CN112941241A - Method for identifying porcine epidemic diarrhea virus type 2a and type 2b - Google Patents
Method for identifying porcine epidemic diarrhea virus type 2a and type 2b Download PDFInfo
- Publication number
- CN112941241A CN112941241A CN202110411684.4A CN202110411684A CN112941241A CN 112941241 A CN112941241 A CN 112941241A CN 202110411684 A CN202110411684 A CN 202110411684A CN 112941241 A CN112941241 A CN 112941241A
- Authority
- CN
- China
- Prior art keywords
- type
- epidemic diarrhea
- diarrhea virus
- porcine epidemic
- pedv
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention relates to the field of veterinary biotechnology, in particular to a method for identifying porcine epidemic diarrhea virus type 2a and type 2 b. Porcine epidemic diarrhea virus can be divided into classical strains and variant strains, and both comprise two subtypes of GII a and GII b. The method provided by the invention uses the primer combination shown as SEQ ID NO.1-3 and the kit containing the primer combination to identify PEDV GII a and PEDV GII b. The method provided by the invention can be used for effectively distinguishing the porcine epidemic diarrhea virus variant strains GIIa and GII b clinically, is used for preventing and controlling the porcine epidemic diarrhea virus in a pig farm, and provides a powerful basis for preventing and controlling the porcine epidemic diarrhea virus in the pig farm.
Description
Technical Field
The invention relates to the field of veterinary biotechnology, in particular to a method for identifying porcine epidemic diarrhea virus type 2a and type 2 b.
Background
Porcine Epidemic Diarrheic (PED) is an acute, highly-contact intestinal infectious disease caused by Porcine Epidemic Diarrheic Virus (PEDV), and is characterized by vomiting, Diarrhea, and dehydration of suckling piglets. Pigs of different ages and breeds can feel, with the morbidity and mortality of suckling piglets being the highest, the "first killer" that leads to high mortality of suckling piglets.
Porcine Epidemic Diarrhea Virus (PEDV) belongs to the family of coronaviridae and the genus of coronavirus, and Virus particles are polymorphic and tend to be circular, have an envelope outside and have a size of 95-190 nm. The nucleic acid is a linear single-stranded positive-strand RNA whose genome encodes 4 structural proteins including spike (S), small membrane (sM), membrane (M) and nucleocapsid (N) proteins and 3 non-structural proteins including replicase 1a, 1b and ORF3 encoded by the ORF3 genes.
Disclosure of Invention
When porcine epidemic diarrhea virus begins to spread widely in a pig farm, the virus strain isolated from the pig farm is porcine epidemic diarrhea virus variant 2b type, but nowadays, most of the virus strains isolated from the pig farm are porcine epidemic diarrhea virus variant 2a type. The method is based on the goal of accurately judging the porcine epidemic diarrhea virus and accurately preventing and controlling the porcine epidemic diarrhea disease.
The invention aims to provide a method for identifying porcine epidemic diarrhea virus type 2a and type 2 b.
In a first aspect, the invention provides a primer combination for detecting porcine epidemic diarrhea virus type 2a and type 2b, as shown in SEQ ID NO. 1-3.
In the primer combination provided by the invention, the primers shown in SEQ ID NO.1 and SEQ ID NO.2 are used for PCR amplification to obtain a nucleotide fragment of porcine epidemic diarrhea virus type 2 a; the primers shown in SEQ ID NO.1 and SEQ ID NO.3 are used for PCR amplification to obtain the nucleotide fragment of the porcine epidemic diarrhea virus type 2 b.
In a second aspect, the invention provides a kit for identifying porcine epidemic diarrhea virus type 2a and type 2b, wherein the kit contains the primer combination.
In a third aspect, the invention provides a method for identifying porcine epidemic diarrhea virus type 2a and type 2b for non-diagnostic purposes, the kit is used for carrying out PCR amplification on a sample to be detected, and the porcine epidemic diarrhea virus type 2a and type 2b are identified according to PCR products.
In the method provided by the invention, the Reaction system for PCR amplification contains one.step Enzyme Mix, 2 xOne.step Reaction Solution, P1 primer, P2 or P3 primer; the amplification procedure of the PCR amplification is 30min at 50 ℃ and 2min at 94 ℃; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 1min, and 34 cycles; fully extend the temperature of 72 ℃ for 10 min.
When the method provided by the invention is used for identifying the porcine epidemic diarrhea virus type 2a and type 2b, the judgment standard is as follows:
if the PCR product has a band at 910bp and the amplification product of the negative control does not have the band, determining that the PEDV GIIa positive reaction is obtained, namely the sample to be detected is the porcine epidemic diarrhea virus variant 2a type;
when the PCR product has a band at 1390bp and the amplified product of the negative control does not have the band, the PEDV GIIb positive reaction is judged, namely the sample to be detected is the porcine epidemic diarrhea virus variant 2b type.
According to the understanding of the person skilled in the art, the invention also claims the use of the above method in the supervision of swine plague, in the quality control of porcine epidemic diarrhea virus vaccines, and in the vaccination of porcine epidemic diarrhea virus.
In a fourth aspect, the invention provides a method for establishing a vaccination scheme of porcine epidemic diarrhea virus in a pig farm, which is used for identifying the strain type of the porcine epidemic diarrhea virus in the pig farm and determining the type of vaccination required by the pig farm.
The invention has the beneficial effects that:
(1) the primer combination provided by the invention has high PCR amplification specificity, and is convenient for development of clinical detection and epidemiological investigation;
(2) the method for detecting the porcine epidemic diarrhea virus type 2a and type 2b is simple and convenient to operate, high in efficiency, good in PCR amplification specificity, high in sensitivity and wide in application prospect;
(3) the method provided by the invention is used for detecting 20 collected suspected piglet epidemic diarrhea virus infection disease materials, and 17 parts of 2a type PEDV and 3 parts of 2b type PEDV are accurately identified.
Drawings
FIG. 1 is an electrophoretogram for detecting the specificity of primers in example 2 of the present invention.
FIG. 2 is an electrophoretogram of clinical samples in example 3 of the present invention.
FIG. 3 is an electrophoretogram of clinical samples in comparative example 1 of the present invention.
FIG. 4 is an electrophoretogram of clinical samples in comparative example 2 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
Unless otherwise specified, test materials, reagents, instruments and the like used in the examples of the present invention are commercially available; all technical measures in the examples of the present invention are conventional measures well known to those skilled in the art, unless otherwise specified.
Example 1 analysis of genome sequences of porcine epidemic diarrhea virus variants 2a and 2b and design of detection primers
This example designed 3 primers for the nucleotide sequences of S1 genes of PEDV GII a (GenBank No. mg020538.1) and PEDV GII b (GenBank No. ky496314), wherein P1 is a common primer, and P2 and P3 are specific primers for PEDV 2a and PEDV 2b, respectively, namely: the P1 and the P2 can amplify 910bp of PEDV GII a type S1 gene, wherein the P1 and the P3 can amplify 1390bp of PEDV GII b type S1 gene, and the primer information is shown in Table 1.
TABLE 1 porcine epidemic diarrhea Virus variant (PEDV) genotyping primers for type 2a and type 2b
| Primer name | Sequence (5-3) |
| P1 | GGAGGTTGTGCTATGCAATATG(SEQ ID NO.1) |
| P2 | GGAGTAACAAAAGAAAT(SEQ ID NO.2) |
| P3 | CTACTACCATCACCTTTG(SEQ ID NO.3) |
Example 2 detection method of porcine epidemic diarrhea Virus variant 2a type and 2b type
In this example, the primers obtained in example 1 were used to identify porcine epidemic diarrhea virus variants 2a and 2b, and the specific steps were as follows:
1. extracting 2a type and 2b type whole genomes of the porcine epidemic diarrhea virus variant strain, wherein the genome RNA extraction method comprises the following steps:
(1) collecting 300 μ l of virus liquid supernatant, adding 1ml trizol, blowing with gun, mixing, and standing on ice for 10 min;
(2) adding 200 μ l chloroform, shaking, mixing, standing on ice for 3min at 13000rpm at 4 deg.C for 15min, and collecting the upper water phase to new EP tube;
(3) adding 500 μ l isopropanol, mixing, and standing on ice for 30 min;
(4)13000rpm, 4 ℃ for 10min, and discarding the supernatant;
(5) adding 1ml of 75% ethanol, washing the precipitate, carrying out 13000rpm at 4 ℃ for 10min, and removing the supernatant;
(6) air dried and 30. mu.l DEPC water was added.
2. One-step PCR amplification of PEDV 2a/2b fragment
PCR amplification System: the P1, P2 and P3 primers were prepared so that the final concentration of each primer was 10 mmol/L.
The PCR reaction system components are shown in Table 2.
TABLE 2 one-step PCR reaction System
| Components | Volume (ul) |
| One·Step Enzyme Mix | 0.8 |
| 2×One·Step |
10 |
| |
1 |
| P2 or |
1 |
| |
2 |
| Sterile water | To 20 |
Amplification procedure for PEDV 2a and PEDV 2 b: 30min at 50 ℃, 2min at 94 ℃ (30 s at 94 ℃, 30s at 50 ℃ and 1min at 72 ℃) for 34 cycles, and 10min at 72 ℃.
3. Determination of detection results of 2a type and 2b type porcine epidemic diarrhea virus variant strains
Taking 10 mul of PCR amplification product, adding into 1% agarose gel containing ethidium bromide for electrophoresis under the condition of 130V for 25 min; and (3) judging the PEDV GIIa and the PEDV GIIb in the sample to be detected according to the electrophoresis result: when a band is evident at 910bp of the PCR product and the amplified product of the negative control (using water as a template) does not have the band, the PCR product shows that the PCR product has a positive reaction on PEDV GIIa, namely the sample to be detected is the porcine epidemic diarrhea virus variant 2a type. When the PCR product has an obvious band at 1390bp and the amplification product of the negative control (using water as a template) does not have the band, the PCR product is judged to have positive reaction to the porcine epidemic diarrhea virus variant 2b, namely the sample to be detected is the porcine epidemic diarrhea virus variant 2b type. The electrophoresis result of the PCR product is shown in FIG. 1, the porcine epidemic diarrhea virus variant 2a type has a specific band at 910bp, the porcine epidemic diarrhea virus variant 2b type has a specific band at 1390bp, and the negative control has no band, which indicates that the primers P1, P2 and P3 can specifically distinguish the porcine epidemic diarrhea virus variant 2a type from the porcine epidemic diarrhea virus variant 2b type.
EXAMPLE 3 detection of clinical samples
Collecting 20 suspected piglet epidemic diarrhea virus infected material, detecting by the detection method provided by the scheme, wherein the result shows that 17 positive material are detected by adopting primers P1, P2 and P3, and can amplify 910bp target strip containing porcine epidemic diarrhea virus type 2a (figure 2); 1390bp of target band can be amplified by 3 disease materials, and the target band contains porcine epidemic diarrhea virus type 2b (figure 2). And (3) carrying out whole genome sequencing on the genome DNA samples which are identified as the 2a and 2b porcine epidemic diarrhea viruses by the PCR, wherein the whole genome gene sequencing result is completely consistent with the PCR identification result, and the primer group and the detection method for detecting and typing the porcine epidemic diarrhea viruses provided by the invention are proved to have high accuracy.
Comparative example 1 results of discriminating 2a and 2b types by designing primers using M Gene as target Gene
This comparative example designed 2 primers for the nucleotide sequences of the M gene of PEDV GII a and PEDV GII b, and the information on the primers is shown in Table 3.
TABLE 3 information Table for primers designed for M Gene nucleotides
| Primer name | Sequence (5-3) |
| P4 | TATGGCTTGCATCATCACTCTTA(SEQ ID NO.4) |
| P5 | TTGACTGAACGACCAACACG(SEQ ID NO.5) |
Using the same detection method as in example 2, bands of 315bp were found in the electrophoresis results of 20 positive specimens to be detected, as shown in FIG. 3. It is proved that designing primers on the M genes of PEDV GII a and PEDV GII b can not distinguish PEDV GIIa and PEDV GIIb in the sample to be detected.
Comparative example 2 primer design for S1 Gene
In this comparative example, 1 common primer was additionally designed for the nucleotide sequence of S1 gene of PEDV GII a and PEDV GII b, and the primers specific to the other two primers 2a and 2b were not changed, and the information on the primers is shown in Table 4.
TABLE 4 common primer information Table designed for the S1 gene
| Primer name | Sequence (5-3) |
| P6 | CTTCTATTTACCTTACCATC(SEQ ID NO.6) |
| P2 | GGAGTAACAAAAGAAAT(SEQ ID NO.2) |
| P3 | CTACTACCATCACCTTTG(SEQ ID NO.3) |
Using the same detection method as in example 2, it was found that 10 samples to be tested selected from positive specimens showed simultaneous appearance of two or more bands with different target band sizes, indicating that the specificity of the fragments amplified using the common primers was not strong, as shown in FIG. 4.
It was confirmed that when the primers shown in SEQ ID NO.6 were used as the common primers, PEDV GIIa and PEDV GIIb could not be distinguished in the sample to be tested.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Wuhan Keshi Probiotics GmbH
<120> a method for discriminating porcine epidemic diarrhea virus type 2a and type 2b
<130> KHP211112098.5
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggaggttgtg ctatgcaata tg 22
<210> 2
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tatggcttgc atcatcactc tta 23
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cttctattta ccttaccatc 20
Claims (8)
1. A primer combination for detecting porcine epidemic diarrhea virus type 2a and type 2b, which is characterized in that the primer combination comprises: primers shown as SEQ ID NO. 1-3.
2. Use of the primer combination of claim 1 for the preparation of a kit for identifying porcine epidemic diarrhea virus type 2a and type 2 b.
3. A kit for identifying porcine epidemic diarrhea virus type 2a and type 2b, comprising the primer combination of claim 1.
4. A method for identifying porcine epidemic diarrhea virus type 2a and type 2b for non-diagnostic purposes, comprising performing PCR amplification on a sample to be tested using the kit of claim 3, and identifying the sample based on the PCR product.
5. The method of claim 4, wherein the PCR amplification system comprises One-Step Enzyme Mix, 2 Xone-Step Reaction Solution, P1 primer, P2 primer, or P3 primer; the amplification procedure of the PCR amplification is 30min at 50 ℃ and 2min at 94 ℃; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 1min, and 34 cycles; fully extending for 10min at 72 ℃.
6. The method of claim 5,
if the PCR product has a band at 910bp and the amplification product of the negative control does not have the band, determining that the PEDV GIIa positive reaction is obtained, namely the sample to be detected is the porcine epidemic diarrhea virus variant 2a type;
when the PCR product has a band at 1390bp and the amplified product of the negative control does not have the band, the PEDV GIIb positive reaction is judged, namely the sample to be detected is the porcine epidemic diarrhea virus variant 2b type.
7. Use of the method of any one of claims 4 to 6 for the quality control of a porcine epidemic diarrhea virus vaccine.
8. A method for preparing a vaccination scheme for porcine epidemic diarrhea virus in a pig farm, which is characterized in that the method of any one of claims 4-6 is used for identifying the strain type of the porcine epidemic diarrhea virus in the pig farm and determining the type of the vaccination required by the pig farm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110411684.4A CN112941241A (en) | 2021-04-16 | 2021-04-16 | Method for identifying porcine epidemic diarrhea virus type 2a and type 2b |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110411684.4A CN112941241A (en) | 2021-04-16 | 2021-04-16 | Method for identifying porcine epidemic diarrhea virus type 2a and type 2b |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112941241A true CN112941241A (en) | 2021-06-11 |
Family
ID=76232823
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110411684.4A Withdrawn CN112941241A (en) | 2021-04-16 | 2021-04-16 | Method for identifying porcine epidemic diarrhea virus type 2a and type 2b |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112941241A (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060474A (en) * | 2013-01-07 | 2013-04-24 | 江苏省农业科学院 | Primers for detecting variants on porcine epidemic diarrhea virus and detection kit thereof |
| CN104498623A (en) * | 2014-11-26 | 2015-04-08 | 江苏省农业科学院 | Primers for differential diagnosis of porcine epidemic diarrhea virus virulent strain and attenuated strain and detection kit thereof |
| CN107557494A (en) * | 2017-09-26 | 2018-01-09 | 华中农业大学 | Differentiate the primer of detection diarrhea of pigs coronavirus and multiple RT PCR methods |
| CN108034765A (en) * | 2017-12-22 | 2018-05-15 | 广东省农业科学院动物卫生研究所 | Primer and probe, the method for quick detection Porcine epidemic diarrhea virus genotype |
| CN109161534A (en) * | 2018-09-14 | 2019-01-08 | 中国农业科学院兰州兽医研究所 | The Porcine epidemic diarrhea virus strain of one pnca gene type 2a and its preparing the application in pig epidemic diarrhea vaccine |
| CN109735662A (en) * | 2019-03-11 | 2019-05-10 | 吉林正业生物制品股份有限公司 | Identify the primer sets and detection kit of Porcine epidemic diarrhea virus classics and variation strain |
| CN109825649A (en) * | 2019-04-09 | 2019-05-31 | 广西大学 | Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic primers group and its diagnostic kit |
| CN111041002A (en) * | 2019-12-23 | 2020-04-21 | 武汉科前生物股份有限公司 | Bivalent inactivated vaccine of porcine epidemic diarrhea virus variant 2a and 2b and preparation method thereof |
-
2021
- 2021-04-16 CN CN202110411684.4A patent/CN112941241A/en not_active Withdrawn
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060474A (en) * | 2013-01-07 | 2013-04-24 | 江苏省农业科学院 | Primers for detecting variants on porcine epidemic diarrhea virus and detection kit thereof |
| CN104498623A (en) * | 2014-11-26 | 2015-04-08 | 江苏省农业科学院 | Primers for differential diagnosis of porcine epidemic diarrhea virus virulent strain and attenuated strain and detection kit thereof |
| CN107557494A (en) * | 2017-09-26 | 2018-01-09 | 华中农业大学 | Differentiate the primer of detection diarrhea of pigs coronavirus and multiple RT PCR methods |
| CN108034765A (en) * | 2017-12-22 | 2018-05-15 | 广东省农业科学院动物卫生研究所 | Primer and probe, the method for quick detection Porcine epidemic diarrhea virus genotype |
| CN109161534A (en) * | 2018-09-14 | 2019-01-08 | 中国农业科学院兰州兽医研究所 | The Porcine epidemic diarrhea virus strain of one pnca gene type 2a and its preparing the application in pig epidemic diarrhea vaccine |
| CN109735662A (en) * | 2019-03-11 | 2019-05-10 | 吉林正业生物制品股份有限公司 | Identify the primer sets and detection kit of Porcine epidemic diarrhea virus classics and variation strain |
| CN109825649A (en) * | 2019-04-09 | 2019-05-31 | 广西大学 | Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic primers group and its diagnostic kit |
| CN111041002A (en) * | 2019-12-23 | 2020-04-21 | 武汉科前生物股份有限公司 | Bivalent inactivated vaccine of porcine epidemic diarrhea virus variant 2a and 2b and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| CHANGHEE LEE: "Porcine epidemic diarrhea virus: An emerging and re-emerging epizootic swine virus", 《VIROLOGY JOURNAL》 * |
| XINSHENG LIU等: "Evaluation and comparison of immunogenicity and cross-protective efficacy of two inactivated cell culture-derived GIIa- and GIIb-genotype porcine epidemic diarrhea virus vaccines in suckling piglets", 《VETERINARY MICROBIOLOGY》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111996191A (en) | Primer group and kit for simultaneously identifying African swine fever wild strain and gene deletion strain based on multiple qPCR technology | |
| WO2018059195A1 (en) | Hrm detection primer, kit, and method for quickly identifying classical strain and variant strain of porcine epidemic diarrhea virus | |
| CN110699489B (en) | Real-time fluorescence PCR detection primer probe set, kit and method for African swine fever virus CD2V gene | |
| CN108950068B (en) | Kit for identifying and detecting QX-type strains of chicken infectious bronchitis viruses | |
| CN110724769A (en) | PCR primer group, kit and detection method for detecting African swine fever virus MGF360-505R gene | |
| CN113564280A (en) | RAA primer for detecting 12 serotypes of avian adenovirus group I and detection method thereof | |
| CN113584227B (en) | Nested PCR (polymerase chain reaction) detection primer group and method for identifying African swine fever gene deletion strain | |
| CN113186312B (en) | Molecular marker for distinguishing Brucella A19 vaccine strain and wild strain | |
| CN111118215A (en) | Fluorescent PCR (polymerase chain reaction) primer, probe and kit for detecting capripoxvirus virus | |
| CN113046489B (en) | Multiple RT-PCR primer group for detecting porcine astrovirus, kit and application thereof | |
| CN108342510B (en) | Multiple RT-PCR kit for BTV-11 type, 17 type, 20 type, 23 type and 24 type genotype typing identification and detection method thereof | |
| CN113174446A (en) | One-step double RT-PCR detection method for bovine viral diarrhea virus typing | |
| CN112662821A (en) | Fluorescent probe primer combination, kit and application of porcine epidemic diarrhea virus M gene | |
| CN112280899A (en) | Porcine astrovirus type 2 TaqMan fluorescent quantitative PCR kit and application thereof | |
| CN109868331B (en) | Dual nested RT-PCR (reverse transcription-polymerase chain reaction) primers of universal porcine epidemic diarrhea virus and porcine coronavirus D, detection method and application | |
| Harada et al. | Molecular typing of Japanese field isolates and live commercial vaccine strain of Mycoplasma synoviae using improved pulsed-field gel electrophoresis and vlhA gene sequencing | |
| CN116656845A (en) | Triple fluorescent quantitative PCR detection kit for diagnosing brucella vaccine immunity and natural infection and detection method thereof | |
| CN112941241A (en) | Method for identifying porcine epidemic diarrhea virus type 2a and type 2b | |
| CN113151586B (en) | Primer combination, kit and method for detecting and identifying porcine pseudorabies virus type I and type II | |
| CN111500774B (en) | Epidemic hemorrhagic disease virus and serotype identification RT-PCR kit | |
| CN113215309A (en) | PCR primer for detecting porcine rotavirus G9 type wild strain and G5 type vaccine strain, detection method and kit | |
| CN112877479A (en) | Primer for rapidly detecting exogenous viruses in porcine pseudorabies live vaccine and application of primer in kit | |
| CN112522446A (en) | Detection primer pair and kit for wild strain of porcine pseudorabies virus | |
| CN110643739A (en) | One-step triple RT-PCR detection primer and kit for distinguishing CHUV, BCV and DAV | |
| CN115074461B (en) | Triple PCR detection method for canine influenza virus, canine coronavirus and canine reovirus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20210611 |
|
| WW01 | Invention patent application withdrawn after publication |