are expensive and the screening cost is very high. And the above method is mainly directed to BVDV-1 and BVDV-2 detection, and has no related detection method of BVDV-3. Therefore, it is highly desirable to provide a specific fragment and detection method that are simple, rapid, and highly sensitive.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a kit and a detection method for detecting bovine viral diarrhea virus type 3.
The technical scheme of the invention is as follows:
a crRNA for detecting bovine viral diarrhea virus type 3, the crRNA being a combination of crRNA-1 and crRNA-2; the sequence of the crRNA-1 is shown as SEQ ID NO. 1; the crRNA-2 sequence is shown as SEQ ID NO. 2.
A crDNA for detecting bovine viral diarrhea virus type 3, the crDNA being a combination of crDNA-1 and crDNA-2; the sequence of the crDNA-1 is shown as SEQ ID NO. 3; the crDNA-2 sequence is shown as SEQ ID NO. 4.
A DNA for detecting bovine viral diarrhea virus type 3, said DNA being a combination of DNA-1 and DNA-2; the sequence of the DNA-1 is shown as SEQ ID NO. 5; the DNA-2 sequence is shown as SEQ ID NO. 6.
The crRNA-1 and the crRNA-2 can be obtained through artificial synthesis or are transcribed by taking crDNA-1 and crDNA-2 as templates, wherein the DNA-1 is a target sequence 1 for identifying and detecting BVDV-3 by using the crRNA-1, and the DNA-2 is a target sequence 2 for identifying and detecting BVDV-3 by using the crRNA-2, and the target sequences are all used for identifying and detecting BVDV-3 strains.
A kit for detecting bovine viral diarrhea virus type 3, the kit comprising the crRNA or crDNA described above.
Further preferred, the kit further comprises a Cas protein;
further preferred, the Cas protein is a Cas13a protein.
According to the invention, the kit comprises a colloidal gold test strip, a colloidal gold test strip card shell and a micropore cup.
According to the invention, the colloidal gold test strip comprises a bottom plate, and a sample pad, a connecting pad, a nitrocellulose membrane and an absorption pad which are sequentially jointed and attached to the bottom plate; the nitrocellulose membrane is sequentially provided with a first detection line, a second detection line and a control line.
According to the present invention, preferably, the FAM monoclonal antibody is immobilized on the first detection line, the Dig monoclonal antibody is immobilized on the second detection line, and the Biotin ligand is immobilized on the control line.
A method for detecting bovine viral diarrhea virus type 3 for non-diagnostic purposes using the above kit, comprising the steps of:
(1) Extracting genome of a sample to be detected;
(2) Taking the extracted genome of the sample to be detected as a template, adding the template into a reaction system containing reaction liquid and polymerase, and carrying out double RT-RPA amplification;
(3) Respectively establishing a T1 detection system and a T2 detection system by taking RT-RPA amplification products, and incubating for 10-60 min at 37 ℃;
(4) Uniformly mixing a T1 detection system and a T2 detection system according to equal volume, adding enzyme-free water to obtain a reaction solution, dripping the reaction solution into a micropore cup, and after 5-10 minutes, displaying a strip on a first detection line or a second detection line or both the first detection line and the second detection line, wherein the first detection line and the second detection line indicate that a sample to be detected is infected with BVDV-3 type strains; and when the first detection line and the second detection line do not display the strip, and the control line displays the strip, the sample to be tested is not infected with BVDV-3 type strains.
According to the present invention, in the step (1), the sample to be tested is a bovine nasal swab or saliva sample, specifically, the sample to be tested is obtained by treating the bovine nasal swab or saliva with a buffer mixture of TCEP (100 mM) and EDTA (1 mM) at 50 ℃ for 5 minutes, and then at 65 ℃ for 5 minutes.
According to a preferred embodiment of the present invention, in step (2), the dual RT-RPA amplification is performed by using two pairs of primers simultaneously, wherein the sequences of the primers are:
forward primer 1:
5'-GAAATTAATACGACTCACTATAGGGAGGAAAGTGAAGCACCAAGGRAACTTAAGAAC -3'；
reverse primer 1:
5'-TTTCTTTTCCAGGAACCCTGCTGCTCCTTT-3'；
forward primer 2:
5'-GAAATTAATACGACTCACTATAGGGTACTAGAAGGAGACMAAATGAAGGTGGCTT-3'；
reverse primer 2:
5'-GGAGGAAGCTGAATGCCACTGCYTTCTCATA-3'。
according to a preferred embodiment of the invention, in step (2), the dual RT-RPA amplification system is: RPA base reaction ball one tube, rehydration buffer 29.5. Mu.L, magnesium acetate buffer 2.5. Mu.L, forward primer 1 and forward primer 2 together 2.5. Mu.L, reverse primer 1 and reverse primer 2 together 2.5. Mu.L, reverse transcriptase 1. Mu.L and the rest 7. Mu.L with enzyme-free water, then 5 aliquots were made, one portion per 9. Mu.L, 1. Mu.L of sample to be tested was added per portion, and total volume 10. Mu.L.
The amplification conditions were: the reaction temperature is 39-42 ℃, and the incubation time is 5-20 min.
Wherein, the RPA basic reaction ball, the rehydration buffer solution and the magnesium acetate buffer solution are all from a Twitter AmpR basic kit, and the RPA basic reaction ball is spherical solid and contains components such as recombinase, polymerase and the like.
According to the present invention, in the step (3), the T1 detection system and the T2 detection system are the same, specifically: tris-HCl (400 mM), mgCl2 (120 mM), ssRNA reporter probe (2. Mu.M), crRNA 0.5. Mu.L, RNase Inhibitor 0.5. Mu. L, cas13a protein 1. Mu. L, T7 Polymerase 0.25. Mu. L, rNTP 0.4.4. Mu.L, RT-RPA amplification product 1. Mu.L, and total volume 10. Mu.L.
It is further preferred that the composition of the present invention,
the ssRNA reporter probe is a ssRNA1 reporter group and a ssRNA2 reporter group;
the ssRNA1 reporter group is: 5'-FAM-UUUUUUUUUUUUUU-Biotin-3';
the ssRNA2 reporter group is: 5'-Dig-UUUUUUUUUUUUUU-Biotin-3'.
According to a preferred embodiment of the invention, in step (4), the volume ratio of the enzyme-free water to the first detection system is 1:6.
The invention is not described in detail in the prior art.
The invention has the technical characteristics that:
after mixing the T1 and T2 detection systems, the Cas13a protein binds to crRNA-1 and crRNA-2 to form Cas13a-crRNA-1 and Cas13a-crRNA-2 complexes, respectively, and then Cas13a-crRNA-1 and Cas13a-crRNA-2 are able to specifically recognize transcripts of DNA-1 and DNA-2 in RT-RPA amplification products. If the RT-RPA amplification product contains a DNA-1 transcription fragment or a DNA-2 transcription fragment, the Cas13a protein is activated, and the ssRNA-1 and the ssRNA-2 reporter group are cut, wherein the cut reporter group and colloidal gold of the first detection line and the second detection line form a display band, namely the first detection line display band or the second detection line display band or both the first detection line and the second detection line display band, so that the sample is positive, and BVDV-3 infection exists. If the RT-RPA amplification product has no DNA-1 transcription fragment or DNA-2 transcription fragment, the Cas13a cannot be activated, the reporter group cannot be combined with the detection line ligand, the first detection line and the second detection line cannot form a display band, and at the moment, the control line displays a band, so that the sample is negative, and BVDV-3 type infection does not exist.
The beneficial effects are that:
1. the kit for detecting bovine viral diarrhea virus type 3 provided by the invention effectively solves the problem of identifying and detecting bovine viral diarrhea virus type 3 strains in the prior art, and provides clear guidance for the subsequent treatment of bovine viral diarrhea. Compared with the traditional detection method, the detection method provided by the invention has the advantages of strong relative bit variability, low detection limit, high sensitivity, low price, simplicity and rapidness in operation and convenience in carrying equipment and instruments.
2. The detection method provided by the invention combines the RT-RPA amplification technology, the CRISPR system and the rapid colloidal gold detection technology, reduces the mutual interference through reasonable proportion, ensures that the respective functions of the reaction reagents can still be accurately and specifically realized, obtains the result through the combination of amplification and detection, ensures that the whole detection process takes very short time to complete in 5-30 min during qualitative detection, has simple operation steps, can visually distinguish whether the bovine viral diarrhea virus type 3 infection is realized through the presentation of the detection line, and provides a brand-new thought and method for prevention, control and treatment of bovine viral diarrhea.
3. The detection method provided by the invention uses two sections of BVDV-3 highly conserved gene fragments (DNA-1 and DNA-2), and greatly enhances the detection efficiency for detecting BVDV-3 by combining the two sections of genes. Then, the crRNA-1, crRNA-2, crDNA-1 and crDNA-2 for detecting the two fragments are designed, and then the detection of bovine viral diarrhea virus type 3 is realized through a test strip, so that the result is clear and definite.
Drawings
FIG. 1 is a diagram showing the conservation analysis of bovine viral diarrhea virus type 3 target DNA-1 gene.
FIG. 2 is a diagram showing the conservation analysis of bovine viral diarrhea virus type 3 target DNA-2 gene.
FIG. 3 is a graph showing the results of detection and identification of bovine viral diarrhea virus type 3.
In the figure: t1 is a first detection line, T2 is a second detection line, and C is a control line.
FIG. 4 is a graph showing the results of a specificity analysis experiment.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
Cas13a protein, beijing family Biotech Co., ltd, is sold in the examples below.
TwistAmp R Basic kit, available from Twitteddx-merry biotechnology Co.
Example 1 design and acquisition of crRNA and DNA for detection of bovine viral diarrhea Virus type 3
The gene sequences of bovine viral diarrhea virus type 3 were downloaded from GenBank as registered with NCBI, including: JX985409.1, AB8719531, KJ627179.1, FJ040215.1, HQ231763.1, JX469119.1, KC788748.1, JQ612704.1, JQ612705.1, KJ627180.1, KU563115.1, KC299709.1, KY683847.1, KY762287.2, KY767958.1, NH410812.1, NH410813.1, NH410814.1, NH410815.1, NH410816.1, nc_012812.1, OP210314.1.
Then comparing the 22 gene sequences by a MAFFT method (https:// MAFFT. Cbrc. Jp/alignment/server/index. Html), and finding that two identical conserved sequences, namely DNA-1 and DNA-2, exist in the 22 gene sequences, wherein the sequences are respectively shown as SEQ ID NO.5 and SEQ ID NO.6, and the specific pair is shown as figure 1 and figure 2. Then, according to the gene sequences of the DNA-1 and the DNA-2, the crRNA-1 and the crRNA-2 are designed, the sequences of the crRNA-1 and the crRNA-2 are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, the DNA-1 is a target sequence 1 for identifying and detecting BVDV-3 by using the crRNA-1, and the DNA-2 is a target sequence 2 for identifying and detecting BVDV-3 by using the crRNA-2, and the target sequences can be used for identifying and detecting BVDV-3 strains.
The specific procedures for obtaining crRNA-1 and crRNA-2 are as follows: and (3) taking artificially synthesized crDNA-1 and crDNA-2 (SEQ ID NO.3 and SEQ ID NO. 4) as templates, respectively carrying out annealing reaction to form double-stranded DNA, then carrying out agarose gel electrophoresis, recovering and purifying DNA fragments by gel, transcribing under the action of T7 RNA polymerase to generate RNA, recovering and purifying to obtain crRNA-1 and crRNA-2, and sub-packaging and freezing the purified crRNA-1 and crRNA-2 to the temperature of-80 ℃. Or directly synthesized artificially according to the sequence.
Annealing system: the DNA oligo to be annealed (synthetic primer) was formulated to 50. Mu.M with sterilized Milli-Q water or redistilled water. Dissolving Annealing Buffer for DNA Oligos (5X), and mixing.
Adding various reagents in the above sequence, and mixing.
The sequence of the T7 primer is as follows:
5’- GAAATTAATACGACTCACTATAGGG-3’。
the annealing reaction was performed by setting a PCR instrument as follows:
the transcription system is as follows: template DNA 1. Mu.g, T7 RNA polymerase mixture 2. Mu.L, NTP Buffer Mix 10. Mu.L, no enzyme water make up, total volume 30. Mu.L.
The transcription conditions were: 37℃for 16h.
Example 2 kit for detection of bovine viral diarrhea Virus type 3
The kit of the embodiment comprises a colloidal gold test strip, a colloidal gold test strip card shell, a micropore cup and a TwitAmp R Basic reagent, crRNA-1, crRNA-2, cas13a protein, RPA amplification primer, tris-HCl (400 mM), mgCl 2 (120 mM), ssRNA reporter probe (0.1. Mu.M), RNase Inhibitor, ssRNA (2. Mu.M), T7 Polymerase, rNTP.
The colloidal gold test strip comprises a bottom plate, and a sample pad, a connecting pad, a nitrocellulose membrane and an absorption pad which are sequentially connected and attached to the bottom plate; the nitrocellulose membrane is sequentially provided with a first detection line, a second detection line and a control line. FAM monoclonal antibody is fixed on the first detection line, dig monoclonal antibody is fixed on the second detection line, and Biotin ligand is fixed on the control line.
The ssRNA reporter probe comprises a ssRNA1 reporter group and a ssRNA2 reporter group;
the ssRNA1 reporter group is: 5'-FAM-UUUUUUUUUUUUUU-Biotin-3';
the ssRNA2 reporter group is: 5'-Dig-UUUUUUUUUUUUUU-Biotin-3'.
Example 3 method of bovine viral diarrhea Virus type 3 detection Using the kit described in example 2
The gene sequence of bovine viral diarrhea virus type 3 (NCBI ID is AB 8719531) is directly synthesized by the Hua big gene, then the bovine viral diarrhea virus type 3 gene sequence and 20 mu L of healthy bovine saliva are mixed to be used as samples to be tested, wherein the samples to be tested are 3 groups, the concentration of the bovine viral diarrhea virus type 3 in the group 1 is 20fM, the concentration of the bovine viral diarrhea virus type 3 in the group 2 is 30fM, the concentration of the bovine viral diarrhea virus type 3 in the group 3 is 10fM, and simultaneously 20 mu L of enzyme-free water is used as a control group to detect the bovine viral diarrhea virus type 3. Meanwhile, 20 mu L of enzyme-free water is used as a control group to detect bovine viral diarrhea virus type 3.
The specific method comprises the following steps:
(1) The sample to be tested is obtained by firstly carrying out 50 ℃ treatment for 5 minutes and then carrying out 65 ℃ treatment for 5 minutes by using a mixed buffer solution of TCEP (100 mM) and EDTA (1 mM);
(2) Taking the extracted genome of the sample to be detected as a template, adding the template into a reaction system containing reaction liquid and polymerase, and carrying out double RT-RPA amplification;
the dual RT-RPA amplification is that two pairs of primers are simultaneously used for RT-RPA amplification, and the sequences of the primers are as follows:
forward primer 1:
5'-GAAATTAATACGACTCACTATAGGGAGGAAAGTGAAGCACCAAGGRAACTTAAGAAC -3'；
reverse primer 1:
5'-TTTCTTTTCCAGGAACCCTGCTGCTCCTTT-3'；
forward primer 2:
5'-GAAATTAATACGACTCACTATAGGGTACTAGAAGGAGACMAAATGAAGGTGGCTT-3'；
reverse primer 2:
5'-GGAGGAAGCTGAATGCCACTGCYTTCTCATA-3'；
the double RT-RPA amplification system is as follows: RPA base reaction ball one tube, rehydration buffer 29.5. Mu.L, magnesium acetate buffer 2.5. Mu.L, forward primer 1 and forward primer 2 together 2.5. Mu.L, reverse primer 1 and reverse primer 2 together 2.5. Mu.L, reverse transcriptase 1. Mu.L, the remainder made up with 7. Mu.L of enzyme free water, then 5 aliquots were made, one portion per 9. Mu.L, 1. Mu.L of sample to be tested was added per portion, and the total volume was 10. Mu.L.
The amplification conditions were: the reaction temperature is 40 ℃ and the incubation time is 15 min;
wherein, the RPA basic reaction ball, the rehydration buffer solution and the magnesium acetate buffer solution are all from a Twitter AmpR basic kit, and the RPA basic reaction ball is spherical solid and contains components such as recombinase, polymerase and the like;
(3) Respectively establishing a T1 detection system and a T2 detection system by taking RT-RPA amplification products, and incubating for 40min at 37 ℃;
the T1 detection system comprises: tris-HCl (400 mM), mgCl 2 (120 mM), ssRNA1 reporter probe (2. Mu.M), crRNA-1.5. Mu.L, RNase Inhibitor 0.5. Mu. L, cas13a protein 1. Mu. L, T7 Polymerase 0.25. Mu. L, rNTP 0.4.4. Mu.L, RPA amplification product 1. Mu.L, total volume 10. Mu.L;
the T2 detection system comprises: tris-HCl (400 mM), mgCl 2 (120 mM), ssRNA2 reporter probe (2. Mu.M), crRNA-2.5. Mu.L, RNase Inhibitor 0.5. Mu. L, cas13a protein 1. Mu. L, T7 Polymerase 0.25. Mu. L, rNTP 0.4.4. Mu.L, RPA amplification product 1. Mu.L, total volume 10. Mu.L;
the ssRNA1 reporter probe is a ssRNA1 reporter group, and the ssRNA1 reporter group is: 5'-FAM-UUUUUUUUUUUUUU-Biotin-3';
the ssRNA2 reporter probe is a ssRNA2 reporter group, and the ssRNA2 reporter group is: 5'-Dig-UUUUUUUUUUUUUU-Biotin-3'.
(4) Uniformly mixing 10 mu L of T1 detection system and 10 mu L of T2 detection system according to equal volume, adding 60 mu L of enzyme-free water to obtain reaction liquid, dripping the reaction liquid into a micropore cup, and after 10 minutes, when a first detection line displays a strip or a second detection line displays a strip or both the first detection line and the second detection line display a strip, indicating that a sample to be tested is infected with BVDV-3 strain; when the first detection line and the second detection line do not display the strip, and the control line displays the strip, the sample to be tested is indicated to not infect BVDV-3 type strains; the results of the test sample and the control group are shown in fig. 3.
As can be seen from fig. 3, when the test sample is in group 1, the first detection line shows red bands, indicating that the test sample is infected with BVDV-3 type strain. When the sample to be tested is in group 2, the first detection line shows a red band, and the second detection line shows a blue band, which indicates that the sample to be tested is infected with BVDV-3 type strain. When the sample to be tested is in group 3, the second detection line shows a blue band, which indicates that the sample to be tested is infected with BVDV-3 type strain. When the sample to be tested is a control group, the first detection line and the second detection line do not display strips, and the control line displays purple strips, so that the sample to be tested is not infected with BVDV-3 type strains.
Example 4 assay specificity experiments
DNA-1 and DNA-2 sequences shown as SEQ ID NO.5 and SEQ ID NO.6 are directly synthesized from the Huada genes, and the two sequences are respectively mixed with 20 mu L of healthy cow saliva to be used as a to-be-detected sample BVDV-3 DNA1 and BVDV-3DNA2. BVDV-3 DNA1, BVDV-3DNA2, pasteurella, bovine respiratory syncytial virus, mycoplasma bovis and mycoplasma ovipneumoniae were then separately tested and specifically analyzed using the kit described in example 2 and the method described in example 3, and the results are shown in FIG. 4.
As shown in FIG. 4, only the sample to be tested containing bovine viral diarrhea virus 3 is in a strip form and positive on the test strip detection line, and other related pathogens causing bovine and sheep respiratory diseases are not in a strip form and negative on the test strip detection line, which indicates that the target DNA-1 and DNA-2 of bovine viral diarrhea virus 3 and the detection method provided by the invention have better specificity, and can accurately identify and detect bovine viral diarrhea virus 3 in a plurality of pathogens causing bovine and sheep respiratory syndrome, such as viruses, bacteria, mycoplasma and the like.
Claims (6)
1. A crRNA for detecting bovine viral diarrhea virus type 3, wherein the crRNA is a combination of crRNA-1 and crRNA-2; the sequence of the crRNA-1 is shown as SEQ ID NO. 1; the crRNA-2 sequence is shown as SEQ ID NO. 2.
2. A crDNA for detecting bovine viral diarrhea virus type 3, wherein the crDNA is a combination of crDNA-1 and crDNA-2; the sequence of the crDNA-1 is shown as SEQ ID NO. 3; the crDNA-2 sequence is shown as SEQ ID NO. 4.
3. A DNA for detecting bovine viral diarrhea virus type 3, wherein the DNA is a combination of DNA-1 and DNA-2; the sequence of the DNA-1 is shown as SEQ ID NO. 5; the DNA-2 sequence is shown as SEQ ID NO. 6.
4. A kit for detecting bovine viral diarrhea virus type 3, the kit comprising the crRNA of claim 1 or the crDNA of claim 2.
5. The kit for detecting bovine viral diarrhea virus type 3 of claim 4 further comprising a Cas13a protein.
6. The kit for detecting bovine viral diarrhea virus type 3 according to claim 4, wherein the kit comprises a colloidal gold test strip, a colloidal gold test strip cartridge, and a microwell cup;
the colloidal gold test strip comprises a bottom plate, and a sample pad, a connecting pad, a nitrocellulose membrane and an absorption pad which are sequentially connected and attached to the bottom plate; the nitrocellulose membrane is sequentially provided with a first detection line, a second detection line and a control line;
FAM monoclonal antibody is fixed on the first detection line, dig monoclonal antibody is fixed on the second detection line, and Biotin ligand is fixed on the control line.
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| CN111979357A (en) * | 2020-09-01 | 2020-11-24 | 石河子大学 | Detection method of bovine viral diarrhea virus based on CRISPR-Cas13a |
| CN112430686A (en) * | 2020-11-24 | 2021-03-02 | 内蒙古农业大学 | Kit, primer and probe for simultaneously detecting BVDV-1, BVDV-2 and BVDV-3 |
| CN113174446A (en) * | 2021-01-19 | 2021-07-27 | 开江县动物疫病预防控制中心 | One-step double RT-PCR detection method for bovine viral diarrhea virus typing |
| CN115851719A (en) * | 2021-06-11 | 2023-03-28 | 山东舜丰生物科技有限公司 | Method for detecting pathogenic microorganisms based on CRISPR technology |
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| CN111979357A (en) * | 2020-09-01 | 2020-11-24 | 石河子大学 | Detection method of bovine viral diarrhea virus based on CRISPR-Cas13a |
| CN112430686A (en) * | 2020-11-24 | 2021-03-02 | 内蒙古农业大学 | Kit, primer and probe for simultaneously detecting BVDV-1, BVDV-2 and BVDV-3 |
| CN113174446A (en) * | 2021-01-19 | 2021-07-27 | 开江县动物疫病预防控制中心 | One-step double RT-PCR detection method for bovine viral diarrhea virus typing |
| CN115851719A (en) * | 2021-06-11 | 2023-03-28 | 山东舜丰生物科技有限公司 | Method for detecting pathogenic microorganisms based on CRISPR technology |
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