CN113817869B - Primer probe group for detecting mink astrovirus based on qPCR and application thereof - Google Patents
Primer probe group for detecting mink astrovirus based on qPCR and application thereof Download PDFInfo
- Publication number
- CN113817869B CN113817869B CN202110967485.1A CN202110967485A CN113817869B CN 113817869 B CN113817869 B CN 113817869B CN 202110967485 A CN202110967485 A CN 202110967485A CN 113817869 B CN113817869 B CN 113817869B
- Authority
- CN
- China
- Prior art keywords
- primer
- probe
- seq
- astrovirus
- qpcr
- 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.)
- Active
Links
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
- C12Q1/6851—Quantitative amplification
Abstract
A primer probe group for detecting mink astrovirus based on qPCR and application thereof belong to the technical field of pathogen detection. In order to solve the problem that the sensitivity, the specificity and the speed of detecting mink astrovirus by using a common PCR method are insufficient, the invention designs and obtains the primer probe group based on qPCR, and the primer probe group is used for detecting the mink astrovirus, so that the sensitivity, the specificity and the speed of detecting the mink astrovirus can be improved, and the method has the characteristics of good repeatability and stability. The qPCR-based primer probe set and the kit containing the primer probe set can be applied to detection of mink astrovirus and have important significance for improvement of mink astrovirus detection technology.
Description
Technical Field
The invention belongs to the technical field of pathogen detection, and particularly relates to a primer probe group for detecting mink astrovirus based on qPCR and application thereof.
Background
In 2014, china first found that mink with encephalitis symptoms (mink tremor syndrome) caused by infection of mink astrovirus has a litter infectivity, which brings great harm to the mink breeding industry. Astrovirus is a non-enveloped, single-stranded RNA virus of the astroviridae family, has higher genetic diversity and recombination potential, and is difficult to detect because polymerase has no proofreading function in the replication process, and replication is extremely easy to mutate.
At present, the clinical detection of the mink astrovirus is mainly a common PCR method, no related patent of the real-time fluorescence quantitative detection method of the mink astrovirus exists in China, and the real-time fluorescence quantitative PCR detection technology is developed on the basis of the common PCR technology, has high sensitivity, high specificity and high speed, and has better effect on detection of various pathogens, so that a real-time fluorescence quantitative PCR detection method for detecting the rice astrovirus is needed.
Disclosure of Invention
The invention provides a primer probe group for detecting mink astrovirus based on qPCR and application thereof, aiming at solving the problems of insufficient sensitivity, specificity and speed of detecting mink astrovirus by using a common PCR method, and the specific technical scheme is as follows:
the first object of the invention is to provide a primer probe group for detecting mink astrovirus based on qPCR, which consists of an upstream primer, a downstream primer and a probe, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID No.1 or SEQ ID No.2, the nucleotide sequence of the downstream primer is shown as SEQ ID No.3, and the nucleotide sequence of the probe is shown as SEQ ID No. 4.
In one embodiment of the invention, the probe is labeled with a reporter group at the 5 'end and a fluorescence quenching group at the 3' end.
In one embodiment of the invention, the reporter is 6-carboxyfluorescein and the fluorescence quenching group is TAMR-N.
The second object of the invention is to provide the application of the primer probe set in preparing a reagent or a kit for detecting mink astrovirus.
The third object of the invention is to provide a kit for detecting mink astrovirus based on qPCR, which comprises an upstream primer with a nucleotide sequence shown as SEQ ID No.1 or SEQ ID No.2, a downstream primer shown as SEQ ID No.3 and a probe shown as SEQ ID No. 4.
In one embodiment of the present invention, the reaction system for amplifying a target gene using the kit is: 2 Xfluorescent quantitative PCR premix 10. Mu.L, upstream primer 0.4. Mu.L, downstream primer 0.4. Mu.L, probe 0.2. Mu.L, DNA 2. Mu.L, deionized water 7.0. Mu.L.
In one embodiment of the invention, the concentration of the upstream primer, the downstream primer and the probe is 10. Mu.M.
In one embodiment of the present invention, the reaction conditions for amplifying the target gene using the kit are 95℃for 30s;95℃for 5s,60℃for 1min,40 cycles.
The invention has the beneficial effects that:
the invention designs a primer probe group based on real-time fluorescence quantitative PCR according to the mink astrovirus ORFla gene by comparing nucleotide sequences of the astrovirus ORFla genes from different hosts and selecting a relative conservation region of the ORFla genes. The primer probe set is applied to detection of mink astrovirus, and the minimum detection amount is 6.274 multiplied by 10 through optimizing a fluorescent quantitative PCR reaction system and conditions 4 The sensitivity, specificity, accuracy, repeatability and stability of the copies/. Mu.L are good and the mink astrovirus detection method is rapid.
Drawings
FIG. 1 is a qPCR standard curve of astrovirus;
FIG. 2 is a graph of the sensitivity detection results of a fluorescent quantitative PCR method; in the graph, the concentration of mink astrovirus in the amplification curve from left to right is 6.274 multiplied by 10 in sequence 10 copies/μL，6.274×10 9 copies/μL，6.274×10 8 copies/μL，6.274×10 7 copies/μL，6.274×10 6 copies/μL，6.274×10 5 copies/μL，6.274×10 4 copies/μL，6.274×10 3 copies/μL；
FIG. 3 is a diagram of the result of specific detection by the fluorescent quantitative PCR method; in the figure, the amplification curves are sequentially from left to right mink astrovirus (6.274×10) 5 cobies/. Mu.L), mink astrovirus (6.274X 10) 4 cobies/. Mu.L), canine distemper virus, canineParvovirus, mink parvovirus.
Detailed Description
The process, conditions, reagents, experimental methods, etc. for carrying out the present invention will be described in further detail below with reference to specific examples and drawings, and are general knowledge and common knowledge in the art, except for the following specific references, and the present invention is not limited in any way.
The instrument used in the invention is a LightCycler 96 fluorescence PCR instrument manufactured by Roche company
The viral RNA extraction kit, RNA reverse transcription kit and PCR premix used in the present invention were purchased from Takara, and the primer probes were synthesized by the company Shanghai, inc.
Example 1: primer probe group for detecting mink astrovirus based on qPCR
Primer and probe design:
the astrovirus genome is 6.8kb in size and comprises 3 open reading frames, the orflia and orflib encoding the nonstructural proteins and the ORF2 encoding the capsid protein, wherein the orflia encoding nonstructural proteins have a gene size of less than 2.8kb, the orflia encoding 110kDa amino acids, the polypeptide comprises a plurality of conserved gene sequences, serine proteases constituting the virus, and the orflib encoding the nonstructural proteins of the virus. The nucleotide sequences of ORFla genes of astroviruses (such as human, pig, cow and the like) from different hosts are compared, the relative conservation region of the ORFla genes is selected, and primers and probes are designed in the conservation region of the sequence of Astv, and the NCBI accession number of a reference sequence is NC_004579.
The sequence of the upstream primer is shown in SEQ ID No. 1: 5'-GCTTGAGGTCTTAGCCAG-3' or as shown in SEQ ID No. 2: 5'-GCTTGAGGTCTTAGCTAG-3';
the sequence of the downstream primer is shown in SEQ ID No. 3: 5'-TCCCTCCAAAGTTTATC-3';
the probe sequence is shown in SEQ ID No. 4: 5'FAM-CAGTCGCTCCGTGACATGGAC-3' TAMR-N.
The sequence amplified by the primer probe group is shown as SEQ ID No. 5:
5’-CTTGAGGTCTTAGCCAGGCACATCCGCAACTTGGGCGGCGAGGAGCCCGTCCATGTCACGGAGCGACTGCTTGATAAACTTTGGAGGGGA-3’
example 2: method for detecting mink astrovirus based on fluorescent quantitative PCR
(1) Taking brain tissue of mink as a detection sample, extracting sample RNA by using a QUAGEN RNA extraction kit, and then reversely transcribing the RNA into cDNA by using the QUAGEN RNA reverse transcription kit.
(2) The ORFla gene was amplified using the primer probe set described in example 1, with a reaction system of 30. Mu.L, comprising: 15. Mu.L of PCR premix (2X), 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, 1. Mu.L of probe, 10. Mu.L of deionized water, and 2. Mu.L of DNA. The reaction conditions are as follows: melting at 94 ℃ for 30s, annealing at 52 ℃ for 30s, and extending at 72 ℃ for 30s, wherein the steps are 37 cycles, and extending at 72 ℃ for 10min.
Optimization of fluorescent quantitative PCR reaction conditions:
by comparing the primer concentration, the probe concentration and the annealing temperature experimental results in the reaction system, a reaction system with high reaction sensitivity, low background fluorescence signal, typical S-shaped amplification fluorescence signal curve and amplification efficiency close to 1 is selected. The reaction system obtained by optimization is as follows: fluorescent quantitative PCR premix (2X) 10. Mu.L, upstream primer (10. Mu.M) 0.4. Mu.L, downstream primer (10. Mu.M) 0.4. Mu.L, probe (10. Mu.M) 0.2. Mu.L, DNA 2. Mu.L, deionized water 7.0. Mu.L; the reaction conditions obtained by optimization are as follows: 95℃for 30 seconds → (95℃for 5 seconds → 60℃for 20 seconds). Times.40.
Evaluation of the method for detecting mink astrovirus according to the present invention:
1. calculating the plasmid copy number corresponding to the optimized fluorescent quantitative PCR reaction condition:
(1) taking brain tissue of mink as a detection sample, extracting sample RNA by using a QUAGEN RNA extraction kit, and then reversely transcribing the RNA into cDNA by using the QUAGEN RNA reverse transcription kit.
(2) The ORFla gene was amplified using the primer probe set described in example 1, with a reaction system of 30. Mu.L, comprising: 10. Mu.L of PCR premix (2X), 0.4. Mu.L of upstream primer, 0.4. Mu.L of downstream primer, 0.2. Mu.L of probe, 7.0. Mu.L of deionized water, and 2. Mu.L of DNA. The reaction conditions are as follows: 95℃for 30 seconds → (95℃for 5 seconds → 60℃for 20 seconds). Times.40.
(3) 2.0% agarose gel electrophoresis detection amplified products, cutting gel, and recovering target fragments with the size of 497bp by using a Takara gel recovery kit. Cloning the amplified target gene to pMD18-T vector with TA cloning kit, converting to colibacillus DH5 alpha strain, screening positive clone, PCR identification, sequencing and culturing the correct monoclonal bacteria.
(4) Plasmid extraction and purification were performed on the bacterial solution using a Takara plasmid extraction kit, and the plasmid concentration was measured using an ultraviolet spectrophotometer, the mass concentration was 192ng/mL, the DNA length was 2791, and the plasmid copy number= (6 02×10) 23 )×(ng/μL×10 -9 )/(DNAlength×660)＝6.274×1010copies/μL。
2. Establishment of a fluorescent quantitative PCR standard curve:
the quantitative standard plasmid obtained was diluted 10-fold with deionized water at a concentration of 6.274X 10 10 ，6.274×10 9 ，6.274×108，6.274×10 7 ，6.274×10 6 copies/. Mu.L, three replicates were made for each dilution. The standard curve (shown in figure 1) is drawn, and the result shows that the detection method has good linear relation, and the correlation coefficient (R2) of the standard curve reaches 0.99. According to the established standard curve, the fluorescence quantitative PCR is 6.274×10 4 -6.274×10 8 The copy copies/uL showed good linearity over the dilution range (see standard curve extremum in figure 1).
3. Method sensitivity rating
The quantitative standard plasmid was diluted 10-fold with deionized water at a concentration of 6.274×10, respectively 10 ，6.274×10 9 ，6.274×10 8 ，6.274×10 7 ，6.274×10 6 ，6.274×10 5 ，6.274×10 4 ，6.274×10 3 The sensitivity of the method was measured using an optimized system and reaction conditions, with the results shown in FIG. 2. As can be seen from the results, the minimum detection amount of the fluorescent quantitative PCR was 6.274×10 4 copies/μL。
4. Method specificity evaluation
Selecting existing cell strain Canine Distemper Virus (CDV), canine Parvovirus (CPV), mink parvovirus (MEV), mink astrovirus (6.274×10) 5 cobies/. Mu.L), mink astrovirus (6.274X 10) 4 cobies/. Mu.L) and negative controls were specifically tested using the method, and the results of the specific test were shown in FIG. 3 using an optimized system and reaction conditions. As can be seen from the results, the Cq values amplified by other pathogens (CDV, CPV, MEV) possibly carried by the mink and the negative control are countless, and can be judged as negative, and the method only has specific amplification on nucleic acid of the mink astrovirus. In FIG. 3, the amplification curves were mink astrovirus (6.274×10 in order from left to right 5 cobies/. Mu.L), mink astrovirus (6.274X 10) 4 cobies/. Mu.L), canine distemper virus, canine parvovirus, mink parvovirus.
5. Method reproducibility and stability evaluation
The 5 dilutions of recombinant plasmids were used as templates for the batch and batch-to-batch reproducibility tests, respectively, which showed that the intraclass Cq value Coefficient of Variation (CV) was 0.21% -3.38% and the interclass Cq value Coefficient of Variation (CV) was 2.76% -5.24% (see Table 1). The result shows that the established mink astrovirus fluorescence quantitative PCR detection method has good repeatability and can be used for stable and reliable detection.
TABLE 1 repeatability and stability evaluation results of fluorescent quantitative PCR
6. Clinical sample detection
44 mink brain tissue samples are detected by a common PCR method and a fluorescent quantitative PCR method respectively, the positive detection rate of the mink astrovirus by the common PCR method is 36.36% (16/44), and the positive detection rate of the established real-time fluorescent quantitative method of the mink astrovirus is 45.45% (20/44). The sensitivity of the established fluorescent quantitative PCR method is higher than that of the conventional PCR method.
Example 3: kit for detecting mink astrovirus based on qPCR
A kit for detecting mink astrovirus based on qPCR comprises an upstream primer (10 mu M) with a nucleotide sequence shown as SEQ ID No.1 or SEQ ID No.2, a downstream primer (10 mu M) with a nucleotide sequence shown as SEQ ID No.3 and a probe (10 mu M) with a nucleotide sequence shown as SEQ ID No. 4.
The method for detecting mink astrovirus by using the kit comprises the following steps:
using mink brain tissue DNA as a detection sample, amplifying ORFla gene by using the upstream primer, the downstream primer and the probe, wherein a reaction system is 30 mu L, and the method comprises the following steps: 10. Mu.L of PCR premix (2X), 0.4. Mu.L of upstream primer, 0.4. Mu.L of downstream primer, 0.2. Mu.L of probe, 7. Mu.L of deionized water and 2. Mu.L of DNA. The reaction conditions are as follows: 95 ℃ for 30s;95℃for 5s,60℃for 20s,40 cycles.
SEQUENCE LISTING
<110> institute of specialty of national academy of agricultural sciences
<120> a primer probe set for detecting mink astrovirus based on qPCR and application thereof
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> Synthesis
<400> 1
gcttgaggtc ttagccag 18
<210> 2
<211> 18
<212> DNA
<213> Synthesis
<400> 2
gcttgaggtc ttagctag 18
<210> 3
<211> 17
<212> DNA
<213> Synthesis
<400> 3
tccctccaaa gtttatc 17
<210> 4
<211> 21
<212> DNA
<213> Synthesis
<400> 4
cagtcgctcc gtgacatgga c 21
<210> 5
<211> 90
<212> DNA
<213> 5
<400> 5
cttgaggtct tagccaggca catccgcaac ttgggcggcg aggagcccgt ccatgtcacg 60
gagcgactgc ttgataaact ttggagggga 90
Claims (8)
1. A primer probe group for detecting mink astrovirus based on qPCR, which is characterized by comprising an upstream primer, a downstream primer and a probe; the nucleotide sequence of the upstream primer is shown as SEQ ID No.1 or SEQ ID No.2, the nucleotide sequence of the downstream primer is shown as SEQ ID No.3, and the nucleotide sequence of the probe is shown as SEQ ID No. 4.
2. The primer probe set of claim 1, wherein the probe is labeled with a reporter group at the 5 'end and a fluorogenic quenching group at the 3' end.
3. The primer probe set of claim 2, wherein the reporter is 6-carboxyfluorescein and the fluorogenic quenching group is TAMR-N.
4. Use of a primer-probe set according to any one of claims 1-3 for the preparation of a reagent or kit for detecting mink astrovirus.
5. A kit for detecting mink astrovirus based on qPCR is characterized by comprising an upstream primer with a nucleotide sequence shown as SEQ ID No.1 or SEQ ID No.2, a downstream primer shown as SEQ ID No.3 and a probe shown as SEQ ID No. 4.
6. The kit according to claim 5, wherein the reaction system for amplifying the target gene using the kit is: 2 Xfluorescent quantitative PCR premix 10. Mu.L, upstream primer 0.4. Mu.L, downstream primer 0.4. Mu.L, probe 0.2. Mu.L, DNA 2. Mu.L, deionized water 7.0. Mu.L.
7. The kit of claim 6, wherein the concentration of each of the upstream primer, the downstream primer and the probe is 10. Mu.M.
8. The kit according to claim 5, wherein the reaction conditions for amplifying the target gene using the kit are 95℃for 30s;95℃for 5s,60℃for 20s,40 cycles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110967485.1A CN113817869B (en) | 2021-08-23 | 2021-08-23 | Primer probe group for detecting mink astrovirus based on qPCR and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110967485.1A CN113817869B (en) | 2021-08-23 | 2021-08-23 | Primer probe group for detecting mink astrovirus based on qPCR and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113817869A CN113817869A (en) | 2021-12-21 |
| CN113817869B true CN113817869B (en) | 2023-09-12 |
Family
ID=78913544
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110967485.1A Active CN113817869B (en) | 2021-08-23 | 2021-08-23 | Primer probe group for detecting mink astrovirus based on qPCR and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113817869B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017905A (en) * | 2014-06-25 | 2014-09-03 | 长兴县人民医院 | Probe, primer and kit for detecting human astrovirus and human noroviruses through real-time fluorescent RT-PCR and application of probe, primer and kit |
| CN105256067A (en) * | 2015-08-07 | 2016-01-20 | 青岛农业大学 | RT-PCR primers for detecting mink astroviruses and detection method using the same |
| CN107299155A (en) * | 2017-08-18 | 2017-10-27 | 福建省农业科学院畜牧兽医研究所 | A kind of primer and probe of goose astrovirus real-time fluorescence quantitative PCR detection |
| CN112280899A (en) * | 2020-10-27 | 2021-01-29 | 广西中医药大学第一附属医院 | Porcine astrovirus type 2 TaqMan fluorescent quantitative PCR kit and application thereof |
-
2021
- 2021-08-23 CN CN202110967485.1A patent/CN113817869B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017905A (en) * | 2014-06-25 | 2014-09-03 | 长兴县人民医院 | Probe, primer and kit for detecting human astrovirus and human noroviruses through real-time fluorescent RT-PCR and application of probe, primer and kit |
| CN105256067A (en) * | 2015-08-07 | 2016-01-20 | 青岛农业大学 | RT-PCR primers for detecting mink astroviruses and detection method using the same |
| CN107299155A (en) * | 2017-08-18 | 2017-10-27 | 福建省农业科学院畜牧兽医研究所 | A kind of primer and probe of goose astrovirus real-time fluorescence quantitative PCR detection |
| CN112280899A (en) * | 2020-10-27 | 2021-01-29 | 广西中医药大学第一附属医院 | Porcine astrovirus type 2 TaqMan fluorescent quantitative PCR kit and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Molecular characterization of a novel astrovirus associated with disease in mink;Christian Mittelholzer 等;《J Gen Virol .》;3087-3094 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113817869A (en) | 2021-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108676920B (en) | Primer and kit for rapidly detecting mouse norovirus and RT-RPA method thereof | |
| CN110760620A (en) | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method | |
| CN111020062A (en) | Triple real-time fluorescent quantitative PCR kit for detecting African swine fever wild strain and gene deletion strain | |
| CN111996191A (en) | Primer group and kit for simultaneously identifying African swine fever wild strain and gene deletion strain based on multiple qPCR technology | |
| CN111304371B (en) | Locked nucleic acid probe fluorescent quantitative PCR detection composition, detection method and detection kit for African swine fever virus wild strain | |
| CN107988340B (en) | PCR amplification primer for rapidly detecting mycoplasma ovipneumoniae and application thereof | |
| CN112795706A (en) | Fluorescent probe primer group and kit for African swine fever virus P72 gene and application of fluorescent probe primer group and kit | |
| CN113403430A (en) | Triple fluorescent quantitative PCR primer group for detecting different types of porcine circovirus, kit and application | |
| CN108588276B (en) | Fluorescent quantitative PCR (polymerase chain reaction) primer pair and kit for D-type influenza virus | |
| CN113817869B (en) | Primer probe group for detecting mink astrovirus based on qPCR and application thereof | |
| CN112280899A (en) | Porcine astrovirus type 2 TaqMan fluorescent quantitative PCR kit and application thereof | |
| CN111500767A (en) | Microfluid chip for simultaneously detecting 9 porcine pathogens | |
| CN115725788A (en) | Primer and TaqMan probe for detecting feline parvovirus and application thereof | |
| CN114622041A (en) | Primer and TaqMan probe for detecting canine torque teno virus and application thereof | |
| CN111996294A (en) | Primer pair and kit for quantitatively detecting eel herpesvirus | |
| CN112410466A (en) | Primer, probe and detection method for porcine circovirus type 2 and porcine circovirus type 4 dual real-time fluorescent quantitative PCR detection | |
| CN110885908A (en) | Real-time fluorescent quantitative RT-PCR detection method of norovirus | |
| CN116144836B (en) | Quadruple fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer set for PRRSV (porcine reproductive and respiratory syndrome virus) and detection method | |
| CN117821661A (en) | Novel coronavirus dual rapid fluorescence PCR detection primer combination, kit and detection method | |
| CN116411132A (en) | Method for rapidly detecting porcine pseudorabies | |
| CN116334316A (en) | MDV TaqMan fluorescent quantitative PCR detection method and composition | |
| CN116024384A (en) | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method for panda parvovirus | |
| CN106048067B (en) | Kit for quantitative detection of Lanzhou lily PR1 gene and detection method thereof | |
| CN106048068B (en) | Kit for quantitative detection of Lanzhou lily PR5 gene and detection method thereof | |
| CN117230256A (en) | Application of primer probe group/detection reagent/triple fluorescence PCR method in ASF strain detection and/or identification |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |