CN107488749A - A kind of LAMP primer and detection method for detecting the type of pig circular ring virus 3 - Google Patents
A kind of LAMP primer and detection method for detecting the type of pig circular ring virus 3 Download PDFInfo
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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
- C12Q1/701—Specific hybridization probes
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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
The present invention relates to virus detection techniques field, more particularly to a kind of LAMP primer for detecting the type of pig circular ring virus 3, sequence 16 in sequence table are seen.Expanded using LAMP primer, if any amplification, then contain the type of pig circular ring virus 3 in measuring samples, such as without amplification, then the type of pig circular ring virus 3 is not contained in measuring samples.This method is sensitive special, easy to be quick, available for PCV3 detections and clinical diagnosis.
Description
Technical field
The present invention relates to virus detection techniques field, more particularly to a kind of LAMP primer for detecting the type of pig circular ring virus 3, also
It is related to the LAMP detection method of the type of pig circular ring virus 3.
Background technology
Pig circular ring virus (Porcine circovirus, PCV) belongs to PCV-II section Circovirus member.The virus
There are two serotypes, i.e. PCV1 and PCV2.PCV2 infection can cause pmws (PMWS), pigskin scorching
Nephrotic syndrome (PDNS), porcine respiratory syndrome (PRDC), sow breeding difficulty syndrome, diarrhoea and congenital tremors etc. are more
Kind clinical disease, huge economic loss is brought to global aquaculture.But the cause of disease one of pigskin inflammation nephrotic syndrome (PDNS)
Directly disputed on.In recent years, American scholar etc. finds breeding difficulty sow, PDNS, porcine myocarditis and multisystem inflammation disease first
Become in tissue and a kind of new pig circular ring virus be present, its Cap gene order and PCV2 homologys only 24%-26%, be named as
PCV3.Chinese scholar detects PCV3 using PCR method from clinical onset pig lungs and lymph node tissue sample, it was demonstrated that China is present
The virus.
The detection of existing pig circular ring virus, is concentrated mainly on 1 type and 2 types, method have PCR method, ELISA antibody tests,
Mediated isothermal amplification fast detection method, as CN 104725490A disclose a kind of porcine circovirus 2 type (PCV2) total length Cap
The preparation method of albumen and the ELISA antibody assay kits using the Cap protein as antigen.The kit contains the above-mentioned pig of coating
The ELISA Plate of circovurus type 2 nucleocapsid protein, and contain sample diluting liquid, confining liquid, cleaning solution, enzyme conjugates, enzyme bottom
Thing solution, terminate liquid and positive and negative control serum, not only high specificity, sensitiveness are high during for detecting PCV2, while can advise greatly
Mould popularization and application.CN 101307367 discloses a kind of ring on porcine circovirus 2 type the invention belongs to field of animal medicine
Mediated isothermal amplification fast detection method, by using specific primer, utilize loop-mediated isothermal amplification technique (LAMP) platform
The specific region of target sequence is expanded, under a series of Quality Controls and negative, positive control auxiliary, from molecular level to justifying including pig
The viral nucleic acid of the type of circovirus virus 2 is detected, and method of the invention has the characteristics of easy, economic, quick, sensitive and special,
Have broad application prospects.
And to the detection of the type of pig circular ring virus 3, mainly there are PCR detections, such as fluorescence quantitative PCR detection, CN107083450A
Disclose the type PCR detection kit of pig circular ring virus 3 and detection method.The type PCR detection kit of pig circular ring virus 3, bag
Include：DNA extracts reagents, 2 × TaqPCR Mix, sense primer and anti-sense primer；The sequence of the sense primer is SEQ ID
Shown in No.1, the sequence of the anti-sense primer, can using the PCR detection method of the kit for shown in SEQ ID No.2
Quickly, the special type of detection pig circular ring virus 3.The invention discloses one kind detection pig circular ring virus 3 type disease by CN 106929607A
Primer pair, method and the kit of poison.The primer pair is made up of sense primer and anti-sense primer, and sequence is respectively such as SEQ ID NO:
1 and SEQ ID NO:Shown in 2.Kit includes the primer pair, can also include other detection reagents.The detection pig annulus
The method of viral 3 types virus, it is using the genomic DNA of testing sample as template, is carried out using primer pair or kit glimmering in real time
Fluorescent Quantitative PCR is reacted, and result interpretation is carried out according to melting curve and amplification curve Cq values.Accuracy of the present invention is high, specificity is good,
It is reproducible, easy to operate, can accurately, fast and efficiently carry out identification PCV3, being advantageous to promote in clinical practice should
With.The ring mediated isothermal amplification detection for not having the type of pig circular ring virus 3 is recorded.
Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) has warp
Help the features such as practical, efficient, quick and easy, a variety of pig Pathogen tests are had been used to, such as swine fever, porcine reproductive and respiratory syndrome
With PRV etc., but there is no PCV3 LAMP detection methods to report.This research is special according to PCV3 gene orders, design
Primer, has been successfully established a kind of PCV3 LAMP detection methods, can be directly by visually sentencing in 35min under 56 DEG C of constant temperatures
It is fixed, and with compared with high specific and sensitiveness.Therefore, a kind of ring mediated isothermal amplification detection side of the type of pig circular ring virus 3 is studied
Method, in the detection of the type of pig circular ring virus 3, it is very important.
The content of the invention
In order to solve the problems, such as loop-mediated isothermal amplification detection method of the above in the prior art without the type of pig circular ring virus 3,
This application discloses a kind of LAMP primer for detecting the type of pig circular ring virus 3.
Present invention also offers the LAMP detection method of the type of pig circular ring virus 3.
What the present invention was obtained through the following steps：
A kind of LAMP primer for detecting the type of pig circular ring virus 3, base sequence are as follows：
F3：5 '-TCCAGTTTTTTCCGGGACAT-3 ', (see sequence 1 in sequence table)
B3：5 '-AACACTTGGCTCCAAGACG-3 ', (see sequence 2 in sequence table)
FIP：5 '-CTTTTTCTCCAGACCCACCCCA-AAAGCAGTGCTCCCCATTG-3 ', (see sequence in sequence table
3)
BIP：5 '-TTCCCGCCAGAATTGGTTTCGG-GCGGAAAGTTCCACTCGTAA-3 ', (see sequence in sequence table
4)
LF：5 '-ACACATATGACCCCACCGT-3 ', (see sequence 5 in sequence table)
LB：5’-GGTGAAGTAACGGCTGTGTTT-3’.(see sequence 6 in sequence table)
A kind of detection method of the type of pig circular ring virus 3, extract measuring samples DNA, the LAMP primer in usage right requirement 1
Expanded, if any amplification, then contain the type of pig circular ring virus 3 in measuring samples DNA, such as without amplification, then treat sample
The type of pig circular ring virus 3 is not contained in product DNA.
Described detection method, preferably 25 μ L optimal reaction system compositions are：The μ L of 10 × Thermopol Buffer 2.5,
MgSO4(100mmol·L-1) 1.5 μ L, dNTP (10mmolL-1) 3.5 μ L, outer primer F3/B3 (4 μm of ol/L) each 1 μ L, inside draw
Thing FIP/BIP (40 μm of ol/L) is respectively 1 μ L, and ring primer LF/LB (10 μm of ol/L) is respectively 1 μ L, Bst archaeal dna polymerases (8000U/
ML) 1.0 μ L, the μ L of glycine betaine (Betaine) 2, sterilize ddH2The μ L of O 6.5 μ L, template cDNA 1.5.
Described detection method, preferable reaction temperature are 59 DEG C, reaction time 36min.
Described detection method, preferably developed the color using the dyestuffs of SYBR Green I.
Described detection method, detection are limited to 1.0 × 101copies/μL。
Beneficial effects of the present invention：
This method lowest detection is limited to 1.0 × 101Copies/ μ L, with porcine reproductive and respiratory syndrome virus (PRRSV),
The equal no cross reactions such as PRV (PRV), CSFV (CSFV) and the type (PCV1, PCV2) of pig circular ring virus 1 and 2.
This method is used to detect 68 parts of respiratory tract sick pig node sample, PCV3 positive rates 30.9%, is with PCR testing result coincidence rates
95.6% (65/68).This method is sensitive special, easy to be quick, available for PCV3 detections and clinical diagnosis.
Brief description of the drawings
Fig. 1 is PCV3 LAMP reaction systems and condition optimizing result, A:DNTP concentration optimizations；B:Mg2+ optimizes；C:Beet
Alkali optimizes；D:Inside and outside primer concentration is than optimization；E:Temperature optimization；F:It is time-optimized
Fig. 2 tests for LAMP reaction sensibilities, A:LAMP electrophoresis results；B:RT-PCR；C:LAMP-SYBR Green I are aobvious
Color.M:DL2000 DNA molecular quality standards；1~9:107~100.01 gene copies/mL；10:Negative control
Fig. 3 is LAMP specific test results, A：LAMP detects Different Kinds of Pathogens electrophoresis result：M:DNA marker
DL2000；1-7:1.Negative control；2.CSFV；3.PEDV；4:PCV1；5:PCV2a+2b；6:PRV；7:PCV3,
8.PRRSV, respectively, B.LAMP reaction product endonuclease digestion product electrophoresis result.
Embodiment
The present invention is further described with reference to specific embodiment：
Embodiment 1
1 material and method
1.1 viruses, recombinant plasmid and pathological material of disease
Virus：PCV3, PCV1, PCV2a, PCV2b, PRV (PRV), CSFV (CSFV), pig are popular
Diarrhea virus (PEDV) etc. is separated by this laboratory and preserved.
Recombinant plasmid：PMD19-T-Cap, gene containing PCV3ORF2, concentration are 1.89 × 10-7Ng/ μ L, gene copy number are
5.22×1010Copies/ μ L, built and provided by Nanjing Jin Sirui Bioisystech Co., Ltd.
Pig clinical sample：From 2016-2017 Jiangsu, Shandong, Zhejiang and 38, Fujian Province pig farm, clinical signs
68 parts of respiratory symptom, lymph node and lung tissue sample.- 20 DEG C save backup.
1.2 viral nucleic acids extract
It is stripped according to DNA extraction kit specification, viral nucleic acid is extracted using TRIZOL reagent specifications method,
Put -20 DEG C of preservations.
1.3LAMP design of primers and synthesis
According to PCV3 strain Cap gene conserved sequences, with online biosoftware (https://primere
Explorer.jp/ specific primer) is designed, primer sequence is shown in Table 1, including a pair of outer primers F3 and B3, a pair of inner primer FIP
Synthesized with BIP and a pair of rings primer LoopF and LoopB, primer by Invitrogen companies.
The LAMP amplimers of table 1
1.4LAMP reaction system
The μ L of LAMP reaction systems 25, containing 10 × Thermopol Buffer (NEB companies) 2.5 μ L, MgSO4(100mmol·
L-1) (NEB companies) 1.5 μ L, dNTP (10mmolL-1) (NEB companies) 3.5 μ L, inner primer FIP/BIP (40 μm of ol/L) be respectively
1 μ L, outer primer F3/B3 (5 μm of ol/L) each 1 μ L, ring primer LF/LB (10 μm of ol/L) are respectively 1 μ L, Bst archaeal dna polymerases
(8000U/mL) (NEB companies) 1.5 μ L, the μ L of glycine betaine (Betaine) (Sigma companies) 2.0, sterilize ddH2The μ L of O 6.5, template
DNA 1.5μL。
1.5LAMP reaction systems and condition optimizing
Reaction system optimization is carried out by the concentration of following ingredients：DNTP concentration gradients 4,6,8,10 and 12mM；Mg2+Concentration
120th, 100,80,60 and 40mM；Beet alkali concn 4.0,3.0,2.0,1.0 and 0 μ L；Inside and outside primer is than 36 μM:3μM、40:4、
40:5、36:6 and 32:8.
Reaction temperature optimizes：Temperature setting (67,65,63,61,59 and 57 DEG C)；Reaction time be arranged to (40,38,36,
34 and 32min) 5 gradients, reaction takes 5 μ L reaction products in 1.5% agarose gel electrophoresis qualification result after terminating, according to bar
Band expanding effect determines optimum reaction condition.
Using various concentrations component in LAMP systems, reaction condition optimization is carried out, Fig. 1 is as a result seen, determines that 25 μ L are most preferably anti-
The system composition of answering is：10 × Thermopol Buffer 2.5 μ L, MgSO4(100mmol·L-1) 1.5 μ L, dNTP (10mmol
L-1) 3.5 μ L, outer primer F3/B3 (4 μm of ol/L) each 1 μ L, inner primer FIP/BIP (40 μm of ol/L) be respectively 1 μ L, ring primer LF/LB
(10 μm of ol/L) is respectively 1 μ L, Bst archaeal dna polymerases (8000U/mL) 1.0 μ L, the μ L of glycine betaine (Betaine) 2, and sterilize ddH2O
The μ L of 6.5 μ L, template cDNA 1.5.Optimal reaction temperature is 59 DEG C, and most short reaction time is 36min.
Reaction product uses HNB (source leaf biology Co., Ltd) and two kinds of SYBR Green I (Invitrogen companies) respectively
Dyestuff carries out colour developing comparison, as a result shows：Using the LAMP high sensitivities of the dyestuffs of SYBR Green I in HNB dyestuffs, Er Qieyan
Colour contrast is more obvious.
In LAMP courses of reaction, the Mg in pyrophosphate ion and reaction solution that dNTP is separated out2+With reference to generation pyrophosphoric acid
Magnesium white precipitate.SYBR Green I then produce green fluorescence by being attached to the ditch of double-stranded DNA, and yellow is presented in feminine gender.
HNB is as a kind of Metal ion indicator, Mg2+Combined with HNB so that reaction system priming color is purple, with entering for reaction
OK, Mg2+Reacted with the pyrophosphate of precipitation so that system color is changed into blueness.Three kinds of reaction results compare, SYBR Green I
Color developing effect is more preferable.
1.6LAMP sensitivity tests
Recombinant plasmid pMD19-T-Cap positive plasmid standard items are diluted to 106Copies/ μ L, by 10 times of doubling dilutions
To 100.1Copies/ μ L, it is same with the PCV3-LAMP methods and fluorescence quantifying PCR method and conventional RT-PCR method that have optimized
Step carries out sensitiveness comparison.And result above is developed the color with SYBR Green two kinds of dyestuffs of I and HNB respectively, screening is optimal
Dyestuff.
Taking PCV3 recombinant plasmids pMD19-T-Cap, (concentration is 1.89 × 10-7Ng/ μ L, gene copy number be 5.22 ×
1010Copies/ μ L), do 10 times of gradient dilutions with distilled water, PCV3 detected using LAMP and PCR, as a result for：Conventional PCR method
Detectable 102The plasmid of copies/ μ L concentration, the plasmid of the detectable 10copies/ μ L concentration of LAMP reactions, compares Standard PCR
Method is sensitive 10 times.See Fig. 2.
1.7LAMP specific test
PRRSV, PRV, PCV1, PCV2 and CSFV sample are taken, viral genome is extracted, is detected with LAMP method, is used
Electroresis appraisal in 1.5% Ago-Gel.
With the restriction enzyme site in Olige software analysis LAMP purpose fragments, single Hae II (identification GCGC) are screened, are entered
Row endonuclease reaction.20 μ L endonuclease reaction systems include：1.5 μ L, LAMP products of Hae II (Takara companies) 1 μ L, 10 ×
Buffer 2 μ L, 15.5 μ L ddH2O.37 DEG C of reaction 2h, take 15 μ L digestion products to be identified in 1.5% Ago-Gel,
Digestion products size should be 184bp.
Take PCV3, PRRSV, PRV, PCV1, PCV2 and CSFV sample to carry out PCV3 LAMP detections, as a result show,
PCV3 samples LAMP expands visible trapezoid-shaped strips, and other Pathogen tests are showed no band (A in Fig. 3).
PCV3 LAMP amplified productions are taken, using the digestions of Hae II, electrophoresis result is shown on 1.5% Ago-Gel, is occurred
About 184bp bands (B in Fig. 3), it is consistent with expected result of calculation, it was demonstrated that amplification is correct.
1.8PCR methods detect PCV3
Carried out by the methods of Palinski.PCR reaction systems are：94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 20s, 50 DEG C are moved back
Fiery 30s, 72 DEG C of extension 1min, carries out 40 circulations；Last 72 DEG C of extensions 5min.Primer is 5 '-
CCACAGAAGGCGCTATGTC-3 ' and 5 '-CCGCATAAGGGTCGTCTT G-3 ', PCR primer is through 1.5% sepharose electrophoresis
Observation.
Clinical sample testing result
68 parts of clinical onset porcine tissue samples are taken, is detected simultaneously with Standard PCR using LAMP method, be the results are shown in Table 2, show
LAMP detection PCV3 positive rates are 30.9% (21/68), and Standard PCR positive rate is 26.5% (18/68), and LAMP method is examined
Extracting rate is higher than Standard PCR testing result, and LAMP and Standard PCR coincidence rate are 95.6% (65/68).
The comparison of the LAMP of table 2 and PCR detection clinical sample PCV3 results
Table 2 Comparison of LAMP with PCR results for PCV3 in clinical
samples
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not limited by embodiment
System, it is other it is any without departing from the present invention Spirit Essences with made under principle change, modification, combine, replacement, simplification should be
Equivalence replacement mode, is included within protection scope of the present invention.
<110>Agricultural University Of Nanjing
<120>A kind of LAMP primer and detection method for detecting the type of pig circular ring virus 3
<160>6
<210>1
<211>20
<212>DNA
<213>It is artificial synthesized
<400>1
TCCAGTTTTT TCCGGGACAT 20
<210>2
<211>19
<212>DNA
<213>It is artificial synthesized
<400>2
AACACTTGGC TCCAAGACG 19
<210>3
<211>41
<212>DNA
<213>It is artificial synthesized
<400>3
CTTTTTCTCC AGACCCACCC CAAAAGCAGT GCTCCCCATT G 41
<210>4
<211>42
<212>DNA
<213>It is artificial synthesized
<400>4
TTCCCGCCAG AATTGGTTTC GGGCGGAAAG TTCCACTCGT AA 42
<210>5
<211>19
<212>DNA
<213>It is artificial synthesized
<400>5
ACACATATGA CCCCACCGT 19
<210>6
<211>21
<212>DNA
<213>It is artificial synthesized
<400>6
GGTGAAGTAA CGGCTGTGTT T 21
Claims (6)
1. a kind of LAMP primer for detecting the type of pig circular ring virus 3, it is characterised in that base sequence is as follows：
F3：5 '-TCCAGTTTTTTCCGGGACAT-3 ',
B3：5 '-AACACTTGGCTCCAAGACG-3 ',
FIP：5 '-CTTTTTCTCCAGACCCACCCCA-AAAGCAGTGCTCCCCATTG-3 ',
BIP：5 '-TTCCCGCCAGAATTGGTTTCGG-GCGGAAAGTTCCACTCGTAA-3 ',
LF：5 '-ACACATATGACCCCACCGT-3 ',
LB：5’-GGTGAAGTAACGGCTGTGTTT-3’.
2. a kind of detection method of the type of pig circular ring virus 3, it is characterised in that measuring samples, the LAMP in usage right requirement 1 draw
Thing is expanded, and if any amplification, then contains the type of pig circular ring virus 3 in measuring samples, such as without amplification, then measuring samples
In do not contain the type of pig circular ring virus 3.
3. detection method according to claim 2, it is characterised in that 25 μ L optimal reaction system compositions are： 10×
Thermopol Buffer 2.5 μ L, 100 mmolL-1M gSO41.5 μ L, 10 mmolL-1The μ of dNTP 3.5
L, 4 μm of ol/L outer primer F3/B3 each 1 μ L, 40 μm of ol/L inner primer FIP/BIP are respectively 1 μ L, 10 μm of ol/L ring primer
LF/LB is respectively 1 μ L, the 8000U/mL μ L of Bst archaeal dna polymerases 1.0, the μ L of glycine betaine 2, and sterilize ddH2The μ L of O 6.5, mould
The μ L of plate cDNA 1.5.
4. detection method according to claim 2, it is characterised in that reaction temperature is 59 DEG C, reaction time 36min.
5. detection method according to claim 2, it is characterised in that developed the color using the dyestuffs of SYBR Green I.
6. detection method according to claim 2, it is characterised in that detection is limited to 1.0 × 101 copies/µL。
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Cited By (6)
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| CN107881261A (en) * | 2017-12-23 | 2018-04-06 | 广东省农业科学院动物卫生研究所 | Detect LAMP primer group, kit and the application of the type of pig circular ring virus 3 |
| CN107937616A (en) * | 2017-12-28 | 2018-04-20 | 广州维佰生物科技有限公司 | Detect the LAMP primer composition thing and its kit and method of PCV3 |
| CN108531660A (en) * | 2018-05-31 | 2018-09-14 | 广西壮族自治区兽医研究所 | A kind of 3 type real-time quantitative LAMP primer of detection pig circular ring virus, kit and application |
| CN109182468A (en) * | 2018-08-24 | 2019-01-11 | 暨南大学 | Primer and its kit and method based on digital LAMP technology detection 3 type of pig circular ring virus |
| CN110423846A (en) * | 2019-07-26 | 2019-11-08 | 博奥生物集团有限公司 | A kind of primer combination, detection method and the kit of the LAMP for the parting detection that can distinguish porcine circovirus 2 type and 3 types |
| CN112176104A (en) * | 2020-10-09 | 2021-01-05 | 中国农业科学院兰州兽医研究所 | Visual LAMP (loop-mediated isothermal amplification) detection kit for porcine circovirus type 3 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881261A (en) * | 2017-12-23 | 2018-04-06 | 广东省农业科学院动物卫生研究所 | Detect LAMP primer group, kit and the application of the type of pig circular ring virus 3 |
| CN107937616A (en) * | 2017-12-28 | 2018-04-20 | 广州维佰生物科技有限公司 | Detect the LAMP primer composition thing and its kit and method of PCV3 |
| CN108531660A (en) * | 2018-05-31 | 2018-09-14 | 广西壮族自治区兽医研究所 | A kind of 3 type real-time quantitative LAMP primer of detection pig circular ring virus, kit and application |
| CN109182468A (en) * | 2018-08-24 | 2019-01-11 | 暨南大学 | Primer and its kit and method based on digital LAMP technology detection 3 type of pig circular ring virus |
| CN110423846A (en) * | 2019-07-26 | 2019-11-08 | 博奥生物集团有限公司 | A kind of primer combination, detection method and the kit of the LAMP for the parting detection that can distinguish porcine circovirus 2 type and 3 types |
| CN112176104A (en) * | 2020-10-09 | 2021-01-05 | 中国农业科学院兰州兽医研究所 | Visual LAMP (loop-mediated isothermal amplification) detection kit for porcine circovirus type 3 |
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Application publication date: 20171219 |