CN103820580B - Porcine circovirus 2 type LAMP diagnostic kit - Google Patents
Porcine circovirus 2 type LAMP diagnostic kit Download PDFInfo
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Classifications
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- 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
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- 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
Abstract
Does the present invention disclose a kind of porcine circovirus 2 type LAMP diagnostic kit, and it comprises 250 �� L? LAMPmix damping fluid, 20 �� L?<i>bst</i>archaeal dna polymerase, 150 �� L ultrapure waters, the outer primer 1 of mixing and outer primer 2,20 �� L negative controls and the 20 �� L positive controls of 40 �� L concentration to be the mixing inner primer 1 of 40pmol/L and inner primer 2 and 40 �� L concentration be 5pmol/L; According to porcine circovirus 2 type (PCV-2) the ORF2 gene order in GenBank, design and synthesis 2 is to primer, by the optimization to LAMP condition, susceptibility, specificity are tried, set up porcine circovirus 2 type LAMP diagnostic method, for the on-the-spot quick diagnosis of porcine circovirus 2 type provides a kind of simple and rapid method, thus meet the on-the-spot rapid quarantine needs of basic unit.
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
Porcine circovirus 2 type is the minimum animal virus found at present, pmws, farrowing sow breeding difficulty, wean pig and growing and fattening pigs respiratory tract disease, pigskin inflammation and the disease such as nephritic syndrome, children's piglet congenital tremors in age can be caused, cause bigger financial loss to China's Swine Production.
At present, main detection method has Virus Isolation, agar diffusion test, Small Volume Serum neutralization test, ELISA, PCR method. And the etiology of routine is separated and Serology test, deficiencies such as there is again trivial operations, waste time and energy, susceptibility is low, PCR method then needs expensive PCR instrument. Ring mediated isothermal amplification, for 6 zone design 4 species-specific primers of target gene, utilizes a kind of strand displacement archaeal dna polymerase to be incubated 30��60 minutes in isothermal condition (about 63 DEG C), can complete nucleic acid amplification reaction. Compared with Standard PCR, it is not necessary to the processes such as the thermally denature of template, temperature cycle, electrophoresis and ultraviolet observation, do not need expensive PCR instrument, it is only necessary to very simple equipment, such as water-bath or thermos cup yet.
Summary of the invention
It is an object of the invention to: providing a kind of porcine circovirus 2 type LAMP diagnostic kit, it can detect porcine circovirus 2 type fast, and easy, sensitive, accurate, high specificity.
The present invention is achieved in that porcine circovirus 2 type LAMP diagnostic kit, it comprises the LAMPmix damping fluid of 250 �� L, 20 �� L concentration are the BstDNA polysaccharase of 0.5U/ �� L, 150 �� L ultrapure waters, the volume of inner primer 1 and inner primer 2 is 40 �� L, and concentration is 40pmol/L, and the volume of outer primer 1 and outer primer 2 is 40 �� L, concentration is 5pmol/L, 20 �� L negative controls and 20 �� 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.
LAMPmix damping fluid comprises the MgCl of 3mmol/ul2, the Tris-HCl of the dNTP2.0 �� L and 25mmol/ �� L of 500pmol/ul, its PH value is 8.3.
This test kit is according to porcine circovirus 2 type (PCV-2) the ORF2 gene order in GenBank, design and synthesis 2 is to primer, by the optimization to LAMP condition, susceptibility, specificity are tried, 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, thus meet the on-the-spot rapid quarantine needs of basic unit.
In order to verify the 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 it is viral
PRV Fujian-1 strain, PCV-1, PCV-2, PPV virulent strain 7909, CFSV crossdrift system virulent strain F114, PRRSV America strain CH-1a strain preserve by the great epidemic disease Fang Zhi key lab of Guizhou Province livestock and poultry.
Main agents
TIANamp virus genom DNA/RNA extracts test kit, silica gel modelTMPCR 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 compose bio tech ltd purchased from Peking blue; Goldview, Tris, EDTA, DL2000, TaqDNAPolymerase(1U/ �� L) and corresponding 10 �� TaqBuffer, dNTP etc. purchased from precious biological (Dalian) Engineering Co., Ltd.
Design of primers
According to porcine circovirus 2 type (PCV-2) the ORF2 gene order in GenBank, principle according to LAMP primer design, design 4 Auele Specific Primers with online PrimerExplorer4.0 software at the conservative region of these sequences, comprise outer primer and inner primer (F3, B3, FIP, BIP). Primer is by the synthesis of precious biological (Dalian) Engineering Co., Ltd.
F3:5,-TGGAGCTCCTCGATCTCAAG-3,; B3:5,-GCCCCACAATGACGTGTAC-3,
FIP:5,-GCAACGGTCACCAGACTCCCGACAACGGAGTGACCTGTC-3,
BIP:5,-AGAGCAGCACCCTGTAACGTTTACGCTTCTGCATTTTCCCG-3,
1.4 the extracting of viral nucleic acid
Clinical sample: getting the sick pig lymphoglandula of 0.1g and add 0.5mL0.1mol/LPBS-80 DEG C of multigelation 3 times, suspension is made in grinding, is then transferred in 1.5mL centrifuge tube. The centrifugal 5min of 12000r/min, gets the extraction of supernatant liquor for viral nucleic acid. The extraction of PRV, PCV-1, PCV-2, PPV, CFSV, PRRSV America strain virus DNA/RNA is extracted test kit specification sheets with reference to TIANamp virus genom DNA/RNA and is carried out, by DNA and RNA of extraction in-70 DEG C of preservations.
The optimization of reaction conditions
To LAMP reaction conditions, temperature of reaction (60 DEG C, 63 DEG C, 65 DEG C), reaction times (30min, 45min, 60min), F3, B3 primer concentration (5pmol/L, 10pmol/L, 20pmol/L), FIP, BIP primer concentration (20pmol/L, 40pmol/L, 80pmol/L), it is optimized, to determine optimum reaction condition, simultaneously with ddH2O is as blank. LAMP reaction carries out in 25 �� L reaction systems, primers F 3, B32 �� L(5pmol/L, 10pmol/L, 20pmol/L), primer 4 �� LFIP, BIP(20pmol/L, 40pmol/L, 80pmol/L), BstDNA polysaccharase (0.5U, 1U, 2U), LAMP reaction buffer 12.5 �� L, template 1 �� L, add fluorescence developing liquid 1 �� L in ring mediated isothermal amplification method fluorescence detection reagent kit, use ddH2O complements to 25 �� L. Reaction conditions is: temperature of reaction (60 DEG C, 63 DEG C, 65 DEG C) 30min, 94 DEG C of deactivation 2min.Amplified production naked eyes are directly observed in the sunlight, or observe under ultraviolet light. Amplified production send the order-checking of precious biological (Dalian) Engineering Co., Ltd.
Sensitivity test
Application PCV-2ORF2 gene F3, B3 primer carries out pcr amplification, by the PCR primer silica gel model of amplificationTMPCR purification kit reclaims DNA fragmentation, is connected with pMD18-T cloning vector, transformed competence colibacillus cell DH5 ��, recombinant chou called after pMD18T-PCV-2. Extracting restructuring thalline plasmid by plasmid DNA Mini Kit, enzyme is cut and is identified with PCR. After measuring concentration with this restructuring thalline plasmid nucleic acid nucleic acid-protein instrument, the viral nucleic acid through 10 times of serial dilutions is used for LAMP, PCR reaction, to determine the susceptibility that the LAMP set up reacts.
Specificity is tested
Taking PRV, PCV-1, PCV-2, PPVDNA as template carries out LAMP reaction respectively, CFSV, PRRSV virus reverse transcription is that cDNA carries out LAMP reaction, uses ddH2O as blank, with determine set up LAMP reaction specificity.
Replica test
LAMP method is set up in application, and duplicate detection PCV-2DNA3 time is with the reliability of assay.
To the detection of clinical sample
The LAMP diagnostic method that the 95 parts of pathological material of disease application gathered to pig farm, Guiyang City, Guizhou Province in 2013 and self-employed pig raiser 2012 are set up detects, and applies PCR diagnostic method simultaneously and detects.
Result
The foundation of 2.1LAMP detection method
LAMP detection method reaction optimum reaction condition in 25 �� L reaction systems is, temperature of reaction 63 DEG C, reaction 30min, 94 DEG C of deactivation 2min. Primers F 3, B3(5pmol/L) each 1 �� L, primers F IP, BIP(40pmol/L) each 2 �� L, BstDNA polysaccharase (1U) 1 �� L, LAMP reaction buffer 12.5 �� L, template 1 �� L, adds fluorescence developing liquid 1 �� L in ring mediated isothermal amplification method fluorescence detection reagent kit, uses ddH2O complements to 25 �� L. After reaction terminates, under ultraviolet light, positive reaction pipe color turns into green, and negative reaction pipe does not occur color to change, and is colourless (Fig. 1). Amplified production send the order-checking of the precious biotechnology company limited in Dalian, and result shows, amplified fragments is the specific band of PCV-2.
Sensitivity test
After measuring concentration with this restructuring thalline plasmid nucleic acid nucleic acid-protein instrument, viral nucleic acid through 10 times of serial dilutions is used for LAMP, PCR reaction, the minimum detection of nucleic acids amount of LAMP diagnostic method set up is 0.30pg/L(Fig. 2), and the minimum detection of nucleic acids amount of PCR diagnostic method is 30pg/L(Fig. 3).
Specificity is tested
Taking PRV, PCV-1, PCV-2, PPVDNA as template carries out LAMP reaction respectively, CFSV, PRRSV virus reverse transcription is that cDNA carries out LAMP reaction, PRV, PCV-1, PPVDNA; CFSV, PRRSVcDNA are colourless under UV-light, and PCV-2DNA is shown as green (see figure 4).
Replica test
The LAMP method that application is set up, duplicate detection PCV-2DNA sample 3 times, result is all consistent.
To the detection of clinical sample
The LAMP diagnostic method that the 95 parts of pathological material of disease application gathered to pig farm, Guiyang City, Guizhou Province in 2013 and self-employed pig raiser for 2012 are set up detects, and has 24 parts for positive in 95 parts of pathological material of diseases, and positive rate is 25.2%(24/95). Applying PCR diagnostic method to detect simultaneously, have 23 parts for positive in 95 parts of pathological material of diseases, positive rate is 22.1%(23/95). PCV-2LAMP diagnostic method and the PCR diagnostic method coincidence rate set up are 95.8%.
Discuss
The method of detection 2 porcine circovirus C-type virus C is a lot of at present, such as electron microscopic observation, virus purification, inoculation test, serology, agar immunodiffusion method, but the bothersome effort of these methods. Traditional PCR, quantitative fluorescent PCR need expensive professional equipment, although China animal and veterinary station at county level is provided with PCR substantially detects related equipment, and operative technique requires height. The technical superiority of ring mediated isothermal amplification method is except high specific, highly sensitive, simple to operate, it may also be useful to water-bath or thermos cup just can realize reaction, it is not necessary to carry out gel electrophoresis as PCR.
Its susceptibility of porcine circovirus 2 type loop-mediated isothermal amplification detection method what voter etc. set up can reach the DNA molecular of 10 copies, the positive rate of LAMP and the positive rate coincidence rate of PCR are 94%, it is after the completion of reaction, add 2 �� LSYBRGreenI fluorescence dyes, carry out result judgement under ultraviolet light. But after the reaction product of LAMP is uncapped, easily form aerosol, it is easy to polluting laboratory, if reagent, pipettor, rifle head, super clean bench etc. are polluted, will there will be false positive, this is to causing disadvantageous effect for the production of practice, limits its range of application. So applicable dyestuff need to be added before the reaction. SYBRGreenI adds before the reaction easily affect LAMP reaction, and fluorexon dyestuff is added in reaction system by this research before the reaction, has reacted the rear positive and has managed color under ultraviolet light and become green, and feminine gender pipe is not owing to having product, still changes without color.
Set up LAMP diagnostic method it is crucial that the design of primer. Primer G/C content is preferably between 50%��60%. The 3 of all primers,The 5 of FIP, BIP,It is more little that free energy changes values (�� G) need to be less than 4, �� G, and the reaction between primer and template more easily occurs. The present invention to primer, by the optimization of reaction conditions, sets up LAMP diagnostic method according to porcine circovirus 2 type (PCV-2) ORF2 gene order, design 2. Result shows, susceptibility results shows, and the minimum detection of nucleic acids amount of the LAMP diagnostic method of foundation is 0.3pg/L, and the minimum detection of nucleic acids amount of PCR diagnostic method is 30pg/L, more highly sensitive than regular-PCR 100 times of LAMP. The detected result of pig sample of 95 parts of natural senses being caught an illness shows, this LAMP method detected result and PCR detected result coincidence rate are 95.8%, shows that the diagnostic method set up is applicable to the on-the-spot rapid quarantine of basic unit.
Accompanying drawing explanation
Fig. 1 is the foundation of LAMP detection method;
In Fig. 1,1:PCV2LAMP product; 2: negative control;
Fig. 2 is PCV-2LAMP sensitization test;
In Fig. 2,1��6:3.0ng/L �� 100��10-5The viral DNA LAMP result of dilution;
Fig. 3 is PCV-2PCR sensitization test;
In Fig. 3, M:D2000; 1��8:30mg/L �� 100��10-7The viral DNA PCR result of dilution;
Fig. 4 is the test of PCV2LAMP specificity;
In Fig. 4,1:PCV-2LAMP product; 2:PRVLAMP product; 3:PCV-1LAMP product; 4:PPVLAMP product;
5:CFSVLAMP product; 6:PRRSVLAMP product.
Embodiment
Embodiments of the invention: porcine circovirus 2 type LAMP diagnostic kit, it comprises the LAMPmix damping fluid of 250 �� L, 20 �� L concentration are the BstDNA polysaccharase of 0.5U/ �� L, 150 �� L ultrapure waters, the volume of inner primer 1 and inner primer 2 is 40 �� L, and concentration is 40pmol/L, and the volume of outer primer 1 and outer primer 2 is 40 �� L, concentration is 5pmol/L, 20 �� L negative controls and 20 �� 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; LAMPmix damping fluid comprises the MgCl of 3mmol/ul2, the Tris-HCl of the dNTP2.0 �� L and 25mmol/ �� L of 500pmol/ul, its PH value is 8.3.
SEQUENCELISTING
Sequence table
<110>Guizhou Province's animal and veterinary institute
<120>porcine circovirus 2 type LAMP diagnostic kit
<130>nm:
<160>4
<170>PatentInversion
<210>1
<211>20
<212>DNA
<213>artificial sequence
<220>
<223>according to porcine circovirus 2 type (PCV-2) the ORF2 gene order in GenBank, design and synthesis 2 to primer, for setting up porcine circovirus 2 type LAMP diagnostic method.
<400>1
TGGAGCTCCTCGATCTCAAG20
<210>2
<211>19
<212>DNA
<213>artificial sequence
<220>
<223>according to porcine circovirus 2 type (PCV-2) the ORF2 gene order in GenBank, design and synthesis 2 to primer, for setting up porcine circovirus 2 type LAMP diagnostic method.
<400>2
GCCCCACAATGACGTGTAC19
<210>3
<211>39
<212>DNA
<213>artificial sequence
<220>
<223>according to porcine circovirus 2 type (PCV-2) the ORF2 gene order in GenBank, design and synthesis 2 to primer, for setting up porcine circovirus 2 type LAMP diagnostic method.
<400>3
GCAACGGTCACCAGACTCCCGACAACGGAGTGACCTGTC39
<210>4
<211>41
<212>DNA
<213>artificial sequence
<220>
<223>according to porcine circovirus 2 type (PCV-2) the ORF2 gene order in GenBank, design and synthesis 2 to primer, for setting up porcine circovirus 2 type LAMP diagnostic method.
<400>4
AGAGCAGCACCCTGTAACGTTTACGCTTCTGCATTTTCCCG41
Claims (4)
1. a porcine circovirus 2 type LAMP diagnostic kit, it is characterized in that: it comprises the LAMPmix damping fluid of 250 �� L, 20 �� L concentration are the BstDNA polysaccharase of 0.5U/ �� L, 150 �� L ultrapure waters, the volume of inner primer 1 and inner primer 2 is 40 �� L, and concentration is 40pmol/L, and the volume of outer primer 1 and outer primer 2 is 40 �� L, concentration is 5pmol/L, 20 �� L negative controls and 20 �� 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, it is characterised in that: negative control is ultrapure water.
3. porcine circovirus 2 type LAMP diagnostic kit according to claim 1, it is characterised in that: positive control is porcine circovirus type 2 strain DNA.
4. porcine circovirus 2 type LAMP diagnostic kit according to claim 1, it is characterised in that: LAMPmix damping fluid comprises the MgCl of 3mmol/ul2, the Tris-HCl of the dNTP2.0 �� L and 25mmol/ �� L of 500pmol/ul, its PH value is 8.3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410094855.5A CN103820580B (en) | 2014-03-16 | 2014-03-16 | Porcine circovirus 2 type LAMP diagnostic kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410094855.5A CN103820580B (en) | 2014-03-16 | 2014-03-16 | Porcine circovirus 2 type LAMP diagnostic kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103820580A CN103820580A (en) | 2014-05-28 |
| CN103820580B true CN103820580B (en) | 2016-06-08 |
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| 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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| 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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| 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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