CN106119410A - Tobacco vein banding mosaic virus RT LAMP detection kit and detection method - Google Patents
Tobacco vein banding mosaic virus RT LAMP detection kit and detection method Download PDFInfo
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Abstract
A kind of tobacco vein banding mosaic virus RT LAMP detection kit and detection method, described detection method comprises the following steps: S1 uses the rapid extraction liquid being made up of dimethyl sulfoxide and 1M Tris HClpH7.5, total serum IgE is extracted from testing sample, and as RNA template；S2 uses forward primer, downstream primer and the ring primer of design, builds RT LAMP reaction system；S3, by described RT LAMP reaction system, is placed in 65 DEG C of waters, reacts 60 minutes, obtains reactant liquor；Described reactant liquor is detected by S4.
Description
Technical field
The present invention relates to a kind of tobacco vein banding mosaic virus RT-LAMP detection reagent box and detection method, be specifically related to horse
Bell potato Y virus belongs to tobacco vein banding mosaic virus (Tobacco vein banding mosaic virus, TVBMV) RT-LAMP and draws
The screening of thing group, the application of TVBMV RT-LAMP detection reagent box.
Background technology
Tobacco vein banding mosaic virus (TVBMV) is a member of Potyvirus, for positive single strand RNA virus, virus full length
About 9.6kb, the polyprotein that coding is made up of 3080 amino acid residues, is cut by three protease of self coding
Becoming 10 maturation proteins, research recently finds that P3 gene frameshit can translate into new albumen P3N-PIPO.Mainly infect Nicotiana tabacum L., kind
The solanaceous crops such as eggplant, Rhizoma Solani tuber osi, produces light floral leaf in crop upper blade, is one of main virus on these crops.
TVBMVX is mainly propagated with non-persistent manner etc. by frictional inoculation, aphid.Have between TVMBV and potexvirus virus
Synergism, once Combined Infection can cause the serious symptoms of host, causes a large amount of underproduction even to be had no harvest.
The isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) of ring mediation is root
According to 6 specific regions of genes of interest, design 4-6 bar specific primer, utilize Bst archaeal dna polymerase at 60～65 DEG C of isothermal bars
Amplifying target genes specifically under part.LAMP amplified reaction can complete within 60 minutes, and its amplified production is reverse with target
The loop-stem structure repeated and multi-ring cauliflower-like structure, produce a large amount of pyrophosphate ion, and form burnt phosphorus in course of reaction
Acid magnesium white precipitate.LAMP method can be by multiple method judged result, as whether there is white precipitate in system after reaction
Whether thing, electrophoresis result have scalariform band, add whether SYBR Green I becomes green etc., have detection sensitivity high,
Detection method is simple and quick, instrument requirements not high.But with regard to design of primers aspect, at present the document of report be mostly with
Line mode (Primer Explore) design primer, range of application is restricted, according to LAMP principle designed, designed many groups primer
Group, chooses the suitableeest primer sets by experimental result, builds LAMP detection method, so will be greatly increased the applicable model of LAMP
Enclose, there is the prospect that is more widely applied.
Since LAMP technology is set up, this technology has been widely used for the detection to pathogenic bacteria, parasite etc., exists in recent years
The detection research of animal virus gradually is promoted, however also the most corresponding for plant virus such as tobacco vein banding mosaic virus
The report of detection kit, therefore develop a kind of RT-LAMP test kit for tobacco vein banding mosaic virus and have important
Using value.
Summary of the invention
TVBMV detection mainly has the sides such as DTBIA, ELSA, RT-PCR, RT-nested PCR, real-time RT-PCR
Method.DTBIA sensitivity is relatively low；And RT-PCR, RT-nested PCR and real-time RT-PCR nucleic acid detection technique needs high
Expensive special instrument, and the time of nucleic acid amplification is longer.
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, it is provided that a kind of quickly Nicotiana tabacum L. arteries and veins band mosaic disease
Poison RT-LAMP detection method.The present inventor, under persevering endeavors, has invented a kind of rapid extraction RNA testing sample
Method, and combine self-designed primer, use RT-LAMP detection method, it is possible to fast and effeciently detect Nicotiana tabacum L. arteries and veins band
Mosaic virus.
The present invention provides
(1) a kind of tobacco vein banding mosaic virus RT-LAMP detection reagent box, it is characterised in that include RT-LAMP primer,
Described RT-LAMP primer is:
(2) test kit as described in (1), also includes the rapid extraction liquid of the RNA of tobacco vein banding mosaic virus, described quickly
Extracting solution is made up of dimethyl sulfoxide and 1M Tris-HCl (pH7.5).
(3) test kit as described in (2), wherein, the volumetric concentration of described dimethyl sulfoxide is 2～10%.
The present invention also provides for the method detecting tobacco vein banding mosaic virus as follows,
(4) a kind of tobacco vein banding mosaic virus RT-LAMP detection method, it is characterised in that comprise the following steps
S1 uses the rapid extraction liquid being made up of dimethyl sulfoxide and 1M Tris-HCl (pH7.5), carries from testing sample
Take total serum IgE, and as RNA template；
S2 uses test kit as described in (1), builds RT-LAMP reaction system, and described reaction system is 20 μ L, including: 2 ×
Reaction buffer (RM) 10 μ L, enzymatic solution (EM) 0.8 μ L, 40pmol/ μ L forward primer FIP 0.8 μ L, 40pmol/ μ L draws upstream
Thing BIP0.8 μ L, 10pmol/ μ L downstream primer F3 0.4 μ L, 10pmol/ μ L downstream primer B3 0.4 μ L, concentration is
Ring primer LB0.8 μ L, the RNA template 1.6 μ L of 20pmol/ μ L, and deionized water (DW) 4.4 μ L；
S3, by described RT-LAMP reaction system, is placed in 65 DEG C of waters, reacts 60 minutes, obtains reactant liquor；
Described reactant liquor is detected by S4.
(5) the RT-LAMP detection method as described in (4), wherein, the volumetric concentration of described dimethyl sulfoxide is 2～10%.
(6) the RT-LAMP detection method as described in (4), is to add in described reactant liquor in wherein said S4 step
Enter calcein fluorescent dye, judge testing result by color change produced in reaction tube.
The present invention designs RT-LAMP primer according to the gene order of TVBMV, is finally established the RT-of TVBMV by experiment
LAMP detection method, for the detection of TVBMV provide a kind of rapidly and efficiently, special sensitive new method, highly sensitive, ratio is commonly
Highly sensitive 10 times of RT-PCR；And turbidity can be observed by the naked eye or color reaction directly carries out result judgement.The method is suitable for
Quick detection in on-the-spot TVBMV.Object of this investigation be exactly develop this virus rapidly and efficiently, sensitive special detection skill
Art, and the detection system that the method for in the past research forms different levels, make the testing staff can according to different condition and purpose
To carry out the most effective selection, for Rhizoma Solani tuber osi inspection and quarantine, potato seed certification and the disease monitoring effective skill of offer producing field
Art is supported.
Accompanying drawing explanation
In Fig. 1 RT-LAMP reaction system, the total serum IgE extracted with the RNA extraction method 2 of the present invention as template, the present invention
After 4 groups of primer reactions of design, the gel electrophoresis figure of reactant liquor.
Fig. 2 optimal reaction temperature amplification curve.
Amplification curve result in Fig. 3 specificity experiments.
Color reaction result in Fig. 4 specific test.
Detailed description of the invention
For solving above-mentioned technical problem, design 4 set is for detecting the primer sets of TVBMV, including the most outwards drawing of primer sets 1
The sequence of thing F3-1 is SEQ ID NO.1, and the sequence of reverse outer primer B3-1 is SEQ ID NO.2；Forward inner primer FIP-1's
Sequence is SEQ ID NO.3, and the sequence of reverse inner primer BIP-1 is SEQ ID NO.4；The forward outer primer F3-2 of primer sets 2
Sequence be SEQ ID NO.5, the sequence of reverse outer primer B3-2 is SEQ ID NO.6；The sequence of forward inner primer FIP-2 is
SEQ ID NO.7, the sequence of reverse inner primer BIP-2 is SEQ ID NO.8；The sequence of the forward outer primer F3-3 of primer sets 3
For SEQ ID NO.9, the sequence of reverse outer primer B3-3 is SEQ ID NO.10；The sequence of forward inner primer FIP-3 is SEQ
ID NO.11, the sequence of reverse inner primer BIP-3 is SEQ ID NO.12；The sequence of the forward outer primer F3-4 of primer sets 4 is
SEQ ID NO.13, the sequence of reverse outer primer B3-4 is SEQ ID NO.14；The sequence of forward inner primer FIP-4 is SEQ ID
NO.15, the sequence of reverse inner primer BIP-4 is SEQ ID NO.16；The sequence of the forward outer primer F3-5 of primer sets 5 is SEQ
ID NO.17, the sequence of reverse outer primer B3-5 is SEQ ID NO.18；The sequence of forward inner primer FIP-5 is SEQ ID
NO.19, the sequence of reverse inner primer BIP-5 is SEQ ID NO.20, and ring primer LB, and particular sequence is shown in Table 1.
Table 1 primer sequence
Loopamp RNA amplification reaction kit, Loopamp reaction tube, Loopamp FD luciferase assay reagent are purchased from day
This Eiken Chemical, RNA extracts test kit and is purchased from Beijing hundred Tyke Bioisystech Co., Ltd, and other reagent are often
Rule analytical reagent.Use the real-time transmissometer of LA-320C that Eiken Chemical of Japan produces.
Test example 1 nucleic acid extraction
Weigh tobacco plant upper blade 0.1g of (comparison with) infecting TVBMV virus or being uninfected by respectively, or use
Tweezers obtain and are equivalent to the tobacco plant upper blade of 0.1g size as experiment material.
RNA extraction method 1: using RNA rapid extraction test kit (centrifugal column type) to extract sample total serum IgE, concrete operations are pressed
Test kit explanation is carried out.This test is specifically used, and QIAGEN Rneasy Mini kit extracts RNA, it is also possible to take other to try
Agent box extracts, or generally laboratory extracting method extracts RNA.
RNA extraction method 2: use the test kit of the present invention, extract RNA crude extract more quickly and easily.Specifically use
Tweezers take tobacco plant upper leaf agreement that contracts a film or TV play to an actor or actress 0.1g, are immediately placed in grinding, be previously added in grinding containing 2～10% dimethyl sub-
1M Tris-HCl (pH7.5) 5mL of sulfone, and grind rapidly 3min, the most static 2min, goes 1.6 μ L as RNA template.Described
Tweezers, grind, after grinding rod and other test tools need to soak 12 hours in DEPC water, 150 degree bakings 6 hours, then
It is standby that taking-up is positioned over 4 DEG C of cold preservations.Containing 2～10% the 1M Tris-HCl (pH7.5) of dimethyl sulfoxide for prepare in advance, high
After temperature autoclave sterilization processes, it is positioned over 4 DEG C of cold preservations standby.Rifle head and various reaction tube that shifting liquid is robbed all use RNase free
Product.
The foundation of test example 2RT-LAMP system and optimization
RT-LAMP reaction system illustrates according to Loopamp RNA amplification test kit, and reaction system is 20 μ L, including: 2 ×
Reaction buffer (RM) 10 μ L, enzymatic solution (EM) 0.8 μ L, 40pmol/ μ L primers F IP0.8 μ L, 40pmol/ μ L BIP0.8 μ L,
10pmol/ μ L F3 0.4 μ L, 10pmol/ μ L B3 0.4 μ L, LB (concentration is 20pmol/ μ L) 0.8 μ L, RNA template 1.6 μ
L, and deionized water (DW) 4.4 μ L.Reaction temperature is set to 59,61,63 and 65 DEG C, and the response time is 60min.Amplified reaction
Carrying out on real-time transmissometer, the shortest time according to occurring amplification curve in 60min determines optimal reaction temperature.
Tobacco vein banding mosaic virus (TVBMV), marmor upsilon (PVY), cucumber mosaic virus (CMV), Rhizoma Solani tuber osi X are sick
Poison (PVX) and the total serum IgE of negative control (NC) and water (as blank, represent with BC) are that template carries out RT-LAMP reaction,
System is simultaneously introduced 1.0 μ L calcein fluorescent dyes (i.e. Loopamp FD luciferase assay reagent), the spy of checking the method
The opposite sex.
RT-LAMP amplified production (1) real-time amplification curve detection: when amplification is carried out, if there being product to occur, reactant liquor meeting
Produce certain turbidity；Turbid ity signal can be collected by transmissometer, reflects with the form of amplification curve, thus sentences result
Disconnected.(2) dye colour reaction detection: add 1 μ L Loopamp FD reagent in LAMP reaction system, after reaction terminates, naked eyes
The fluorescence color reaction of observing response liquid.Aobvious green is judged as positive reaction；If showing orange to be judged as negative reaction.
Result and analysis
The determination of 1.RNA extracting method
Preparation dimethyl sulfoxide concentration is 1M Tris-HCl (pH7.5) solution of 2%, 4%, 6%, 8%, 10% respectively,
Compound method is in the volumetric flask of 100mL, adds 10g dimethyl sulfoxide, is subsequently adding the 1M Tris-HCl prepared
(pH7.5) and constant volume, 1M Tris-HCl (pH7.5) solution of dimethyl sulfoxide concentration 10% is obtained.Other concentration can use 1M
Tris-HCl (pH7.5) is diluted, it is also possible to again prepare.With RNA extraction method 2 is extracted, and use spectrophotometric
Measurement amount A260 and the value of A280, calculate A260/A280 ratio, Rneasy Mini kit extract RNA as positive control, its
The results are shown in Table 2.
The A260/A280 ratio of the RNA solution that table 2 distinct methods obtains
Therefore, 1M Tris-HCl (pH7.5) solution all using dimethyl sulfoxide concentration to be 10% in the present embodiment extracts
Sample RNA, but this is not limited to this concentration, it should be appreciated by those skilled in the art that what the solution of other concentration was extracted
RNA can be equally used for the detection of the present invention, and obtains same Detection results.
2. the determination of optimal primer sets
The RNA crude extract obtained with RNA extraction method 1 and RNA extraction method 2 respectively is as template, and in use table 1,5 are drawn
Thing group, joins the RT-LAMP reaction system in test example 2, and reaction temperature is set to 61 DEG C, reacts 60 minutes.Result is either
The RNA which kind of method is extracted as template, the scalariform band of primer sets 4 amplification the most clearly, the brightest, therefore select the primer sets 4 to be
Optimal primer sets, it is the result that template obtains that Fig. 1 is shown that the RNA crude extract that RNA extraction method 2 obtains, symbol M in Fig. 1
It it is labelling；1 to 4 represents the 1st to 4 primer sets respectively.
3. the determination of optimal reaction temperature
The total serum IgE obtained with RNA extraction method 2, as template, carries out RT-LAMP reaction, the amplification of real-time transmissometer output
Time graph result such as Fig. 2.By curve in figure it can be seen that RT-LAMP is under the reaction temperature of 59,61,63 and 65 DEG C, respectively
Amplification curve is created when about 40,36,48 and 32min.According to the requirement that the Site Detection time is the shortest, determine that 65 DEG C are
Optimal reaction temperature.
4.RT-LAMP specificity experiments
Tobacco vein banding mosaic virus (TVMBV), marmor upsilon (PVY), cucumber mosaic virus (CMV), Rhizoma Solani tuber osi X are sick
Poison (PVX) positive material and health tobacco plant upper blade (negative control represents with NC) are in Linyi University's biologic test
The specimen that the heart preserves, takes out these specimen from-30 DEG C of refrigerators, directly takes out about 0.1g blade, according to the present invention's with tweezers
RNA extraction method 2 obtains total serum IgE.Carrying out RT-LAMP reaction with total serum IgE for template, temperature is 65 DEG C, and the time is 60min.Body
System adds calcein fluorescent dye so that reaction can be judged by color change produced in reaction tube after terminating
Whether expand, result is as shown in Figure 3.As seen from Figure 3, only TVBMV is able to detect that amplification curve, and other samples are instead
Amplification curve all it is not detected by Ying Shi.After reaction terminates, add the aobvious green of reaction tube of TVBMV total serum IgE, it is judged that for sun
Property；And other reaction tubes all show orange, it is judged that for feminine gender, result is as shown in Figure 4.Amplification curve result and the result of color reaction
Unanimously, showing that the RT-LAMP detection method that this institute is set up has the specificity of height, testing result is the most accurate.
The present invention, during setting up the RT-LAMP detection method of TVBMV, have employed Eiken Chemical company of Japan
The LoopampRNA amplification kit of research and development preparation, simplifies the preparation before experiment reaction, ensure that the matter of experiment simultaneously
Amount.In terms of interpretation of result, research employs the real-time transmissometer of LA-320C, can be to experimental result quantitative analysis.It addition, pass through
The supporting calcein fluorescent dye of test kit is added so that covered also observe that reaction in RT-LAMP reaction system
Produced color change, it is to avoid secondary pollution, it is ensured that the accuracy of result.It addition, use the RNA of the present invention quickly to carry
Take liquid, can the most quickly detect, and scene obtains result, use Rneasy Mini kit without entering laboratory
Extract RNA.Containing 2～10% dimethyl sulfoxide in the RNA rapid extraction liquid of the present invention, although can quickly carry in the present invention
The mechanism taking TVBMV viral RNA is the most indefinite, but according to analysis, owing to it has highly polar, it is possible to make TVBMV viral RNA
Quickly release, and join RT-LAMP reaction system before RNA decomposes such that it is able to smoothly as template, carry out RT-LAMP
Amplification.This mechanism further will illustrate in test from now on.
When setting up for certain virus LAMP detection method, it is most important that the design of special primer, and whether primer
Special it is decided by that the comparison result of selected sequence is the most special.Therefore, the standard of abundance must be carried out in the early stage of design of primers
Standby work, utilizes sequence analysis software to be screened by aim sequence, is uploaded to primer after then sequence being carried out manual handle again
Design server design primer, can obtain ideal primer sets.Carrying out result context of detection, if having ready conditions to the greatest extent
Amount uses real-time transmissometer, result can be carried out quantitative analysis, it is ensured that the accuracy of result；Meanwhile, result detection is being carried out
Time, avoid detection of uncapping the most as far as possible, prevent that secondary pollution causes false positive results to occur.
Claims (6)
1. a tobacco vein banding mosaic virus RT-LAMP detection reagent box, it is characterised in that include RT-LAMP primer, described
RT-LAMP primer is:
Upstream outer primer FIP sequence SEQ ID NO.15；
Upstream inner primer BIP sequence SEQ ID NO.16；
Downstream outer primer F3 sequence SEQ ID NO.13；
Downstream outer primer B3 sequence SEQ ID NO.14；
And ring primer LB sequence SEQ ID NO.21.
2. test kit as claimed in claim 1, also includes the rapid extraction liquid of the RNA of tobacco vein banding mosaic virus,
Described rapid extraction liquid is made up of dimethyl sulfoxide and 1M Tris-HCl (pH7.5).
3. test kit as claimed in claim 2, wherein, the volumetric concentration of described dimethyl sulfoxide is 2～10%.
4. a tobacco vein banding mosaic virus RT-LAMP detection method, it is characterised in that comprise the following steps
S1 uses the rapid extraction liquid being made up of dimethyl sulfoxide and 1M Tris-HClpH7.5, extracts total from testing sample
RNA, and as RNA template；
S2 uses test kit as claimed in claim 1, builds RT-LAMP reaction system, and described reaction system is 20 μ L, including: 2
× reaction buffer 10 μ L, enzymatic solution 0.8 μ L, 40pmol/ μ L forward primer FIP 0.8 μ L, 40pmol/ μ L forward primer
BIP0.8 μ L, 10pmol/ μ L downstream primer F3 0.4 μ L, 10pmol/ μ L downstream primer B3 0.4 μ L, 20pmol/ μ L ring primer
LB0.8 μ L, RNA template 1.6 μ L, and deionized water 4.4 μ L；
S3, by described RT-LAMP reaction system, is placed in 65 DEG C of waters, reacts 60 minutes, obtains reactant liquor；
Described reactant liquor is detected by S4.
5. RT-LAMP detection method as claimed in claim 4, wherein, the volumetric concentration of described dimethyl sulfoxide be 2～
10%.
6. RT-LAMP detection method as claimed in claim 4, wherein, is to add in described reactant liquor in described S4 step
Add calcein fluorescent dye, judge testing result by color change produced in reaction tube.
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Citations (4)
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|---|---|---|---|---|
| CN1133065A (en) * | 1993-07-09 | 1996-10-09 | 阿斯格罗种子公司 | Lettuce infectious yellows virus genes |
| CN102286637A (en) * | 2011-07-21 | 2011-12-21 | 陕西省烟草研究所 | Method for detecting tobacco viruses by reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technology |
| CN103146847A (en) * | 2013-03-22 | 2013-06-12 | 南京农业大学 | RT-LAMP (loop-mediated isothermal amplification) rapid detection kit for cucumber green mottle mosaic virus (CGMMV) and detection method |
| CN105331746A (en) * | 2015-12-10 | 2016-02-17 | 山东出入境检验检疫局检验检疫技术中心 | Duck flavivirus RT-LAMP detection kit |
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|---|---|---|---|---|
| CN1133065A (en) * | 1993-07-09 | 1996-10-09 | 阿斯格罗种子公司 | Lettuce infectious yellows virus genes |
| CN102286637A (en) * | 2011-07-21 | 2011-12-21 | 陕西省烟草研究所 | Method for detecting tobacco viruses by reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technology |
| CN103146847A (en) * | 2013-03-22 | 2013-06-12 | 南京农业大学 | RT-LAMP (loop-mediated isothermal amplification) rapid detection kit for cucumber green mottle mosaic virus (CGMMV) and detection method |
| CN105331746A (en) * | 2015-12-10 | 2016-02-17 | 山东出入境检验检疫局检验检疫技术中心 | Duck flavivirus RT-LAMP detection kit |
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