CN106011311A - Kit and method for detecting watermelon mosaic virus RT-LAMP - Google Patents
Kit and method for detecting watermelon mosaic virus RT-LAMP Download PDFInfo
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- CN106011311A CN106011311A CN201610486394.5A CN201610486394A CN106011311A CN 106011311 A CN106011311 A CN 106011311A CN 201610486394 A CN201610486394 A CN 201610486394A CN 106011311 A CN106011311 A CN 106011311A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a kit and method for detecting watermelon mosaic virus RT-LAMP. The detection method comprises the steps of S1, extracting total RNA from a sample to be tested by means of a fast extraction solution composed of dimethyl sulfoxide and 1M Tris-HClpH7.5, and taking total RNA as an RNA template; S2, establishing an RT-LAMP reaction system by means of designed sense primer, reverse primer and loop primer; S3, placing the RT-LAMP reaction system in a water area which is 59 DEG C for reaction lasting 60 min, so that reaction liquid is obtained; S4, detecting the reaction liquid.
Description
Technical field
The present invention relates to a kind of watermelon mosaic virus RT-LAMP detection reagent box and detection method, be specifically related to Citrullus vulgaris flower
The screening of mosaic virus (Watermelon mosaic virus, WMV) RT-LAMP primer sets, WMV RT-LAMP detection reagent box
Application.
Background technology
Watermelon mosaic virus (WMV) is a member of Potyvirus, and for positive single strand RNA virus, virus full length is about
10kb, mainly infects the Cucurbitaceae such as Citrullus vulgaris, Fructus Melo section crop, produces light floral leaf in crop upper blade, be on these crops
One of main virus.WMV is mainly propagated with non-persistent manner by frictional inoculation, aphid.Watermelon mosaic virus (WMV) is at me
State's each ground family crop producing region presents ascendant trend year by year, has become as the major issue affecting the melon production of China.
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, but plant virus such as watermelon mosaic virus is examined the most accordingly
The report of test agent box, therefore develops a kind of RT-LAMP test kit for watermelon mosaic virus and has important using value.
Summary of the invention
WMV detection mainly has the methods such as DTBIA, ELSA, RT-PCR, RT-nested PCR, real-time RT-PCR.
DTBIA sensitivity is relatively low；And RT-PCR, RT-nested PCR and real-time RT-PCR nucleic acid detection technique needs costliness
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 watermelon mosaic virus
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 that watermelon mosaic is sick
Poison.
The present invention provides
(1) a kind of watermelon 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 watermelon mosaic virus, described rapid extraction
Liquid 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 watermelon mosaic virus as follows,
(4) a kind of watermelon 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 F30.4 μ L, 10pmol/ μ L downstream primer B3 0.4 μ L, concentration is 20pmol/
Ring primer LB0.8 μ L, the RNA template 1.6 μ L of μ L, and deionized water (DW) 4.4 μ L；
S3, by described RT-LAMP reaction system, is placed in 59 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 WMV, is finally established the RT-LAMP of WMV by experiment
Detection method, for the detection of WMV provide a kind of rapidly and efficiently, special sensitive new method, highly sensitive, compare conventional RT-PCR
Highly sensitive 10 times；And turbidity can be observed by the naked eye or color reaction directly carries out result judgement.The method is applicable to scene
The quick detection of WMV.Object of this investigation be exactly develop this virus rapidly and efficiently, sensitive special detection technique, with in the past
The method of research forms the detection system of different levels, makes testing staff can carry out extensively according to different condition and purpose
Effective selection, for Citrullus vulgaris, Fructus Melo Seedling inspection and quarantine and the disease monitoring effective technical support of offer producing field.
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 WMV, including the forward outer primer of primer sets 1
The sequence of F3-1 is SEQ ID NO.1, and the sequence of reverse outer primer B3-1 is SEQ ID NO.2；The sequence of forward inner primer FIP-1
Being classified as SEQ ID NO.3, the sequence of reverse inner primer BIP-1 is SEQ ID NO.4；The forward outer primer F3-2's of primer sets 2
Sequence is SEQ ID NO.5, and 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, 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 Citrullus vulgaris upper blade 0.1g of (comparison with) infecting WMV virus or being uninfected by respectively, or obtain with tweezers
Take and be equivalent to the Citrullus vulgaris 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 Citrullus vulgaris upper leaf agreement that contracts a film or TV play to an actor or actress 0.1g, be immediately placed in grinding, and are previously added containing 2～10% dimethyl sulfoxide in grinding
1M Tris-HCl (pH7.5) 5mL, and grind rapidly 3min, then stand 2min, take 1.6 μ L as RNA template.Described tweezer
Son, grind, after grinding rod and other test tools need to soak 12 hours in DEPC water, 150 degree bakings 6 hours, then take
Go out to be positioned over 4 DEG C of cold preservations standby.Containing 2～10% the 1M Tris-HCl (pH7.5) of dimethyl sulfoxide for prepare in advance, high temperature
After 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 to produce
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 DEG C, 61 DEG C, 63 DEG C and 65 DEG C, and the response time is 60min.Expand
Increasing reaction to carry out on real-time transmissometer, the shortest time according to occurring amplification curve in 60min determines optimal reaction temperature.
Watermelon mosaic virus (WMV), marmor upsilon (PVY), cucumber mosaic virus (CMV), tobacco vein banding mosaic virus
(TVBMV) 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 6% 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 1,4 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 2 amplification the most clearly, the brightest, therefore select the primer sets 2 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 34,36,48 and 42min.According to the requirement that the Site Detection time is the shortest, determine that 59 DEG C are
Optimal reaction temperature.
3.RT-LAMP specificity experiments
Watermelon mosaic virus virus (WMV), marmor upsilon (PVY), cucumber mosaic virus (CMV), Nicotiana tabacum L. arteries and veins band floral leaf
Virus (TVBMV) positive material and healthy Citrullus vulgaris plant upper blade (negative control represents with NC) are Linyi University's biology examination
Test the specimen that center preserves, from-30 DEG C of refrigerators, take out these specimen, directly take out about 0.1g blade, according to this with tweezers
Bright RNA extraction method 2 obtains total serum IgE.Carrying out RT-LAMP reaction with total serum IgE for template, temperature is 59 DEG C, and the time is
60min.System adds calcein fluorescent dye so that reaction can be by color produced in reaction tube after terminating
Change judges whether amplification, and result is as shown in Figure 3.As seen from Figure 3, only WMV is able to detect that amplification curve, other samples
Product are all not detected by amplification curve within the response time.After reaction terminates, add the aobvious green of reaction tube of WMV total serum IgE, it is judged that for
Positive；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 knot of color reaction
Fruit is consistent, shows that the RT-LAMP detection method that this institute is set up has the specificity of height, and testing result is the most accurate.
The present invention, during setting up the RT-LAMP detection method of WMV, have employed Eiken Chemical company of Japan and grinds
It is transported to the LoopampRNA amplification kit of system, simplifies the preparation before experiment reaction, ensure that the quality of experiment simultaneously.
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
RT-LAMP reaction system adds the calcein fluorescent dye that test kit is supporting so that covered also observing that reacts institute
The color change produced, it is to avoid secondary pollution, it is ensured that the accuracy of result.It addition, use the RNA rapid extraction of the present invention
Liquid, can the most quickly detect, and scene obtains result, uses Rneasy Mini kit to carry without entering laboratory
Take RNA.Containing 2～10% dimethyl sulfoxide in the RNA rapid extraction liquid of the present invention, although in the present invention can rapid extraction
The mechanism of WMV viral RNA is the most indefinite, but according to analysis, owing to it has highly polar, it is possible to make the fast rapid release of WMV viral RNA
Put, and join RT-LAMP reaction system before RNA decomposes such that it is able to smoothly as template, carry out RT-LAMP amplification.Should
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 as far as possible
Use real-time transmissometer, result can be carried out quantitative analysis, it is ensured that the accuracy of result；Meanwhile, when carrying out result detection,
Avoid detection of uncapping the most as far as possible, prevent that secondary pollution causes false positive results to occur.
Claims (6)
1. a watermelon 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.7；
Upstream inner primer BIP sequence SEQ ID NO.8；
Downstream outer primer F3 sequence SEQ ID NO.5；
Downstream outer primer B3 sequence SEQ ID NO.6；
And ring primer LB sequence SEQ ID NO.17.
2. test kit as claimed in claim 1, also includes the rapid extraction liquid of the RNA of watermelon 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 watermelon 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 59 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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112266979A (en) * | 2020-10-30 | 2021-01-26 | 安徽省农业科学院作物研究所 | RPA detection primer based on watermelon mosaic virus conserved region, detection method and application thereof |
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| CN103382506A (en) * | 2013-05-20 | 2013-11-06 | 广西大学 | RT-LAMP technology for rapidly detecting sorghum mosaic virus |
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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 |
| CN101906485A (en) * | 2010-07-21 | 2010-12-08 | 西北农林科技大学 | Primer set and kit for quickly detecting various melon viruses |
| 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 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112266979A (en) * | 2020-10-30 | 2021-01-26 | 安徽省农业科学院作物研究所 | RPA detection primer based on watermelon mosaic virus conserved region, detection method and application thereof |
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