CN103060474A - Primers for detecting variants on porcine epidemic diarrhea virus and detection kit thereof - Google Patents
Primers for detecting variants on porcine epidemic diarrhea virus and detection kit thereof Download PDFInfo
- Publication number
- CN103060474A CN103060474A CN2013100125591A CN201310012559A CN103060474A CN 103060474 A CN103060474 A CN 103060474A CN 2013100125591 A CN2013100125591 A CN 2013100125591A CN 201310012559 A CN201310012559 A CN 201310012559A CN 103060474 A CN103060474 A CN 103060474A
- Authority
- CN
- China
- Prior art keywords
- diarrhea virus
- epidemic diarrhea
- porcine epidemic
- primer
- 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.)
- Pending
Links
Images
Abstract
The invention provides primers for detecting variants on porcine epidemic diarrhea virus and a detection kit thereof, which belongs to the technical field of biotechnology. The primers include a forward primer PEDV-VF(SEQ ID No.1) which is 5'-TTGGTGAAAACCAGGGTFTC-3' and a reverse primer PEDV-VR(SEQ ID N0.2) which is 5'-CAACACTATGTTCACTCA-3'. The invention further provides the detection kit for detecting the variants on the porcine epidemic diarrhea virus. The detection method comprises the following steps: adopting total RNA as a template, adopting the former primers to carry out inverse transcription PCR(RT-PCR) amplification, and judging according to the positions of the amplified fragments after the reaction is ended. The primer provided by the invention has good specificity. The detection method is simple and quick, has high accuracy, and provides a guarantee for detection of the variants on the porcine epidemic diarrhea virus.
Description
Technical field
The present invention relates to the Porcine epidemic diarrhea virus variant detects with primer and detection kit thereof.Belong to technical field of biological.
Background technology
Porcine epizootic diarrhea (Porcine Epidemic Diarrhea, PED) be by Porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus, PEDV) cause a kind of acute, the height contact transmissible disease, often take acute enteritis and with the watery diarrhea of dehydration as feature, the equal susceptible of the pig at various ages.This disease at first was found in Britain in 1971, after this, Europe and countries in Asia also reported should disease break out.China was separated to the PEDV(classical strains in 1973).
Year spring winter to 2011 in 2010 pig farm in all parts of the country swinery in serious infectious diarrhea disease successively occurs, morbidity is propagated very fast suddenly, Epidemic Scope is wide, and the popular time is long, uses the control of various microbiotic invalid, its incidence and mortality is very high, causes great financial loss.Show after deliberation, the Porcine epidemic diarrhea virus variant is arch-criminal (the Complete Genome Sequence of a Porcine Epidemic Diarrhea Virus Variant. J. Virol such as Jianfei Chen of this epidemic situation, 2012,86:3408).Porcine epidemic diarrhea virus variant (the Complete Genome Sequence of a Virulent Porcine Epidemic Diarrhea Virus Strain. J. Virol such as Yanjun Zhou, 2012,86:13862) with original strain PEDV(classical strains) (the Complete Genome Sequence of a Chinese Virulent Porcine Epidemic Diarrhea Virus Strain. J. Virol such as Jianfei Chen, 2012, genomic homology 86:3408) is about 97%, what variation was maximum is the S gene, homology is below 94%, and sequence signature (the New variants of porcine epidemic diarrhea virus such as Wentao Li that has amino acid generation deletion and insertion, China, 2011. Emerg. Infect. Dis, 2011,85:11538).
Before this, China there is no the specific detection technology of PEDV variant (called after PEDV-V).The conventional RT-PCR technology can not be distinguished the PEDV(classical strains) and the PEDV-V(PEDV variant), therefore can not detect specifically the PEDV variant.Nowadays, can only determine this Viral infection by gene sequencing to the detection of Porcine epidemic diarrhea virus variant, trivial operations, length consuming time, cost are higher.
Therefore, based on above research and current demand, the applicant is according to the feature of Porcine epidemic diarrhea virus variant S gene generation nucleotide deletion and insertion, designed and synthesized the primer of a pair of detection Porcine epidemic diarrhea virus variant, adopt RT-PCR method amplification S gene, by the molecular size range of amplified fragments, judge whether to exist the Porcine epidemic diarrhea virus variant, thereby special, responsive, save time, laborsaving, detect the Porcine epidemic diarrhea virus variant at low cost.Other RT-PCR methods can not address this problem at present; The gene sequencing Technology Need time is longer, and time-consuming, effort, cost are high.Therefore, this technology can play an important role to diagnosis and the monitoring of Porcine epidemic diarrhea virus variant.
Summary of the invention
Technical problem
The object of the invention is to be provided for primer sequence and the detection kit thereof that the PEDV variation strain RT-PCR detects.
Technical scheme
The present invention is by analyzing the S gene order of PEDV variant, at the sequence place design primer that S gene generation Nucleotide inserts and lacks.Described primer pair is comprised of forward and reverse primer, and its nucleotide sequence is shown in SEQ ID NO. 1 and SEQ ID NO. 2 in the sequence table.
Particularly, the invention provides a kind of PEDV variant detection and use primer, the nucleotides sequence of the forward primer of this primer (PEDV-VF) sequence SEQ ID NO.1 and reverse primer (PEDV-VR) sequence SEQ ID NO.2 is classified as:
SEQ ID NO.1：5’-TTGGTGAAAACCAGGGTGTC-3’
SEQ ID NO.2：5’-CAACACTATGTTCACTCA-3’。This primer amplifies 327 bp dna fragmentations from total RNA of the pathological material of disease extraction of infected pigs's epidemic diarrhea virus variant.
The present invention also provides a kind of test kit for detection of the Porcine epidemic diarrhea virus variant, and this test kit contains above-mentioned primer SEQ ID NO.1 and SEQ ID NO.2.
Concrete, this test kit comprises:
(1) negative control:Get the DEPC water of sterilization, put-20 ℃ of Refrigerator stores;
(2) RT reaction solution:
Aseptic DEPC water 4.75 μ L
10μmol/L dNTP 1μL
20μmol/L PEDV-VR 1μL
5 * reverse transcription damping fluid, 4 μ L
40 U/ μ L RNA enzyme inhibitorss, 1 μ L
0.1 mmol/L DTT 2μL
200U/ μ L ThermoScript II 0.25 μ L
-20 ℃ of preservations behind the mixing;
(3) PCR reaction solution:
Aseptic DEPC water 32.5 μ L
20 μ mol/LPCR primer mixed solutions, 2 μ L
10 * PCR damping fluid, 5 μ L
2.5 mmol/L dNTP 8μL
5U/ μ L archaeal dna polymerase 0.5 μ L
-20 ℃ of preservations behind the mixing.
Described test kit comprises that also positive control is the faecal samples of Porcine epidemic diarrhea virus variant JS-HZ2012 diarrhea.
Beneficial effect
The present invention is the primer according to the design of Porcine epidemic diarrhea virus variant S gene order, primer of the present invention is to compare and screen through the S gene order of a large amount of PEDV to obtain, and whether the detection kit of using among this primer and the present invention contain the Porcine epidemic diarrhea virus variant in the judgement sample rapidly, for the dysentery of pig provides testing tool.
Simultaneously, advanced the comparison of a large amount of clinical detection and sequencing analysis, confirm that all detection primer of the present invention and detection kit can detect the popular Porcine epidemic diarrhea virus variant of present China accurately, and can not detect the Porcine epidemic diarrhea virus classical strains, can reach the purpose of distinguishing variant and classical strain, and detection method can't be distinguished variant and classical strain in the past.Primer of the present invention and detection kit can provide effective instrument for detecting clinically the diarrhea of pigs disease.
Description of drawings
Fig. 1 is the RT-PCR detected result of Porcine epidemic diarrhea virus variant (PEDV-V), wherein M:DL2000 DNA ladder; 1, Porcine epidemic diarrhea virus variant; 2, Porcine epidemic diarrhea virus classical strains (PEDV); 3, transmissible gastro-enteritis virus (TGVE); 4, porcine rotavirus (PRV); 5, porcine reproductive and respiratory syndrome virus (PRRSV); 6, negative control.
Fig. 2 is the sensitivity experiment result of detection method of the present invention.M:DL2000 DNA ladder wherein; 1-11 represents the RNA template of continuous 10 times of dilutions of adding in the reverse transcription reaction system; 12, negative control.
Embodiment
Following embodiment is used for further specifying of the present invention, but is not used for limiting the scope of the invention.
1, the design of primer and synthetic
According to the nucleotide sequence of Porcine epidemic diarrhea virus variant S gene, adopt Primer 5.0 design primers, obtain the primer sequence of following sequence through a large amount of screenings:
The sequence of forward primer (PEDV-VF) is SEQ ID NO.1, and reverse primer (PEDV-VR) sequence is SEQ ID NO.2, and specifically sequence is composed as follows:
SEQ ID NO.1：5’- TTGGTGAAAACCAGGGTGTC -3’
SEQ ID NO.2：5’- CAACACTATGTTCACTCA-3’。
According to the synthetic forward primer PEDV-VF of above-mentioned nucleotide sequence information and reverse primer PEDV-VR.Above-mentioned primer PEDV-VF and PEDV-VR are configured to respectively the solution that concentration is 20 μ mol/L, for subsequent use.
2, a kind of test kit that detects the Porcine epidemic diarrhea virus variant, the according to the form below assembling:
Table 1 test kit forms
| Composition (10 parts/box) | Content |
| |
1 pipe |
| |
1 pipe |
| |
1 pipe |
| The |
1 pipe |
|
The |
1 pipe |
| Detect primer (forward primer, reverse primer) | Each 1 |
| Working instructions | |
| 1 part |
3, the preparation of negative control sample
Get the DEPC water of sterilization, put-20 ℃ of Refrigerator stores.
4, the preparation of positive control sample
This laboratory is carried out the RT-PCR detection from the faecal samples (coming from pig farm, Hongze, Jiangsu Province) of the diarrhea collected clinically with primer of the present invention, special amplified band (327 bp) appears, and by gene clone, determining nucleic acid sequence and analysis, determine its make a variation regional S gene and Porcine epidemic diarrhea virus variant (CH/FJND-3/2011, the PEDV variation strains such as ZJCZ4) (the Complete Genome Sequence of a Porcine Epidemic Diarrhea Virus Variant. J. Virol such as Jianfei Chen, 2012,86:3408; The Complete Genome Sequence of a Virulent Porcine Epidemic Diarrhea Virus Strain. J. Virol such as Yanjun Zhou, 2012,86:13862) the S gene homology is 100%, confirm that this sample is that the Porcine epidemic diarrhea virus variant is positive, called after JS-HZ2012(GenBank accession number: KC210-147).Put-20 ℃ of Refrigerator stores.
5, the preparation of RT reaction solution
Prepare the RT reaction solution by following prescription
Aseptic DEPC water 4.75 μ L
10μmol/L dNTP 1μL
20μmol/L PEDV-VR 1μL
5 * reverse transcription damping fluid, 4 μ L
40 U/ μ L RNA enzyme inhibitorss, 1 μ L
0.1 mmol/L DTT 2μL
200U/ μ L ThermoScript II 0.25 μ L
-20 ℃ of preservations behind the mixing, after the assay was approved packing is used for the finished product test kit.
6, the preparation of PCR reaction solution
Prepare the PCR reaction solution by following prescription
Aseptic DEPC water 32.5 μ L
20 μ mol/LPCR primer mixed solutions, 2 μ L
10 * PCR damping fluid, 5 μ L
2.5 mmol/L dNTP 8μL
5U/ μ L archaeal dna polymerase 0.5 μ L
-20 ℃ of preservations behind the mixing, after the assay was approved packing is used for the finished product test kit.
7, detect the method for Porcine epidemic diarrhea virus variant, comprising:
1) sample preparation:Tissue sample: gather enteron aisle and the faecal samples of 46 the doubtful porcine epizootic diarrheas morbidity pigs in East China, take by weighing the 0.5g of organizing to be checked and put and shred in the mill and grind, add 1.5mL PBS and continue to grind.Get ground tissue juice to be checked, put in the 1.5mL sterilization centrifuge tube, centrifugal 5 min of 8000g get supernatant 100 μ L, put in the 1.5mL sterilization centrifuge tube stand-by.
Positive control sample: get positive control sample 100 μ L, put in the 1.5mL sterilization centrifuge tube stand-by.
Negative control sample: get negative control sample 100 μ L, put in the 1.5mL sterilization centrifuge tube stand-by.
2) virus is extracted RNA:Get the Trizol reagent that then sample to be checked, positive control sample, the negative control sample processed add 1ml, concuss shakes up; Keep 5 min under the room temperature, add the 0.2ml chloroform, concuss 15 s, then at room temperature keep 15 min after, 4 ℃, centrifugal 15 min of 12000 g; The upper water phase transition in new 1.5ml centrifuge tube, is added the 0.5ml Virahol, put upside down mixing, keep 15 min under the room temperature; 4 ℃, centrifugal 10 min of 12000 g, RNA will form precipitation in sidewall and the bottom of pipe; Outwell supernatant liquor, add 75% washing with alcohol precipitation, then 4 ℃, centrifugal 5 min(of 7500 g suspend such as precipitation, then use 12000 g), discard ethanol; After being deposited under the room temperature fully drying, be dissolved in 40 μ LdH
2O(DEPC processes) in ,-20 ℃ save backup.
3) RT reaction:In 20 μ L reaction systems, add 4.75 μ LDEPC and process water, the total RNA of 6 μ L, 1 μ L PEDV-VR(20 μ mol/L), 1 μ L dNTP(10 μ mol/L), in 65 ℃ of maintenance 5 min, transfer to rapidly 5 min of quenching on ice behind the mixing; Then in reaction tubes, add 2 μ L DTT(0.1 mmol/L), 4 μ L, 5 * reverse transcription damping fluid, 1 μ LRNA enzyme inhibitors (40 U/ μ L), 0.25 μ L ThermoScript II (200U/ μ L), 42 ℃ of reaction 50 min; Obtain reverse transcription product cDNA after 72 ℃ of 15 min deactivation ThermoScript II.
4) pcr amplification
In the reaction system of 50 μ L, add 30.5 μ LDEPC and process water, 2 μ L cDNA, 2 μ L PEDV-VF(20 μ mol/L), 2 μ L PEDV-VR(20 μ mol/L), 10 * PCR damping fluid, 5 μ L, 8 μ L dNTP(2.5 mmol/L), 0.5 μ L archaeal dna polymerase (5U/ μ L).The reaction parameter of PCR is: 94 ℃, and 5min; 94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 1min, totally 35 circulations; 72 ℃ are extended 5min.
5), judge the method for Porcine epidemic diarrhea virus variant
An amplified fragments (327 bp) identical with the positive control size appears in the test sample electrophoresis, is judged to be the Porcine epidemic diarrhea virus variant positive;
The test sample electrophoresis amplified fragments different from positive control size (327 bp) occur or amplified fragments do not occur, is judged to be the Porcine epidemic diarrhea virus variant negative.
6) result
After testing, it is positive that the enteron aisle of 46 the doubtful porcine epizootic diarrhea morbidity pigs in East China and faecal samples are the Porcine epidemic diarrhea virus variant, and the amplified fragments in the same size of its RT-PCR amplified production and positive control is 372 bp.
8, specificity experiment
Detect other viruses that may cause diarrhea of pigs (Complete Genome Sequence of a Highly Prevalent Porcine Circovirus 2 Isolated from Piglet Stool Samples in China. J. Virol such as Porcine epidemic diarrhea virus classical strains, TGEV, PRV, PRRSV(Bin Li with Porcine epidemic diarrhea virus variation strain RT-PCR detection kit, 2012,86:4716) with the health pig sample.The result shows, may cause that with other of Porcine epidemic diarrhea virus variation strain RT-PCR detection kit detection of the present invention virus of diarrhea of pigs, health pig sample are all negative, the results are shown in accompanying drawing 1.The Porcine epidemic diarrhea virus variation strain RT-PCR detection kit that shows foundation has good specificity, can accurately judge whether there is the Porcine epidemic diarrhea virus variant in the sample.
9, sensitivity test
In the reverse transcription reaction system, add total RNA of different concns, carry out sensitivity test.Particularly, in 20 μ L reverse transcription systems, add the total RNA after the continuous 10 times of dilutions of 1 μ L.According to method of the present invention, the cDNA that adds 2 μ L in the PCR reaction system, utilize SEQ ID NO.1 and SEQ ID NO.2 to increase, the result as shown in Figure 2, utilize the inventive method, Porcine epidemic diarrhea virus variant positive can detect 327 bpDNA fragments, and the inventive method is highly sensitive for detection of the Porcine epidemic diarrhea virus variant, minimum detection quantity is 82fg/ μ L sample RNA, and effect is very good.
SEQUENCE LISTING
＜110〉Jiangsu Province Agriculture Science Institute
＜120〉the Porcine epidemic diarrhea virus variant detects with primer and detection kit thereof
<130> 0
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 20
<212> DNA
＜213〉artificial
<220>
<221> PEDV-VF
<222> (1)..(20)
<223>
<400> 1
ttggtgaaaa ccagggtgtc 20
<210> 2
<211> 18
<212> DNA
＜213〉artificial
<220>
<221> PEDV-VR
<222> (1)..(18)
<223>
<400> 2
caacactatg ttcactca 18
Claims (4)
1. the Porcine epidemic diarrhea virus variant detects the primer of usefulness, comprises
Forward primer PEDV-VF(SEQ ID NO.1): 5 '-TTGGTGAAAACCAGGGTGTC-3 '
With reverse primer PEDV-VR(SEQ ID NO.2): 5 '-CAACACTATGTTCACTCA-3 ';
This primer amplifies 327 bp dna fragmentations from total RNA of the pathological material of disease extraction of infected pigs's epidemic diarrhea virus variant.
2. test kit for detection of the Porcine epidemic diarrhea virus variant, this test kit contains primer claimed in claim 1.
, test kit as claimed in claim 2, it is characterized in that, comprising:
(1) negative control:Get the DEPC water of sterilization, put-20 ℃ of Refrigerator stores;
(2) RT reaction solution:
Aseptic DEPC water 4.75 μ L
10μmol/L dNTP 1μL
20μmol/L PEDV-VR 1μL
5 * reverse transcription damping fluid, 4 μ L
40 U/ μ L RNA enzyme inhibitorss, 1 μ L
0.1 mmol/L DTT 2μL
200U/ μ L ThermoScript II 0.25 μ L
-20 ℃ of preservations behind the mixing;
(3) PCR reaction solution:
Aseptic DEPC water 32.5 μ L
20 μ mol/LPCR primer mixed solutions, 2 μ L
10 * PCR damping fluid, 5 μ L
2.5 mmol/L dNTP 8μL
5U/ μ L archaeal dna polymerase 0.5 μ L
-20 ℃ of preservations behind the mixing.
4. test kit as claimed in claim 2 or claim 3, also comprise: positive control is the faecal samples of Porcine epidemic diarrhea virus variant JS-HZ2012 diarrhea.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100125591A CN103060474A (en) | 2013-01-07 | 2013-01-07 | Primers for detecting variants on porcine epidemic diarrhea virus and detection kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100125591A CN103060474A (en) | 2013-01-07 | 2013-01-07 | Primers for detecting variants on porcine epidemic diarrhea virus and detection kit thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103060474A true CN103060474A (en) | 2013-04-24 |
Family
ID=48103365
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013100125591A Pending CN103060474A (en) | 2013-01-07 | 2013-01-07 | Primers for detecting variants on porcine epidemic diarrhea virus and detection kit thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103060474A (en) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320452A (en) * | 2013-07-08 | 2013-09-25 | 江苏省农业科学院 | Amplification method of N gene complete sequence of PEDV (Porcine Epidemic Diarrhea Virus) variant |
| CN103509882A (en) * | 2013-06-28 | 2014-01-15 | 广东温氏食品集团股份有限公司 | Fluorescent quantitative PCR primers and probes for porcine epidemic diarrhea viruses |
| CN103667531A (en) * | 2013-12-06 | 2014-03-26 | 湖北省农业科学院畜牧兽医研究所 | FQ-PCR detection method for swine epidemic diarrhea virus (PEDV) and primer pair used therein |
| CN104152578A (en) * | 2013-06-03 | 2014-11-19 | 中国农业科学院上海兽医研究所 | RT-PCR kit for identification and diagnosis of porcine epidemic diarrhea virus and application of RT-PCR kit |
| CN104531902A (en) * | 2014-12-31 | 2015-04-22 | 洛阳普莱柯万泰生物技术有限公司 | Kit and manufacturing method thereof |
| CN105695635A (en) * | 2016-03-31 | 2016-06-22 | 广西壮族自治区兽医研究所 | Multiplex RT-PCR (reverse transcription-polymerase chain reaction) detection kit for porcine epidemic diarrhea virus |
| CN107475450A (en) * | 2017-09-13 | 2017-12-15 | 河南省农业科学院畜牧兽医研究所 | A kind of primer sets and kit for the strain of Porcine epidemic diarrhea virus novel variant and vaccine strain antidiastole |
| 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 |
| CN108441579A (en) * | 2017-12-15 | 2018-08-24 | 广州市瑞品生物技术有限公司 | The real-time fluorescent RT-PCR method for detecting and kit of Porcine epidemic diarrhea virus |
| CN109666766A (en) * | 2019-01-30 | 2019-04-23 | 珠海出入境检验检疫局检验检疫技术中心 | Fluorescence RT-PCR primer sets, kit and the method for SADS-CoV |
| 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 |
| CN112941241A (en) * | 2021-04-16 | 2021-06-11 | 武汉科前生物股份有限公司 | Method for identifying porcine epidemic diarrhea virus type 2a and type 2b |
| CN114317815A (en) * | 2021-09-30 | 2022-04-12 | 江苏蓬祥畜禽生态养殖有限公司 | PCR method for amplifying N-terminal hypervariable region of S gene of porcine epidemic diarrhea virus |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851301A (en) * | 2012-07-06 | 2013-01-02 | 广东大华农动物保健品股份有限公司 | Pig epidemic diarrhea virus M gene full sequence amplification method |
-
2013
- 2013-01-07 CN CN2013100125591A patent/CN103060474A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851301A (en) * | 2012-07-06 | 2013-01-02 | 广东大华农动物保健品股份有限公司 | Pig epidemic diarrhea virus M gene full sequence amplification method |
Non-Patent Citations (4)
| Title |
|---|
| LI ET AL: "New Variants of Porcine Epidemic Diarrhea Virus,China,2011", 《EMERGING INFECTIOUS DISEASES》, vol. 18, no. 8, 31 August 2012 (2012-08-31) * |
| PAN ET AL: "Isolation and characterization of a variant porcine epidemic diarrhea virus in China", 《VIROLOGY JOURNAL》, vol. 195, no. 9, 12 September 2012 (2012-09-12) * |
| 王树坤等: "猪流行性腹泻病毒S基因片段的克隆及原核表达", 《生物技术进展》, vol. 2, no. 4, 31 December 2012 (2012-12-31) * |
| 陈建飞: "猪流行性腹泻病毒全基因组序列分析及感染性cDNA克隆的构建", 《中国博士学位论文全文数据库 农业科技辑》, no. 10, 15 October 2012 (2012-10-15) * |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152578A (en) * | 2013-06-03 | 2014-11-19 | 中国农业科学院上海兽医研究所 | RT-PCR kit for identification and diagnosis of porcine epidemic diarrhea virus and application of RT-PCR kit |
| CN104152578B (en) * | 2013-06-03 | 2016-03-30 | 中国农业科学院上海兽医研究所 | Porcine epidemic diarrhea virus RT-PCR differential diagnosis kit and application thereof |
| CN103509882A (en) * | 2013-06-28 | 2014-01-15 | 广东温氏食品集团股份有限公司 | Fluorescent quantitative PCR primers and probes for porcine epidemic diarrhea viruses |
| CN103509882B (en) * | 2013-06-28 | 2015-09-23 | 广东温氏食品集团股份有限公司 | The fluorescence quantification PCR primer of Porcine epidemic diarrhea virus and probe |
| CN103320452A (en) * | 2013-07-08 | 2013-09-25 | 江苏省农业科学院 | Amplification method of N gene complete sequence of PEDV (Porcine Epidemic Diarrhea Virus) variant |
| CN103667531A (en) * | 2013-12-06 | 2014-03-26 | 湖北省农业科学院畜牧兽医研究所 | FQ-PCR detection method for swine epidemic diarrhea virus (PEDV) and primer pair used therein |
| CN104531902A (en) * | 2014-12-31 | 2015-04-22 | 洛阳普莱柯万泰生物技术有限公司 | Kit and manufacturing method thereof |
| CN105695635A (en) * | 2016-03-31 | 2016-06-22 | 广西壮族自治区兽医研究所 | Multiplex RT-PCR (reverse transcription-polymerase chain reaction) detection kit for porcine epidemic diarrhea virus |
| CN107475450A (en) * | 2017-09-13 | 2017-12-15 | 河南省农业科学院畜牧兽医研究所 | A kind of primer sets and kit for the strain of Porcine epidemic diarrhea virus novel variant and vaccine strain antidiastole |
| 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 |
| CN108441579A (en) * | 2017-12-15 | 2018-08-24 | 广州市瑞品生物技术有限公司 | The real-time fluorescent RT-PCR method for detecting and kit of Porcine epidemic diarrhea virus |
| CN109666766A (en) * | 2019-01-30 | 2019-04-23 | 珠海出入境检验检疫局检验检疫技术中心 | Fluorescence RT-PCR primer sets, kit and the method for SADS-CoV |
| 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 |
| CN112941241A (en) * | 2021-04-16 | 2021-06-11 | 武汉科前生物股份有限公司 | Method for identifying porcine epidemic diarrhea virus type 2a and type 2b |
| CN114317815A (en) * | 2021-09-30 | 2022-04-12 | 江苏蓬祥畜禽生态养殖有限公司 | PCR method for amplifying N-terminal hypervariable region of S gene of porcine epidemic diarrhea virus |
| CN114317815B (en) * | 2021-09-30 | 2023-10-20 | 江苏蓬祥畜禽生态养殖有限公司 | PCR method for amplifying N-terminal hypervariable region of porcine epidemic diarrhea virus S gene |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103060474A (en) | Primers for detecting variants on porcine epidemic diarrhea virus and detection kit thereof | |
| CN101736094B (en) | Variant porcine reproductive and respiratory syndrome virus (PRRSV) TaqMan fluorescence quantitative RT-PCR detecting kit and application thereof | |
| CN103509882B (en) | The fluorescence quantification PCR primer of Porcine epidemic diarrhea virus and probe | |
| CN105483291B (en) | Pig Delta coronavirus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus multiple RT-PCR detection primer | |
| CN103255229B (en) | One tube PCR type kit for discriminating and diagnosing goose parvovirus and Muscovy duck parvovirus | |
| CN102618667B (en) | Method for detecting five types of potato viruses through polyfunctional real-time fluorescent PCR (polymerase chain reaction) | |
| CN103243179B (en) | Shell-type PCR (polymerase chain reaction) amplification primer of porcine epidemic diarrhea virus and application thereof | |
| CN110229932A (en) | African swine fever virus nucleic acid is hands-free to take fluorescence isothermal amplification detection kit | |
| CN107557494A (en) | Differentiate the primer of detection diarrhea of pigs coronavirus and multiple RT PCR methods | |
| CN104498623B (en) | Primers for differential diagnosis of porcine epidemic diarrhea virus virulent strain and attenuated strain and detection kit thereof | |
| CN104561368A (en) | Multiple real-time fluorescent quantative PCR rapid diagnosis kit for six porcine viruses | |
| CN105506182A (en) | Quintuple RT-PCR detection kit for porcine viral diarrhea viruses | |
| CN101294223B (en) | Primer and probe sequence for testing type II pig circular ring virus nucleotide fragment | |
| CN105624329A (en) | Real-time fluorescence nucleic acid isothermal amplification detection kit for human herpesvirus 1 | |
| CN105400906A (en) | Triple PCR primer set capable of simultaneously detecting porcine circovirus II, porcine pseudorabies virus and porcine parvovirus and application thereof | |
| CN102212617B (en) | Primer pair, probe and kit for detecting classical swine fever virus wild strain | |
| CN104099428B (en) | A kind of triple real-time fluorescence PCR primers, probe and test kit differentiating Ranavirus virus | |
| CN103725793A (en) | Multiple fluorescent quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) and application thereof | |
| CN102888471B (en) | Primer for detecting porcine circovirus P1 by using SYBR GreenI fluorescent quantitative polymerase chain reaction (PCR) | |
| CN105002298A (en) | Fluorescent quantitative PCR detection method of spring viraemia of carp virus | |
| CN103509880A (en) | LAMP detection kit of highly-pathogenic porcine reproductive and respiratory syndrome viruses | |
| CN105695635A (en) | Multiplex RT-PCR (reverse transcription-polymerase chain reaction) detection kit for porcine epidemic diarrhea virus | |
| CN103205507A (en) | RT-PCR primers and kit for detecting high-pathopoiesia porcine reproductive andrespiratory syndrome and classic porcine reproductive andrespiratory syndrome | |
| CN102653798A (en) | Fluorescent PCR (polymerase chain reaction) detection method for identifying traditional strain and highly pathogenic strain of PRRSV (porcine reproductive and respiratory syndrome virus) | |
| CN102634605B (en) | Method for detecting egg drop syndrome viruses and kit for method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130424 |