CN105861744A - Primer combination used for rapid isothermal detection of porcine deltacoronavirus, and method thereof - Google Patents
Primer combination used for rapid isothermal detection of porcine deltacoronavirus, and method thereof Download PDFInfo
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- 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
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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 invention discloses a primer combination used for rapid isothermal detection of porcine deltacoronavirus, and a method thereof. The method using a loop-mediated isothermal amplification (LAMP) technology to detect the porcine deltacoronavirus, disclosed in the invention, is used to detect the porcine deltacoronavirus in an environment sample, a medical sample and the sanitation and epidemic prevention field. The method comprises the following steps: designing specific primers by adopting an N segment encoding region gene sequence in a porcine deltacoronavirus genome as a target sequence, optimizing a reaction system, and carrying out target gene specific amplification. The method only needs a constant temperature device, does not need thermotropy and long-time temperature circulation of a template, allows a result to be directly observed, and has the characteristics of low cost, high efficiency and simple operation. The porcine deltacoronaviru nucleic acid LAMP detection method established in the invention also has the characteristics of strong specificity, high sensitivity, high convenience and fastness, can be implemented in a base laboratory and a miniature experiment station, and provides a new technology and method for detection of the porcine deltacoronaviru.
Description
Technical field
The present invention relates to virus examination technology, specifically use ring mediated isothermal amplification method to detect pig Delta coronavirus.
Background technology
Grice diarrhoea is the one common property multiple diseases affecting Swine herd production, transmissible gastro-enteritis virus (Transmissible
Gastroenteritis virus, TGEV), Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV), pig
Rotavirus (Porcine rotovirus, PRoV) is the main pathogen of China's epizootic disease toxicity grice diarrhoea, wherein TGEV
It is coronavirus with PEDV.Within 2012, Hong Kong University scholar detects Novel pig coronavirus-pig Delta coronavirus
((porcine deltacoronavirus, PDCoV)), gene sequencing is shown to be δ coronavirus.Patrick in 2012 etc.
The result detecting 169 parts of diarrhea of pigs samples shows that pig Delta coronavirus positive rate is 10%；Marthaler etc. are to U.S.
The pig farm sample in state some areas in January, 2014 to February detects, and result PDCoV positive rate is 30%, shows PDCoV
Generally exist in the U.S. central and east.Causing the new discovery pathogen of grice diarrhoea, PDCoV has caused European Union and world animal
Whether the attention of health organization (OIE), contain the crown disease of pig Delta in detection domestic animal blood serum sample the most fast and accurately
Poison will assist in the propagation controlling virus, and the normal trade for the aquaculture animals and animal product of China provides strong technology
Support.
At present, the detection method of pig Delta coronavirus is neutralization test and fluorescence quantitative RT-RCR.The required detection of neutralization test
Overlong time, wastes time and energy；Fluorescence quantitative RT-RCR is higher to personnel and equipment requirement, it is difficult to popularizes at laboratories and pushes away
Extensively.Notomi etc. develop a kind of constant temperature nucleic acid amplification method, i.e. ring mediated isothermal amplification method (loop-mediated
Isothermal amplification, LAMP), it is characterized under isothermy (about 65 DEG C) acting on about 60min
Completing nucleic acid amplification reaction, what is more important the method need not valuable instrument and reagent, just can complete anti-in water-bath
Should, it is particularly suitable for on-the-spot in the wild and department of basic unit application.In recent years, LAMP method has been successfully used to diagnosis and has betided people
Class and the viral disease of animal, become the effective tool detecting multiple Causative virus.We establish a kind of utilization ring mediation etc.
The method of temperature amplification technique (LAMP) detection pig Delta coronavirus, for food and raw material, environmental samples, enter the territory boar blood
The detection of pig Delta coronavirus in final proof product.Its technical scheme is to encode N section district in pig Delta coronavirus gene group
Sequence for the purpose of domain gene sequence, designs specific primer, optimizing reaction system, carries out genes of interest specific amplification, this
One thermostat of bright need, it is not necessary to the thermal denaturation of template, long-time temperature cycles, and can direct observed result, tool
There are low cost, high efficiency, feature simple to operate.
The pig Delta coronavirus nucleic acid LAMP detection method that the present invention sets up has high specificity, highly sensitive, convenient and swift
Etc. feature, can carry out in small test base, provide a kind of new technology and method for the detection of pig Delta coronavirus.
Summary of the invention
In order to improve animal epidemic detection efficiency, save the time, Imported and exported animals cannot be met day by day solving traditional detection method
The present situation increased, the present invention, through experiment detection, finally explores one and uses ring mediated isothermal amplification method detection pig Delta hat
The method of shape virus, the method has quick, convenient, the low cost of detection, is suitable for the spies such as on-the-spot in the wild and department of basic unit application
Point.
It is an object of the invention to provide a kind of method applying ring mediated isothermal amplification method detection pig Delta coronavirus.
The present invention is achieved by the following technical solutions:
The primer of a set of inspection pig Delta coronavirus, it is characterised in that: detect pig Dare by loop-mediated isothermal amplification technique
The primer sets of tower coronavirus, sequence is: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
Above-mentioned primer can detect at least 10 pig Delta coronavirus copied.
Above-mentioned primer is used for the application checked in the kit of pig Delta coronavirus in preparation.
A kind of for checking the kit of pig Delta coronavirus, including:
(1) loop-mediated isothermal amplification reaction reagent, including the composition of composition each in amplification system；
(2) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 are proportionally mixed.
(3) positive sample comparison, ' negative ' specimens comparison；
(4) operation instructions.
Also including SYBR Green I dyestuff in mentioned reagent box, joining can be anti-at uviol lamp and observed under daylight in product
Should the change of pipe color.
Mentioned reagent box is utilized to carry out ring mediated isothermal amplification detection pig Delta coronavirus method, including:
(1) extraction of RNA and the preparation of cDNA
Sampling originally, after inactivation of virus, carries out reverse transcription reaction, obtains cDNA, and-20 DEG C standby；
(2) detection in pig Delta coronavirus N fragment gene district
2 μ L cDNA samples, 5 μ L Bst DNA polymerase buffer liquid (10 ×), 2 μ L Bst archaeal dna polymerase (8000U/L),
5 μ L dNTP (10mmol/L), 3 μ L Betaine (5mol/L), 6 μ L MgSO4 (25mmol/L), corresponding to N section base
The primer SEQ as claimed in claim 1 ID NO:1 of cause, each 4 μ L of SEQ ID NO:2, SEQ ID NO:3, SEQ
The each 0.5 μ L of IDNO:4,18 μ LH2O, mixing；
(3) water bath with thermostatic control or hot block expand or any insulation material 60 DEG C～65 DEG C, 30min～90min；
(4) amplified production uses fluorescent dye determination to synchronize detection.
Said method is able to detect that at least 10 pig Delta coronavirus copied.
The method is in detection food and raw material, environmental samples, kind of the pig anteserum sample of entering the territory in terms of pig Delta coronavirus
Purposes.Can carry out in basic unit or small test base, also apply be applicable to the aspect such as Site Detection, evaluation of hygiene simultaneously.
Accompanying drawing explanation
Fig. 1 N fragment gene LAMP expands electrophoresis result (1,100bp Marker；2, N genes；3, negative control)；
Fig. 2 N fragment gene LAMP amplified production cleavage map (1,100bp Marker；2, N genes；3, it is digested result)；
Fig. 3 LAMP expands visible ray result (1, N gene；2, negative control)；
Fig. 4 LAMP expands ultraviolet light result (1, N gene；2, negative control)；
Fig. 5 N genetic test sensitivity (1,100bp Marker；2,107Copies/ μ L,；3,106copies/μL；
4,105copies/μL；5,104copies/μL；6,103copies/μL；7,102copies/μL；8,101Copies/ μ L 9,100)copies/μL；
Fig. 6 specific test (1,100bp Marker；2, negative control；3, N genes；4, transmissible gastroenteritis of swine
Virus；5, Porcine epidemic diarrhea virus；6, porcine rotavirus；7, foot and mouth disease virus；8 vesicular stomatitis virus).
Detailed description of the invention
Only further describe the present invention by the mode with reference to following non-limiting embodiment now.It is understood that below
Embodiment is merely possible to illustration, should be by any way when making the restriction overall to the invention described above.Unless otherwise noted,
Embodiments of the invention use the molecular biology routine techniques in this area.These technology are known to technical staff, and at literary composition
Explain in detail in offering.
Material and method
1.1 material
Total RNA extraction reagent box, DNA extraction kit, Bst archaeal dna polymerase, M-MULV reverse transcriptase, RNase
Inhibitor, Dpn II restriction endonuclease are purchased from NEB company；Plasmid Mini Kit, Escherichia coli TOP10 competent cell
Purchased from TIANGEN company；Betaine (glycine betaine), MgSO4 are purchased from Sigma company.
The design of 1.2 primers and synthesis
According to logging in the pig Delta coronavirus N fragment gene sequence of GenBank database, applied biology software carries out sequence
Comparison, in the conserved regions design 2 pairs of primers (outer primer and inner primers) of N fragment gene, the synthetic modification of primer is by handsome company
Completing, sequence is shown in Table 1.
Table 1 primer and probe sequence
The extraction of 1.3 RNA and the preparation of cDNA
EP pipe water-treated for the DEPC that learns from else's experience, adds 1mL TRIzol and 200 μ L virus liquids, and after mixing, room temperature places 5
min；Adding 200 μ L chloroforms, firmly rock 30s, room temperature stands 3min, 4 DEG C of centrifugal 15min, obtains being layered liquid；Take
Upper liquid moves into clean EP pipe, adds 500 μ L isopropanols, and-20 DEG C stand 30min, 4 DEG C of centrifugal 15min；Remove
Upper strata suspension；Add the DEPC ethanol of 1mL 75%, vortex oscillation, 4 DEG C of centrifugal 10min；Remove supernatant, air drying
5min；50 μ L DEPC water are added in test tube.Carry out reverse transcription with reference to M-MuLV reverse transcriptase process, obtain
CDNA ,-20 DEG C standby.
1.4 the structure of positive recombinant plasmid
Utilize the specific primer for pig Delta coronavirus N fragment gene, cDNA is carried out PCR expansion in 50 μ L systems
Increasing, the PCR primer obtained is cloned into after purification in pGEM-T easy carrier and converts to Escherichia coli TOP10 competence
In cell, from the recombinant bacterium purifying amplification, extract plasmid, carry out being digested qualification, choose positive recombinant plasmid and check order, from
And be that detection provides positive plasmid, and utilize this positive plasmid that LAMP method is optimized.
1.5 pig Delta coronavirus loop-mediated isothermal amplification method detections
After groping and optimizing reaction condition, set up following amplification reaction system: 2 μ L cDNA samples, 5 μ L Bst DNA are poly-
Synthase buffer solution (10 ×), 2 μ L Bst archaeal dna polymerase (8000U/L), 5 μ L dNTP (10mmol/L), 3 μ L Betaine
(5mol/L), 6 μ L MgSO4 (25mmol/L), corresponding to primers F IP of N fragment gene and each 4 μ L, F3 and the B3 of BIP
Each 0.5 μ L, 18 μ LH2O.Mixing, 65 DEG C, water-bath 1.5h.
The detection of 1.6 amplified productions
After LAMP reaction terminates, take 5 μ L amplified productions and carry out 1.5% agarose gel electrophoresis；Remaining amplified production uses fluorescence
Dye method synchronizes detection, adds the SYBR Green I dyestuff of 2 μ L 50 times dilution, in amplification pipe under uviol lamp and daylight
Observing response pipe color changes.Reclaim the positive products of N fragment gene, carry out being digested qualification, reaction system: 8 μ LN genes return
Receive product, 1 μ L Dpn II restriction endonuclease, 2 μ L restriction endonuclease buffer solution (10 ×), 9 μ LH2O；Mixing, 37 DEG C, water-bath 8h.
Recovery product with N gene carries out regular-PCR amplification for template simultaneously, and amplified production serves sea raw work order-checking.
The detection sensitivity of 1.7 pig Delta coronavirus N fragment genes
Take positive plasmid DNA, measure concentration through ultraviolet specrophotometer, after 10 times of serial dilutions, carry out the crown disease of pig Delta
The sensitivity technique test of poison N fragment gene.Concrete testing program is carried out with reference to the routine operation program of LAMP.
1.8 specific test
Respectively with transmissible gastro-enteritis virus, Porcine epidemic diarrhea virus, porcine rotavirus, foot and mouth disease virus, vesiculovirus mouth
Scorching viral nucleic acid is as testing sample, with the pig Delta coronavirus N fragment gene DNA that extracted as positive control,
With the water that processes through DEPC as negative control, the method for inspection specific.
Embodiment 1
The amplification of pig Delta coronavirus N fragment gene
Sampling originally, after extracting viral genome, carries out reverse transcription with reference to M-MuLV reverse transcriptase process, obtains cDNA,
Utilize specific primer that pig Delta coronavirus N fragment gene is expanded, after amplification, obtain S fragment gene 1029bp, with
Theoretical amplification value is consistent.
Embodiment 2
The detection of pig Delta coronavirus LAMP method
1.1 Gel electrophoresis results are observed
After groping and optimizing reaction condition, set up the LAMP detection method for pig Delta coronavirus N fragment gene,
Testing result is as it is shown in figure 1, the amplification stepped distribution of amplifying nucleic acid electrophoretic band in N fragment gene district, with notional result phase
Symbol, negative control has no that specific product occurs.
1.2 amplified productions be digested qualification
Reclaiming the positive amplification product of N fragment gene, application Dpn II restriction endonuclease carries out being digested qualification.Result is as in figure 2 it is shown, N
Gene is 164bp and 45bp band through being digested rear electrophoresis result, is consistent with notional result, without the positive amplification product being digested
It it is still stepped band.
1.3 dye method results are observed
LAMP method amplified production adds the SYBR Green I dyestuff of 2 μ L 50 times dilution, sees under uviol lamp and daylight
Examine the change of reaction tube color.Shown in Fig. 3, N fragment gene positive amplification pipe is under visible light in yellow green, and negative control is Exocarpium Citri Rubrum
Look.In Fig. 4, N fragment gene positive amplification pipe visible fluorescence under ultraviolet light produces, and in negative control pipe, unstressed configuration produces.Return
Receiving the positive products of N fragment gene, after carrying out regular-PCR amplification, order-checking is learnt, extension increasing sequence and the pig in GenBank database
Delta coronavirus N fragment gene is consistent, shows LAMP method specific augmentation detection pig Delta coronavirus N Duan Ji
Cause.
Embodiment 3
The detection sensitivity of pig Delta coronavirus N fragment gene
Take positive plasmid DNA, measure concentration through ultraviolet specrophotometer, after 10 times of serial dilutions, carry out pig Delta crown
The sensitivity technique test of virus N fragment gene.The coronavirus N fragment gene of pig Delta shown in Fig. 5 10 copies above dilute
Degree of releasing all shows that typical LAMP expands banding pattern, illustrates that the sensitivity of this detection method can reach the level of 10 copies.
Embodiment 4
Specific test
Respectively with transmissible gastro-enteritis virus, Porcine epidemic diarrhea virus, porcine rotavirus, foot and mouth disease virus, vesiculovirus mouth
Scorching viral nucleic acid is as testing sample, with the pig Delta coronavirus N fragment gene DNA that extracted as positive control,
With the water that processes through DEPC as negative control, the method for inspection specific.As shown in Figure 6, positive control and feminine gender are right for result
According to all setting up, being positive with the DNA constructed by pig Delta coronavirus, other test specimens are negative, and show examination
Test the primer good to pig Delta coronavirus specific.
Loop-mediated isothermal amplification technique (LAMP) is a kind of new nucleic acid isothermal amplification technology, and principle is in genes of interest fragment
Multiple sites on design 4 or 6 primers, as long as generally 4 primers can complete the amplification of genes of interest, if added
Two other ring primer, can effectively accelerate whole course of reaction, but increasing of reaction system inner primer also can increase primer two
The formation of aggressiveness.LAMP reaction result method of discrimination is various, and the most conventional has electrophoresis, fluorescence method, nephelometry and calcium yellow
Green element method.Nephelometry and calcein method are the methods that a large amount of pyrophosphate accessory substances produced based on LAMP reaction carry out detecting,
Therefore all there is a certain degree of ambient interferences in these 2 kinds of methods, and interference strength extends with the reaction time and increases；Electrophoresis and
Fluorescence method utilizes double-strand interca-lating dyes directly to detect amplified production, and therefore background is lower, and wherein the positive of fluorescence method, feminine gender can use
Naked eyes are distinguished, and require low to the technology of operating personnel, are more suitable for field quick detection.
LAMP technology is used to establish the detection method of detection pig Delta coronavirus nucleic acid herein, by reporting with document
Real-time fluorescence RT-PCR method compares, and the detection sensitivity of two kinds of methods is suitable, by 107The virus quantity plasmid replicated is carried out
After 10 times of serial dilutions, the LAMP method detection sensitivity that application is set up herein is 10 copies；From the detection time
LAMP completes 1 detection and can complete in 1.5h, and real-time fluorescence RT-PCR rule needs 3h.Reality from required instrument
Time fluorescence RT-PCR need quantitative real time PCR Instrument, and LAMP only needs thermostat water bath.
In sum, the pig Delta coronavirus nucleic acid LAMP detection method that this research is set up has high specificity, sensitivity
The feature such as high, convenient and swift, can be carried out in basic unit or small test base, also apply be applicable to simultaneously Site Detection, evaluation of hygiene,
The aspects such as clinical diagnosis, provide a kind of new technology and method for the detection of pig Delta coronavirus.
Claims (8)
- The primer of the most a set of inspection pig Delta coronavirus, it is characterised in that: detect pig moral by loop-mediated isothermal amplification technique The primer sets of your tower coronavirus, sequence is: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, Above-mentioned primer can detect at least 10 pig Delta coronavirus copied.
- 2. the primer described in claim 1 is used for the application checked in the kit of pig Delta coronavirus in preparation.
- 3. for checking a kit for pig Delta coronavirus, including:(1) loop-mediated isothermal amplification reaction reagent, including the composition of composition each in amplification system；(2) SEQ ID NO:1 described in the claim 1 proportionally mixed, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4.(3) positive sample comparison, ' negative ' specimens comparison；(4) operation instructions.
- Kit the most according to claim 3, the most also includes SYBR Green I dyestuff, joins in product permissible Change at uviol lamp and observed under daylight reaction tube color.
- 5. utilize the primer of claim 1 or the kit of claim 3 to carry out ring mediated isothermal amplification detection pig Delta crown Viral methods, including:(1) extraction of RNA and the preparation of cDNASampling originally, after inactivation of virus, carries out reverse transcription reaction, obtains cDNA, and-20 DEG C standby；(2) detection of pig Delta coronavirus N gene regions2 μ L cDNA samples, 5 μ L Bst DNA polymerase buffer liquid (10 ×), 2 μ L Bst archaeal dna polymerase (8000U/L), 5 μ L dNTP (10mmol/L), 3 μ L Betaine (5mol/L), 6 μ L Mg SO4 (25mmol/L), corresponding to N The primer SEQ as claimed in claim 1 ID NO:1 of fragment gene, each 4 μ L of SEQ ID NO:2, SEQ ID NO:3, SEQ The each 0.5 μ L of ID NO:4,18 μ L H2O, mixing；(3) water bath with thermostatic control or hot block expand or any insulation material 60 DEG C～65 DEG C, 30min～90min；(4) amplified production uses fluorescent dye determination to synchronize detection.
- 6. method is able to detect that at least 10 pig Delta coronavirus copied as claimed in claim 5.
- 7. method is detecting food and raw material, environmental samples, is entering the territory and plant pig Dare in pig anteserum sample as claimed in claim 5 Purposes in terms of tower coronavirus.
- 8. method can be carried out in basic unit or small test base as claimed in claim 5, can be applicable to Site Detection, health The aspects such as evaluation.
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Cited By (8)
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| CN107326101A (en) * | 2017-07-19 | 2017-11-07 | 湖北出入境检验检疫局检验检疫技术中心 | The RT LAMP detection primers of crucian coronavirus HB93 a kind of and application |
| CN107460255A (en) * | 2017-08-30 | 2017-12-12 | 广西壮族自治区兽医研究所 | A kind of RT LAMP primers group, kit and application for detecting pig fourth type coronavirus |
| CN109536642A (en) * | 2018-12-19 | 2019-03-29 | 长江大学 | A kind of universal pig fourth type coronavirus RT-Nested PCR detection method |
| CN110982935A (en) * | 2019-12-13 | 2020-04-10 | 华南农业大学 | LAMP primer composition for detecting porcine delta coronavirus by adopting microfluidic chip technology and kit thereof |
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| CN112410465A (en) * | 2020-03-27 | 2021-02-26 | 大连民族大学 | Novel coronavirus SARS-CoV-2ORF1ab and N gene constant temperature amplification primer group and kit |
| CN113981140A (en) * | 2021-10-09 | 2022-01-28 | 广州达安基因股份有限公司 | Detection method of novel coronavirus delta mutant strain and nucleic acid detection kit |
| CN113981149A (en) * | 2021-11-23 | 2022-01-28 | 广东省农业科学院动物卫生研究所 | Porcine delta coronavirus detection primer group, probe, kit and application |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107326101A (en) * | 2017-07-19 | 2017-11-07 | 湖北出入境检验检疫局检验检疫技术中心 | The RT LAMP detection primers of crucian coronavirus HB93 a kind of and application |
| CN107460255A (en) * | 2017-08-30 | 2017-12-12 | 广西壮族自治区兽医研究所 | A kind of RT LAMP primers group, kit and application for detecting pig fourth type coronavirus |
| CN109536642A (en) * | 2018-12-19 | 2019-03-29 | 长江大学 | A kind of universal pig fourth type coronavirus RT-Nested PCR detection method |
| CN110982935A (en) * | 2019-12-13 | 2020-04-10 | 华南农业大学 | LAMP primer composition for detecting porcine delta coronavirus by adopting microfluidic chip technology and kit thereof |
| CN112410465A (en) * | 2020-03-27 | 2021-02-26 | 大连民族大学 | Novel coronavirus SARS-CoV-2ORF1ab and N gene constant temperature amplification primer group and kit |
| CN111471802A (en) * | 2020-05-29 | 2020-07-31 | 华南农业大学 | Porcine delta coronavirus rapid detection primer, kit and application thereof |
| CN113981140A (en) * | 2021-10-09 | 2022-01-28 | 广州达安基因股份有限公司 | Detection method of novel coronavirus delta mutant strain and nucleic acid detection kit |
| CN113981140B (en) * | 2021-10-09 | 2024-02-27 | 广州达安基因股份有限公司 | Novel coronavirus delta mutant strain detection method and nucleic acid detection kit |
| CN113981149A (en) * | 2021-11-23 | 2022-01-28 | 广东省农业科学院动物卫生研究所 | Porcine delta coronavirus detection primer group, probe, kit and application |
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