CN103820580A - Loop-mediated isothermal amplification (LAMP) diagnostic kit for porcine circovirus type 2 - Google Patents
Loop-mediated isothermal amplification (LAMP) diagnostic kit for porcine circovirus type 2 Download PDFInfo
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Abstract
The invention discloses a loop-mediated isothermal amplification (LAMP) diagnostic kit for porcine circovirus type 2. The diagnostic kit comprises 250muL of LAMPmix buffer solution, 20muL of (i)Bst(/i) deoxyribonucleic acid (DNA) polymerase, 150muL of ultrapure water, 40muL of mixed inner primer 1 and inner primer 2, of which the concentration is 40pmol/L, 40muL of mixed outer primer 1 and outer primer 2, of which the concentration is 5pmol/L, 20muL of negative control and 20muL of positive control. Two pairs of primers are designed and synthetized according to a porcine circovirus type 2(PCV-2) ORF2 gene sequence in GenBank, an LAMP diagnostic method for the porcine circovirus type 2 is built by optimization of the LAMP condition, and the sensitivity and specificity tests, and a simple and fast method is provided for rapid diagnosis of the porcine circovirus type 2 on the spot. Thus, quick quarantine needs of the basic level on the spot are met.
Description
Technical field
The present invention relates to a kind of test kit, especially a kind of giant salamander pathogenic hydrophila gingivalis PCR diagnostic kit.
Background technology
At present, main detection method has Virus Isolation, agar diffusion test, Small Volume Serum neutralization test, ELISA, PCR method.And conventional etiology separates and Serology test, deficiencies such as having again trivial operations, waste time and energy, susceptibility is low, PCR method needs expensive PCR instrument.Ring mediated isothermal amplification, for 4 kinds of special primers of 6 zone design of target gene, utilizes a kind of strand displacement archaeal dna polymerase isothermal condition (63 ℃ of left and right) insulation 30～60 minutes, can complete nucleic acid amplification reaction.Compared with conventional PCR, do not need the process such as thermally denature, temperature cycle, electrophoresis and ultraviolet visualization of template, do not need expensive PCR instrument yet, only need very simple equipment, as water-bath or thermos cup.
Summary of the invention
The object of the invention is: a kind of porcine circovirus 2 type LAMP diagnostic kit is provided, and it can rapid detection porcine circovirus 2 type, and easy, sensitive, accurate, high specificity.
The present invention is achieved in that porcine circovirus 2 type LAMP diagnostic kit, and it comprises the LAMPmix damping fluid of 250 μ L, and 20 μ L concentration are 0.5U/ μ L's
bstarchaeal dna polymerase, 150 μ L ultrapure waters, the volume of inner primer 1 and inner primer 2 is 40 μ L, and concentration is 40pmol/L, and the volume of outer primer 1 and outer primer 2 is 40 μ L, and concentration is 5pmol/L, 20 μ L negative controls and 20 μ L positive controls; The sequence of outer primer 1 is: 5
,-TGGAGCTCCTCGATCTCAAG-3
,; The sequence of outer primer 2 is: 5
,-GCCCCACAATGACGTGTAC-3
,; The sequence of inner primer 1 is: 5
,-GCAACGGTCACCAGACTCCCGACAACGGAGTGACCTGTC-3
,; The sequence of inner primer 2 is: 5
,-AGAGCAGCACCCTGTAACGTTTACGCTTCTGCATTTTCCCG-3
,.
Negative control is ultrapure water.
Positive control is porcine circovirus type 2 strain DNA.
LAMP mix damping fluid comprises the MgCl of 3 mmol/ul
2, the dNTP 2.0 μ L of 500pmol/ul and the Tris-HCl of 25mmol/ μ L, its pH value is 8.3.
This test kit is according to the porcine circovirus 2 type in GenBank (PCV-2) ORF2 gene order, the synthetic 2 pairs of primers of design, by the optimization to LAMP condition, susceptibility, specificity examination, research and development porcine circovirus 2 type LAMP diagnostic kit, for the on-the-spot quick diagnosis of porcine circovirus 2 type provides a kind of simple and rapid method, thereby meet the on-the-spot rapid quarantine needs of basic unit.
In order to verify technique effect of the present invention, carry out following experiment:
the foundation of porcine circovirus 2 type LAMP diagnostic method
1 materials and methods
1.1 viral
The strain of PRV Fujian-1, PCV-1, PCV-2, PPV virulent strain 7909, CFSV crossdrift are that virulent strain F114, PRRSV America strain CH-1a strain are preserved by the great epidemic disease Fang Zhi of Guizhou Province livestock and poultry key lab.
main agents
tIANampvirus genom DNA/RNA extracts test kit, silica gel model
tMpCR purification kit is purchased from Tian Gen biochemical technology company limited; Ring mediated isothermal amplification method DNA cloning test kit, ring mediated isothermal amplification method fluorescence detection reagent kit are composed bio tech ltd purchased from Peking blue; Goldview,
tris, EDTA,
dL2000,
taqdNA Polymerase(1U/ μ L) and corresponding 10 ×
taqbuffer,
dNTPdeng purchased from precious biological (Dalian) Engineering Co., Ltd.
design of primers
According to the porcine circovirus 2 type in GenBank (PCV-2) ORF2 gene order, according to the principle of LAMP design of primers, 4 Auele Specific Primers of conservative region design with online PrimerExplorer 4.0 softwares in these sequences, comprise outer primer and inner primer (F3, B3, FIP, BIP).Primer is synthetic by precious biological (Dalian) Engineering Co., Ltd.
F3：5
，-TGGAGCTCCTCGATCTCAAG-3
，; B3：5
，-GCCCCACAATGACGTGTAC-3
，
FIP：5
，-GCAACGGTCACCAGACTCCCGACAACGGAGTGACCTGTC-3
，
BIP：5
，-AGAGCAGCACCCTGTAACGTTTACGCTTCTGCATTTTCCCG-3
，
the extracting of 1.4 viral nucleic acids
Clinical sample: get the sick pig lymphoglandula of 0.1g and add 0.5mL 0.1mol/L PBS-80 ℃ multigelation 3 times, grind and make suspension, be then transferred in 1.5 mL centrifuge tubes.The centrifugal 5min of 12000 r/ min, gets the extraction of supernatant liquor for viral nucleic acid.The extraction reference of PRV, PCV-1, PCV-2, PPV, CFSV, PRRSV America strain virus DNA/RNA
tIANampvirus genom DNA/RNA extracts test kit specification sheets to carry out, by the DNA extracting and RNA in-70 ℃ of preservations.
the optimization of reaction conditions
To LAMP reaction conditions, temperature of reaction (60 ℃, 63 ℃, 65 ℃), reaction times (30min, 45min, 60min), F3, B3 primer concentration (5pmol/L, 10pmol/L, 20 pmol/L), FIP, BIP primer concentration (20pmol/L, 40pmol/L, 80 pmol/L), be optimized, to determine optimum reaction condition, simultaneously with ddH
2o is as blank.LAMP reaction is carried out in 25 μ L reaction systems, primers F 3, B3 2 μ L(5pmol/L, 10pmol/L, 20 pmol/L), primer 4 μ L FIP, BIP(20pmol/L, 40pmol/L, 80 pmol/L),
bstarchaeal dna polymerase (0.5U, 1U, 2U), LAMP reaction buffer 12.5 μ L, template 1 μ L, adds fluorescence developing liquid 1 μ L in ring mediated isothermal amplification method fluorescence detection reagent kit, uses ddH
2o complements to 25 μ L.Reaction conditions is: temperature of reaction (60 ℃, 63 ℃, 65 ℃) 30min, 94 ℃ of deactivation 2min.Amplified production is with the naked eye directly observed under daylight, or observes under UV-light.Amplified production send the order-checking of precious biological (Dalian) Engineering Co., Ltd.
sensitivity test
Application PCV-2 ORF2 gene F3, B3 primer carry out pcr amplification, by the PCR product silica gel model of amplification
tMpCR purification kit reclaims DNA fragmentation, is connected transformed competence colibacillus cell with pMD18-T cloning vector
dH5 α, recombinant chou called after pMD18T-PCV-2.Extract in a small amount test kit by plasmid DNA and extract recombinant bacterium physique grain, enzyme is cut with PCR and is identified.Measure after concentration with this recombinant bacterium physique grain nucleic acid nucleic acid-protein instrument, react for LAMP, PCR through the viral nucleic acid of 10 times of serial dilutions, with the susceptibility of determining that the LAMP of foundation reacts.
specific test
Carry out respectively LAMP reaction take PRV, PCV-1, PCV-2, PPV DNA as template, the reverse transcription of CFSV, PRRSV virus is that cDNA carries out LAMP reaction, uses ddH
2o is as blank, with the specificity of determining that the LAMP of foundation reacts.
replica test
LAMP method, 3 reliabilities with assay of duplicate detection PCV-2 DNA are set up in application.
to the detection of clinical sample
Applied to 95 parts of pathological material of diseases of pig farm, Guiyang City, Guizhou Province in 2013 and self-employed pig raiser's collection the LAMP diagnostic method of setting up to 2012 and detect, apply PCR diagnostic method simultaneously and detect.
result
the foundation of 2.1 LAMP detection methods
LAMP detection method reaction optimum reaction condition in 25 μ L reaction systems is that 63 ℃ of temperature of reaction, react 30min, 94 ℃ of deactivation 2min.Primers F 3, B3(5pmol/L) each 1 μ L, primers F IP, BIP(40pmol/L) each 2 μ L,
bstarchaeal dna polymerase (1U) 1 μ L, LAMP reaction buffer 12.5 μ L, template 1 μ L, adds fluorescence developing liquid 1 μ L in ring mediated isothermal amplification method fluorescence detection reagent kit, uses ddH
2o complements to 25 μ L.After reaction finishes, under UV-light, positive reaction pipe color becomes green, and colour-change does not occur negative reaction pipe, is colourless (Fig. 1).Amplified production send the order-checking of Dalian precious biotechnology company limited, and result shows, the specific band that amplified fragments is PCV-2.
sensitivity test
With after this recombinant bacterium physique grain nucleic acid nucleic acid-protein instrument mensuration concentration, viral nucleic acid through 10 times of serial dilutions reacts for LAMP, PCR, the minimum detection of nucleic acids amount of LAMP diagnostic method of setting up is 0.30 pg/ L(Fig. 2), and the minimum detection of nucleic acids amount of PCR diagnostic method is 30 pg/L(Fig. 3).
specific test
Carry out respectively LAMP reaction take PRV, PCV-1, PCV-2, PPV DNA as template, the reverse transcription of CFSV, PRRSV virus is that cDNA carries out LAMP reaction, PRV, PCV-1, PPV DNA; CFSV, PRRSV cDNA be under UV-light, be colourless, and the shown in green (see figure 4) of PCV-2 DNA.
replica test
The LAMP method that application is set up, duplicate detection PCV-2 DNA sample 3 times, result is all consistent.
to the detection of clinical sample
The LAMP diagnostic method that 95 parts of pathological material of diseases application that gather to pig farm, Guiyang City, Guizhou Province in 2013 and self-employed pig raiser for 2012 are set up detects, in 95 parts of pathological material of diseases, have 24 parts positive, positive rate is 25.2%(24/95).Apply PCR diagnostic method simultaneously and detect, in 95 parts of pathological material of diseases, have 23 parts positive, positive rate is 22.1%(23/95).PCV-2 LAMP diagnostic method and the PCR diagnostic method coincidence rate set up are 95.8%.
discuss
The method that detects at present 2 porcine circovirus C-type virus C is a lot, as electron microscopic observation, viral separation, inoculation test, serology, agar immunodiffusion method, but the bothersome effort of these methods.Traditional PCR, quantitative fluorescent PCR need expensive professional equipment, detect pertinent instruments equipment, and operative technique require although China's animal and veterinary at county level station has substantially all been equipped with PCR high.The technical superiority of ring mediated isothermal amplification method is except high specific, highly sensitive, simple to operate, uses the just energy realization response of water-bath or thermos cup, does not need to carry out gel electrophoresis as PCR.
Its susceptibility of porcine circovirus 2 type loop-mediated isothermal amplification detection method of the foundation such as what voter can reach the DNA molecule of 10 copies, the positive rate coincidence rate of the positive rate of LAMP and PCR is 94%, it is after reaction finishes, add 2 μ L SYBR Green I fluorescence dyes, under UV-light, carry out result judgement.But after the reaction product of LAMP is uncapped, easily form aerosol, easily pollute laboratory, if reagent, pipettor, rifle head, super clean bench etc. are polluted, will there will be false positive, this has limited its range of application to cause disadvantageous effect for the production of practice.So need add applicable dyestuff before reaction.SYBR Green I adds the easy LAMP of impact reaction before reaction, this research adds fluorexon dyestuff in reaction system before reaction, reacted rear positive pipe color under UV-light and become green, and negative tube is owing to there is no product, still without colour-change.
The key of setting up LAMP diagnostic method is the design of primer.Primer GC content is preferably between 50%～60%.3 of all primers
,5 of FIP, BIP
,free energy change value (△ G) need be less than ﹣ 4, and △ G is less, and reacting more easily between primer and template occurs.The present invention, according to porcine circovirus 2 type (PCV-2) ORF2 gene order, designs 2 pairs of primers, by the optimization of reaction conditions, sets up LAMP diagnostic method.Result shows, the demonstration of susceptibility result, and the minimum detection of nucleic acids amount of LAMP diagnostic method of foundation is 0.3 pg/ L, and the minimum detection of nucleic acids amount of PCR diagnostic method is 30pg/L, highly sensitive 100 times than regular-PCR of LAMP.Detected result to the sick pig samples of 95 parts of natural infections shows, this LAMP method detected result and PCR detected result coincidence rate are 95.8%, show that the diagnostic method of setting up is applicable to basic unit's scene rapid quarantine.
Accompanying drawing explanation
Fig. 1 is the foundation of LAMP detection method;
In Fig. 1,1:PCV2 LAMP product; 2: negative control;
Fig. 2 is PCV-2 LAMP sensitization test;
In Fig. 2,1～6:3.0 ng/L × 10
0～10
-5the viral DNA LAMP result of dilution;
Fig. 3 is PCV-2 PCR sensitization test;
In Fig. 3, M:D2000; 1～8:30 mg/L × 10
0～10
-7the viral DNA PCR result of dilution;
Fig. 4 is PCV2 LAMP specific test;
In Fig. 4,1:PCV-2 LAMP product; 2:PRV LAMP product; 3:PCV-1 LAMP product; 4:PPV LAMP product;
5:CFSV LAMP product; 6:PRRSV LAMP product.
Embodiment
Embodiments of the invention: porcine circovirus 2 type LAMP diagnostic kit, it comprises the LAMPmix damping fluid of 250 μ L, 20 μ L concentration are 0.5U/ μ L's
bstarchaeal dna polymerase, 150 μ L ultrapure waters, the volume of inner primer 1 and inner primer 2 is 40 μ L, and concentration is 40pmol/L, and the volume of outer primer 1 and outer primer 2 is 40 μ L, and concentration is 5pmol/L, 20 μ L negative controls and 20 μ L positive controls; The sequence of outer primer 1 is: 5
,-TGGAGCTCCTCGATCTCAAG-3
,; The sequence of outer primer 2 is: 5
,-GCCCCACAATGACGTGTAC-3
,; The sequence of inner primer 1 is: 5
,-GCAACGGTCACCAGACTCCCGACAACGGAGTGACCTGTC-3
,; The sequence of inner primer 2 is: 5
,-AGAGCAGCACCCTGTAACGTTTACGCTTCTGCATTTTCCCG-3
,; Negative control is ultrapure water; Positive control is porcine circovirus type 2 strain DNA; LAMP mix damping fluid comprises the MgCl of 3 mmol/ul
2, the dNTP 2.0 μ L of 500pmol/ul and the Tris-HCl of 25mmol/ μ L, its pH value is 8.3.
SEQUENCE LISTING
sequence table
<110> Guizhou Farming Animal Science and Veterinary Research Institute
<120> porcine circovirus 2 type LAMP diagnostic kit
<130> nm:
<160> 4
<170> PatentIn version
<210> 1
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> is according to the porcine circovirus 2 type in GenBank (PCV-2) ORF2 gene order, and the synthetic 2 pairs of primers of design, for setting up porcine circovirus 2 type LAMP diagnostic method.
<400> 1
TGGAG CTCCT CGATC TCAAG 20
<210> 2
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> is according to the porcine circovirus 2 type in GenBank (PCV-2) ORF2 gene order, and the synthetic 2 pairs of primers of design, for setting up porcine circovirus 2 type LAMP diagnostic method.
<400> 2
GCCCC ACAAT GACGT GTAC 19
<210> 3
<211> 39
<212> DNA
<213> artificial sequence
<220>
<223> is according to the porcine circovirus 2 type in GenBank (PCV-2) ORF2 gene order, and the synthetic 2 pairs of primers of design, for setting up porcine circovirus 2 type LAMP diagnostic method.
<400> 3
GCAAC GGTCA CCAGA CTCCC GACAA CGGAG TGACC TGTC
39
<210> 4
<211> 41
<212> DNA
<213> artificial sequence
<220>
<223> is according to the porcine circovirus 2 type in GenBank (PCV-2) ORF2 gene order, and the synthetic 2 pairs of primers of design, for setting up porcine circovirus 2 type LAMP diagnostic method.
<400> 4
AGAGC AGCAC CCTGT AACGT TTACG CTTCT GCATT TTCCC G 41
Claims (4)
1. a porcine circovirus 2 type LAMP diagnostic kit, is characterized in that: it comprises the LAMPmix damping fluid of 250 μ L, and 20 μ L concentration are 0.5U/ μ L's
bstarchaeal dna polymerase, 150 μ L ultrapure waters, the volume of inner primer 1 and inner primer 2 is 40 μ L, and concentration is 40pmol/L, and the volume of outer primer 1 and outer primer 2 is 40 μ L, and concentration is 5pmol/L, 20 μ L negative controls and 20 μ L positive controls; The sequence of outer primer 1 is: 5
,-TGGAGCTCCTCGATCTCAAG-3
,; The sequence of outer primer 2 is: 5
,-GCCCCACAATGACGTGTAC-3
,; The sequence of inner primer 1 is: 5
,-GCAACGGTCACCAGACTCCCGACAACGGAGTGACCTGTC-3
,; The sequence of inner primer 2 is: 5
,-AGAGCAGCACCCTGTAACGTTTACGCTTCTGCATTTTCCCG-3
,.
2. porcine circovirus 2 type LAMP diagnostic kit according to claim 1, is characterized in that: negative control is ultrapure water.
3. porcine circovirus 2 type LAMP diagnostic kit according to claim 1, is characterized in that: positive control is porcine circovirus type 2 strain DNA.
4. porcine circovirus 2 type LAMP diagnostic kit according to claim 1, is characterized in that: LAMP mix damping fluid comprises the MgCl of 3 mmol/ul
2, the dNTP 2.0 μ L of 500pmol/ul and the Tris-HCl of 25mmol/ μ L, its pH value is 8.3.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651534A (en) * | 2015-03-04 | 2015-05-27 | 广西壮族自治区兽医研究所 | Porcine circovivus loop-mediated isothermal amplification kit and application thereof |
| 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 |
| CN105950764A (en) * | 2016-06-30 | 2016-09-21 | 贵州省畜牧兽医研究所 | Actinobacillus pleuropneumoniae LAMP diagnostic kit |
| CN109055612A (en) * | 2018-08-24 | 2018-12-21 | 暨南大学 | Primer and its kit and method based on digital LAMP technology detection porcine circovirus 2 type |
| CN113337641A (en) * | 2021-06-16 | 2021-09-03 | 龙岩学院 | Porcine circovirus type 2 LAMP (loop-mediated isothermal amplification) detection primer group and kit |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307367A (en) * | 2008-02-20 | 2008-11-19 | 中国农业科学院兰州兽医研究所 | Technology for rapidly detecting porcine circovirus type2 |
| CN101586169A (en) * | 2009-06-30 | 2009-11-25 | 中国兽医药品监察所 | Porcine circovirus 2 LAMP detection kit and detecting method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101307367A (en) * | 2008-02-20 | 2008-11-19 | 中国农业科学院兰州兽医研究所 | Technology for rapidly detecting porcine circovirus type2 |
| CN101586169A (en) * | 2009-06-30 | 2009-11-25 | 中国兽医药品监察所 | Porcine circovirus 2 LAMP detection kit and detecting method |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651534A (en) * | 2015-03-04 | 2015-05-27 | 广西壮族自治区兽医研究所 | Porcine circovivus loop-mediated isothermal amplification kit and application thereof |
| 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 |
| CN105950764A (en) * | 2016-06-30 | 2016-09-21 | 贵州省畜牧兽医研究所 | Actinobacillus pleuropneumoniae LAMP diagnostic kit |
| CN109055612A (en) * | 2018-08-24 | 2018-12-21 | 暨南大学 | Primer and its kit and method based on digital LAMP technology detection porcine circovirus 2 type |
| CN113337641A (en) * | 2021-06-16 | 2021-09-03 | 龙岩学院 | Porcine circovirus type 2 LAMP (loop-mediated isothermal amplification) detection primer group and kit |
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