CN101451166B - Method for detecting tomato aspermy virus - Google Patents
Method for detecting tomato aspermy virus Download PDFInfo
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- CN101451166B CN101451166B CN2008102404165A CN200810240416A CN101451166B CN 101451166 B CN101451166 B CN 101451166B CN 2008102404165 A CN2008102404165 A CN 2008102404165A CN 200810240416 A CN200810240416 A CN 200810240416A CN 101451166 B CN101451166 B CN 101451166B
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Abstract
The invention aims to provide a method for detecting a tomato aspermy virus (TAV). The method utilizes loop-mediated isothermal amplification (LAMP) to detect the tomato aspermy virus (TAV). The method comprises the steps of preparation of a reaction system, incubation treatment, inactivation treatment and product detection in turn, wherein the reaction system comprises 0.5 to 1.5 mol/L of Betaine, 2 to 25 mmol/L of dNTP, 2 to 15 mmol/L of Mg<2+>, an outer primer and an inner primer, and the concentration ratio of the inner primer to the outer primer is 1:1-1:8. The method has the advantages that the method does not need a special instrument (such as a PCR instrument), and is quicker and more economic than the prior laboratory method; and the method is simple and convenient to identify, has high specificity, can judge whether a target fragment is amplified or not by observing with the naked eye or detecting the turbidity of deposit through a turbidity meter and an ultraviolet imaging system, so as to judge whether a sample plant to be detected is infected with the tomato aspermy virus or not, and does not need to use a gel electrophoresis method for observation.
Description
Technical field:
The present invention relates to the detection range of plant virus, particularly a kind of method that detects tomato aspermy virus, this method utilize loop-mediated isothermal amplification (LAMP) to detect tomato aspermy virus.
Background technology:
Chrysanthemum is Chinese traditional famous flower, also is one of the world's four big cut-flowers, but because the harm of virus disease descends chrysanthemum quality, output, deterioration of strains is very serious.Harm chrysanthemum viral species a lot, reported have 20 surplus kind.(tomato aspermy virus is a kind of important cause of disease of harm chrysanthemum TAV) to tomato aspermy virus, generally takes place on ornamental chrysanthemum.The tomato aspermy virus ownership is Cucumovirus, and ill chrysanthemum floral leaf, distortion occur when sick, the plant atrophy, and pattern is undesired.Juice, black peach aphid and some other aphid make perishability and propagate.Minority can be planted biography.Be with the malicious bar head also can transmitted virus.Flower virus disease is a class special disease of the harm flower plant that caused by virus, and this disease differs greatly with general disease at aspects such as symptom feature, pests occurrence rule and prophylactico-therapeutic measuress.Along with the development of domestic and international flowers trade, the circulation of seedling and seedling from numerous, it is very outstanding that the virus disease problem also becomes, and risen to public status inferior to fungal disease in recent years, it has influenced the yield and quality of flowers, has also influenced the foreign exchange earning of flowers.And requirements such as customs quarantine control during overseas introduction, stem apex detoxify detection detect when organizing viral level extremely low early.Therefore, development quick and precisely, detection easy and simple to handle and the method for identifying virus be the focus of correlative study always.Therefore, the method for the detection of development and identifying virus is the focus of correlative study always.At present, generally the polymerase chain reaction of Cai Yonging (PCR) detection method needs special instrument, detects the cost height, and has the shortcoming of easy crossed contamination, complex operation, and is unfavorable for on-the-spot rapid detection, and it is bigger to apply difficulty.So, all need in scientific research and the production practice a kind of quick and precisely, the detection method of tomato aspermy virus easy and simple to handle.
Summary of the invention:
The purpose of this invention is to provide a kind of method that detects tomato aspermy virus, this method utilizes loop-mediated isothermal amplification (LAMP) to detect tomato aspermy virus (TAV).
The steps in sequence that this method comprises is:
A. prepare reaction system;
B. described reaction system is carried out incubation and handle, obtain the incubation product; The temperature that this incubation is handled is 65 ℃, and the time is 30-80min;
C. described incubation product is carried out inactivation treatment, obtain the deactivation product; The temperature of this inactivation treatment is 80 ℃, and the time is 5-15min;
D. described deactivation product is detected, obtain detected result.
Comprise in the described reaction system:
Betaine0.5-1.5mol/L，
dNTP2-25mmol/L，
Mg
2+2-15mmol/L，
Outer primer, inner primer; The ratio of the concentration of described inner primer and outer primer is 1: 1-1: 8;
In the above-mentioned reaction system, according to the specific LAMP primer of the design of the conserved sequence in the tomato aspermy virus of GenBank announcement, this conserved sequence is TAV coat protein (the Genbank number of landing EU499736); This primer comprises two outer primer f3, B3 and two inner primer FIP, BIP.Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.The nucleotide sequence of this primer is as follows:
F3：5’-AAGCCGATGAAATGGTGATC-3’，
B3：5’-GTTCAAAGGCACCTCATCGA-3’；
FIP：5’-TTCAAGCGTAGTTTAACGTCTCCGCTTCAACGCTCCCGGT-3’，
BIP：5’-GCTCTAAACTATCTGAAGTCTTTTAGCCGTTAGCTGGATG-3’；
The chrysanthemum kind that this method is suitable for is: the little chrysanthemum that crawls, as the powder carpet; Big chrysanthemum is as refreshing horse.
Beneficial effect of the present invention is:
1. the present invention does not need specific apparatus (as the PCR instrument), and is faster than the conventional rule method in laboratory, economy.
2. the present invention identifies easy, high specificity is arranged, as long as detect by an unaided eye or turbidimeter ultraviolet imagery system detect the precipitation turbidity and just whether can judge the purpose fragment amplification, whether infect tomato aspermy virus is arranged thereby judge the sample plant that is used to detect, and needn't observe with the method for gel electrophoresis.
Description of drawings:
Fig. 1: the electrophorogram of the LAMP method reaction product of tomato aspermy virus and PCR method reaction product.
Embodiment:
Embodiment 1:
A kind of method that detects tomato aspermy virus, this method utilize loop-mediated isothermal amplification (LAMP) to detect tomato aspermy virus (TAV).
The steps in sequence that this method comprises is:
A. in the Eppendoff of 1.5-2.0ml pipe, prepare reaction system; Used solvent is redistilled water (ddH
2O);
B. described reaction system is carried out incubation and handle, obtain the incubation product; The temperature that this incubation is handled is 65 ℃, and the time is 30-80min, is preferably 60min; This incubation is handled and is carried out in metal constant temperature mixing instrument, also can carry out in water-bath or metal bath;
C. described incubation product is carried out inactivation treatment, obtain reaction product; The temperature of this inactivation treatment is 80 ℃, and the time is 5-15min, is preferably 10min; This inactivation treatment is being carried out in metal constant temperature mixing instrument;
D. described reaction product is detected, obtain detected result;
The method that detects is for being the detection of 1.0% agarose gel electrophoresis; Also can use the turbidity that detects this reaction product than turbid instrument to change, also direct viewing with the naked eye; Also can be to the processing of dyeing in this reaction product, the method that this dyeing is handled is: the nucleic acid combination dye SYBR Green I that adds 0.1 μ l in this deactivation product, under ultraviolet lamp, observe:, then prove and contain tomato aspermy virus in the sample if reaction product presents fluorescence.
Metal constant temperature mixing instrument described in the present embodiment is available from German Eppendorf company.
The chrysanthemum kind that this method is suitable for is: the little chrysanthemum that crawls, as the powder carpet; Big chrysanthemum is as refreshing horse.
What adopt in the present embodiment is that (Loop-mediatedisothermalamplification LAMP), is the constant temperature nucleic acid amplification method of a kind of novelty of exploitation such as Japanese scholar Notomi to loop-mediated isothermal amplification.Be characterized in 4 kinds of special primers of 6 zone design at target gene, the LAMP reaction produces the band of many different sizes, it is the Cauliflower spline structure that constitutes by ring texture, stripe size differs about 100bp, utilize strand displacement archaeal dna polymerase (Bst DNA poLymerase) in isothermal condition (about 65 ℃) insulation dozens of minutes, can finish nucleic acid amplification reaction.This method RT-PCR commonly used than the laboratory is faster, and economy does not need specific apparatus, and its result can utilize the magnesium pyrophosphate precipitation that produces in instrument detecting or the visual inspection amplification process to judge.At present, external existing this type of more report, as detect in uncommon (family name) intestinal bacteria of tomato yellow leaf curl virus, tomato spotted wilt virus, intestines aggressive dust, the egg in Salmonellas, the river prawn white spot syndrome virus etc., and set up special official website (http://loopamp.eiken.co.jp/e/tech/index.htm).And domestic starting late, 2002 and Beijing Milk Cow Center in 2003 introduce the sex that the LAMP method detects the embryo 2 times.Simultaneously, people are also constantly improveing the LAMP technology, and it can be played a role better in fields such as disease gene diagnosis, food analysis and environmental monitorings.
The LAMP method that adopts in the present embodiment has following characteristics: (1) high specific: use 6 sections, 4 kinds of primers, and the order of these 6 sections also has regulation.Therefore the specificity of L AMP method amplification is very high, and whether the existence that can just can judge target gene according to whether increasing, promptly can carry out bacterium or viral qualitative detection.(2) only need just energy amplified reaction of a steady temperature.Do not need special reagent and expensive instrument, do not need to carry out in advance the sex change of double-stranded DNA.(3) fast efficient amplification: whole amplification can be finished less than one hour, and productive rate is very high.If on primer, further improve again, can improve its amplification efficiency greatly, proliferation time reduce on the original basis 1/3～1/2 (4) highly sensitive: amplification template can reach 1～10 copy (5) and identify easy: when nucleic acid is synthetic in a large number, pyrophosphate ion of separating out from dNTP and the Mg ionic bond the reaction soln produce by product---the magnesium pyrophosphate precipitation.It has high specificity, as long as detect by an unaided eye or turbidimeter ultraviolet imagery system detects the precipitation turbidity and just can whether judge amplification.Though for the check of some virus, the sensitivity of LAMP method and specificity do not surmount the PCR of special methods as yet; Its sensitivity of turbidity detection method of magnesium pyrophosphate also is lower than the agarose gel electrophoresis analytical method; But to a great extent, the LAMP method has met clinical required requirement.If to LAMP method method carry out further perfect, optimize and promote, this technology will have broad application prospects aspect viral detecting.
Embodiment 2:
Present embodiment is the composition of the reaction system described in the embodiment 1.Comprise in this reaction system:
Betaine?0.5-1.5mol/L，
dNTP?2-25mmol/L，
Mg
2+?2-15mmol/L，
Outer primer, inner primer, the ratio of the concentration of described inner primer and outer primer is 1: 1-1: 8;
Introduce Mg in the present embodiment
2+Method be: adding concentration in this reaction system is the MgCl of 2-15mmol/L;
The consumption of this inner primer is 0.2 μ mol/L, and the consumption of this outer primer is 0.8 μ mol/L; With volume is that 25 μ l systems are example, also comprises in the described system: the big fragment 1 μ l of 1 U Bst archaeal dna polymerase, 10 * Bst polymerse buffer, 1 μ l, dna profiling (concentration is 15 μ g/ml) 1 μ l, the ddH of capacity
2O;
The volume of this reaction system can also can be 50 μ l.
The volume of this reaction system can be 25 μ l, also can be 50 μ l.
Described dna profiling extracts the chrysanthemum plant that self-infection has tomato aspermy virus, and the preparation method of this dna profiling is:
At first, method according to the product description of TRIZOL Reagent (available from Invitrogen company) is extracted chrysanthemum blade RNA with the Trizol method, method in the product description of according to the M-MLV first chain cDNA synthetic agent box (available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) obtains described dna profiling by the RT-PCR amplification again;
In the above-mentioned reaction system, according to the specific LAMP primer of the design of the conserved sequence in the tomato aspermy virus of GenBank announcement, this conserved sequence is TAV coat protein (the Genbank number of landing EU499736); This primer comprises two outer primer f3, B3 and two inner primer FIP, BIP.Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.The nucleotide sequence of primer is as follows:
F3：5’-AAGCCGATGAAATGGTGATC-3’，
B3：5’-GTTCAAAGGCACCTCATCGA-3’；
FIP：5’-TTCAAGCGTAGTTTAACGTCTCCGCTTCAACGCTCCCGGT-3’，
BIP：5’-GCTCTAAACTATCTGAAGTCTTTTAGCCGTTAGCTGGATG-3’；
The reagent and the equipment that use in the present embodiment are conventional products, all can buy to obtain on market.
Bst DNA PoLymerase described in the present embodiment purchases the company in New England Biolabs, and described Betaine purchases the company in Solarbio, and TRIZOL Reagent is available from Invitrogen company, and SYBR Green I dyestuff is purchased in hundred Tyke biologies;
In the present embodiment, each component in the described reaction system can be in the ratio range that provides flexible combination, do not enumerate one by one at this.
Embodiment 3:
Present embodiment is the preferred version on embodiment 2 bases, and the quality of each component is identical with embodiment 2 with the source in the used reaction system.Comprise in this reaction system:
Betaine?0.6mol/L，
dNTP?3mmol/L，
Mg
2+?3mmol/L，
Outer primer, inner primer, the ratio of the concentration of described inner primer and outer primer is 1: 1;
Introduce Mg in the present embodiment
2+Method be: adding concentration in this reaction system is the MgCl of 3mmol/L;
The consumption of this inner primer is 0.2 μ mol/L, and the consumption of this outer primer is 0.2 μ mol/L; With volume is that 25 μ l systems are example, also comprises in the described system: the big fragment 1 μ l of 1 U Bst archaeal dna polymerase, 10 * Bst polymerse buffer, 1 μ l, dna profiling (concentration is 15 μ g/ml) 1 μ l, the ddH of capacity
2O;
The volume of this reaction system can also can be 50 μ l.
Identical among used primer and source thereof and the embodiment 1 in the present embodiment.
Embodiment 4:
Present embodiment is the preferred version on embodiment 2 bases, and the quality of each component is identical with embodiment 2 with the source in the used reaction system.Comprise in this reaction system:
Betaine?1mol/L，
dNTP?12mmol/L，
Mg
2+?9mmol/L，
Outer primer, inner primer, the ratio of the concentration of described inner primer and outer primer is 1: 5;
Introduce Mg in the present embodiment
2+Method be: adding concentration in this reaction system is the MgCl of 9mmol/L;
The consumption of this inner primer is 0.2 μ mol/L, and the consumption of this outer primer is 1 μ mol/L; With volume is that 25 μ l systems are example, also comprises in the described system: the big fragment 1 μ l of 1 U Bst archaeal dna polymerase, 10 * Bst polymerse buffer, 1 μ l, dna profiling (concentration is 15 μ g/ml) 1 μ l, the ddH of capacity
2O;
The volume of this reaction system can also can be 50 μ l.
Identical among used primer and source thereof and the embodiment 1 in the present embodiment.
Embodiment 5:
Present embodiment is the preferred version on embodiment 2 bases, and the quality of each component is identical with embodiment 2 with the source in the used reaction system.Comprise in this reaction system:
Betaine?1.6mol/L，
dNTP?25mmol/L，
Mg
2+?14mmol/L，
Outer primer, inner primer, the ratio of the concentration of described inner primer and outer primer is 1: 8;
Introduce Mg in the present embodiment
2+Method be: adding concentration in this reaction system is the MgCl of 14mmol/L;
The consumption of this inner primer is 0.2 μ mol/L, and the consumption of this outer primer is 1.6 μ mol/L; With volume is that 25 μ l systems are example, also comprises in the described system: the big fragment 1 μ l of 1 U Bst archaeal dna polymerase, 10 * Bst polymerse buffer, 1 μ l, dna profiling (concentration is 15 μ g/ml) 1 μ l, the ddH of capacity
2O;
The volume of this reaction system can also can be 50 μ l.
Identical among used primer and source thereof and the embodiment 1 in the present embodiment.
Embodiment 6:
Present embodiment is made comparison with LAMP method and PCR method to the sensitivity that tomato aspermy virus detects.
The detection steps in sequence is:
A. the dna profiling of getting original concentration and be 15 μ g/ml carries out doubling dilution respectively, and the concentration of dilution rear pattern plate is respectively 10 of original concentration
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6
B. the template after will diluting is carried out LAMP method and the reaction of PCR method respectively; In the described LAMP method reaction, the detection step among reaction system described in the Application Example 4 and the embodiment 1 obtains LAMP method reaction product and PCR method reaction product respectively.
In the described PCR reaction, reaction system is 25 μ l, comprises following component:
The nucleotides sequence of this primer is classified as:
Upstream primer: 5 '-TTCACCACTGTCACACACTCTAGA-3 '
Downstream primer: 5 '-GGTTCAAAGGCACCTCATCGATCA-3 '
Response procedures is:
C. above-mentioned reaction is got 9 μ l LAMP method reaction product and PCR method reaction product respectively after finishing, and carries out 1% agarose gel electrophoresis, after electrophoresis finishes, and the ultraviolet visualization result.
Among Fig. 1, among the swimming lane M DL2000 marker; Swimming lane 1,3 is respectively LAMP method reaction product in 5,7,9,11,13, obtains concentration after the dna profiling dilution of this product and is followed successively by original concentration: 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6 Swimming lane 2,4 is respectively PCR method reaction product in 6,8,10,12, obtains concentration after the dna profiling dilution of this product and is followed successively by original concentration: 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6Negative contrast in the swimming lane 13.
As can be known from Fig. 1: use the LAMP method, when the concentration dilution of described dna profiling to 10 of original concentration
-6Shi Yiran can detect tomato aspermy virus; And use the PCR method, when the concentration dilution of described dna profiling to 10 of original concentration
-2The time just can't detect tomato aspermy virus.So application LAMP method obviously is better than the PCR method to the detection sensitivity of tomato aspermy virus.
Claims (5)
1. method that is used to detect tomato aspermy virus (TAV) is characterized in that: this method utilizes loop-mediated isothermal amplification (LAMP) to detect tomato aspermy virus (TAV);
The steps in sequence that this method comprises is:
A. prepare reaction system;
B. described reaction system is carried out incubation and handle, obtain the incubation product; The temperature that this incubation is handled is 65 ℃, and the time is 30-80min;
C. described incubation product is carried out inactivation treatment, obtain the deactivation product; The temperature of this inactivation treatment is 80 ℃, and the time is 5-15min;
D. described deactivation product is detected, obtain detected result;
Volume is to comprise in the described reaction system of 25 μ l:
Betaine0.5-1.5mol/L，
dNTP2-25mmol/L，
Mg
2+2-15mmol/L，
Outer primer, inner primer; The ratio of the concentration of described inner primer and outer primer is 1: 1-1: 8;
The consumption of this inner primer is 0.2 μ mol/L, and the consumption of this outer primer is 0.8 μ mol/L;
Also comprise in the described reaction system:
The big fragment 1 μ l of 1U Bst archaeal dna polymerase, 10 * Bst polymerse buffer, 1 μ l,
Concentration is the dna profiling 1 μ l of 15 μ g/ml, the ddH of capacity
2O;
The nucleotides sequence of described outer primer is classified as:
5’-AAGCCGATGAAATGGTGATC-3’，
5’-GTTCAAAGGCACCTCATCGA-3’；
The nucleotides sequence of described inner primer is classified as:
5’-TTCAAGCGTAGTTTAACGTCTCCGCTTCAACGCTCCCGGT-3’，
5’-GCTCTAAACTATCTGAAGTCTTTTAGCCGTTAGCTGGATG-3’。
2. the method for detection tomato aspermy virus according to claim 1 (TAV) is characterized in that: comprise in the described reaction system:
Betaine?0.6mol/L，
dNTP?3mmol/L，
Mg
2+?3mmol/L，
Outer primer, inner primer, the ratio of the concentration of described inner primer and outer primer is 1: 1.
3. the method for detection tomato aspermy virus according to claim 1 (TAV) is characterized in that: comprise in the described reaction system:
Betaine?1mol/L，
dNTP?12mmol/L，
Mg
2+?9mmol/L，
Outer primer, inner primer, the ratio of the concentration of described inner primer and outer primer is 1: 5.
4. the method for detection tomato aspermy virus according to claim 1 (TAV) is characterized in that: comprise in the described reaction system:
Betaine?1.6mol/L，
dNTP?25mmol/L，
Mg
2+?14mmol/L，
Outer primer, inner primer, the ratio of the concentration of described inner primer and outer primer is 1: 8.
5. the method for detection tomato aspermy virus according to claim 1 (TAV) is characterized in that: the chrysanthemum kind that this method is suitable for is: powder carpet, refreshing horse.
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| 周广青.《环介导等温基因扩增技术及其在病毒检测中的应用》.《生物技术通讯》.2008,第19卷(第2期),290-292. * |
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