CN103146847B - RT-LAMP (loop-mediated isothermal amplification) rapid detection kit for cucumber green mottle mosaic virus (CGMMV) and detection method - Google Patents
RT-LAMP (loop-mediated isothermal amplification) rapid detection kit for cucumber green mottle mosaic virus (CGMMV) and detection method Download PDFInfo
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Abstract
The invention discloses an RT-LAMP (loop-mediated isothermal amplification) rapid detection kit for cucumber green mottle mosaic virus (CGMMV) and a detection method. The method comprises the steps of extracting plant viruses RNA, detecting an RT-LAMP reaction and electrophoresis detection or fluorochrome, and determining whether a plant carries the viruses. An RT-LAMP primer is designed according to a coat protein gene conserved region of CGMMV, and an RT-LAMP technology is adopted to establish a detection technology which can rapidly and sensitively detect the cucurbitaceous plant CGMMV such as cucumbers, watermelons and calabashes with high specificity and an application method thereof in diagnosing disease. The detection method provided by the invention can utilize the rapid extraction RNA and conventional RNA extraction as an RNA template for an RT-LAMP experiment. The method is utilized to rapidly identify a CGMMV plant sample, the targeted quarantine inspection protective measure is adopted, and the loss caused by the disease is reduced.
Description
Technical field
The invention belongs to agricultural cience and farming techniques field, relate to the Fast Detection Technique of cucumber green mottle mosaic virus on the cucurbitaceous plants such as watermelon, cucumber and cucurbit and the application in disease screening thereof, be specifically related to a kind of RT-LAMP quick detection kit and detection method of cucumber green mottle mosaic virus.
Background technology
Cucumber green mottle mosaic virus (Cucumber green mottle mosaic virus, CGMMV) belongs to Tombusviridae (Tombusviridae), Tobamovirus (Tobamovirus) member.This disease ground family crop of seriously causing harm is the quarantine harmful organisms of China.The catch an illness seedling stage existing BG of 2～3 leaves or the sap green of seedling top tip part of cucumber green mottle mosaic virus is mottled, and blade is more flat, produces the sick portion protuberance that sap green is mottled, young leaves is dark green, and later stage vein is changed thoroughly, and blade diminishes, cause plant dwarfing, the mottled distortion of blade, is systematicness and infects.The sugar-preserved gourd existing strong green piebald of catching an illness, the also generation knob having, causing fruit becomes lopsided melon, affects commodity value, the serious underproduction 25% left and right.Nature mainly infects the multiple ground family crops such as muskmelon, watermelon, cucumber, pumpkin and bottle gourd, propagates by seed and soil, also can pass through in addition juice, and the modes such as farming operation and blade contact are propagated.It is the main path of these virus long-distance communications with malicious seed dispersal.
This disease, since nineteen thirty-five is found, had occurred in Britain, Germany, Denmark, Holland, Russia, India, Japan, Korea S and China Taiwan once and had caused serious harm.Cucumber green mottle virus disease was not recorded in China mainland in the past, and within 2006, domestic indivedual local discovery watermelon green statins are refuted virus disease, and in December, 2006, the Ministry of Agriculture issued bulletin No. 788, was classified as national Plant Quarantine harmful organism.
On plant virus Identification, the normal method adopting has at present: biological experiment, serology detects, electron microscopic observation and molecular Biological Detection.Due to traditional plant virus monitoring method, whether be cucumber green mottle mosaic virus as these methods such as electron microscopic observation, traditional biological inoculation qualification are difficult to go out respectively, therefore need one fast and accurately method detect this virus.Ring mediated constant temperature nucleic acid amplification technology (Loop-mediated isothermal amplification, LAMP), be the constant temperature nucleic acid amplification method in a kind of novelty of invention in 2000 by Notomi etc., he is a kind of highly sensitive strand displacement technology, a kind of novel PCR substitute technology.LAMP feature be for 6 zone design of target gene 4 special primers, utilize a kind of strand displacement archaeal dna polymerase-Bst DNA polymerase under the condition of constant temperature, to react 60min and just can complete nucleic acid amplification reaction, by observing and can observe clearly reaction result under UV-light after fluorescent dye.Do not need long temperature cycle, do not need expensive PCR instrument, do not need the processes such as loaded down with trivial details electrophoresis ultraviolet observation.Disease surveillance and quick diagnosis that the mankind and the various virus of animals and plants, bacterium, parasite etc. cause are widely used at present.But, still RT-LAMP technology is not applied in the detection of cucumber green mottle mosaic virus (CGMMV) at present.
Summary of the invention
The object of the present invention is to provide a kind of RT-LAMP primer sets for cucumber green mottle mosaic virus rapid detection.
Another object of the present invention is to provide a kind of cucumber green mottle mosaic virus RT-LAMP quick detection kit.
Another object of the present invention is to provide a kind of RT-LAMP method for quick of cucumber green mottle mosaic virus.The method is for cucumber green mottle mosaic virus on the Curcurbitaceae such as rapid detection watermelon, cucumber and cucurbit, can quick diagnosis goes out doubtful diseased plant whether carry cucumber green mottle mosaic virus by the method.
Object of the present invention can be achieved through the following technical solutions:
A kind of RT-LAMP primer sets for cucumber green mottle mosaic virus rapid detection, it is that described RT-LAMP primer sets comprises a pair of outer primer and a pair of inner primer, described outer primer is respectively shown in SEQ ID No.1 and SEQ ID No.2 the sequence of F3, B3, and described inner primer is respectively shown in SEQ ID No.3 and SEQ ID No.4 the sequence of FIP, BIP.
The application of above-mentioned primer sets in quick detection kit or the detection reagent of preparing cucumber green mottle mosaic virus.
A kind of cucumber green mottle mosaic virus RT-LAMP quick detection kit that contains above-mentioned primer sets.
Above-mentioned cucumber green mottle mosaic virus RT-LAMP quick detection kit, it is that described test kit comprises the detection system being mainly made up of plant virus RNA rapid extraction liquid, RT-LAMP reaction solution, DEPC water and fluorescence dye SYBR Green I detection liquid.
Above-mentioned cucumber green mottle mosaic virus RT-LAMP quick detection kit, it is that described plant virus RNA rapid extraction liquid comprises 0.5M NaOH and Tris-HCL(pH8.0); Adopt plant virus RNA rapid extraction liquid to extract sample and slightly carry total RNA, the sample of extraction is slightly carried to total RNA and join in RT-LAMP reaction solution and mix, set up amplification system; Taking reaction cumulative volume 10 μ L as example, in described amplification system, the content of each component is: sample is slightly carried total RNA 0.8 μ L, 1 × ThermoPol Buffer, 4mM MgCl
2, 0.8mM dNTP, 0.8M Betaine, the Bst DNA Polymerase of 3.2U, the AMV Polymerase of 0.5U, the BIP of the FIP of 1 μ M and 1 μ M, the F of 0.2 μ M
3b with 0.2 μ M
3, supplement DEPC water to 10 μ L.
A kind of RT-LAMP method for quick of cucumber green mottle mosaic virus, it is to use above-mentioned primer sets to carry out RT-LAMP amplification to testing sample, utilizes electrophoresis detection or fluorescence dye technology to identify and in sample to be detected, whether contains cucumber green mottle mosaic virus.
The RT-LAMP method for quick of above-mentioned cucumber green mottle mosaic virus, specifically comprises the following steps:
(1) extract sample and slightly carry total RNA;
(2) get sample that step (1) extracts and slightly carry total RNA and join in RT-LAMP reaction solution and mix, set up amplification system;
(3) in 63 ~ 64 DEG C of waters bath with thermostatic control, react 60 ~ 75min;
(4) result diagnosis: get detection of fluorescent dyes liquid SYBR Green I and join in step (3) amplified reaction product and diagnose, under UV-light, observe, be positive and in aobvious green expression sample, contain cucumber green mottle mosaic virus, be negative and keep the orange expression sample of dyestuff not contain cucumber green mottle mosaic virus.
The extracting method that the described sample of step (1) is slightly carried total RNA is: in 0.5M NaOH solution, grind and make lapping liquid fast with the sick leaf just having gathered, lapping liquid is joined to Tris-HCL(pH8.0) in make sample and slightly carry total RNA extracting solution.
The mass volume ratio g:mL of described sick leaf and 0.5M NaOH solution is 1:4; Described mixed solution and Tris-HCL(pH8.0) volume ratio be 1:49.
Described in step (2), reacting cumulative volume 10 μ L is example, and in amplification system, the content of each component is: sample is slightly carried total RNA 0.8 μ L, 1 × ThermoPol Buffer, 4mM MgCl
2, 0.8mM dNTP, 0.8M Betaine, the Bst DNA Polymerase of 3.2U, the AMV Polymerase of 0.5U, the BIP of the FIP of 1 μ M and 1 μ M, the F of 0.2 μ M
3b with 0.2 μ M
3, supplement DEPC water to 10 μ L.
The method of cucumber green mottle mosaic virus on rapid detection diseased plant provided by the present invention, specifically comprises following detailed step:
1) the cucumber green mottle mosaic virus nucleotide sequence of having reported according to NCBI, finds out this disease nucleic acid coat protein gene conservative region sequence after analyzing use online LAMP primer-design software Primer Explorer V4 (http://primerexplorer.jp/elamp4.0.0/index.html) design primer as template reference sequences by software DNAstar;
2) rapid fractionation method extracts Plant samples and slightly carries total RNA;
3) different Mg is set
2+concentration, dNTP concentration, inside and outside primer concentration ratio, Betaine concentration, determine optimum response system; Change respectively reaction times and temperature and carry out RT-LAMP amplified reaction, determine optimum reaction condition.
4) judge that by UV-light or naked eyes SYBR Green I dyeing carries out visualization processing to RT-LAMP product.
5), under optimum response Parameter Conditions, compare the sensitivity of checking RT-LAMP with RT-PCR method;
6) specificity taking CMV and TMV as this reaction of contrast checking;
Primer sequence provided by the invention is as follows:
Wherein, described detection kit comprises following solution:
Solution 1: plant virus RNA rapid extraction liquid comprises: 0.5M NaOH and Tris-HCL(pH8.0);
RT-LAMP reaction solution comprises solution 2 and solution 3:
Solution 2:10 × ThermoPol Buffer, MgCl
2(25mM), dNTP(10mM each), Betaine(5M) and, Bst DNA Polymerase(8U/ μ L), AMV Polymerase(10U/ μ L);
Solution 3:RT-LAMP primer mixed solution comprises: inner primer FIP(20 μ M) and BIP(20 μ M), outer primer F
3(10 μ M) and B
3(10 μ M);
Solution 4:DEPC water;
Solution 5: detection of fluorescent dyes liquid SYBR Green I.
Further, the RT-LAMP method for quick of described detection cucumber green mottle mosaic virus, comprises the steps:
1) with the sick leaf 0.1g just having gathered, in the 0.5M NaOH of 400 μ L solution, grind, get 10 μ L lapping liquids and join 490 μ L Tris-HCL(pH8.0) in, the liquid of 0.8 μ L directly got as the total RNA template of thick extraction detecting;
2) preparation RT-LAMP reaction solution: setting up amplification system by cumulative volume 10 μ L is: 5.45 μ L solution 2[10 × ThermoPol Buffer 1 μ L, MgCl
2(25mM) 1.6 μ L, dNTP(10mM each) 0.8 μ L, Betaine(5M) 1.6 μ L, Bst DNA Polymerase(8U/ μ L) 0.4 μ L, AMV Polymerase(10U/ μ L) 0.05 μ L], 1.4 μ L solution 3[FIP(20 μ M) and BIP(20 μ M) each 0.5 μ L, outer primer F
3(10 μ M) and B
3(10 μ M) each 0.2 μ L], 2.35 μ L DEPC water;
3) getting the sample that step 1) extracts slightly carries total RNA 0.8 μ L and joins step 2) in the RT-LAMP reaction solution of preparation, mix;
4) in 63 ~ 64 DEG C of waters bath with thermostatic control, react 60 ~ 75min;
5) result diagnosis: 1 μ L solution 5 is joined in step 4) amplified reaction product and diagnosed, under UV-light, observe, being positive is to contain cucumber green mottle mosaic virus in green expression sample, is negative and keeps the orange expression sample of dyestuff not contain cucumber green mottle mosaic virus.
Compared to existing technology, tool of the present invention has the following advantages:
The gene order of the cucumber green mottle mosaic virus (CGMMV) that the present invention issues according to GenBank, the Auele Specific Primer 2 of RT-LAMP detection method that has designed CGMMV for coat protein gene conservative region is right, a pair of outer primer F3, B3 and a pair of inner primer FIP, BIP, 2 pairs of primer sequences are as implied above.Taking the total RNA of rapid extraction sample as template, set up the RT-LAMP technology of rapid detection CGMMV.
RT-LAMP detection technique of the present invention is utilized Auele Specific Primer (4), and to identify several specific regions of CGMMV virus target gene, specificity is stronger.Reaction is carried out under isothermal condition (63 ~ 64 DEG C), do not need expensive PCR instrument, amplified reaction is exceedingly fast, and generally just can complete at 60 ~ 75min, amplified production amount is large, utilizes electrophoresis detection or fluorescence dye technology to identify sample to be tested and whether contains CGMMV virus; Highly sensitive, high specificity, be applicable to the accurate detection of virus.
RT-LAMP technology of the present invention does not need twice amplification of independent transcriptive process,reversed and RT-PCR, reduces some links of Molecular Detection, has reduced the pollution rate detecting.And experimental results show that detection sensitivity is higher 100 times than RT-PCR, makes the method more accurate to the early diagnosis of CGMMV virus.
That the present invention has advantages of is simple to operate, cost is low, reaction is quick, high specificity, highly sensitive, and the used time is short, and remolding sensitivity conventional RT-PCR is high 100 times, higher to viral early diagnosis accuracy.Can extensively utilize this sample rapid detection, the popular monitoring of CGMMV, quarantine to detect and viral discriminating.
Brief description of the drawings
Fig. 1 is different Mg
2+the optimization electrophorogram of concentration, dNTP concentration, inside and outside primer concentration ratio, Betaine concentration
A is Mg
2+(1 is negative for concentration optimization result figure; 2-6 is Mg
2+2,3,4,5,6mM concentration:; M is Marker);
B is that (1 is negative for dNTP concentration optimization result figure; 0.2,0.4,0.6,0.8,1.0mM 2-6 is dNTP concentration:; M is Marker);
C is that (1 is negative for outer primer F3 and B3 concentration optimization result figure; 2-6 is that F3, B3 are each: 0.05,0.10,0.15,0.20,0.25 μ M; M is Marker);
D is that (1 is negative for FIP and BIP concentration optimization result figure; 2-6 is that FIP, BIP are each: 0.4,0.6,0.8,1.0,1.2 μ M; M is Marker);
E is that (1 is negative for Betaine concentration optimization result figure; 2-6 is Betaine concentration: 0,0.4,0.8,1.2,1.6M)
Fig. 2 is the electrophorogram of RT-LAMP result under differential responses temperature condition
M is Marker; 1 is 61 DEG C of positives, and 2 is 61 DEG C of feminine genders, and 3 is 62 DEG C of positives, and 4 is 62 DEG C of feminine genders, and 5 is 63 DEG C of positives, and 6 is 63 DEG C of feminine genders, and 7 is 64 DEG C of positives, and 8 is 64 DEG C of feminine genders, and 9 is 65 DEG C of positives, and 10 is 65 DEG C of feminine genders.
Fig. 3 is the electrophorogram of RT-LAMP result under differential responses time conditions
1 is negative; 2 is 15min; 3 is 30min; 4 is 45min; 5 is 60min; 6 is 75min; M is Marker.
Fig. 4 is RT-LAMP and RT-PCR sensitivity comparison diagram and fluorescence dye method detection RT-LAMP amplified production figure
A is RT-LAMP reaction result figure; B is RT-PCR reaction result figure; C is SYBR Green I fluorescence dye method detected result figure; M is Marker; 1 is 72ng/ μ L; 2 is 7.2ng/ μ L; 3 is 7.2 × 10
-1ng/ μ L; 4 is 7.2 × 10
-2ng/ μ L; 5 is 7.2 × 10
-3ng/ μ L; 6 is 7.2 × 10
-4ng/ μ L; 7 is 7.2 × 10
-5ng/ μ L; 8 is 7.2 × 10
-6ng/ μ L; 9 negative contrasts.
Detected result: from left to right, 1-4 test tube is green, and 5-9 is orange.
Fig. 5 is for utilizing rapid extraction plant RNA to carry out RT-LAMP reaction result figure as template
1 is negative; 2 is the RNA of rapid extraction health plant; 3 is the RNA of rapid extraction band CGMMV diseased plant; 4 is positive.
Fig. 6 utilizes Quick sampling to detect the specific result figure of RT-LAMP of CGMMV
A is RT-LAMP reaction result figure; B is RT-PCR reaction result figure; M is Marker; 1 is positive; 2 for slightly carrying the sick sample of CGMMV; 3 for slightly carrying the sick sample of CMV; 4 for slightly carrying the sick sample of TMV; 5 for slightly carrying healthy plant; 6 is negative.
Detected result: from left to right, 1-2 test tube is green, and 3-6 is orange.
Embodiment
The optimization of embodiment 1 cucumber green mottle mosaic virus RT-LAMP reaction conditions
1. the design of cucumber green mottle mosaic virus RT-LAMP primer
The cucumber green mottle mosaic virus of having reported according to NCBI
（http://www.ncbi.nlm.nih.gov/nuccore/NC_001
801.1) complete nucleotide gene group sequence, after analyzing by software DNAstar, find out this viral nucleotide coat protein region 5763 ~ 6248bp, utilize corresponding zone 5763 ~ 6248bp on NP_044580.1 as template reference sequences, use online LAMP primer-design software Primer Explorer V4 (http://primerexplorer.jp/elamp4.0.0/index.html) design primer.The corresponding parameter such as 3 ' end or 5 ' end stability and primer dimer of primer providing by software again compares selection to the multipair primer of designing, and finally chooses one group of optimal primer, and sequence is as follows:
F3 CCGTCAGGACTTTACTTA
B3 GAGCTGAGAAGCGAAACGA
FIP ACTCGCGGAAAGAATCTCTTCCTTCTAGTTGCTTCACAAGGTAC
BIP CTGTCGTAGATATTAATTCTAGATTCCAACACAGGACCGTTGAGGAA
2. the Mg of cucumber green mottle mosaic virus RT-LAMP
2+concentration optimization experiment
Obtain diseased plant from Agricultural University Of Nanjing's plant virology laboratory, adopt RT-PCR to filter out cucumber green mottle mosaic virus diseased plant for RT-LAMP test experience, use RNA simple Total RNA Kit total RNA extraction reagent box to extract the total RNA of sample.RT-LAMP system is: 10 × ThermoPol Buffer, 1 μ L, dNTP(10mM each) 0.8 μ L, inner primer FIP(20 μ M) and BIP(20 μ M) each 0.5 μ L, outer primer F
3(10 μ M) and B
3(10 μ M) each 0.2 μ L, Betaine(5M) 1.6 μ L, Bst DNA Polymerase(8U/ μ L) 0.4 μ L, AMV Polymerase(10U/ μ L) 0.05 μ L, MgCl
2(25mM) final concentration, at 2 ~ 6mM, increases progressively with the amount of every 1mM, and the total RNA template of 0.8 μ L is supplemented DEPC water to 10 μ L.63 DEG C of 60min of reaction conditions, 80 DEG C of 10min.1% gel electrophoresis observations.Choose through optimizing minimum final concentration 4mM(Fig. 1 A that amplification efficiency is the highest), as MgCl in reaction system
2consumption.
3. the dNTP concentration optimization of cucumber green mottle mosaic virus RT-LAMP experiment
Employing embodiment 1 2. middle clear, the bright reaction of electrophoretic band organizes corresponding MgCl
2consumption is further made the optimization experiment of dNTP concentration, and reaction conditions is constant, and the final concentration of dNTP, at 0.2 ~ 1mM, increases progressively with the amount of every 0.2mM, 1% gel electrophoresis observations for amplified production.Through optimizing, the final concentration of determining dNTP of the present invention is 0.8mM(Fig. 1 B).
4. the outer primer concentration optimization of cucumber green mottle mosaic virus RT-LAMP experiment
The fixing amount of having optimized, outer primer F3, B3 mix in the ratio of 1:1, and F3, B3 final concentration increase progressively successively with the amount of 0.05mM, 0.10mM, 0.15mM, 0.20mM, 0.25mM, and reaction conditions is constant, 1% gel electrophoresis observations.The final concentration that selects F3 and B3 is respectively 0.20 μ M(Fig. 1 C).
5. the inner primer concentration optimization of cucumber green mottle mosaic virus RT-LAMP experiment
The fixing above amount of having optimized, inner primer FIP, BIP mix in the ratio of 1:1, FIP, BIP final concentration increase progressively successively with the amount of 0.4mM, 0.6mM, 0.8mM, 1.0mM, 1.2mM, and reaction conditions is constant, and therefore after revision test, selecting inner primer final concentration is respectively 1 μ M(Fig. 1 D).
6. the optimization of the Betaine concentration of cucumber green mottle mosaic virus RT-LAMP
On the basis of above optimization Test, carry out the optimization of Betaine consumption, Betaine final concentration increases progressively successively with the amount of 0M, 0.4M, 0.8M, 1.2M, 1.6M, and reaction conditions is constant, reacts at least in triplicate 1% gel electrophoresis observations.Expanding effect best (Fig. 1 E) in the time that the concentration of Betaine is 0.8M.
7. the thermograde of cucumber green mottle mosaic virus RT-LAMP experiment
Although the optimal reactive temperature of Bst DNA polymerase is 65 DEG C, but many reports have confirmed RT-LAMP and have been applicable to temperature of reaction between 60 ~ 65 DEG C, simultaneously because different primers can not ensure that in the time designing the Tm value of all primers is in full accord, and RT-LAMP also will consider the optimal temperature of ThermoScript II, Given this, the present invention is optimized test to RT-LAMP temperature of reaction.Press embodiment 1. ~ amount 6. optimized adds reaction system, system is at 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C, isothermal reaction 1h under 65 DEG C of five kinds of conditions, 80 DEG C of reaction 10min stop RT-LAMP reaction, amplified production loading, on 1% sepharose, carry out leakage of electricity swimming, result is presented under same reaction system condition, more and more brighter 61 ~ 63 DEG C of band brightness, and 64 ~ 65 DEG C time band brightness darkness deepens, and negative control at each temperature is without amplified production (Fig. 2).Therefore the temperature of reaction of RT-LAMP of the present invention is 63 ~ 64 DEG C.
8. the reaction times gradient experiment of cucumber green mottle mosaic virus RT-LAMP
Many reports are verified, and the RT-LAMP reaction times is less than 1h amplified production also can be detected.Be placed under 63 DEG C of constant temperatures and react by the RT-LAMP system application of sample of having optimized, set temperature reaction 15min respectively, 30min, 45min, 60min, five kinds of processing of 75min, complete reaction rear electrophoresis and detect.Result shows amplified production in the time that the reaction times is 45min, but product is less, but in the time that the reaction times is 60 ~ 75min, RT-LAMP amplified production starts to increase, after this in the time continuing to increase the reaction times, the quantitative changeization of amplified production little (Fig. 3), therefore the reaction times of the definite RT-LAMP of the present invention is 60 ~ 75min.
9. RT-LAMP and the comparison of RT-PCR susceptibility
In order to determine the sensitivity of RT-LAMP and two kinds of detection methods of RT-PCR, the total RNA extracting is measured to the rear RNase-free ddH that uses of concentration (72ng/ μ L) with NanoDrop1000
2o carries out 10 doubling dilutions, and-80 DEG C of preservations are as template.Get respectively each concentration RNA diluent 0.8 μ L after 10 doubling dilutions as template, adopt the RT-LAMP reaction system of having optimized to react (63 DEG C are reacted 60min).Get 3 μ L amplified production loadings, leakage of electricity swimming on 1% sepharose.Simultaneously, keep identical RNA ratio with RT-LAMP, getting the total RNA of 10 μ L is template, 1.5 μ L B3 are that reverse transcription primer and 1 μ L dNTP react 5min at 65 DEG C, then ice bath 2min, after ice bath, add 4 μ L 5 × M-MLV buffer, 2 μ L DTT (50mM), 1 μ L M-MLV and 0.5 μ L RRI carry out synthetic cDNA the first chain of reverse transcription.Getting the synthetic cDNA of 1.6 μ L reverse transcription is template, carries out pcr amplification taking F3 and B3 as primer, and use system is that the conventional PCR reaction system of 10 μ L is reacted.After finishing, reaction gets 3 μ L amplified production loadings, leakage of electricity swimming on 1% sepharose.Result shows can detect that by RT-LAMP method concentration is 7.2 × 10
-2total RNA of the CGMMV of ng/ μ L, and RT-PCR also can detect that concentration is total RNA of the CGMMV of 7.2ng/ μ L, RT-LAMP is than highly sensitive 100 times of RT-PCR (Fig. 4 A, B).
10. fluorescence dye method detects RT-LAMP sensitivity amplified production
Press embodiment 1 9. in after RT-LAMP reaction product electrophoresis detection, in PCR pipe, add SYBR Green I 0.7 μ L(1:1000), observations after several minutes, under UV-light, positive amplified production can become green, and keeps orange (Fig. 4 C) of dyestuff without amplified production and negative control.
Embodiment 2, slightly carries cucumber green mottle mosaic virus sample and does specificity and the Visualization that RT-LAMP reacts
1. utilize the sample of rapid extraction slightly to carry total RNA and be RT-LAMP
Fast Coarse is put forward the method for RNA, can make the sick sample of CGMMV RT-LAMP rapid detection in detection, raises the efficiency.Mentioning fast RNA method is: with the sick leaf 0.1g just having gathered, in the 0.5M NaOH of 400 μ L solution, grind, get 10 μ L lapping liquids and join 490 μ L Tris-HCL(pH8.0) in, the liquid of directly getting 0.8 μ L does CGMMV RT-LAMP test experience (Fig. 5) as the total RNA template of thick extraction.
2. cucumber green mottle mosaic virus RT-LAMP specific test
In order to verify the specificity of RT-LAMP method, selection can be infected melon virus, and cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) (TMV) be in contrast in contrast.The total RNA of CGMMV extracting with test kit respectively and the CGMMV of runic, CMV, total RNA of TMV and healthy plant is as template, reacts by the RT-LAMP reaction system of having optimized in embodiment 1.When result shows the RNA template of removing to increase TMV and CMV with RT-LAMP primer, do not amplify band; And can amplify object band when the thick total RNA of CGMMV extracting of amplification kit amplification, this RT-LAMP detection method that shows that the present invention sets up has good specificity (Fig. 6 A).
3. fluorescence dye method detects RT-LAMP specificity result
By example 2 2. in after RT-LAMP reaction product electrophoresis detection, in PCR pipe, add SYBR Green I 0.7 μ L(1:1000), observations after several minutes, under UV-light, positive amplified production can become green, and keeps orange (Fig. 6 B) of dyestuff without amplified production and negative control.
On this detection plant, the application of cucumber green mottle mosaic virus method comprises: in quick diagnosis and the discriminating of the doubtful sick plant of multiple ground family crop such as muskmelon, watermelon, cucumber, pumpkin and bottle gourd.
Above-mentioned enforcement does not limit the present invention in any form.
Claims (6)
1. the RT-LAMP primer sets for cucumber green mottle mosaic virus rapid detection, it is characterized in that described RT-LAMP primer sets is a pair of outer primer and a pair of inner primer, described outer primer is respectively shown in SEQ ID No.1 and SEQ ID No.2 the sequence of F3, B3, and described inner primer is respectively shown in SEQ ID No.3 and SEQ ID No.4 the sequence of FIP, BIP.
2. the application of primer sets claimed in claim 1 in quick detection kit or the detection reagent of preparing cucumber green mottle mosaic virus.
3. a cucumber green mottle mosaic virus RT-LAMP quick detection kit that contains primer sets claimed in claim 1.
4. cucumber green mottle mosaic virus RT-LAMP quick detection kit according to claim 3, is characterized in that described test kit comprises plant virus RNA rapid extraction liquid, RT-LAMP reaction solution, DEPC water and fluorescence dye SYBR Green inspection I and surveys liquid;
Described plant virus RNA rapid extraction liquid comprises 0.5M NaOH and Tris-HCL pH8.0;
Described RT-LAMP reaction solution comprises solution 2 and solution 3, solution 2:10 × ThermoPol Buffer, MgCl
225mM, dNTP 10mM each, Betaine 5M, Bst DNA Polymerase 8U/ μ L, AMV Polymerase 10U/ μ L;
Solution 3:RT-LAMP primer mixed solution comprises: inner primer FIP 20 μ M and BIP 20 μ M, outer primer F
310 μ M and B
310 μ M.
5. the RT-LAMP method for quick of a cucumber green mottle mosaic virus, it is characterized in that right to use requires the primer sets described in 1 to carry out RT-LAMP amplification to testing sample, utilize electrophoresis detection or fluorescence dye technology to identify and in sample to be detected, whether contain cucumber green mottle mosaic virus.
6. the RT-LAMP method for quick of cucumber green mottle mosaic virus according to claim 5, is characterized in that specifically comprising the following steps:
(1) extract sample and slightly carry total RNA;
(2) get sample that step (1) extracts and slightly carry total RNA and join in RT-LAMP reaction solution and mix, set up amplification system;
(3) in 63～64 DEG C of waters bath with thermostatic control, react 60～75min;
(4) result diagnosis: get detection of fluorescent dyes liquid SYBR Green and add I and enter in step (3) amplified reaction product to diagnose, under UV-light, observe, be positive and in aobvious green expression sample, contain cucumber green mottle mosaic virus, be negative and keep the orange expression sample of dyestuff not contain cucumber green mottle mosaic virus;
The extracting method that the described sample of step (1) is slightly carried total RNA is: in 0.5M NaOH solution, grind and make lapping liquid fast with the sick leaf just having gathered, lapping liquid is joined and in Tris-HCL pH8.0, makes sample and slightly carry total RNA extracting solution;
The mass volume ratio g:mL of described sick leaf and 0.5M NaOH solution is 1:4; The volume ratio of described mixed solution and Tris-HCL pH8.0 is 1:49;
Described in step (2), reacting cumulative volume 10 μ L is example, and in amplification system, the content of each component is: sample is slightly carried total RNA 0.8 μ L, 1 × ThermoPol Buffer, 4mM MgCl
2, 0.8mM dNTP, 0.8M Betaine, the Bst DNA Polymerase of 3.2U, the AMV Polymerase of 0.5U, the BIP of the FIP of 1 μ M and 1 μ M, the F of 0.2 μ M
3b with 0.2 μ M
3, supplement DEPC water to 10 μ L.
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| CN103484569A (en) * | 2013-09-24 | 2014-01-01 | 广西大学 | Method for quickly detecting sorghum mosaic virus by RT-LAMP (Reverse transcription loop-mediated isothermal amplification) |
| CN103558379A (en) * | 2013-11-07 | 2014-02-05 | 曹冬梅 | Rapid detection kit for cucumber green mottle mosaic viruses |
| CN105177187B (en) * | 2015-10-14 | 2018-08-14 | 浙江大学 | Detect sample preparation methods and purposes that Curcurbitaceae seed carries cucumber green mottle mosaic virus |
| CN106011311A (en) * | 2016-06-28 | 2016-10-12 | 临沂大学 | Kit and method for detecting watermelon mosaic virus RT-LAMP |
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| CN106167835A (en) * | 2016-06-29 | 2016-11-30 | 临沂大学 | Cucumber green mottle viral RT LAMP detection kit and detection method |
| CN105969911A (en) * | 2016-06-29 | 2016-09-28 | 临沂大学 | RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection reagent kit and detection method for cucumber mosaic viruses |
| CN106282406A (en) * | 2016-08-09 | 2017-01-04 | 陈定虎 | Cucumber green mottle mosaic LAMP primer, test kit and detection method |
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