CN113046489B - Multiple RT-PCR primer group for detecting porcine astrovirus, kit and application thereof - Google Patents
Multiple RT-PCR primer group for detecting porcine astrovirus, kit and application thereof Download PDFInfo
- Publication number
- CN113046489B CN113046489B CN202110550666.4A CN202110550666A CN113046489B CN 113046489 B CN113046489 B CN 113046489B CN 202110550666 A CN202110550666 A CN 202110550666A CN 113046489 B CN113046489 B CN 113046489B
- Authority
- CN
- China
- Prior art keywords
- primers
- pcr
- multiplex
- astrovirus
- pastv4
- 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
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]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a multiplex RT-PCR primer group for detecting porcine astrovirus, which comprises five pairs of specific primers, namely primers PAStV1-F and PAStV1-R, primers PAStV2-F and PAStV2-R, primers PAStV3-F and PAStV3-R, primers PAStV4-F and PAStV4-R and primers PAStV5-F and PAStV5-R, wherein the primers respectively have base sequences of sequence tables SEQ ID No.1 to SEQ ID No. 10. Accordingly, the inventor also prepares a corresponding RT-PCR kit and establishes a corresponding RT-PCR detection method. The method is simple and convenient in actual operation, reduces the operation times, greatly avoids cross contamination, and saves the detection time and the reagent consumption. The established detection method aims at the gene amplification of the virus conserved region, and has strong pertinence and high sensitivity. Can meet the requirements of simple, convenient, economic, convenient and accurate inspection in clinical experiments.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a multiple RT-PCR primer group for detecting porcine astrovirus, a kit and application thereof.
Background
Porcine astrovirus was first discovered in pig manure by electron microscopy in 1980. Astrovirus is associated with diarrhea in the gut of a variety of animals, ranging from birds to mammals to humans to infections. Porcine astrovirus often infects young, old and immunocompromised animals. Meanwhile, rotavirus, coronavirus, calicivirus and other enterovirus mixed infection are often detected in positive samples of the porcine astrovirus. The porcine astrovirus has various genotypes, and at present, 5 genotypes (PAStV 1-PAStV 5) are found, and the sequence homology of each genotype is not high, which indicates that the genotypes are derived from different clades. Although the genetic background of each genotype is obviously different, the porcine astrovirus of different genotypes can be detected in the same area and even the same pig body, and the phenomenon of mixed infection of PAStVs is common. In addition, co-infection with different genotypes of PAstV often leads to genetic recombination phenomena with the potential risk of interspecies transmission and even zoonosis. The mixed infection of multiple genotypes is frequently generated among the genomes of the PAStV, and the infection of two or more genotypes is generated, so that the genetic variation of the virus is further accelerated, and the monitoring of the porcine astrovirus is challenged. It is worth mentioning that the porcine astrovirus interspecies barrier may not be strict, and the results of genetic evolution analysis suggest that the porcine astrovirus may cross the interspecies barrier of humans and other animals. Researchers find that recombination phenomenon exists between human astrovirus and partial ORF2 gene of porcine astrovirus, which indicates that the porcine astrovirus has close relationship with the human astrovirus in the evolution process. Therefore, the establishment of an efficient and rapid detection method can monitor the infection condition of the porcine astrovirus in a swinery and analyze the recombination and variation rules among strains, which has important public health significance for the biological characteristics and pathogenic mechanism of the astrovirus and the prevention of the infection of the human and animal co-suffering astrovirus.
In the current intensive management production, the condition of polygenic mixed infection of the porcine astrovirus is common, the epidemiological monitoring of the porcine astrovirus is a long-term and very necessary work, and the establishment of a rapid and accurate diagnostic method becomes a powerful tool for monitoring the PAStVs. In contrast, the establishment of multiplex PCR detection technology can amplify multiple gene fragments simultaneously, and is low in cost, rapid, cheap and undoubtedly the best detection means at present.
Disclosure of Invention
The invention provides a multiplex RT-PCR primer group, a kit and application thereof for detecting porcine astrovirus, which are simple and convenient to operate, avoid cross contamination, save detection time, consume reagents, have strong pertinence and high sensitivity, and solve the problems of complicated operation and high cross contamination risk of single RT-PCR, and the specific scheme is as follows:
a multiplex RT-PCR primer group for detecting porcine astrovirus comprises five pairs of specific primers, namely primers PAStV1-F and PAStV1-R, primers PAStV2-F and PAStV2-R, primers PAStV3-F and PAStV3-R, primers PAStV4-F and PAStV4-R and primers PAStV5-F and PAStV5-R, which have base sequences of sequence tables SEQ ID No.1 to SEQ ID No.10 respectively.
A multiplex RT-PCR kit for detecting porcine astrovirus, the kit comprising a multiplex RT-PCR primer set.
The kit also comprises 5 x buffer and dNTPMix, M-MLVRevereTranscriptae, RNase inhibitor, RNA, tarakaEx XTaq, 10 XExTaqbuffer, dNTPmix, templatecDNA and ddH 2 O。
The application of the multiplex RT-PCR primer group for detecting the porcine astrovirus in RT-PCR amplification.
The RT-PCR amplification reaction system is that each of multiple RT-PCR primer groups is 0.8 mu mol/L, tarakaEx XTaq0.25 mu L and 10 XExTaqBuffer 5 mu L, dNTPMix mu L, cDNA template is 1 mu L, and ddH after sterilization 2 The content of O is filled to 50 mu L.
The RT-PCR amplification reaction conditions are denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min and 30 cycles.
THE ADVANTAGES OF THE PRESENT INVENTION
1. Aiming at the lack of effective and reliable technology for simultaneously detecting and diagnosing multi-genotype porcine astrovirus, the inventor researches and designs five pairs of specific primers, namely primers PAStV1-F and PAStV1-R, primers PAStV2-F and PAStV2-R, primers PAStV3-F and PAStV3-R, primers PAStV4-F and PAStV4-R and primers PAStV5-F and PAStV5-R, establishes a corresponding RT-PCR kit and establishes a corresponding RT-PCR detection method. The RT-PCR detection method can be used for carrying out PCR amplification on cDNA (complementary deoxyribonucleic acid) obtained from the total RNA of the same detection sample as a template, judging the genotype of the sample to be detected infected with the virus through agarose gel electrophoresis according to the size of an amplification product, identifying five genotypes of the virus amplified simultaneously in the same reaction system, and having important practical effects on early detection and effective prevention and control of the occurrence and spread of the porcine astrovirus.
2. The method is simple and convenient in actual operation, reduces the operation times, greatly avoids cross contamination, and saves the detection time and the reagent consumption. The established detection method aims at the gene amplification of the virus conserved region, and has strong pertinence and high sensitivity. Can meet the requirements of simple, convenient, economic, convenient and accurate inspection in clinical experiments. Provides technical support for understanding the prevalence of the porcine astrovirus and preventing and controlling diseases. Lays a foundation for monitoring the prevalence and mixed infection conditions of the porcine astrovirus types 1-5 and the rapid diagnosis of molecular biology.
Drawings
FIG. 1 is a diagram showing the detection of specific primers corresponding to PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5, wherein the lanes in the diagram are sequentially M from left to right: m is DL2000 DNA Marker; A. b, C, D, E is sequentially PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5;
FIG. 2 is a graph of the optimization of primer concentrations for multiplex RT-PCR, where M is DL2000 DNA Marker; the primer concentration of 1,2,3,4,5 is 0.2. Mu. Mol/L, 0.4. Mu. Mol/L, 0.6. Mu. Mol/L, 0.8. Mu. Mol/L and 1.0. Mu. Mol/L in sequence.
FIG. 3 is a graph of the optimization of the annealing temperature for multiplex RT-PCR, wherein M is DL2000 DNA Marker; the annealing temperatures of 1,2,3,4,5 are 53 ℃; at 54 ℃;55 ℃;56 ℃; and 57 ℃.
FIG. 4 is a diagram showing the detection results of the specificity of the primers for simultaneously detecting PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 according to the present invention, wherein the lanes from left to right are: m is DL2000 DNA Marker;1: PAStV1;2: PAStV2;3: PAStV3;4: PAStV4;5: PAStV5;6: PAStV1, PAStV2, PAStV3, PAStV4, and PAStV5;7-13 non-target genes; 14: and (5) negative control.
FIG. 5 is a graph showing the results of single sensitivity experiments for simultaneously detecting PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 according to the present invention, wherein the lanes sequentially show from left to right: 1;2, 1ng; 3; 4; 5;6, 0.1pg;7: negative control positive plasmid.
FIG. 6 is a graph showing the results of detection of multiple RT-PCR sensitivities simultaneously, wherein the lanes from left to right are: 1; 2; 3; 4; 5;6, 0.1pg;7: negative control positive plasmid.
And (3) positive plasmids.
Detailed Description
The invention will be further explained and illustrated with reference to the drawings and the following examples, which do not limit the scope of the invention. Unless otherwise specified, the technical means in the examples are conventional means well known to those skilled in the art.
Example 1
1.1 Strain for test and clinical specimens
Positive nucleic acid samples of porcine astrovirus type 1, porcine astrovirus type 2, porcine astrovirus type 3, porcine astrovirus type 4, porcine astrovirus type 5, porcine enterovirus, porcine senecio virus, porcine pseudorabies virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus are identified by the laboratory and stored at-80 ℃. Porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus triple live vaccine (Harbin Vitaceae Biotechnology development Co.), and porcine Japanese encephalitis B virus (Wuhanke department Probiotics GmbH). The clinical stool samples are all from a certain pig farm in Guangxi of 2020, and the total amount is 275.
1.2 primer design and Synthesis
According to the reported whole genome sequences of PASTV1, PASTV2, PASTV3, PASTV4 and PASTV5 on GenBank, the genomes of different strains are compared and analyzed, conservative nucleic acid fragments are screened, five pairs of specific primers aiming at the five viruses are designed by using Pirmer 5.0 primer design software, and the five pairs of primers are analyzed on line through NCBI. Five pairs of primers (Table 1) synthesized by Biotechnology, shanghai, inc. were finally screened. The five pairs of primers can specifically amplify specific fragments of 125 (PAStV 1), 573 (PAStV 2), 175 (PAStV 3), 485 (PAStV 4) and 305 (PAStV 5).
TABLE 1 sequence Listing of multiplex RT-PCR primers
EXAMPLE 2cDNA template preparation
2.1 extraction of RNA of sample to be detected: collecting pig feces, diluting the feces sample with PBS by vortex oscillation, centrifuging at 8000r/min at 4 deg.C, collecting supernatant, and storing at-80 deg.C.
2.2 reverse transcription reaction conditions: the reaction was carried out at 42 ℃ for 1h, and the resulting cDNA was stored at-20 ℃ for PCR amplification.
2.3 TABLE 2 reverse transcription PCR System
2.4 extraction of cDNA: DNAs of PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 were extracted according to the conventional method and stored at-20 ℃.
Example 3 Single PCR amplification sequence validation
The cDNA of PAstV1, PAstV2, PAstV3, PAstV4 and PAstV5 stored in example 2 was used as a template for single PCR amplification using the five pairs of specific primers designed in example 1. The single PCR amplification system is shown in Table 3. The reaction condition is denaturation at 98 ℃ for 10s; annealing at 55 ℃ for 30s; extending for 1min at 72 ℃;30 cycles. RT-PCR products were detected by 1.5% agarose gel electrophoresis. As shown in FIG. 1, the PCR product was purified to obtain the target gene by a purification kit, and the target gene was sequenced by Biotechnology engineering (Shanghai) Ltd. The sequencing result shows that the nucleotide sequence of the strain is consistent with that of the corresponding strain.
TABLE 3RT-PCR reaction System
| Reagent | 25 μ L reaction System |
| Taraka Ex×Taq | 0.25μL |
| 10×Ex Taq Buffer | 5μL |
| dNTP Mix | 4μL |
| Upstream primer | 0.8μmol/L |
| Downstream primer | 0.8μmol/L |
| Template cDNA | Each 1 mu L |
| ddH 2 O | Make up to 50 μ L |
EXAMPLE 4 multiplex RT-PCR reaction
4.1 multiple RT-PCR reaction system and optimized condition;
under the condition that the reaction conditions are unchanged (denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles), mixing the PAStV1, the PAStV2, the PAStV3, the PAStV4 and the PAStV5 with the same concentration to serve as a multiple RT-PCR template, and optimizing the multiple RT-PCR reaction conditions including primer concentration and a reaction system.
And (3) primer concentration screening: the reaction system used was: 1.0 mu L of each upstream primer and downstream primer; tarakaEx × Taq0.25 μ L;10 XExTaqBuffer 5. Mu.L; 4 μ L of dNTPmix; 2.5. Mu.L each of cDNA templates; sterilized ddH 2 Supplementing O to 50 μ L; 5 different concentration gradients of 0.2. Mu. Mol/L, 0.4. Mu. Mol/L, 0.6. Mu. Mol/L, 0.8. Mu. Mol/L and 1.0. Mu. Mol/L were selected for PCR amplification, and the results of primer concentration screening were shown in FIG. 2.
Annealing stability optimization: the annealing temperature is selected to be 53 ℃; at 54 ℃;55 ℃;56 ℃;5: PCR amplification was performed at 57 ℃ with 5 different gradients and the optimal annealing temperature was chosen, the results are shown in FIG. 3.
4.2 multiplex RT-PCR specificity;
and (3) amplifying the mixed cDNA template of the PAStV1, the PAStV2, the PAStV3, the PAStV4 and the PAStV5 and the cDNA of the positive sample of the PEV, the PRRSV, the SVV, the CSFV, the JEV and the triple live vaccine (PEDV, RV and TGEV) of the pig and the DNA template of the positive sample of the PRV under the condition of establishing a multiple RT-PCR reaction, wherein the amplification result shows (as shown in figure 4) that only the single cDNA of the PAStV1, the PAStV2, the PAStV3, the PAStV4 and the PAStV5 and the mixed cDNA of the PAStV1, the PAStV2, the PAStV3, the PAStV4 and the PASV 5 amplify a band which is in accordance with the size of a target fragment.
4.3 Single/multiplex RT-PCR sensitivity assay
The concentrations of the cDNAs of PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 were respectively adjusted to 1:10ng by a spectrophotometer; 2; 3;4: 0.1pg; 6; 7, PCR amplification was performed using cDNA of each concentration as a template, and the minimum amount detected was detected.
As shown in FIG. 5, 5 virus genotype plasmids were singly subjected to sensitive amplification, the method showed minimum detection amounts of cDNA of PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 of 0.1pg, 1pg and 0.1pg, respectively, as shown in FIG. 6, and 5 virus genotype plasmids were mixed and subjected to sensitive amplification, the method showed minimum detection amounts of cDNA of PAStV1, PAStV2, PAStV3, PAStV4 and PAStV5 of 10.
EXAMPLE 5 preliminary application of clinical samples
The multiplex RT-PCR primer of the invention is used for RT-PCR detection of the following samples, 275 dejecta collected in each pig farm in Guangxi in 2020 is detected.
TABLE 4 clinical sample test results
Astrovirus has been prevalent in many countries around the world since its first discovery, and for a considerable period of time, its clinical symptoms have been considered to be only sporadic, mild diarrhoea, and the pathogenicity of astrovirus has not been appreciated. With the development of detection means, astrovirus is known more deeply, and the astrovirus infection is accompanied by nephritis, hepatitis, nervous symptoms and other phenomena. Further, it has been found that the astrovirus has a cross-species gene recombination phenomenon.
Pork is one of the main meat foods in China, and with the rapid development of intensification, the price of the pork is continuously increased. The distribution range of the breeding industry is wide, the breeding scale is large, and the like, and the astrovirus infectivity is high due to the large-scale breeding mode. Although the cause of porcine diarrhea virus is not only astrovirus, astrovirus is often co-infected with other diarrhea virus and often plays a "culprit" role in diarrhea virus. Moreover, the astrovirus has polygenic mixed infection, so that diarrhea symptoms are aggravated, or nervous or encephalitis symptoms are caused, and economic losses are often caused to farmers. At present, diagnosis and detection aiming at astrovirus mostly stay in the detection level of single RT-PCR or double RT-PCR, and other detection methods such as: direct immunofluorescence tests, electron microscopy, agar immunodiffusion tests are expensive and not suitable for large-scale clinical testing. Therefore, the method for establishing the multiple RT-PCR detection aiming at different genotypes has high efficiency and saves cost.
The invention aims at the mixed infection condition of distinguishing all genotypes of the porcine astrovirus and establishes a multiple RT-PCR detection kit. The detection objects relate to AstV1, PAstV2, PAstV3, PAstV4 and PAstV5. The partial genotypes of the genes have great harm in swinery, the potential public health significance is particularly important, and the genes have important values for detecting epidemic situations and preventing epidemic diseases.
The multiplex RT-PCR established by the invention has high sensitivity, and the minimum detection amount reaches 10pg; and can specifically amplify the target gene; the requirement on the content of the extracted RNA is low, and the reaction system is simple and easy to operate.
In conclusion, the invention provides a detection and identification method which can specifically and sensitively detect the genotypes of the PAStV1, the PAStV2, the PAStV3, the PAStV4 and the PAStV5 and the mixed infection condition, and has important application value in public epidemiological investigation and the like.
The above-described embodiments are merely illustrative of the present invention, and not restrictive, and it should be understood that various changes and modifications in the spirit and scope of the invention may be made by those skilled in the art from the disclosure herein without departing from the spirit and scope of the invention.
Sequence listing
<110> Guangxi university
<120> multiple RT-PCR primer group and kit for detecting porcine astrovirus and application thereof
<130> LWY2021009F
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggccgtggca ggagcagatc 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gactgaggtt taccccgtct 20
<210> 3
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
accaccgcgc aggagg 16
<210> 4
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tgttgytcaa grgcagc 17
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gatgtgatga ccctctatgg g 21
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gccggtcaag catctcatca g 21
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tggggtcctg aagcatttgc 20
<210> 8
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
aatggggacc atccaca 17
<210> 9
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
aatgtgcgkg tgaaaga 17
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tgaaatgtga cttcacctga 20
Claims (6)
1. The multiplex RT-PCR primer group for detecting the porcine astrovirus is characterized by comprising five pairs of specific primers, namely primers PAStV1-F and PAStV1-R, primers PAStV2-F and PAStV2-R, primers PAStV3-F and PAStV3-R, primers PAStV4-F and PAStV4-R and primers PAStV5-F and PAStV5-R, wherein nucleotide sequences of the primers are respectively base sequences shown in a sequence table SEQ ID No.1 to SEQ ID No. 10.
2. A multiplex RT-PCR kit for detecting porcine astrovirus, the kit comprising the multiplex RT-PCR primer set of claim 1.
3. The multiplex RT-PCR kit for detecting porcine astrovirus according to claim 2, wherein the kit further comprises 5 xbuffer, dNTPmix, M-MLV Reverse transcriptase, RNase inhibitor, RNA, taraka Ex xTaq, 10 xExTaq Buffer, dNTPmix, template cDNA and ddH 2 O。
4. Use of the multiplex RT-PCR primer set according to claim 1 for RT-PCR amplification of porcine astrovirus for non-diagnostic purposes.
5. The use of claim 4, wherein the RT-PCR amplification reaction system comprises multiple RT-PCR primer sets of 0.8 μmol/L each, 0.25 μ L of Taraka Ex xTaq, 5 μ L of 10 xEx Taq Buffer, 1 μ L of dNTP Mix4 μ L, cDNA template each, and sterilized ddH 2 The content of O is filled to 50 mu L.
6. The use of claim 4, wherein the RT-PCR amplification reaction conditions are 98 o C modified 10s,55 o C anneal 30s,72 o C extension is 1min,30 cycles.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2021102372206 | 2021-03-03 | ||
| CN202110237220.6A CN112831606A (en) | 2021-03-03 | 2021-03-03 | Multiplex RT-PCR (reverse transcription-polymerase chain reaction) primer group of porcine astrovirus, kit and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113046489A CN113046489A (en) | 2021-06-29 |
| CN113046489B true CN113046489B (en) | 2022-12-06 |
Family
ID=75934488
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110237220.6A Pending CN112831606A (en) | 2021-03-03 | 2021-03-03 | Multiplex RT-PCR (reverse transcription-polymerase chain reaction) primer group of porcine astrovirus, kit and application |
| CN202110550666.4A Active CN113046489B (en) | 2021-03-03 | 2021-05-17 | Multiple RT-PCR primer group for detecting porcine astrovirus, kit and application thereof |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110237220.6A Pending CN112831606A (en) | 2021-03-03 | 2021-03-03 | Multiplex RT-PCR (reverse transcription-polymerase chain reaction) primer group of porcine astrovirus, kit and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN112831606A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113186359B (en) * | 2021-06-21 | 2022-07-15 | 浙江理工大学 | Multiplex PCR rapid diagnosis kit for detecting and parting pig astrovirus |
| CN113388632B (en) * | 2021-06-29 | 2022-06-28 | 广西大学 | Primer and kit for detecting bovine astrovirus ELISA antibody and application of primer and kit |
| CN116574846A (en) * | 2023-05-24 | 2023-08-11 | 河南农业大学 | Real-time fluorescent quantitative PCR reagent and method for detecting porcine astrovirus type 5 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014153168A2 (en) * | 2013-03-14 | 2014-09-25 | The Trustees Of Columbia University In The City Of New York | Porcine astrovirus sequences and uses thereof |
| CN105506182A (en) * | 2016-01-12 | 2016-04-20 | 中国农业科学院兰州兽医研究所 | Quintuple RT-PCR detection kit for porcine viral diarrhea viruses |
| CN109880937A (en) * | 2019-04-24 | 2019-06-14 | 四川大学 | A kind of pig virus diarrhoea multiple RT-PCR detection method |
| CN110512027A (en) * | 2019-08-27 | 2019-11-29 | 南京农业大学 | Five heavy RT-PCR detection methods of pig virus diarrhoea disease cause of disease |
| CN110592278A (en) * | 2019-09-04 | 2019-12-20 | 广西大学 | Multiplex RT-PCR kit for PRoV, PoSaV and PAStV |
| CN113186359A (en) * | 2021-06-21 | 2021-07-30 | 浙江理工大学 | Multiplex PCR rapid diagnosis kit for detecting and parting pig astrovirus |
-
2021
- 2021-03-03 CN CN202110237220.6A patent/CN112831606A/en active Pending
- 2021-05-17 CN CN202110550666.4A patent/CN113046489B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014153168A2 (en) * | 2013-03-14 | 2014-09-25 | The Trustees Of Columbia University In The City Of New York | Porcine astrovirus sequences and uses thereof |
| CN105506182A (en) * | 2016-01-12 | 2016-04-20 | 中国农业科学院兰州兽医研究所 | Quintuple RT-PCR detection kit for porcine viral diarrhea viruses |
| CN109880937A (en) * | 2019-04-24 | 2019-06-14 | 四川大学 | A kind of pig virus diarrhoea multiple RT-PCR detection method |
| CN110512027A (en) * | 2019-08-27 | 2019-11-29 | 南京农业大学 | Five heavy RT-PCR detection methods of pig virus diarrhoea disease cause of disease |
| CN110592278A (en) * | 2019-09-04 | 2019-12-20 | 广西大学 | Multiplex RT-PCR kit for PRoV, PoSaV and PAStV |
| CN113186359A (en) * | 2021-06-21 | 2021-07-30 | 浙江理工大学 | Multiplex PCR rapid diagnosis kit for detecting and parting pig astrovirus |
Non-Patent Citations (6)
| Title |
|---|
| Establishment of a Multiplex RT-PCR Method for the Detection of Five Known Genotypes of Porcine Astroviruses;Xin Liu等;《frontiers in Veterinary Science》;20210615;第9卷;第1-9页 * |
| Identification and characterization of multiple porcine astrovirus;Chao-Ting Xiao等;《Archives of virology》;20161218;第162卷(第4期);第943左栏第10-13排、第944页右栏倒数第11排至第945页左栏倒数第5排，表1、2，图1 * |
| Molecular epidemiology and viremia of porcine astrovirus in pigs from Guangxi province of China;Yifeng Qin等;《BMC Veterinary Research》;20191227;第15卷(第1期);第1-9页 * |
| 猪星状病毒3 型TaqMan荧光定量PCR方法的建立及初步应用;刘心等;《中国兽医科学》;20201013;第50卷(第11期);第1374页左栏倒数第10排至倒数第7排、右栏倒数第13排至倒数第7排、第1375页左栏倒数第4排至右栏最后一排，表1、图1-4 * |
| 猪星状病毒分离鉴定及其全基因组序列分析和衣壳蛋白原核表达;刘欢;《中国优秀硕士学位论文全文数据库农业科技辑》;20150215;第2015卷(第2期);D050-301页 * |
| 猪星状病毒实验室检测技术研究进展;庄金秋等;《养猪》;20211010;第2021卷(第5期);第98-100页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112831606A (en) | 2021-05-25 |
| CN113046489A (en) | 2021-06-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113046489B (en) | Multiple RT-PCR primer group for detecting porcine astrovirus, kit and application thereof | |
| CN110760620A (en) | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method | |
| Sun et al. | Sensitive and rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimps by loop-mediated isothermal amplification | |
| CN111020062A (en) | Triple real-time fluorescent quantitative PCR kit for detecting African swine fever wild strain and gene deletion strain | |
| CN108676920B (en) | Primer and kit for rapidly detecting mouse norovirus and RT-RPA method thereof | |
| CN114134252B (en) | Primers and kit for detecting coronavirus | |
| CN115044710B (en) | Primer group and kit for detecting pangolin beta coronavirus and application of primer group and kit | |
| CN113403430A (en) | Triple fluorescent quantitative PCR primer group for detecting different types of porcine circovirus, kit and application | |
| CN113174446A (en) | One-step double RT-PCR detection method for bovine viral diarrhea virus typing | |
| 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 | |
| CN111118215A (en) | Fluorescent PCR (polymerase chain reaction) primer, probe and kit for detecting capripoxvirus virus | |
| Tong et al. | Rapid detection of Decapod iridescent virus 1 (DIV1) by recombinase polymerase amplification | |
| CN117025846A (en) | Primer group for detecting novel coronavirus by multiple ddPCR and application thereof | |
| Zhang et al. | Rapid detection of pigeon Megrivirus using TaqMan real-time PCR technology | |
| CN112280899A (en) | Porcine astrovirus type 2 TaqMan fluorescent quantitative PCR kit and application thereof | |
| CN113046476A (en) | Primer composition and kit for rapidly detecting N501Y mutation of novel coronavirus | |
| CN113355460B (en) | Primer and kit for detecting novel goose reovirus, and detection method and application thereof | |
| CN116144834A (en) | African swine fever virus identification and detection method | |
| CN114058740A (en) | Fluorescent PCR amplification primer probe set capable of identifying genotype I and genotype II African swine fever viruses and detection kit | |
| CN111996294A (en) | Primer pair and kit for quantitatively detecting eel herpesvirus | |
| CN111961757A (en) | Double-gene probe method real-time fluorescence quantitative PCR kit for detecting duck tembusu virus and application | |
| CN111500774A (en) | Epidemic hemorrhagic disease virus and serotype identification RT-PCR kit | |
| KR20080111785A (en) | Primers and probes of polymerase chain reactions for the detection of water-borne viruses, and detection kits and methods thereof | |
| CN111647683A (en) | New coronavirus 2019-nCoV nucleic acid detection kit and application thereof |
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 |