CN106834536A - Method using organizing the direct RT LAMP of juice to detect corium solani - Google Patents
Method using organizing the direct RT LAMP of juice to detect corium solani Download PDFInfo
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Abstract
The invention provides the method using organizing the direct RT LAMP of juice to detect corium solani, it comprises the following steps：(1) juice of potato plant is taken, is placed in RT LAMP reaction systems and is reacted；(2) detected, the detection includes following method：1) agarose gel electrophoresis analytical procedure (1) products therefrom is utilized, waterfall shape band occurs, then there is corium solani, such as without any band, then in the absence of corium solani；2) using visually observing, such as occur muddy or precipitation in reaction solution, then there is corium solani, such as reaction solution is limpid, then in the absence of corium solani.The present invention, without RNA extraction steps, simplifies detection program directly by the use of the juice of potato plant as template；Reaction condition of the present invention is easy, and the reaction time is short；The present invention directly using visually observing can obtain testing result.Due to above-mentioned advantage, instant invention is especially suited for extensive sample detection.
Description
Technical field
The invention belongs to biological technical field, and in particular to one kind is utilized organizes the direct RT-LAMP detections potato of juice
The method of PLRV.
Background technology
Potato is the fourth-largest cereal crops for being only second to rice, wheat and corn in the world.Potato susceptible viral sense
Dye, Potyvirus has a strong impact on its yield and quality.Wherein corium solani is most destructive Potyvirus,
Generally it is distributed in the most of potato planting areas in the world (including China).Corium solani (potato leafroll
Virus, PLRV) be Lutoevirus section (Luteoviridae), the representative of Polerovirus (Polerovirus) into
Member, by aphis propagation.PLRV infects potato to be caused leaf roll, yellow, stunts, ossifys and the symptom such as stem tuber net necrosis, is led
Cause potato Severe Reduction, the severe one underproduction more than 80%.
The extensive virus-free production of seed stock of tissue cultures is the main method for controlling potato virus disease, but excellent potato seed
Production requirement set up quick, accurate, sensitive virus detection techniques.The method of current Potato viruses detection mainly has enzyme-linked
Immune detection (ELISA) and RT-polymerase chain reaction (RT-PCR).ELISA is widely used in horse because of simple to operate
In bell potato Viral diagnosis, however, sensitivity is relatively low and greatly limit the accurate of its detection the shortcomings of the interference of false negative result
Property.RT-PCR is more more sensitive than ELISA, is also commonly used for Potato viruses detection.However, RT-PCR detection process needs four steps
Suddenly, it is respectively successively that (1) extracts RNA from plant tissue, (2) reverse transcription reaction, (3) PCR reactions, (4) are by gel electrophoresis
The result of imaging visual PCR reactions.Therefore, the process of RT-PCR complicated and time consumptions, prevents it to exist from meeting quick virus detection
The need in the virus-free production of seed stock of extensive tissue cultures.Reverse transcription loop-mediated isothermal amplification technique (RT-LAMP) is a kind of
Simply, quickly, sensitivity Potato viruses detection method (Notomi T etc., 2000) high and low cost.However, in RT-
In LAMP detections, the first step that RNA templates are not only detection is prepared, be also the basis for ensureing that whether accurate testing result is.At present
It is main to use the methods such as CTAB methods, SDS methods by extracting the template that RNA is expanded as RT-LAMP, these methods behaviour in testing sample
Make complicated, extraction time is more long, while the reagent such as the phenol contained in extract solution, chloroform is easily polluted to environment.To big
During scale sample carries out Viral diagnosis, the extraction of RNA templates takes time and effort most one in having turned into detection process
Link.
The content of the invention
In view of the shortcomings of the prior art, utilized it is an object of the invention to provide one kind and organize the direct RT-LAMP of juice to examine
The method for surveying corium solani, the method comprises the following steps：
(1) juice of potato plant is taken, is placed in RT-LAMP reaction systems and is reacted；
(2) detected, the detection includes following method：
1) agarose gel electrophoresis analytical procedure (1) products therefrom is utilized, waterfall shape band occurs, then there is potato volume
Mosaic virus, such as without any band, then in the absence of corium solani；
2) using visually observing, such as occur muddy or precipitation in reaction solution, then there is corium solani, such as reaction solution
It is limpid, then in the absence of corium solani；
The corium solani is the coded sequence ORF3 of corium solani coat protein, and length is 627bp.
Preferably, the juice of the potato plant is the juice in Potato Shoot-tips.As long as it should be noted that horse
The tissue juice of bell potato plant is suitable for the present invention, simply using the juice of Potato Shoot-tips in the embodiment of the present invention.
As shown in the embodiment of the present invention, RT-LAMP reactions are directly carried out using Potato Shoot-tips juice, can be to Ma Ling
Potato PLRV is detected.
Those skilled in the art easily knows that the present invention is for the contribution of prior art：Due to directly using Ma Ling
Used as template, the present invention need not carry out the extraction of RNA to the juice of potato plant, the greatly simplified journey of corium solani detection
Sequence, it is to avoid common detection methods are due to the environmental pollution that needs organic reagent to cause and reduce testing cost.
It is worthy of note that, in addition to the detection method cited by step (2), remaining detection means commonly used in the art,
Developer (such as SYBR Green I) is added such as in reactant, the agarose gel electrophoresis imaging of reactant is also suitable for this
Invention.Cited detection means functions only as citing effect, should not be construed limitation of the present invention.
The RT-LAMP reaction systems are (pressing 25 μ l system computings)：1.6 μM of inner primer FIP/BIP, 0.4 μM of outer primer
F3/B3,2.5 μ l 10X ThermoPol buffer solutions, 0.8M betaine, 1.0mM dNTP, 8mM MgSO4, 10U M-MLV are inverse
Transcriptase, 4U RNase inhibitor and 10U Bst archaeal dna polymerases.The formula of the ThermoPol buffer solutions is：20mM
Tris-HCl, 10mM (NH4)2SO4, 10mM KCl, 2mM MgSO4, 0.1%X-100, pH=8.8.
In step (1), reaction temperature is 65 DEG C.
The present invention is also resided in for the contribution of prior art：Reaction condition of the invention is easy, compared to conventional RT-PCR
With RT-LAMP detection methods, the present invention is physically easier to perform.
Preferably, in step (1), the reaction time is 60 minutes.
The present invention also have a contribution be：Reaction time needed for the present invention is very short, it is only necessary to 60 minutes or so；In addition,
Because the present invention is without the preparation of RNA templates, the time of about 2-3 hours is also saved, while the present invention can also be with the naked eye straight
Detection is connect, detection time is also significantly less than conventional RT-PCR and RT-LAMP detection methods.It is readily appreciated that, the present invention is especially suitable for greatly
The detection of scale samples.
The acquisition methods of the juice of the potato plant are：With certain position of aseptic major part needle-penetration potato plant
(such as leaf or stem apex), acquisition sticks to the juice on pin.
For the ease of detection, the juice of potato plant can be obtained with above-mentioned easy method.What deserves to be explained is, should
Method does not apply to the method for unique juice for obtaining potato plant of the invention, other acquisitions beneficial to detection operation
Method belongs to protection scope of the present invention.
The sequence of the inner primer is：
FIP(5'→3')：TCTACGTCTTCGGCGCCTGGACAGAGTTCAGCCAG
TGGT；
BIP(5'→3')：ATCGCCGCTCAAGAAGAACTGGCCCATGAGGTTG
TCCTTTGT。
The sequence of the outer primer is：
F3(5'→3')：AAGGCAATCCCTTCGCAG；
B3(5'→3')：CCCGAAGGTGAAACTTCCTT.
Preferably, used in the step (2), when being detected and visually observed.
The present invention is also resided in for the contribution of prior art：The present invention can carry out direct detection using naked eyes, such as react
Occur muddy or precipitation in liquid, then there is corium solani, such as reaction solution is limpid, then in the absence of corium solani.
Being readily appreciated that is, with the naked eye can directly be detected, for this area, especially for extensive sample detection
Speech, its progressive for bringing is very significant.Or prior art adds developer (such as SYBR in detection in reactant
Green I), otherwise enter row agarose gel electrophoresis detection to reactant.These methods are required to plenty of time and cost, uncomfortable
Preferably extensive sample detection.
Beneficial effects of the present invention：
The present invention directly simplifies juice as template by the use of potato, without RNA extraction steps, simplifies detection program；
Reaction condition of the present invention is easy, and the reaction time is short；The present invention directly using visually observing can obtain testing result.Due to having
Above-mentioned advantage, instant invention is especially suited for extensive sample detection.
Brief description of the drawings
Fig. 1：The schematic diagram of template for needed for preparing RT-LAMP detections.By aseptic major part needle-penetration tissue culture seedlings of potatoes (seedling
6-8cm high) stem apex (being about 0.1cm), juice is sticked on needle point therewith.
Fig. 2：To visually observe the result of the direct RT-LAMP product of Potato Shoot-tips.PCR pipe+：It is positive control
(having infected the tissue culture seedlings of potatoes sample of PLRV), PCR pipe-：For negative control (is uninfected by the tissue culture seedlings of potatoes sample of PLRV
Product)；
Fig. 3：It is to carry out the result that RT-PCR and RT-LAMP is expanded using primer pair PLRV ORF3 sequences；In figure：Swimming lane
M：It is DL2000DNA relative molecular mass standards；Swimming lane W：It is ddH2O template amplification results；Swimming lane-：To be uninfected by PLRV's
The testing result of tissue culture seedlings of potatoes sample；Swimming lane+：To have infected the testing result of the tissue culture seedlings of potatoes sample of PLRV；1：
It is the result of the RNA template amplifications that conventional method is extracted from plant tissue；2：For the result that stem apex juice template is directly expanded.
Specific embodiment
The present invention is specifically described below by embodiment, it is necessary to it is pointed out here that be that following examples are use
It is further detailed in the present invention, it is impossible to be interpreted as limiting the scope of the invention, the field is skilled in technique
Personnel still fall within protection scope of the present invention according to some nonessential modifications and adaptations that foregoing invention content is made.
Experimental plant kind：Potato " pale reddish brown white " kind
The conventional method of embodiment 1 extracts RNA from tissue culture seedlings of potatoes stem-tip tissue
1.1 experiment reagents
Plant tissue total RNA extraction reagent box is purchased from border biological gene Science and Technology Ltd. of Beijing village ally.
It is prepared by 1.2 detection sample stem apexs
Testing sample is the tissue culture seedlings of potatoes of tissue culture room growth, including 6 parts of samples of PLRV have been infected through ELISA method identification
Product and 6 parts of samples without infection PLRV.Positive control：The tissue culture seedlings of potatoes of PLRV is infected；Negative control is 1.：Without sense
Contaminate the tissue culture seedlings of potatoes of PLRV.
Each sample is tissue-cultured seedling (height of seedling 6-8cm) stem-tip tissue (being about 0.1cm).
1.3RNA extraction process is specific as follows：
The stem-tip tissue (being about 0.1cm) that will be cut is put into the mortar for filling liquid nitrogen, is fully ground, then according to Beijing
The specification of the plant tissue Total RNAs extraction that border biological gene Science and Technology Ltd. of village ally provides is operated.
1.4 result interpretations：The quality extracted using micro-spectrophotometer detection RNA.
The direct RT-PCR methods detection corium solani of the stem apex of embodiment 2
2.1 experiment reagents
5X TRUE RT MasterMix reverse transcription reactions kit is purchased from Beijing Ai Delai bio tech ltd；M-
MLV reverse transcriptases are purchased from Promega companies；2X Utaq PCR MasterMix kits are purchased from Beijing village ally border bio-based
Because of Science and Technology Ltd..
Primer：Forward primer F, its nucleotide sequence is as shown in table 1；Reverse primer B, its nucleotide sequence is as shown in table 1.
Primer is synthesized by holding up section's biotechnology (Chengdu) Co., Ltd.
Table 1, RT-PCR and the primer of RT-LAMP detections
It is prepared by 2.2 detection sample stem apexs
Testing sample is the tissue culture seedlings of potatoes of tissue culture room growth, including 6 parts of samples of PLRV have been infected through ELISA method identification
Product and 6 parts of samples without infection PLRV.Positive control：The tissue culture seedlings of potatoes of PLRV is infected；Negative control is 1.：Without sense
Contaminate the tissue culture seedlings of potatoes of PLRV；Negative control is 2.：With ddH2O is template RT-PCR amplified productions.
Each sample pierces through stem apex, stem apex juice in the needle point on tissue-cultured seedling (height of seedling 6-8cm) respectively using aseptic pin
Liquid is sticked on needle point therewith, and the stem apex juice that will be adhered on needle point is placed in RT reaction systems, has at this moment infected the sample of PLRV
The RNA reverse transcriptions for savoring PLRV in juice are the DNA of PLRV.Carried out by template of the RNA of conventional method extraction in embodiment 1 simultaneously
RT-PCR is detected, used as the positive control detected with the direct RT-PCR of group sample stem apex.
2.3RT-PCR reaction systems
RT reverse transcription reactions and PCR reactions are carried out in PCR pipe.
2.3.1RT reaction system and reaction condition are specific as follows：
Conventional RT reaction systems cumulative volume is 20 μ l, wherein each component content is：1 μ g RNA and 4 μ l 5X TRUE RT
MasterMix.After the completion of each composition configuration, with ddH2O polishings are to 20 μ l systems.
Conventional RT reaction conditions：42 DEG C of isothermal reactions 20 minutes, subsequent 85 DEG C of reactions are inactivated for 5 seconds.
In the direct RT-PCR methods of stem apex, RT reaction systems cumulative volume is 20 μ l, composition with conventional RT reaction systems, simply
It is not added with RNA.
In the direct RT-PCR methods of stem apex, RT reaction conditions：The needle point that stem apex juice will have been adhered to is immersed in RT reaction systems,
Reacted 10 minutes in room temperature.In operation, the needle point for having adhered to stem apex juice is set to be completely immersed in RT reaction systems as far as possible, and cover tightly
The lid of PCR pipe, can otherwise influence RT reaction results.
2.3.2PCR reaction system and reaction condition are specific as follows：
PCR reaction systems cumulative volume is 25 μ l, wherein each component content is：12.5μl 2X Utaq PCR
MasterMix, 10 μM of forward and reverse each 1 μ l of primer and 0.5 μ g DNA profilings.After the completion of each composition configuration, with ddH2O polishings are to 25
μ l systems.
Reaction condition：1. -94 DEG C of predegeneration, 3 minutes；2. 30 circulate -94 DEG C, 30 seconds；54 DEG C, 1 minute；72℃,30
Second；3. last extends -72 DEG C, 7 minutes.
2.4 result interpretations：1.5% agarose gel electrophoresis analyzes RT-PCR product, 627bp bands occurs, represents
Result is the positive, and PLRV is contained in sample；Without the band, represent that result is feminine gender, PLRV is free of in sample.Stem apex is direct
The result of RT-PCR methods detection PLRV is consistent with the result of the RNA templates RT-PCR detections PLRV that conventional method in embodiment 1 is extracted,
Illustrate that the method for the direct RT-PCR detections PLRV of stem apex is reliable, feasible.
The direct RT-LAMP methods detection corium solani of the stem apex of embodiment 3
Testing sample is the tissue culture seedlings of potatoes of tissue culture room growth, including 6 parts of samples of PLRV have been infected through ELISA method identification
Product and 6 parts of samples without infection PLRV.Positive control：The tissue culture seedlings of potatoes of PLRV is infected；Negative control is 1.：Without sense
Contaminate the tissue culture seedlings of potatoes of PLRV；Negative control is 2.：With ddH2O is template RT-LAMP amplified productions.
Each sample pierces through stem apex, stem apex juice in the needle point on tissue-cultured seedling (height of seedling 6-8cm) respectively using aseptic pin
Liquid is sticked on needle point therewith, and the stem apex juice that will be adhered on needle point is placed in and amplification is reacted in RT-LAMP reaction systems.While with
The RNA that conventional method is extracted in embodiment 1 carries out RT-LAMP detections for template, is detected as with the direct RT-LAMP of group sample stem apex
Positive control.
3.1 experiment reagents
Bst archaeal dna polymerases and 10 × ThermoPol reaction buffers are purchased from New England Biolabs companies；M-
MLV reverse transcriptases are purchased from Promega companies；Glycine betaine is purchased from Sigma companies.
4 specific primers：Outer primer F3, its nucleotide sequence is as shown in table 1；Outer primer B3, its nucleotide sequence is such as
Shown in table 1；Inner primer FIP, as shown in table 1, inner primer BIP, its nucleotide sequence is as shown in table 1 for its nucleotide sequence.Primer
Synthesized by holding up section's biotechnology (Chengdu) Co., Ltd.
3.2 RT-LAMP reaction systems
RT-LAMP reactions are carried out in PCR pipe.
RT-LAMP reaction systems and reaction condition are specific as follows：
Conventional RT-LAMP reaction systems cumulative volume is 25 μ l, wherein each component content is：1.6 μM of inner primer FIP/
BIP, 0.4 μM of outer primer F3/B3,2.5 μ l 10X ThermoPol buffer solution, 0.8M betaine, 1.0mM dNTP, 8mM
MgSO4, 10U M-MLV reverse transcriptases, 4U RNase inhibitor, 10U Bst archaeal dna polymerases and 0.1 μ g RNA.Each composition is matched somebody with somebody
After the completion of putting, with ddH2O polishings are to 25 μ l systems.RT-LAMP reaction conditions：65 DEG C of isothermal reactions 60 minutes, subsequent 80 DEG C anti-
Answer 5 minutes inactivations.
The direct RT-LAMP reaction systems cumulative volume of stem apex is 25 μ l, wherein each component content is ibid, is simply not added with RNA, and
It is that the needle point that will adhere to stem-tip tissue juice is immersed in RT-LAMP reaction systems, 65 DEG C of isothermal reactions 60 minutes.In operation,
The needle point for having adhered to stem-tip tissue juice is completely immersed in RT-LAMP reaction systems as far as possible, and cover tightly the lid of PCR pipe, it is no
RT-LAMP reaction results can then be influenceed.
3.3 result interpretations：
Method 1, RT-LAMP product is visually observed, such as muddy or precipitation occurs in reaction solution, represents that result is the positive,
Contain PLRV in sample；As reaction solution is limpid, without muddy or precipitation, represent that result is feminine gender, PLRV is free of in sample.
, there is waterfall shape band in method 2,1.5% agarose gel electrophoresis analysis RT-LAMP product, represent result
It is the positive, PLRV is contained in sample；There is no any band, represent that result is feminine gender, PLRV is free of in sample.The direct RT- of stem apex
The result of LAMP methods detection PLRV is consistent with the result of the RNA templates RT-LAMP detections PLRV that conventional method in embodiment 1 is extracted,
Illustrate that the method for the direct RT-LAMP detections PLRV of stem apex is reliable, feasible.Method 1 is consistent with the result of the interpretation of method 2, says
The result of the bright direct RT-LAMP product of naked eyes interpretation stem apex is reliable, feasible.
Claims (10)
1. it is a kind of to utilize the method for organizing the direct RT-LAMP of juice to detect corium solani, it is characterised in that methods described
Comprise the following steps：
(1) juice of potato plant is taken, reaction in RT-LAMP reaction systems is placed directly within；
(2) detected, the detection includes following method：
1) agarose gel electrophoresis analytical procedure (1) products therefrom is utilized, waterfall shape band occurs, then there is potato leaf roll
Poison, such as without any band, then in the absence of corium solani；
2) using visually observing, such as occur muddy or precipitation in reaction solution, then there is corium solani, such as reaction solution is clear
It is clear, then in the absence of corium solani；
The corium solani is the coded sequence ORF3 of corium solani coat protein, and length is 627bp.
2. detection method according to claim 1, it is characterised in that the RT-LAMP reaction systems are (based on 25 μ l systems
Calculate) be：1.6 μM of inner primers FIP/BIP, 0.4 μM of outer primer F3/B3,2.5 μ l 10X ThermoPol buffer solutions, 0.8M
Betaine, 1.0mM dNTP, 8mM MgSO4, 10U M-MLV reverse transcriptases, 4U RNase inhibitor and 10U Bst DNA are poly-
Synthase.
3. detection method according to claim 2, it is characterised in that the formula of the ThermoPol buffer solutions is：20mM
Tris-HCl, 10mM (NH4)2SO4, 10mM KCl, 2mM MgSO4, 0.1%X-100, pH=8.8.
4. the detection method according to claim any one of 1-3, it is characterised in that in step (1), reaction temperature is 65
℃；And/or, the reaction time is 60 minutes.
5. the detection method according to claim any one of 1-3, it is characterised in that in step (1), the potato plant
Juice be the juice in Potato Shoot-tips.
6. detection method according to claim 1, it is characterised in that the juice in Potato Shoot-tips is placed directly within RT-
65 DEG C of isothermal reactions 60 minutes in LAMP reaction systems.
7. detection method according to claim 1, it is characterised in that the acquisition methods of the juice of the potato plant
For：With aseptic major part needle-penetration potato plant, the juice sticked on pin is obtained.
8. detection method according to claim 2, it is characterised in that the sequence of the inner primer is：
FIP(5'→3')：TCTACGTCTTCGGCGCCTGGACAGAGTTCAGCCAGTGGT；
BIP(5'→3')：ATCGCCGCTCAAGAAGAACTGGCCCATGAGGTTGTCCTTTGT.
9. detection method according to claim 2, it is characterised in that the sequence of the outer primer is：
F3(5'→3')：AAGGCAATCCCTTCGCAG；
B3(5'→3')：CCCGAAGGTGAAACTTCCTT.
10. detection method according to claim 1, it is characterised in that in the step (2), uses meat when being detected
Eye observation.
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Cited By (3)
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| CN108728579A (en) * | 2018-06-13 | 2018-11-02 | 四川师范大学 | A method of using the direct RT-PCR detections various plants virus of plant micro-assembly robot |
| CN109355438A (en) * | 2018-12-17 | 2019-02-19 | 甘肃省农业科学院马铃薯研究所 | A kind of reverse transcription ring mediated isothermal amplification RT-LAMP detection reagent of corium solani PLRV |
| CN114015811A (en) * | 2021-12-09 | 2022-02-08 | 贵州大学 | Potato virus one-step triple qualitative detection method without RNA extraction |
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| CN114015811A (en) * | 2021-12-09 | 2022-02-08 | 贵州大学 | Potato virus one-step triple qualitative detection method without RNA extraction |
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