CN101586169A - Porcine circovirus 2 LAMP detection kit and detecting method - Google Patents
Porcine circovirus 2 LAMP detection kit and detecting method Download PDFInfo
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- CN101586169A CN101586169A CNA2009100875623A CN200910087562A CN101586169A CN 101586169 A CN101586169 A CN 101586169A CN A2009100875623 A CNA2009100875623 A CN A2009100875623A CN 200910087562 A CN200910087562 A CN 200910087562A CN 101586169 A CN101586169 A CN 101586169A
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Abstract
The invention relates to a porcine circovirus 2 LAMP detection kit and detecting method. Based on the PCV2 gene sequence disclosed by GenBank, four PCV2 LAMP primers are designed in the sequence conservative region; the set PCV2 LAMPreaction system is adopted to conduct the LAMP reaction by taking the PCV2 HuB strain, PCV2LN strain virus DNA as a formwork. the LA-320 LAMP Tubidimeter is used for analyzing the added SYBRgreenI developer in the reaction process and after the reaction to judge the result, the result shows that the PCV2DNA has high-efficiency specificity amplification in LA-320 LAMP Tubidmeter at 63 DEG for 30 min, the SYBRgreenI developer is added to judge the result is consistent with the result displayed by the instrumental analysis. the sensitivity test proves that in the method, the 50ngPCV2 DNA formwork is diluted by 10<-7>, the efficient amplification still can be conducted, thus the method has high susceptibility. It shows that the method is special, simple and rapid, and is suitable for the PCV2 detecting work by the specificity test and the LAMP detecting of PVC2nucleic acid clinical sample.
Description
Technical field
The present invention relates to porcine circovirus 2 type LAMP detection kit and detection method thereof, belong to the eqpidemic disease diagnostic techniques in the veterinary biologics field.
Background technology
(Porcine circo virus PCV), is to find at present minimum virus to be divided into two hypotypes of PCV1 and PCV2 to pig circular ring virus.Wherein PCV1 does not cause morbidity, the PCV2 type can cause multisystem asthenia syndrome (Postweaning Multisystemic Wasting Syndrome behind the weaned piglet, PMWS), the only upward discovery polyinfection of pig of being everlasting and suffering from the porcine reproductive and respiratory syndrome that causes by PRRSV.Infected pigs can discharge virus in movement, through the oral cavity, the respiratory tract approach infects the pig of different ages.Farrowing sow infects piglet through the placenta vertical transmission.Owing to do not have effective medicine at present, so the control of this disease is still to put prevention first.
PCV2 does not produce CPE in viral separation and Culture, so this method of simple use is difficult to judge whether cause of disease exists.The normal ELASA of use method is measured antibody and PCR method mensuration antigen in the present testing.But all can't overcome determining that the ELASA susceptibility is low, PCR is prone to false positive results at present.
The isothermal amplification (LAMP) of ring mediation is a kind of novel nucleic acids amplification technique (Notomi by inventions such as T.Notomi, T., et al., Loop-mediated isothermal amplification of DNA.Nucleic Acids Res.2000,28, e63.), this technology relies on primer and a kind of archaeal dna polymerase with strand displacement characteristic of 4 special designs, can be efficiently under isothermal condition, high amplified target sequence specifically.In recent years, this technology is widely used in pathogen detection abroad.People (2007) such as Masaki Imai at four kinds of cordiale zymic ITS sequences Design the LAMP Auele Specific Primer, set up LAMP detection architecture efficiently.LAMP can also detect other and human relevant virus, as viral hemorrhagic septicemia (VHS), cytomegalovirus (CMV), Ebola virus (EBOV), chronic burkitt's lymphoma virus (EBV), rainbow virus, human herpes virus type 8, hematopoietic tissue necrosis virus (IHHNV), tomato spotted wilf virus, tomato yellow leaf curl virus etc.There is no both at home and abroad at present and be useful on LAMP detection kit and the application of this method in porcine circovirus 2 type detects that detects porcine circovirus 2 type.
Summary of the invention
The objective of the invention is to adopt the isothermal amplification (LAMP) of ring mediation to set up the LAMP detection method that detects porcine circovirus 2 type, and a kind of porcine circovirus 2 type LAMP detection kit that is used for above purpose is provided.
The present invention utilizes loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification LAMP) sets up detection method at porcine circovirus 2 type.Present method has many primers and increases simultaneously, and formed the ring texture that has the primer function at two ends, this many primers combination and the principle that can produce primer certainly make it have characteristics such as highly sensitive, high specificity, because LAMP operation step simply reaches the precipitation that comprises a large amount of nucleic acid and magnesium pyrophosphate in the reaction product, the judgement reaction result that can detect by an unaided eye after the fluorescent agent colour developing is applicable to the use of testing under the various experiment conditions.Method of the present invention is specifically implemented
The foundation and the checking of 1 porcine circovirus 2 type LAMP detection method
(1) the reaction preparation of reagent
1) PCV2LAMP design of primers
6 strain sequences of the PCV2 that announces according to GenBank are template, design following two pairs of primers, and sequence is as follows:
Sequence 1 (PB1): ATCACAAGGACAACGGAGTG
Sequence 2 (PB2): GCCCCACAATGACGTGTAC
Sequence 3 (PB3): GCTCTGCAACGGTCACCAGA-ACCTCTCTACTGCTGTGAGT
Sequence 4 (PB4): TTGTCAGAAATTTCCGCGGGCT-TTCGTCTTCCAATCACGCTT
2) preparation of solution in the reaction system (50 reacting weights)
1. primer mixed solution (PB): get 100pmol/ μ l PB1 2.5 μ l, 100pmol/ μ l PB2 2.5 μ l, 100pmol/ μ lPB3 20 μ l, add sterilization deionized water 5 μ l after 100pmol/ μ l PB4 20 μ l mix, total system 50 μ l.
2. react buffer mixture (RB): get 4mmol/ μ l sal epsom and get 50 μ l, 1.6mol/ μ l trimethyl-glycine 50 μ l, 10mmol/ μ ldNTPs 50 μ l, be dissolved in the 475 μ l PCR level water fixed molten to 625 μ l.
3. reaction enzymes mixed solution (EB): get 8U/ μ l Bst large fragment DNA polysaccharase 48 μ l, 0.1 μ mol/ μ l DTT, 2 μ l.
4. developer: get 1 μ l SYBRgreenI (available from Invitrogen company) and be dissolved in 49 μ l PCR level water.
(2) viral DNA extracts reagent (LBBI-DNA) preparation:
1) DA: 1%2-mercaptoethanol solution 10ml, 10mmol/mLTris-HCL (pH 8.0) 3ml, 10mmol/mlEDTA 1ml are dissolved in the sterilization distilled water, fixed molten to 30ml.
2) after DB:200mmol/ml 15mlNaOH, 1%SDS solution 15ml mix, fixed molten to 30ml with deionized water.
3) DC:75ml dehydrated alcohol is in the plastic containers of packing into after pH 4.8200mmol sodium-acetate (NaAc) 1ml mixes.
4) DD:50u RNAse A is dissolved in 100ml nuclease free water (DEPC).
(3) extraction of DNA
Sample DNA uses the LBBI-DNA reagent of the present invention's preparation to extract.Add DA 500 μ l in the 100 μ l samples, vortex 10s behind the adding RB 250 μ l, adds isopyknic DC behind the concuss 1min, vortex 10s, and centrifugal 1min, abandoning supernatant, room temperature add DD 10 μ l dissolving after placing 5min.Get 1 μ l and utilize the total DNA amount of spectrophotometric determination.
Utilize LBBI-DNA reagent that PCV2HuB strain, PCV2LN strain isolated viral sample (inventor's collection) by cell cultures are extracted sample total DNA (seeing Table 1).
PCV DNA extraction amount behind table 1 purifying
(4) reaction is carried out
PB 1.0μl，
RB 12.5μl，
EB 1.0μl，
Sample DNA 5.5 μ l
After mixing in above each composition adding reaction tubes, place 65 ℃ of water bath with thermostatic control isothermal duplication 30min, observations behind the developer mixing that adding 1.0 μ l prepared after reaction finished.
(5) checking of detected result
1) the Tubidimeter real-time absorbancy of PCV2LAMP reaction product is identified
Utilize LAMP Tubidimeter LA-320 instrument (Japanese Rong Yuan Co., Ltd. produce) to monitor in real time and carry out response situation in the LAMP reaction tubes, show in the note reaction process speed of reaction and time in each reaction tubes with the form of picture and text.The PCV2DNA sample that extracts in above (3) has been carried out the LAMP detection, and Fig. 1 has shown the Tubidimeter real-time absorbance detection result of PCV2LAMP product.Wherein negative contrast, 1,2 are respectively PCV2HuB strain and PCV2LN strain, 3,4: be masterplate amplification contrast with water, Fig. 1 display result conforms to expection.Red partly expression is positive judges that green portion is represented negative judgement.Blue portion is represented the product light absorption value.
2) the visual evaluation of PCV2LAMP reaction product
After the PCV2LAMP reaction finishes, add the SYBRgreenI of 1 μ L in the product, visual inspection LAMP reaction solution colour-change.The color of the reaction solution of negative reaction is an orange, and the reaction solution of positive is green (see figure 2), conforms to Fig. 1 qualification result, expection, shows that the color reaction that is presented has specificity.
3) PCV2LAMP specificity test
Viral sample DNA such as extracting PPV, PRV according to the PCV2LAMP detection method that the present invention set up, carry out the specificity test.With PCV2 as positive control, the water that DEPC handles is as negative control, the viral DNA template of Detection and Extraction, the specificity of the checking LAMP method of setting up, positive control and negative control are all normal as a result, PCV2 is positive, other test sample (see figure 3) that is negative, and the result shows: this method detects PCV2 has specificity.
4) PCV2LAMP sensitivity test
The dna profiling of extractive PCV2HuB strain virus is carried out 10 times of systems be diluted to 1～1.0 * 10
-7Deng 7 extent of dilution.Carry out the susceptibility test with the PCV2LAMP detection method of being set up.The dna profiling (10ng/ μ l) of the PCV2HuB strain virus that extracts with the PCV2LAMP method amplification of setting up is by 1～10
-7Dilute 10 times of gradient dilutions, Fig. 4 is the dilution analytical resultss of the different dilutions of the right PCV2HuB strain dna profiling of PCV2LAMP Tubidimeter LA-320.From the PCV2LAMP Tubidimeter LA-320 curvature analysis result of Fig. 4 as can be seen, along with the reduction of template concentrations, positive reaction speed slope is constant, and the increase 5min consuming time of reaction is to extent of dilution 10
-7The time (see figure 4) that still can increase fast and effectively.In reaction, when the reaction product amount reach can be determined close on value the time finally be judged as the male time difference between 2min～5min to product, prove that PCV2LAMP has very high efficient at the amplification of PCV2 template.
5) PCV2LAMP detects the application test result
To the morbidity swine disease material of 10 parts of doubtful PCV of clinical collection (every pig got the sample that Lymphoid tissue and serum are used as antigen and antibody test respectively), gather total DNA of 10 duplicate samples (Lymphoid tissues of 10 pigs) according to the extracting method extracting that the present invention proposes, carry out result's comparison with PCV2LAMP detection method of setting up and PCR detection, use ELISA test kit (available from green poem source, Shenzhen bio-engineering corporation) that the serum antibody of corresponding swine disease material is detected simultaneously.Adopt LAMPTubidimeter observation of use instrument result, the result shows (see figure 5): in the doubtful pathological material of disease of 10 pigs, it is positive that the PCV2LAMP method detects 5 duplicate samples nucleic acid, and the pig anteserum sample corresponding with it has 3 parts to be positive through the ELASA TPPA.Pathological material of disease is carried out detecting with PCR after the cell cultures, and 4 parts of pathological material of diseases detect the PCV2 positive (seeing Table 2), and the positive findings that the PCV2LAMP method detects 5 duplicate samples has comprehensively been contained the ELASA TPPA, PCR verifies synthesis result.
Three kinds of methods of table 2. are to the qualification result of PCV2 in 10 parts of pathological material of diseases (positive+/ negative-)
2. the preparation of test kit and assembling
Formulated by following various components becomes various components (50 reacting weights), and divides to install to and also use corresponding plug seal in glass or the plastic small container:
(1)LBBI-DNA：
1) DA: 1%2-mercaptoethanol solution 10ml, 10mmol/mLTris-HCL (pH 8.0) 3ml, 10mmol/mlEDTA 1ml are dissolved in the sterilization distilled water, fixed molten to 30ml, in the rnase-free plastic containers of packing into.
2) after DB:200mmol/ml 15ml NaOH, 1%SDS solution 15ml mix, fixed molten with deionized water to 30ml, in the rnase-free plastic containers of packing into.
3) DC:75ml dehydrated alcohol is in the plastic containers of packing into after pH4.8 200mmol sodium-acetate 1ml mixes.
4) DD:50u RNAse A is dissolved in 100ml nuclease free water (DEPC), in the rnase-free plastic containers of packing into.
(2) preparation of solution in the reaction system
1) PB: get 100pmol/ μ l PB1 2.5 μ l, 100pmol/ μ l PB2 2.5 μ l, 100pmol/ μ l PB3 20 μ l, after mixing, 100pmol/ μ l PB4 20 μ l add sterilization deionized water 5 μ l, total system 50 μ l are filled in the plastic containers of rnase-free.
2) RB: get 4mmol/ μ l sal epsom and get 50 μ l, 1.6mol/ μ l trimethyl-glycine 50 μ l, 10mmol/ μ l dNTPs 50 μ l, be dissolved in the 475 μ l PCR level water fixed moltenly, be filled in the plastic containers of rnase-free to 625 μ l.
3) EB: get 8U/ μ l Bst large fragment DNA polysaccharase 48 μ l, 0.1 μ mol/ μ l DTT, 2 μ l are filled in the plastic containers of rnase-free.
(3) developer
Get 1 μ lSYBRgreenI (available from Invitrogen company) and be dissolved in 49 μ l PCR level water, be filled in the plastic containers of rnase-free.
The use of 3 test kits
(1) extraction of sample DNA
Add 500 μ l DA in the 100 μ l test samples, vortex 15s, the centrifugal 1min of 12000g gets supernatant and places new pipe; The DB solution of 300 μ l is added in the supernatant liquor, vortex or concuss 90s; Add isopyknic DC solution, vortex 1min, the centrifugal 1min of 12000g.Abandoning supernatant; Place 5min in the room temperature, treat to add 10 μ l DD and it is dissolved into sample DNA after the drying precipitate.
(2) preparation of reaction solution
The reactive system that the use of this test kit is adopted is 20 μ l reactive systems.
Get PB 1.0 μ l, RB 12.5 μ l and EB 1.0 μ l,, place little reaction tubes, add sample DNA 5.5 μ l. mixings again.
(3) Fan Ying carrying out
After LAMP Tubidimeter temperature arrives 65 ℃, the reaction tubes that adds each composition is placed 65 ℃ of water bath with thermostatic control isothermal duplication 30min.
(4) result judges
After reaction finishes, add developer (SYBRgreenI) the 1.0 μ l that prepare.Visual inspection: the color of the reaction solution of negative reaction presents orange, and the reaction solution of positive presents green.
Description of drawings
Fig. 1: the Tubidimeter real-time absorbancy of PCV LAMP product is identified column diagram 1 road: PCV2HuB strain, 2 roads: PCV2LN strain, 3,4 roads: water contrast;
Fig. 2: LAMP reaction system colour developing result 1,2,3:PCV2 positive, 4:PCV2 negative sample
Fig. 3: specificity test PCV LAMP Tubidimeter LA-320 analysis chart 1:PCV2HuB strain, 2:PCV2 LN strain, 3,4:DEPC H2O, 5,6:PRV, 7,8:PPV
Fig. 4: PCV210
-1～10
-7Gradient dilution LAMP Tubidimeter LA-320 analyzes 1:10
-1, 2:10
-2, 3:10
-3, 4:10
-4, 5:10
-5, 6:10
-6, 7:10
-710 times of gradient dilutions, 8: blank.
The 10 part samples detected result LAMP Tubidimeter LA-320 instrument cylindricality analysis charts 1 of Fig. 5: PCV2LAMP to gathering: positive control, 2: feminine gender contrast, 3:HuN08,4:SD03,5:TJ13,6:SX23,7:HuB2,8:SX21
Positive effect of the present invention is:
The present invention relates to a kind of porcine circovirus 2 LAMP detection kit and detection method thereof. Announce according to GenBank The PCV2 gene order has designed 4 of PCV2LAMP primers at its sequence conservative region; With PCV2HuB strain, PCV2LN strain Viral DNA is template, and the PCV2LAMP reaction system of using this research to set up is carried out the LAMP reaction. Utilize LA-320LAMP Add SYBRgreenI developer result of determination after Tubidimeter instrument analytical reactions process and reaction finish, the result is presented at 63 ℃ of 30min in the LA-320LAMP Tubidimeter instrument, PCV2DNA has obtained efficient specific amplification, adds The SYBRgreenI colour developing is judged consistent with Instrumental Analysis demonstration result. Prove by sensitivity tests: this method can be to 50 The dna profiling of ngPCV2 carries out 10-7Still can efficiently increase after the dilution, demonstrate the sensitiveness of this method height; By special The property test and clinical sample the LAMP of PCV2 nucleic acid detect, the result shows that this method is special, simple, rapid, is suitable for The testing of PCV2.
Embodiment
Following examples further specify the present invention, but not as limitation of the present invention.
1) design of primers is a template according to the PCV2 strain sequence that GenBank announces, designs following two pairs of primers, primer sequence:
PB1：ATCACAAGGACAACGGAGTG
PB2：GCCCCACAATGACGTGTAC
PB3：GCTCTGCAACGGTCACCAGAACCTCTCTACTGCTGTGAGT
PB4：TTGTCAGAAATTTCCGCGGGCTTTCGTCTTCCAATCACGCTT
The preparation of component in the test kit: the formulated by following various components becomes various components (50 reacting weights), and divides to install to and also use corresponding plug seal in glass or the plastic small container:
1. viral DNA extracts (LBBI-DNA) reagent:
1) DA: 1%2-mercaptoethanol solution 10ml, 10mmol/mLTris-HCL (pH 8.0) 3ml, 10mmol/mlEDTA 1ml are dissolved in the sterilization distilled water, fixed molten to 30ml, in the rnase-free plastic containers of packing into.
2) after DB:200mmol/ml 15ml NaOH, 1%SDS solution 15ml mix, fixed molten with deionized water to 30ml, in the rnase-free plastic containers of packing into.
3) DC:75ml dehydrated alcohol is in the plastic containers of packing into after pH 4.8200mmol sodium-acetate 1ml mixes.
4) DD:50u RNAseA is dissolved in 100ml nuclease free water (DEPC), in the rnase-free plastic containers of packing into.
2. the preparation of solution in the reaction system:
1) PB: get 2.5 μ l 100pmol/ μ l PB1,2.5 μ l 100pmol/ μ l PB2,20 μ l 100pmol/ μ l PB3, add sterilization deionized water 5 μ l after 20 μ l100pmol/ μ l PB4 mix, total system 50 μ l are filled in the plastic containers of rnase-free.
2) RB: get 4mmol/ μ l sal epsom and get 50 μ l, 1.6mol/ μ l trimethyl-glycine 50 μ l, 10mmol/ μ l dNTPs 50 μ l, be dissolved in the 475 μ l PCR level water fixed moltenly, be filled in the plastic containers of rnase-free to 625 μ l.
3) EB: get 8U/ μ l Bst large fragment DNA polysaccharase 48 μ l, 0.1 μ mol/ μ l DTT, 2 μ l are filled in the plastic containers of rnase-free.
3 developers:
Get 1 μ lSYBRgreenI (available from Invitrogen company) and be dissolved in 49 μ l PCR level water, be filled in the plastic containers of rnase-free.
The use of PCV2LAMP test kit
1. the extraction of sample DNA
Add 500 μ l DA in the 100 μ l test samples, vortex 15s, the centrifugal 1min of 12000g gets supernatant and places new pipe; The DB solution of 300 μ l is added in the supernatant liquor, vortex or concuss 90s; Add isopyknic DC solution, vortex 1min, again through the centrifugal 1min of 12000g, abandoning supernatant is placed 5min and is treated to add 10 μ l DD and it is dissolved into sample DNA after the drying precipitate in the room temperature.
2. the preparation of reaction solution
Get PB 1.5 μ l, RB 12.5 μ l and EB 1.0 μ l, place little reaction tubes, add sample DNA 5.5 μ l. mixings again.
3. Fan Ying carrying out
After LAMP Tubidimeter temperature arrives 65 ℃, the reaction tubes that adds each composition is placed 65 ℃ of water bath with thermostatic control isothermal duplication 30min.
4. the result judges
After reaction finishes, add the developer 1 μ l for preparing.Visual inspection: the color of the reaction solution of negative reaction presents orange, and the reaction solution of positive presents green.
Sequence table
＜110〉China Veterinery Drug Inspection Office
＜120〉porcine circovirus 2 type LAMP detection kit and detection method thereof
<160>4
<210>1
<211>20
<212>DNA
＜213〉primer PB1
＜223〉artificial sequence
<400>1
ATCACAAGGA?CAACGGAGTG 20
<210>2
<211>19
<212>DNA
＜213〉primer PB2:
＜223〉artificial sequence
<400>2
GCCCCACAAT?GACGTGTAC
<210>3
<211>40
<212>DNA
＜213〉primer PB3
＜223〉artificial sequence
<400>3
GCTCTGCAAC?GGTCACCAGA?ACCTCTCTAC?TGCTGTGAGT 40
<210>4
<211>42
<212>DNA
＜213〉primer PB4
＜223〉artificial sequence
<400>4
TTGTCAGAAA?TTTCCGCGGG?CTTTCGTCTT?CCAATCACGC?TT 42
Claims (5)
1. a porcine circovirus 2 type LAMP detection kit is characterized in that extracting reagent by viral DNA forms with the LAMP reaction system reagent two portions that contain sequence 1,2,3 and 4 primers.
2. a kind of according to claim 1 porcine circovirus 2 type LAMP detection kit is characterized in that the prescription of viral DNA extraction reagent component wherein is as follows:
1) DA:1%2-mercaptoethanol solution 10ml, pH 8.010mmol/mLTris-HCL 3ml, 10mmol/mlEDTA 1ml, the sterilization distilled water adds to 30ml;
2) DB:200mmol/ml NaOH 15ml, 1%SDS solution 15ml, final volume is 30ml;
3) DC: dehydrated alcohol 75ml, pH 4.8200mmol sodium-acetate 1ml;
4) DD:RNAse A 50u, nuclease free water add to 100ml.
3. a kind of according to claim 1 porcine circovirus 2 type LAMP detection kit is characterized in that the prescription of LAMP reaction system reagent component wherein is as follows:
1) primer mixed solution:
100pmol/ μ l PB1 primer 2 .5 μ l, 100pmol/ μ l PB2 primer 2 .5 μ l, 100pmol/ μ l PB3 primer 20 μ l, 100pmol/ μ l PB4 primer 20 μ l, sterilization deionized water 5 μ l, final volume is 50 μ l;
2) reaction buffer mixture:
4mmol/ μ l sal epsom 50 μ l, 1.6mol/ μ l trimethyl-glycine 50 μ l, 10mmol/ μ l dNTPs 50 μ l, 475 μ l PCR level water, final volume is 625 μ l;
3) reaction enzymes mixed solution: 8U/ μ l Bst large fragment DNA polysaccharase 48 μ l, 0.1 μ mol/ μ l DTT, 2 μ l, final volume is 50 μ l;
4) developer: SYBRgreenI 1 μ l, PCR level water 49 μ l, final volume is 50 μ l.
4. as claim 1,3 described a kind of porcine circovirus 2 type LAMP detection kit is characterized in that the primer of employed sequence 1,2,3 in the LAMP reaction system reagent wherein and 4 is:
PB1：ATCACAAGGACAACGGAGTG
PB2：GCCCCACAATGACGTGTAC
PB3：GCTCTGCAACGGTCACCAGAACCTCTCTACTGCTGTGAGT
PB4：TTGTCAGAAATTTCCGCGGGCTTTCGTCTTCCAATCACGCTT
5. the detection method of a porcine circovirus 2 type LAMP detection kit is characterized in that viral DNA extracting method wherein is:
Add 500 μ l DA in the 100 μ l test samples, vortex 15s, the centrifugal 1min of 12000g gets supernatant and places new pipe; The DB solution of 300 μ l is added in the supernatant liquor, vortex or concuss 90s; Add isopyknic DC solution, vortex 1min, again through the centrifugal 1min of 12000g, abandoning supernatant is placed 5min and is treated to add 10 μ l DD and it is dissolved into sample DNA after the drying precipitate in the room temperature.
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| CN104894293A (en) * | 2015-04-30 | 2015-09-09 | 陕西溯源农业发展有限公司 | Porcine circovirus type 2 isothermal PCR on-site rapid detection kit |
| CN107304454A (en) * | 2016-04-25 | 2017-10-31 | 上海市农业科学院 | A kind of LAMP visual quick detection kit of porcine circovirus 2 type |
| CN106566896A (en) * | 2016-10-25 | 2017-04-19 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Reagent and detection method for detecting porcine circovirus type 2 and application |
| CN107034316A (en) * | 2017-06-19 | 2017-08-11 | 北京博奥晶典生物技术有限公司 | The system and its special LAMP primer of 6 boars virus are detected simultaneously |
| CN113774166A (en) * | 2021-09-13 | 2021-12-10 | 青岛农业大学 | Porcine circovirus type 2, type 3 and type 4 on-site rapid high-sensitivity differential diagnosis kit and use method thereof |
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