CN105063038B - A kind of RPA primers and kit for detecting grape leaf roll associated virus 3 - Google Patents
A kind of RPA primers and kit for detecting grape leaf roll associated virus 3 Download PDFInfo
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- CN105063038B CN105063038B CN201510553597.7A CN201510553597A CN105063038B CN 105063038 B CN105063038 B CN 105063038B CN 201510553597 A CN201510553597 A CN 201510553597A CN 105063038 B CN105063038 B CN 105063038B
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Abstract
The invention discloses a kind of RPA primers and kit for detecting grape leaf roll associated virus 3.The RPA primers of the present invention are made up of the single strand dna shown in SEQ ID No.1 and the single strand dna shown in SEQ ID No.2.Proved by testing：The present invention RPA primer specificities are good, high sensitivity, detection time is short, does not need special instrument and applied widely, and the RPA detection methods that grape leaf roll associated virus 3 is established based on the primer are sensitive, accurate, easy and quick, there is directive significance to inlet and outlet complementary goods and examination and test of products quarantine.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of RPA primers for detecting grape leaf roll associated virus 3 and
Kit.
Background technology
Grapevine leaf-roll virus (Grapevine leafroll-associated virus, GLRaV) is to be only second to grape
A kind of worldwide Grapevine virus disease of Fan Leaf, is distributed also more generally, field susceptible gene is higher, and grape is produced at home
Amount and qualitative effects are very big, and the virus for causing this sick is independent or a variety of grape leaf rolls are with disease by grape leaf roll associated virus
Malicious Combined Infection causes.It has been reported that grape leaf roll associated virus have 11 kinds, respectively GLRaV-1~9, GLRaV-Dr and
GLRaV-De, China have identified that clear and definite grape leaf roll associated virus species shares 6 kinds, i.e. GLRaV-1~5 and 7.GLRaV is
It is proved to belong to typical Clostero viruses, only exists in bast and blade, it is not by machinery, typically by sense that it, which is propagated,
The propagating materials such as the maternal plant of dye or scion.GLRaV have it is half latent, cause weak Growth of Grape, fruit maturation delay,
Fruit size is uneven, coloring is bad, sugar content is reduced and yield declines, and growing for grape is had a significant impact.
At present, detecting the method for grapevine leafroll virus mainly has indicator plant method, enzyme linked immunosorbent assay (ELISA), anti-
Transcriptional polymerase chain reaction (RT-PCR) and real-time fluorescence PCR method.Indicator plant detection method is fairly simple, but its detection speed
It is very slow, and can not determine to infect the species of virus, sensitivity is relatively low.It is existing both at home and abroad much to be detected using ELISA method
The report of grape leaf roll associated virus, this method high sensitivity is simple to operate, but required detection time is also at 1~2 day, and
The Antibody preparation cycle is grown, and costly, sensitivity is not as good as RT-PCR.Using the RT-PCR of Protocols in Molecular Biology foundation, it is immunized
The methods of catching RT-PCR, real-time fluorescence RT-PCR and probe hybridization check so that Grapevine virus A testing result is more fast
It is fast, sensitive, accurate.But the method for traditional PCR-based needs to be denatured, anneals, extends three steps, and the temperature of each step differs
Sample limits its use range, it is necessary to special thermal cycler is carried out.Therefore many isothermal duplication skills independent of PCR instrument
Art is developed, and is especially most widely used with LAMP.
RPA (Recombinase polymerase amplifcation) is a new isothermal amplification technique, and it is led
It is to form microfilament when searching the sequence of complete complementary pairing therewith on template DNA using recombinase and primer to want principle,
Template DNA is set to unwind under the side group of single-stranded DNA binding protein, primer starts to match in template DNA, and in the work of archaeal dna polymerase
Extended with lower replicate.
The content of the invention
A kind of detection of one object of the present invention or auxiliary detect whether virus to be measured is grape leaf roll associated virus 3
RPA primers.
It is provided by the invention detection or auxiliary detection it is to be measured virus whether be grape leaf roll associated virus 3 RPA primers
It is made up of the single strand dna shown in SEQ ID No.1 and the single strand dna shown in SEQ ID No.2.
It is a further object to provide one kind detection or auxiliary to detect whether virus to be measured is that grape leaf roll is adjoint
The RPA reagents of virus 3.
It is provided by the invention detection or auxiliary detection it is to be measured virus whether be grape leaf roll associated virus 3 RPA reagents
Including above-mentioned primer.
In above-mentioned RPA reagents, the final concentration of the primer 1 and the primer 2 in the RPA reagents is 0.4 μm of ol/
L。
It is a still further object of the present invention to provide one kind detection or auxiliary to detect whether virus to be measured is that grape leaf roll is adjoint
The kit of virus 3.
It is provided by the invention detection or auxiliary detection it is to be measured virus whether be grape leaf roll associated virus 3 kit bag
Include above-mentioned primer or above-mentioned RPA reagents.
It is a still further object of the present invention to provide above-mentioned primer or the new application of above-mentioned RPA reagents or mentioned reagent box.
The invention provides above-mentioned primer or above-mentioned RPA reagents or mentioned reagent box to detect or aid in detection grape leaf roll
Application in associated virus 3.
Present invention also offers above-mentioned primer or above-mentioned RPA reagents or mentioned reagent box to prepare detection or auxiliary detection Portugal
Application in No. 3 products of grape leaf roll associated virus.
Present invention also offers above-mentioned primer or above-mentioned RPA reagents or mentioned reagent box to detect or aid in detection to treat test sample
Whether product infect the application in grape leaf roll associated virus 3.
Present invention also offers above-mentioned primer or above-mentioned RPA reagents or mentioned reagent box to prepare detection or aid in detection to treat
Whether test sample product infect the application in No. 3 products of grape leaf roll associated virus.
Present invention also offers above-mentioned primer or above-mentioned RPA reagents or mentioned reagent box to detect or aid in detect disease to be measured
Whether poison is application in grape leaf roll associated virus 3.
Present invention also offers above-mentioned primer or above-mentioned RPA reagents or mentioned reagent box to prepare detection or aid in detection to treat
Survey virus whether be grape leaf roll associated virus 3 product in application.
Final object of the present invention is to provide a kind of detection or auxiliary detects whether virus to be measured is grape leaf roll companion
With the virus method of No. 3.
Whether detection or auxiliary detection virus to be measured provided by the invention is that the method for grape leaf roll associated virus 3 includes
Following steps：
(1) RPA amplifications are carried out with above-mentioned primer pair virus to be measured, obtains RPA amplified productions；
(2) size of the RPA amplified productions is detected；
If the RPA amplified productions contain the fragment that size is 380bp, the virus to be measured is or candidate is grape volume
Leaf associated virus 3；
If the RPA amplified productions do not contain the fragment that size is 380bp, the virus to be measured is not or candidate is not
Grape leaf roll associated virus 3.
In the above method, the template of the RPA amplifications is viral cDNA to be measured.
In the above method, the temperature of the RPA amplifications is 37 DEG C, and the time of the RPA amplifications is 40min.
Application of the above method in detecting or aiding in detection grape leaf roll associated virus 3 falls within the protection of the present invention
Scope.
The method of the present invention mainly possesses advantages below：
(1) 1 pair of primer is only needed to complete to expand, it is not necessary to complicated design of primers process；
(2) isothermal reaction at 37 DEG C is only needed, it is not necessary to special heat circulating equipment；
(3) reaction time only needs 40min；
(4) result is easy to judge, unlike the dispersion plating of LAMP products, RPA amplified productions have according to design of primers site
The band of particular size.
The present invention designs specific RPA amplifications for the conserved sequence of the HSP70 genes of grape leaf roll associated virus 3
Primer, and the RPA detection methods of grape leaf roll associated virus 3 are established based on the primer, can be to grape leaf roll associated virus 3
Number carry out qualitative detection.Proved by testing：The present invention RPA amplimers specificity is good, high sensitivity, detection time are short,
Special instrument and applied widely is not needed, and the RPA detection sides of grape leaf roll associated virus 3 are established based on the primer
Method is sensitive, accurate, easy and quick, has directive significance to inlet and outlet complementary goods and examination and test of products quarantine.
Brief description of the drawings
Fig. 1 is the foundation of RPA detection methods.Wherein, 1：Grape leaf roll associated virus 3；2：Negative control.
Fig. 2 is specificity experiments.Wherein, 1：Grape leaf roll associated virus 3；2：Grape leaf roll associated virus 2；3：It is husky
Ground grape stem acne associated virus；4：Grapevine fleck virus；5：Grapevine virus A；6：Negative control.
Fig. 3 is RPA sensitivity experiments.1-6：Template cDNA dilution factor is respectively 100、10-1、10-2、10-3、10-4With
10-5。
Fig. 4 is PCR sensitivity experiments.1-6：Template cDNA dilution factor is respectively 100、10-1、10-2、10-3、10-4With
10-5。
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
RPA amplification kit TwistAmp Basic kits in following embodiments are the products of TwistDX companies, production
Product catalog number (Cat.No.) is TABAS03KIT.
(the Grapevine leafroll-associated virus of grape leaf roll associated virus 3 in following embodiments
3) in document, " Wang Zhong, Liu order China, Li Jie, Li Mingjun, Cheng Yu qin .3 kind grape leaf roll associated virus RT-PCR detection [J] China
Fruit tree, 2012,04:Mistake disclosed in 43-46. ", the public can obtain from Yi Li Entry-Exit Inspection and Quarantine Bureau.
(the Grapevine leafroll-associated virus of grape leaf roll associated virus 2 in following embodiments
2) in document, " Zhao Jingjing, Qian Yike, a word used in person's names surpass, Qingyuan Guo, Hu Baishi, and Lu Ping cause the virus and class disease of grape yellow class symptom
Malicious RT-PCR detections [J] Xinjiang Agricultural Sciences, 2015,06:Mistake disclosed in 1099-1104 ", the public can enter and leave the border from Yi Li and examine
Test Quarantine Bureau's acquisition.
Sand grape stem acne associated virus (Grapevine rupestris stem pitting in following embodiments
Associated virus), Grapevine fleck virus (Grapevine fleck virus) and Grapevine virus A (Grapevine
Virus A) document " the three kinds of Grapevine virus RT-PCR in the refined Xinjiang of Liang Qiaoling, Qian Yike, Zhang Na, Lu Ping, Liu Xu detections and
Sequence analysis [J] plant protection journals, 2015,03:Mistake disclosed in 376-381. ", the public can be from Yi Li inspection and quarantining for import/export
Office obtains.
Embodiment 1, RPA primers and its RPA kits for detecting grape leaf roll associated virus 3
First, the design of RPA primers
For the conserved sequence of the grape leaf roll associated virus HSP70 genes of No. 3, design detects grapevine leafroll virus 3
RPA primers, primer size 380bp.Particular sequence is as follows：
Sense primer GLRaV3-F：5 '-ATTGAGGAACAATTTGGGGGACATCCGGCAATACT-3 ' (sequence 1)；
Anti-sense primer GLRaV3-R：5 '-GAACCGACGTATCAATATCTAACGGCTGCCGACTA-3 ' (sequence 2).
2nd, for detecting the RPA kits and its application method of grape leaf roll associated virus 3
1st, for detecting the RPA kits of grape leaf roll associated virus 3
RPA kits for detecting grape leaf roll associated virus 3 include the sense primer that above-mentioned steps one design
GLRaV3-F and anti-sense primer GLRaV3-R, tube cell containing lyophozyme, rehydration buffer solution (Rehydration Buffer), acetic acid
Magnesium solution (280mmol/L).Above-mentioned tube cell containing lyophozyme, rehydration buffer solution (Rehydration Buffer) and magnesium acetate are molten
Liquid (280mmol/L) derives from RPA amplification kit TwistAmp Basic kits.
2nd, for the application method for the RPA kits for detecting grape leaf roll associated virus 3
(1) RPA is expanded
Using the cDNA of testing sample as template, RPA amplifications are carried out using GLRaV3-F and GLRaV3-R primers, obtain RPA
Amplified production.Blank control (DNA profiling is ultra-pure water) is set simultaneously.
The compound method of RPA amplification systems is as follows：Added into the 0.2mL TwistAmp reaction tubes containing lyophilized enzyme powder
Rehydration buffer solution (Rehydration Buffer) 29.5 μ L, μ L of deionized water 12.5, each 2 μ L of upstream and downstream primer be (primer
Final concentration is 0.4 μm of ol/L), the μ L of template DNA 1, finally add the μ L (280mmol/L) of magnesium acetate solution 2.5.
RPA amplification reaction conditions：Above-mentioned RPA amplification systems are fully mixed, is placed on 37 DEG C of metal bath and reacts
40min, obtain RPA amplified productions.
(2) electrophoresis detection of RPA amplified productions
After RPA reactions terminate, 50 μ L phenol/chloroforms (1 are added into above-mentioned RPA amplified productions:1) solution, fully mix
12000rpm centrifuges 2min afterwards, takes 5 μ L of supernatant liquid to observe result on gel imaging system in 1.5% agarose gel electrophoresis.
And RPA amplified productions are sequenced.
If RPA amplified productions contain the fragment that size is 380bp, virus to be measured is or candidate is the adjoint disease of grape leaf roll
Poison 3；
If RPA amplified productions do not contain the fragment that size is 380bp, virus to be measured is not or candidate is not grape leaf roll
Associated virus 3.
3rd, for the application for the RPA kits for detecting grape leaf roll associated virus 3
1st, RNA extraction and cDNA synthesis
With reference to kit operation (plant total RNA extraction reagent box, article No. DP432, Tiangeng bio tech ltd)
The RNA of the grape leave of grape leaf roll associated virus 3 has been infected in extraction.And with reference to kit (PrimeScript RT-PCR
Kit, article No. RR014A, TaKaRa) operation, using the RNA of acquisition as template, reverse transcription obtain cDNA.By the cDNA of acquisition
Be stored in -20 DEG C it is standby.
2nd, RPA is expanded
The cDNA obtained using step 1 carries out RPA amplifications as template, using GLRaV3-F and GLRaV3-R primers.Set simultaneously
Put blank control (DNA profiling is ultra-pure water).
The compound method of RPA amplification systems is as follows：Added into the 0.2mL TwistAmp reaction tubes containing lyophilized enzyme powder
Rehydration buffer solution (Rehydration Buffer) 29.5 μ L, the μ L of deionized water 12.5, each 2 μ L of upstream and downstream primer, template
The μ L of DNA 1, finally add the μ L (280mmol/L) of magnesium acetate solution 2.5.
RPA amplification systems are fully mixed, is placed on 37 DEG C of metal bath and reacts 40min, obtain RPA amplified productions.
3rd, the electrophoresis detection of RPA amplified productions
After RPA reactions terminate, 50 μ L phenol/chloroforms (1 are added into RPA amplified productions:1) solution, after fully mixing
12000rpm centrifuges 2min, takes the μ L of supernatant 5 to observe result on gel imaging system in 1.5% agarose gel electrophoresis.And
RPA amplified productions are sequenced
As a result it is as shown in Figure 1：The RPA amplified productions of grape leaf roll associated virus 3 contain 1 band, and size is
380bp, and negative control is without band.Illustrate the RPA primers and RPA that are used to detect grape leaf roll associated virus 3 of the present invention
Kit can be No. 3 with effective detection grape leaf roll associated virus.
The specific detection of embodiment 2, RPA primers
1st, RNA extraction and cDNA synthesis
RNA extraction and cDNA synthetic method in reference to the step of embodiment 1 three, respectively from having infected grape leaf roll
Associated virus 3 grape leave, the grape leave of grape leaf roll associated virus 2 infected, infected sand grape stem acne
In the grape leave of associated virus, the grape leave for having infected Grapevine fleck virus and the grape leave for having infected Grapevine virus A
It is adjoint that the cDNA of grape leaf roll associated virus 3, the cDNA of grape leaf roll associated virus 2, sand grape stem acne is prepared
The cDNA of virus, the cDNA and Grapevine virus A of Grapevine fleck virus cDNA.
2nd, RPA is expanded
The cDNA of grape leaf roll associated virus 3 that is obtained respectively with step 1, the cDNA of grape leaf roll associated virus 2,
The cDNA of sand grape stem acne associated virus, the cDNA and Grapevine virus A of Grapevine fleck virus cDNA are template, are used
GLRaV3-F and GLRaV3-R carries out RPA amplifications, respectively obtains grape leaf roll associated virus 3, grape leaf roll associated virus 2
Number, sand grape stem acne associated virus, Grapevine fleck virus and Grapevine virus A RPA amplified productions.Blank control is set simultaneously
(DNA profiling is ultra-pure water).
The compound method of RPA amplification systems is as follows：Added into the 0.2mL TwistAmp reaction tubes containing lyophilized enzyme powder
Rehydration buffer solution (Rehydration Buffer) 29.5 μ L, the μ L of deionized water 12.5, each 2 μ L of upstream and downstream primer, template
The μ L of DNA 1, finally add the μ L (280mmol/L) of magnesium acetate solution 2.5.
RPA amplification systems are fully mixed, is placed on 37 DEG C of metal bath and reacts 40min, obtain RPA amplified productions.
3rd, the electrophoresis detection of RPA amplified productions
After RPA reactions terminate, 50 μ L phenol/chloroforms (1 are added into RPA amplified productions:1) solution, after fully mixing
12000rpm centrifuges 2min, takes 5 μ L of supernatant liquid to observe result on gel imaging system in 1.5% agarose gel electrophoresis.
As a result it is as shown in Figure 2：As can be seen from the figure：The amplified production of only grape leaf roll associated virus 3 contains 1
Size be 380bp band, grape leaf roll associated virus 2, sand grape stem acne associated virus, Grapevine fleck virus and grape
Viral A is without band.Illustrate that the RPA primer specificities of the present invention are high.
Embodiment 3, the sensitivity technique of RPA primers and the contrast with regular-PCR sensitivity
1st, RNA extraction and cDNA synthesis
RNA extraction and cDNA synthetic method in reference to the step of embodiment 1 three, respectively from having infected grape leaf roll
The cDNA of grape leaf roll associated virus 3 is prepared in the grape leave sample of associated virus 3, and grape leaf roll is adjoint
The cDNA of virus 3 carries out gradient dilution, respectively obtains dilution factor as 100、10-1、10-2、10-3、10-4With 10-5Grape leaf roll
The cDNA that associated virus 3.
2nd, RPA is expanded
Respectively using the dilution factor that above-mentioned steps 1 obtain as 100、10-1、10-2、10-3、10-4With 10-5Grape leaf roll it is adjoint
The cDNA of virus 3 is template, and RPA amplifications are carried out using GLRaV3-F and GLRaV3-R.
The compound method of RPA amplification systems is as follows：Added into the 0.2mL TwistAmp reaction tubes containing lyophilized enzyme powder
Rehydration buffer solution (Rehydration Buffer) 29.5 μ L, the μ L of deionized water 12.5, each 2 μ L of upstream and downstream primer, template
The μ L of DNA 1, finally add the μ L (280mmol/L) of magnesium acetate solution 2.5.RPA amplification systems are fully mixed, are placed in 37 DEG C
40min is reacted on metal bath, obtains RPA amplified productions.
3rd, PCR is expanded
Respectively using the dilution factor that above-mentioned steps 1 obtain as 100、10-1、10-2、10-3、10-4With 10-5Grape leaf roll it is adjoint
The cDNA of virus 3 is template, enters performing PCR amplification using primer P3U and P3D, obtains pcr amplification product.
Primer sequence is as follows：
P3U：5′-CGCTCATGGTGAAAGCAGACG-3′；
P3D：5′-CTTAGAACAAAAATATGGAGCAG-3′.
The system and reaction condition bibliography " Genetic variability and population of PCR amplifications
Method in structure of Grapevine leafroll-associated virus 3isolates ".
4th, electrophoresis detection
1) after RPA reactions terminate, 50 μ L phenol/chloroforms (1 are added into RPA amplified productions respectively:1) solution, it is fully mixed
It is even, 12000rpm centrifugation 2min, take 5 μ L of supernatant liquid to observe knot on gel imaging system in 1.5% agarose gel electrophoresis
Fruit.RPA electrophoresis results are as shown in Figure 3.
2) after PCR reactions terminate, 5 μ L pcr amplification product is taken in 1.5% agarose gel electrophoresis, in gel imaging system
Result is observed on system.PCR electrophoresis results are as shown in Figure 4.
It is 10 as can be seen that the RPA methods of the present invention are suitable with the sensitivity of regular-PCR from Fig. 3 and Fig. 4-3Dilution
Degree.
Claims (10)
1. it is a kind of detect or auxiliary detection it is to be measured virus whether be grape leaf roll associated virus 3 RPA primers, by SEQ ID
Single strand dna composition shown in single strand dna and SEQ ID No.2 shown in No.1.
2. it is a kind of detect or auxiliary detection it is to be measured virus whether be grape leaf roll associated virus 3 RPA reagents, including right will
Seek the primer described in 1.
3. RPA reagents according to claim 2, it is characterised in that：The primer 1 and the primer 2 are in the RPA reagents
In final concentration be 0.4 μm of ol/L.
4. it is a kind of detect or auxiliary detection it is to be measured virus whether be grape leaf roll associated virus 3 kit, including right will
Ask the primer described in 1 or the RPA reagents described in Claims 2 or 3.
5. the RPA reagents described in primer or Claims 2 or 3 described in claim 1 or the kit described in claim 4 exist
Application in detection or auxiliary detection grape leaf roll associated virus 3；
Or the primer described in claim 1 or the RPA reagents described in Claims 2 or 3 or the kit described in claim 4 exist
Prepare the application in detection or auxiliary detection grape leaf roll No. 3 products of associated virus.
6. the RPA reagents described in primer or Claims 2 or 3 described in claim 1 or the kit described in claim 4 exist
Whether detection or auxiliary detection testing sample infect the application in grape leaf roll associated virus 3；
Or the primer described in claim 1 or the RPA reagents described in Claims 2 or 3 or the kit described in claim 4 exist
Prepare detection or aid in whether detection testing sample infects the application in No. 3 products of grape leaf roll associated virus；
Or the primer described in claim 1 or the RPA reagents described in Claims 2 or 3 or the kit described in claim 4 exist
Whether detection or auxiliary detection virus to be measured are application in grape leaf roll associated virus 3；
Or the primer described in claim 1 or the RPA reagents described in Claims 2 or 3 or the kit described in claim 4 exist
Prepare detection or auxiliary detection it is to be measured virus whether be grape leaf roll associated virus 3 product in application.
7. it is a kind of detect or auxiliary detection it is to be measured virus whether be grape leaf roll associated virus 3 method, comprise the following steps：
(1) virus to be measured of the primer pair described in claim 1 carries out RPA amplifications, obtains RPA amplified productions；
(2) size of the RPA amplified productions is detected；
If the RPA amplified productions contain the fragment that size is 380bp, the virus to be measured is or candidate is grape leaf roll companion
With viral No. 3；
If the RPA amplified productions do not contain the fragment that size is 380bp, the virus to be measured is not or candidate is not grape
Leaf roll associated virus 3.
8. according to the method for claim 7, it is characterised in that：The template of the RPA amplifications is viral cDNA to be measured.
9. the method according to claim 7 or 8, it is characterised in that：The temperature of the RPA amplifications is 37 DEG C, and the RPA expands
The time of increasing is 40min.
10. any described method answering in detecting or aiding in detection grape leaf roll associated virus 3 in claim 7-9
With.
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| CN107254556A (en) * | 2017-08-10 | 2017-10-17 | 中国检验检疫科学研究院 | Method, RPA IAC primers and kit based on RPA IAC technology examination bean mosaic virus 4s |
| CN109295259A (en) * | 2018-11-02 | 2019-02-01 | 中国水产科学研究院淡水渔业研究中心 | A kind of primer and detection method being used to detect river crab bunyavirus based on RPA technology |
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| CN101978051A (en) * | 2008-01-31 | 2011-02-16 | 俄勒冈州高教委暨俄勒冈州大学 | Closterovirus vectors and methods |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101978051A (en) * | 2008-01-31 | 2011-02-16 | 俄勒冈州高教委暨俄勒冈州大学 | Closterovirus vectors and methods |
Non-Patent Citations (5)
| Title |
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| Brief Report of a New Highly Divergent Variant of Grapevine leafroll-associated virus 3 (GLRaV-3);Dariusz E. Goszczynski;《J Phytopathol》;20131231;874-879 * |
| Rapid and sensitive detection of Little cherry virus 2 using isothermal reverse transcription-recombinase polymerase amplification;Tefera A.Mekuria et al.;《Journal of Virological Methods》;20140504;24-30 * |
| Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification;Goncalo Silva et al.;《Journal of Virological Methods》;20150623;138-144 * |
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