CN102080132B - Seminested-RT (reverse transcription)-Realtime PCR (polymerase chain reaction) kit for detecting potato virus X - Google Patents
Seminested-RT (reverse transcription)-Realtime PCR (polymerase chain reaction) kit for detecting potato virus X Download PDFInfo
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Abstract
The invention discloses a seminested-RT (reverse transcription)-Realtime PCR (polymerase chain reaction) kit for detecting potato virus X. The method for detecting potato virus X comprises the following steps: design of a TaqMan probe and primers, extraction of plant total RNA (ribose nucleic acid) and quality control, synthesis of cDNA (complementary deoxyribonucleic acid) by reverse transcription of RNA, and real-time fluorescent PCR detection. The method organically combines real-time fluorescent PCR technology and nested PCR technology. A forward primer is added to the outer side of a real-time fluorescent PCR primer according to the design principle of nested PCR, and a reverse primer is respectively paired with two forward primers to form two sets of independent PCR systems, which share one TaqMan probe. The high amplification efficiency of the primers ensures the high detection sensitivity, and effectively enhances the detection sensitivity; the two sets of PCR systems formed by the three primers authenticate each other, thereby greatly enhancing the detection accuracy, simplifying the operation and shortening the detection cycle; and the totally-enclosed operating system reduces the pollution.
Description
Technical field
The present invention relates to a kind of detection method of virus, specifically half-nest type-RT-Realtime PCR detects the test kit of potato virus X, belongs to the plant virus detection range.
Background technology
(Potato virus X PVX) is one of of paramount importance disease on the yam to potato virus X, and the serious susceptible yam ability underproduction is more than 80%.The potato virus X particle is a bending, long 470-580nm, and diameter 13nm, the spiral symplex structure is single strand RNA virus.Potato virus X has medium pathogenic usually, causes the system floral leaf and ring spot symptom of many monocotyledonss and dicotyledons.Propagate through mechanical contact in natural condition, do not have known amboceptor, this virus is propagated through artificial inoculation easily.The host range of single virus is narrower, and potato virus X only limits to contaminate plant of Solanaceae.Though when being the single dip-dye of potato virus X symptom smaller or too high in temperature, cross very easily latent disease under the low or high fertilizer and water condition; But when this virus and other can be assisted the yam kind of the anti-anti-difference of sick viral MOI; Often cause serious underproduction phenomenon, even reach more than 80%.Therefore, potato virus X is one of main virus that causes the potato virus sexual involution.
At present, the method for detection potato virus X mainly contains differential host's inoculation method, enzyme-linked immunosorbent assay (ELISA), RT-PCR technology etc.The electron microscopy apparatus expensive, detection sensitivity is low; The elisa technique complex operation step, sense cycle is long, sensitivity is low, be prone to false positive; The PCR gel electrophoresis technology increases in many aspects than ELISA, but is easy to pollute; Do not see the institute road that adopts real-time fluorescence PCR to detect PVX.
Summary of the invention
The objective of the invention is to; To the problems referred to above; The PVX coat protein of giving chapter and verse (CP) gene conservative sequence has designed and synthesized three nest-type PRC primers and a TaqMan fluorescent probe, has set up the novel method of half-nest type-RT-Realtime PCR detection PVX.This method is organic to have combined real-time fluorescence PCR and nest-type PRC technology: a positive strand primer has been set up according to the principle of design of nest-type PRC in the outside at real-time fluorescence PCR primer; The anti-chain primer forms two with two positive strand primer pairings respectively and overlaps independently PCR reaction, and a shared TaqMan probe.The function that the real-time fluorescence PCR technology adopts TaqMan fluorescent probe signal to amplify has effectively improved the sensitivity that detects; Article three, two cover PCR systems of primer formation are verified each other, have greatly improved the accuracy that detects; Totally enclosed os has reduced pollution, and PCR result's Real Time Observation has greatly shortened sense cycle.The accuracy that this method detects, sensitivity geometric ratio nest-type PRC, Real-time PCR and other method are high.
Technical scheme of the present invention is:
Half-nest type-RT-Realtime PCR detects the test kit of potato virus X, may further comprise the steps to be prepared from:
(1) TaqMAN probe and primer design are synthetic, according to CP gene order conserved regions in the full genome of PVX in the nucleic acid database, design three primers and a TaqMan probe;
(2) total RNA extracts and quality control, from plant leaf, extracts total RNA, measures its OD value;
(3) the synthetic cDNA of reverse transcription carries out the synthetic cDNA of reverse transcription with the total RNA of plant that extracts;
(4) real-time fluorescence PCR detects, and comprises that specific detection and sensitivity detect;
Described method for detecting specificity is: from infect PVX virus and other control plant blade, extract total RNA; The total RNA of plant that extracts is carried out the synthetic specificity template cDNA of reverse transcription respectively, synthetic cDNA is carried out the special real-time fluorescence PCR test of primer probe;
Described sensitivity detection method is: with the total rna ladder degree dilution of the plant leaf of the potato-infecting X virus of extracting in the above-mentioned steps (2), and carry out the synthetic cDNA of reverse transcription respectively, carry out the real-time fluorescence PCR check then.
Described three primers and a TaqMan probe sequence are respectively: a upstream primer sequence is: 5 '-TGCCCAAACTGCTGCCTT-3 '; Another upstream primer sequence is: 5 '-GGCCAGGGCACAATCCA-3 '; The downstream primer sequence is: 5 '-CAGTGATACGACCTCGAGTGACA-3 '; Probe sequence is: 5 ' (FAM)-CGACTTTGCCAGCCTAGATGCAG-3 ' (TAMRA).
The method of the synthetic cDNA of described reverse transcription is: in reaction system, add the total RNA of damping fluid, ThermoScript II and damping fluid thereof, random primer and plant; Add diethylpyrocarbonate water constant volume; Reaction conditions is: 37 ℃, 15 minutes, and 85 ℃, 5 seconds, synthetic cDNA.
Described damping fluid is 5 PrimeScript
TMBuffer contains four kinds of dNTP mixtures and Mg in the damping fluid
2+Described ThermoScript II and damping fluid thereof are PrimeScript
TMRT Enzyme Mix I, described random primer are Oligo dT Primer and Random 6 mers.
Described specific detection, reaction system is replenished DEPC water constant volume for adding PCR SuperMix-UDG, optical dye, upstream primer, downstream primer, probe and cDNA respectively; Cycling condition is followed successively by: 45-55 ℃, 1-3min; 90-100 ℃, 1-3min; 90-100 ℃, 10-20s; 55-65 ℃, 25-35s.
Described PCR SuperMix-UDG is
quantitative PCR SuperMix-UDG, mainly comprises
Taq archaeal dna polymerase, Tris-HCl, KCl, 6mM MgCl2,400 μ M dGTP, 400 μ M dATP, 400 μ M dCTP, 800 μ M dUTP, ura DNA transglucosylase and stablizer.
Described cycling condition the best is followed successively by: 50 ℃, 2 minutes; 95 ℃, 2 minutes; 95 ℃, 15 seconds, 60 ℃, 30 seconds, 45 circulations.
Beneficial effect of the present invention is: the present invention is organic to have combined real-time fluorescence PCR and nest-type PRC technology: a positive strand primer has been set up according to the principle of design of nest-type PRC in the outside at real-time fluorescence PCR primer; The anti-chain primer forms two with two positive strand primer pairings respectively and overlaps independently PCR reaction, and a shared TaqMan probe.Three high amplification efficiencies of primer designing among the present invention have guaranteed the highly sensitive that detects, adopt simultaneously that TaqMan fluorescent probe signal amplifying function has also effectively improved the sensitivity that detects in the real-time fluorescence PCR technology; Article three, two cover PCR systems of primer formation are verified each other, have greatly improved the accuracy that detects; Amplification procedure and the result of ability Real Time Observation PCR need not to carry out follow-up a large amount of and loaded down with trivial details work, have simplified operation and have shortened sense cycle; Totally enclosed os has reduced pollution.Compare with other method of having reported, this method is easier, quick, accurate and sensitive.Test-results shows that the detection sensitivity of the inventive method can reach the total RNA of 0.071fg/ μ l plant.
Description of drawings
Fig. 1 is cover 1 (PVXF1/PVXR/PVXP) primer probe specificity test pcr amplification result;
Fig. 2 is cover 2 (PVXF2/PVXR/PVXP) primer probe specificity test pcr amplification result;
Fig. 3 is cover 1 (PVXF1/PVXR/PVXP) primer probe sensitivity test pcr amplification result;
Fig. 4 is cover 2 (PVXF1/PVXR/PVXP) primer probe sensitivity test pcr amplification result;
Fig. 5 is cover 1 combination (PVXF1/PVXR/PVXP) and cover 2 combination (PVXF2/PVXR/PVXP) primer probe amplification efficient comparing results;
The longitudinal axis is Δ Rn (a fluorescence information increased value, down together) among Fig. 1, and transverse axis is Cycle number (cycle number, down together); A is PVX, and B, C, D, E, F are followed successively by and are ArMV, PVYn, PBRSV, healthy leaf (CK1) and DEPC water contrast (CK2);
The longitudinal axis is Δ Rn among Fig. 2, and transverse axis is Cycle number; A is PVX, and B, C, D, E, F are followed successively by and are ArMV, PVYn, PBRSV, healthy leaf (CK1) and DEPC water contrast (CK2);
The longitudinal axis is Δ Rn among Fig. 3, and transverse axis is Cycle number; A, B, C, D, E, F, G, H, I, J, the pairing RNA concentration of K are followed successively by 7.1ng/ μ l, 710pg/ μ l, 71pg/ μ l, 7.1pg/ μ l, 710fg/ μ l, 71fg/ μ l, 7.1fg/ μ l, 0.71fg/ μ l, 0.071fg/ μ l, 0.0071fg/ μ l, DEPC water;
The longitudinal axis is Δ Rn among Fig. 4, and transverse axis is Cycle number; A, B, C, D, E, F, G, H, I, J, the pairing RNA concentration of K are followed successively by 7.1ng/ μ l, 710pg/ μ l, 71pg/ μ l, 7.1pg/ μ l, 710fg/ μ l, 71fg/ μ l, 7.1fg/ μ l, 0.71fg/ μ l, 0.071fg/ μ l, 0.0071fg/ μ l, DEPC water;
The longitudinal axis is Δ Rn among Fig. 5, and transverse axis is Cycle number; A is cover 1 combination (PVXF1/PVXR/PVXP), and B is for being cover 2 combinations (PVXF2/PVXR/PVXP).
Embodiment
Below the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein only is used for explanation and explains the present invention, and be not used in qualification the present invention.
1 raw material and source
1.1 material
Potato virus X (PVX), arabis mosaic virus (ArMV), yam black ring spot poison (PBRSV), the downright bad strain (PVY of system of marmor upsilon arteries and veins
N) malicious source provides by Institute of quarantine of animals and plants, Chinese Academy of Inspection and.
1.2 main instruments and reagent
1.2.1 key instrument
1.2.1.1 the real-time fluorescence PCR gene-amplificative instrament is ABI Prism 7000 (an American AB I company);
1.2.1.2 the nucleic acid-protein analyser is BioPhotometer (German eppendorf company).
1.2.2 main agents
1.2.2.1 plant total RNA extraction reagent box (trade(brand)name E.Z.N.A
TM, U.S. Omega Company products, article No.: R2867-01) available from Beijing doulbe-sides' victory biotech firm, its composition and operation steps reference reagent box carry specification sheets.
1.2.2.2 (article No.: DRR037A) available from the precious biotech firm (TaKaRa) in Dalian, its composition comprises the reverse transcription test kit: (1) 5 * PrimerScript
TMBuffer (mainly comprises 4 kinds of dNTP (G, A, T, C) mixture and Mg
2+); (2) PrimerScript
TMEnzyme Mix I (mainly comprising ThermoScript II and damping fluid thereof); (3) Oligo dT Primer (a kind of primer of reverse transcription at random); (4) Random 6 mers (a kind of primer of reverse transcription at random).
1.2.2.3 real-time fluorescent PCR reagent case (
Quantitative PCR SuperMix-UDG, article No.: C11730-025) available from American I nvitrogen company, its composition comprises: (1)
Quantitative PCR SuperMix-UDG (mainly comprises
Taq archaeal dna polymerase, Tris-HCl, KCl, 6mM MgCl
2, 400 μ M dGTP, 400 μ MdATP, 400 μ M dCTP, 800 μ M dUTP, ura DNA transglucosylase and stablizer); (2) ROX (optical dye).
2. detection method
2.1TaqMan probe and primer design are synthetic
According to CP gene order conserved regions in the full genome of PVX in the U.S. NCBI nucleic acid database, design three primers and a TaqMan probe, it is synthetic that primer and probe are given birth to the worker by Shanghai.
Design three primers (2 upstream primer, 1 downstream primer) and 1 TaqMan probe in this research altogether; Upstream primer is represented with PVXF; Wherein upstream primer PVXF1 sequence is: 5 '-TGCCCAAACTGCTGCCTT-3 '; The PVXF2 sequence is: 5-GGCCAGGGCACAATCCA-3, downstream primer represent that with PVXR sequence is: 5 '-CAGTGATACGACCTCGAGTGACA-3 '; Probe representes that with PVXP sequence is: 5 ' (FAM)-CGACTTTGCCAGCCTAGATGCAG-3 ' (TAMRA); PCR product length PVXF1/PVXR is that 98bp, PVXF1/PVXR are 65bp; Article three, primer and probe can be formed the different reaction combination of two covers, and cover 1 is PVXF1/PVXR/PVXP, and cover 2 is PVXF2/PVXR/PVXP.
2.2 total RNA extracts and quality control
With plant total RNA extraction reagent box E.Z.N.A
TMFrom sick blade and healthy potato leaf, extract total RNA, the total RNA extraction step of plant reference reagent box carries specification sheets; Measure its OD value with nucleic acid-protein analyser BioPhotometer, draw its concentration and Reinheitszahl, and control the nucleic acid quality with this.
2.3 cDNA is synthesized in reverse transcription
The total RNA of plant that extracts is carried out the reverse transcription operation with reverse transcription test kit DRR037A, with synthetic cDNA.Reaction system is following: in 25 μ l reaction systems, add 5 * PrimerScript
TMBuffer, 5 μ l, PrimerScript
TMEnzyme Mix I, 1.25 μ l, Oligo dT Primer (50 μ M), 1.25 μ l, Random 6 mers (100 μ M), 1.25 μ l, an amount of total RNA (10 μ l reaction systems can maximum can be used for 500ng) adds DEPC water to 25 μ l.Reaction conditions is: 37 ℃, 15 minutes, and 85 ℃, 5 seconds, synthetic cDNA.
2.4 real-time fluorescence PCR test
2.4.1 specificity test
2.4.1.1 the total RNA of plant extracts
With plant total RNA extraction reagent box E.Z.N.A
TM, with reference to method described in " 1.4 ", from infecting PVX, ArMV, PBRSV, PVY respectively
NExtract total RNA in the blade of 4 kinds of viruses and the healthy potato leaf (CK1).
2.4.1.2cDNA it is synthetic
5 total RNA of plant that " 1.6.1.1 " extracted and DEPC water contrast (CK2) are carried out reverse transcription respectively with reverse transcription test kit DRR037A and are synthesized cDNA.Reaction system and condition are said with reference to " 1.5 ".
2.4.1.3 specific PCR test
With 6 cDNA of " 1.6.1.2 " synthetic; Carry out the special real-time fluorescence PCR test of primer probe: (1) reaction system: in 25 μ L systems, add
quantitative PCR SuperMix-UDG, 12.5 μ l respectively; ROX, 0.5 μ l; Upstream primer PVXF1 (or PVXF2) (15 μ M), 1 μ l; Downstream primer (PVXR) (15 μ M), 1 μ l, probe (PVXP) (10 μ M), 1 μ l, cDNA, 2.0 μ l; Replenish DEPC water to 25 μ l, each cDNA is provided with 2 repetitions at least; (3) in 96 orifice plates of ABI Prism 7000 instruments, react; (4) cycling condition is: 50 ℃, 2 minutes; 95 ℃, 2 minutes; 95 ℃, 15 seconds, 60 ℃, 30 seconds, 45 circulations.
2.4.2 sensitivity test
With the total RNA of plant leaf of the potato-infecting X virus of extracting in " 1.6.1.1 " (PVX), using the dilution of DEPC water is 10
-1～10
-10, 11 gradients of DEPC water, and carry out the synthetic cDNA of reverse transcription respectively, carry out the real-time fluorescence PCR test then.Reverse transcription and real-time fluorescence PCR operation are with reference to " 1.5 " and " 1.6.1.1 ".
2.4.3 the amplification efficiency simultaneous test of two cover primer probes
Use same PVX virus cDNA to be template, respectively with overlapping 1 combination (PVXF1/PVXR/PVXP) and the test of cover 2 combinations (PVXF1/PVXR/PVXP) carrying out real-time fluorescence PCR, to compare the amplification efficiency of this two covers primer probe.Real-time fluorescence PCR is with reference to " 1.6.1.1 ".
3 results and analysis
3.1 specificity test pcr amplification result
Cover 1 (PVXF1/PVXR/PVXP) result sees that Fig. 1, cover 2 (PVXF2/PVXR/PVXP) result see Fig. 2.Fig. 1,2 result show, overlaps 1 primer probe and cover 2 primer probes and only in the cDNA of PVX is the reaction of template, amplification curve occurs, and amplified signal does not appear in all the other 5 kinds of cDNA, shows that the specificity of cover 1, cover 2 primer probes is fine, and the result fulfills the expectation.
3.2 sensitivity test pcr amplification result
From the blade of band PVX, extract the total RNA of plant, using the dilution of DEPC treating water is 10
-1～10
-10, 11 gradients of DEPC water.Extracting RNA concentration and Reinheitszahl in this test is: 71.2 (about 71ng/ μ l), OD260/280=2.07, OD260/230=2.21.The pairing RNA concentration of dilution each gradient of back is respectively: 7.1ng/ μ l, 710pg/ μ l, 71pg/ μ l, 7.1pg/ μ l, 710fg/ μ l, 71fg/ μ l, 7.1fg/ μ l, 0.71fg/ μ l, 0.071fg/ μ l, 0.0071fg/ μ l, DEPC water.Behind the synthetic respectively cDNA of reverse transcription, carry out the real-time fluorescence PCR amplification.Overlap 1 result and see that Fig. 3, cover 2 results see Fig. 4.The result of pcr amplification shows, when RNA is diluted to 10
-10When (being 0.0071fg/ μ l), detect, so the sensitivity that this method detects can reach 10 less than signal
-9, i.e. the total RNA of 0.071fg/ μ l plant.
3.3 the amplification efficiency comparative test result of two cover primer probes
The amplification efficiency comparative test result of cover 1 combination (PVXF1/PVXR/PVXP) and cover 2 combination (PVXF2/PVXR/PVXP) primer probes is seen Fig. 5.The result of Fig. 5 shows that the amplification efficiency of this two covers primer probe is about the same.
4 conclusions and discussion
Potato virus X is a kind of very important yam disease, and the serious susceptible yam ability underproduction is more than 80%.For the prophylactico-therapeutic measures of this disease most importantly early find, control early, the advanced person's of in order to protect the sound development of China yam and related industries, develop fast, Sensitive Detection should virus technology has more great realistic meaning.
Diverse ways, its detection sensitivity differs greatly, and like ELISA and PCR gel electrophoresis method, the sensitivity of detection differs more than 100 times, and the PCR gel electrophoresis is relevant with the real-time fluorescence PCR sensitivity also more than 100 times.This testing laboratory also carried out a large amount of testing laboratories to be proved, adopts the real-time fluorescence PCR technology of different primer probes, because the amplification efficiency of primer is different, sensitivity difference also can reach more than 1000 times.In actual detected evaluation work; In order to improve result's accuracy, often need carry out plural validation test to a result, the different methods detection sensitivity is different; Be difficult to guarantee that the result of two tests is just the same; This is particularly detecting under the few situation of target content with regard to for result's judgement has increased difficulty, and this difficulty will further strengthen.If two cover method detection sensitivities are the same or very approaching, this difficult problem just is readily solved.
The real-time fluorescence PCR detection technique is the high-new detection technique that grows up international recent years, and this technology has organically combined fluorescence detecting system pcr amplification system.Compare with other detection technique of having reported at present; This technology has huge meliority; Be mainly reflected in: fluorescent signal is passed through in (1); Can Real Time Observation detected sample template DNA (or cDNA) amplification situation in the PCR process, need not to dye, electrophoresis and ultraviolet detection, simplified trace routine greatly and shortened detection time; (2) adopt the fluorescent signal enlarging function, the sensitivity of its detection has obtained improving greatly; (3) totally enclosed os has reduced the PCR product staining with the intersection of sample room of testing laboratory's environment has been stained, and effectively reduces and has avoided false positive results.Real-time fluorescence PCR technology, be at present the most accurately, sensitive technology, its meliority makes it in Plant Quarantine, to have broad application prospects.
The method of " half-nest type-RT-Realtime PCR " detection potato virus X (PVX) has been set up in this research.This method had both been utilized easy, the quick and highly sensitive characteristic of real time fluorescent PCR method institute inherent, simultaneously dexterously with the principle of nest-type PRC, applied in the real-time fluorescence PCR technology.Only on the basis of general Realtime PCR, set up a primer in the design, just formed fully independently detection architecture of two covers, two cover primer probes are verified each other, have further improved accuracy again.The amplification efficiency of this two covers primer probe is high and about the same, thereby has guaranteed the consistence of two cover system detected results.Test-results shows that the sensitivity that they detect all can reach the total RNA of 0.071fg/ μ l plant.
What should explain at last is: the above is merely the preferred embodiments of the present invention; Be not limited to the present invention; Although the present invention has been carried out detailed explanation with reference to previous embodiment; For a person skilled in the art, it still can be made amendment to the technical scheme that aforementioned each embodiment put down in writing, and perhaps part technical characterictic wherein is equal to replacement.All within spirit of the present invention and principle, any modification of being done, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. half-nest type-RT-Realtime PCR detects the method for potato virus X, it is characterized in that may further comprise the steps: according to coat protein gene sequence conserved regions in the full genome of potato virus X, design three primers and a TaqMan probe;
(1) from plant leaf, extracts total RNA and measure its OD value;
(2) with the synthetic cDNA of the total RNA reverse transcription of plant of extracting;
(3) real-time fluorescence PCR detects, and comprises that the specific detection of primer probe and sensitivity detect;
Described three primers and a TaqMan probe, wherein a upstream primer sequence is:
5 '-TGCCCAAACTGCTGCCTT-3 '; Another upstream primer sequence is:
5 '-GGCCAGGGCACAATCCA-3 '; The downstream primer sequence is:
5 '-CAGTGATACGACCTCGAGTGACA-3 '; Probe sequence is: 5 ' FAM-CGACTTTGCCAGCCTAGATGCAG-3 ' TAMRA.
2. half-nest type according to claim 1-RT-Realtime PCR detects the method for potato virus X, it is characterized in that described method for detecting specificity is: with synthetic cDNA in the step (2) is that template is carried out the test of primer probe specificity real-time fluorescence PCR; Wherein, plant leaf described in the step (1) is plant leaf and other corresponding control plant blade of potato-infecting X virus.
3. half-nest type according to claim 1-RT-Realtime PCR detects the method for potato virus X; It is characterized in that described sensitivity detection method is: the total RNA of plant leaf of the potato-infecting X virus of extracting in the above-mentioned steps (2) is carried out gradient dilution; And cDNA is synthesized in reverse transcription respectively, carries out the real-time fluorescence PCR check then.
4. half-nest type according to claim 1-RT-Realtime PCR detects the method for potato virus X; The method that it is characterized in that the synthetic cDNA of described reverse transcription is: in reaction system, add the total RNA of damping fluid, ThermoScript II and damping fluid thereof, random primer and plant; Add diethylpyrocarbonate water constant volume; Reaction conditions is: 37 ℃, 15 minutes, and 85 ℃, 5 seconds, synthetic cDNA; Described damping fluid is 5 * PrimeScript
TMBuffer; Described ThermoScript II and damping fluid thereof are PrimeScript
TMRT Enzyme Mix I, described random primer are Oligo dT Primer and Random 6 mers.
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| CN105936947A (en) * | 2016-06-28 | 2016-09-14 | 临沂大学 | Potato X virus RT-LAMP detection kit and detection method |
| CN106702001B (en) * | 2016-12-30 | 2020-07-24 | 北京泰格瑞分子检验有限公司 | Nested PCR with degradable outer primer |
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| WO2007130519A2 (en) * | 2006-05-02 | 2007-11-15 | Government Of The Usa, As Represented By The Secretary, Department Of Health And Human Services | Viral nucleic acid microarray and method of use |
| CN101849509A (en) * | 2010-06-29 | 2010-10-06 | 浙江省农业科学院 | Root tip detoxification and rapid propagation technology for purple potatoes |
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| WO2007130519A2 (en) * | 2006-05-02 | 2007-11-15 | Government Of The Usa, As Represented By The Secretary, Department Of Health And Human Services | Viral nucleic acid microarray and method of use |
| CN101849509A (en) * | 2010-06-29 | 2010-10-06 | 浙江省农业科学院 | Root tip detoxification and rapid propagation technology for purple potatoes |
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| Title |
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| 耿金培等.半巢式RT- Realtime PCR检测马铃薯Y病毒脉坏死株系.《植物检疫》.2009,(第3期),17－20. * |
| 耿金培等.半巢式反转录实时荧光PCR检测马铃薯黑环斑病毒.《植物检疫》.2009,24-26. * |
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