CN105969911A - RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection reagent kit and detection method for cucumber mosaic viruses - Google Patents
RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection reagent kit and detection method for cucumber mosaic viruses Download PDFInfo
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Abstract
The invention discloses an RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection reagent kit and a detection method for cucumber mosaic viruses. The detection method includes steps of S1, extracting total RNA (ribonucleic acid) from to-be-detected samples by the aid of quick extract liquid and utilizing the total RNA as an RNA template; S2, constructing RT-LAMP reaction systems by the aid of designed upstream primers, downstream primers and loop primers; S3, arranging the RT-LAMP reaction systems in water areas at the temperatures of 65 DEG C and carrying out reaction for 60 minutes to obtain reaction liquid; S4, detecting the reaction liquid. The quick extract liquid comprises 10% of ethyl acetate, 2-10% of dimethyl methionine and 1M Tris-HClpH 7.5.
Description
Technical field
The present invention relates to a kind of cucumber mosaic virus RT-LAMP detection reagent box and detection method, be specifically related to Herba bromi japonici flower
Mosaic virus section Cucumovirus cucumber mosaic virus (Cucumber mosaic virus, CMV) RT-LAMP primer sets
Set up, the application of CMV RT-LAMP detection reagent box.
Background technology
Cucumber mosaic virus (CMV) is Bromoviridae Cucumovirus representative species, for triad genome
With a sub-gene group, it is positive single strand RNA virus, the long 3357bp of RNA1, RNA2 long 3050bp, RNA3 long 2216bp, RNA4
For the sub-gene group of RNA3, long 1000bp.CMV is the most serious a kind of virus disease, and virus can reach in addition to growing point
Any position.This virus host scope is many, distribution is wide, one of plant virus of tool Economic Importance, can infect kind more than 1000
Dicotyledonous and monocotyledon.CMV can be propagated in non-persistent mode by more than 60 kinds of aphids, and easy mechanical inoculation is propagated, and is China ten
On Zi Hua section, Solanaceae, pulse family and cucurbitaceae vegetable, topmost specialized hospital virus, is also the important disease of Nicotiana tabacum L., Fructus Musae, Herba Passiflorae Caeruleae
Former, China is separated to CMV from 120 various plants of 38 sections.
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 cucumber mosaic virus is examined the most accordingly
The report of test agent box, therefore develops a kind of RT-LAMP test kit for cucumber mosaic virus and has important using value.
Summary of the invention
CMV 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 cucumber 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 cucumber mosaic virus
Poison.
The present invention provides
(1) a kind of cucumber 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 cucumber mosaic virus, described rapid extraction
Liquid is made up of the dimethyl methyl thiamine of ethyl acetate, 2～10% and the 1M Tris-HClpH7.5 of 10%.
(3) test kit as described in (2), wherein, the volumetric concentration of described dimethyl methyl thiamine is 6%.
The present invention also provides for the method detecting cucumber mosaic virus as follows,
(4) a kind of cucumber mosaic virus RT-LAMP detection method, it is characterised in that comprise the following steps
S1 uses the dimethyl methyl thiamine of ethyl acetate, 2～10% by 10% and 1M Tris-HClpH7.5 to constitute
Rapid extraction liquid, extracts total serum IgE from testing sample, 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 methyl thiamine is 6%.
(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 CMV, is finally established the RT-LAMP of CMV by experiment
Detection method, for the detection of CMV 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 CMV.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, the disease monitoring for Fructus Cucumidis sativi seedling inspection and quarantine and production field provides effective technical support.
Accompanying drawing explanation
Fig. 1 optimal reaction temperature amplification curve.
Amplification curve result in Fig. 2 specificity experiments.
Color reaction result in Fig. 3 specific test.
Detailed description of the invention
For solving above-mentioned technical problem, design 1 set is for detecting the primer sets of CMV, including the sequence of forward outer primer F3-1
Being classified as SEQ ID NO.1, the sequence of reverse outer primer B3-1 is SEQ ID NO.2；The sequence of forward inner primer FIP-1 is SEQ
ID NO.3, the sequence of reverse inner primer BIP-1 is SEQ ID NO.4；And ring primer LB, 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 the cucumber leaves 0.1g of (comparison with) infecting CMV virus or being uninfected by respectively, or obtain phase with tweezers
When the cucumber leaves in 0.1g size is 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 Fructus Cucumidis sativi Newborn Leaves 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 the ethyl acetate containing 10%, 2～
The extracting solution 5mL that the dimethyl methyl thiamine of 10% and 1M Tris-HClpH7.5 are constituted, and grind rapidly 3min, then stand
2min, takes 1.6 μ L as RNA template.Described tweezers, grind, grinding rod and other test tools need to soak in DEPC water
After 12 hours, 150 degree are toasted 6 hours, then take out that to be positioned over 4 DEG C of cold preservations standby.Ethyl acetate, 2～10% containing 10%
The 1M Tris-HClpH7.5 solution of dimethyl methyl thiamine for prepare in advance, after autoclave sterilization processes, be positioned over 4 DEG C cold
Hide standby.Rifle head and various reaction tube that shifting liquid is robbed all use RNase free product.
The concentration of the ethyl acetate of described 10% refers in 100ml solution, containing 10g ethyl acetate, dimethyl methyl thiamine
Concentration same.
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 IP 0.8 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.
Cucumber mosaic virus (CMV), marmor upsilon (PVY), potato virus X (PVX), 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
1. the determination of optimum extraction liquid
Respectively preparation dimethyl methyl Thiamine concentrations be 2%, 4%, 6%, 8%, 10% and contain 10% ethyl acetate
1M Tris-HCl (pH7.5) solution, compound method is in the volumetric flask of 100mL, addition 10g ethyl acetate, and 2 or 4,
Or the dimethyl methyl thiamine of 6 or 8 or 10g, it is subsequently adding the 1M Tris-HCl (pH7.5) constant volume prepared, obtains two
1M Tris-that is that methyl first Thiamine concentrations is 2% (or 4% or 6% or 8% or 10%) and that contain 10% ethyl acetate
HCl (pH7.5) solution.Extract according to above-mentioned RNA extraction method 2, and use spectrophotometer measurement A260's and A280
Value, calculates A260/A280 ratio, and Rneasy Mini kit extraction RNA is as positive control, and it the results are shown in Table 2.
The A260/A280 ratio of the RNA solution that table 2 distinct methods obtains
Therefore, the present embodiment all use dimethyl methyl Thiamine concentrations to be 6% and contain the 1M of 10% ethyl acetate
Tris-HCl (pH7.5) solution extracts sample RNA, but this is not limited to this concentration, it should be appreciated by those skilled in the art that it
The RNA that the solution of its concentration is extracted can be equally used for the detection of the present invention, and obtains same Detection results.
2. 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. 1.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 46,44,42 and 38min.According to the requirement that the Site Detection time is the shortest, determine that 65 DEG C are
Optimal reaction temperature.
3.RT-LAMP specificity experiments
Cucumber mosaic virus (CMV), marmor upsilon (PVY), potato virus X (PVX), tobacco vein banding mosaic virus
(TVBMV) positive material and healthy cucumber leaves (negative control represents with NC) are that Linyi University's biologic test center preserves
Specimen, takes out these specimen from-30 DEG C of refrigerators, directly takes out about 0.1g blade with tweezers, according to the RNA extraction side of the present invention
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.System adds calcium
Yellowish green element fluorescent dye is so that reaction can judge whether amplification, knot by color change produced in reaction tube after terminating
Fruit is as shown in Figure 2.As seen from Figure 2, only CMV is able to detect that amplification curve, other samples within the response time the most not
Amplification curve detected.After reaction terminates, add the aobvious green of reaction tube of CMV total serum IgE, it is judged that for the positive；And other reaction tubes
All show orange, it is judged that for feminine gender, result is as shown in Figure 3.Amplification curve result is consistent with the result of color reaction, shows this research
The RT-LAMP detection method 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 CMV, 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.The RNA rapid extraction liquid of the present invention is that dimethyl methyl Thiamine concentrations is 6% and contains the 1M of 10% ethyl acetate
Tris-HCl (pH7.5) solution, although in the present invention can the mechanism of rapid extraction CMV viral RNA the most indefinite, but root
According to analysis, owing to it has highly polar, it is possible to make CMV viral RNA quickly discharge, and it is anti-to join RT-LAMP before RNA decomposes
Answer system such that it is able to smoothly as template, carry out RT-LAMP amplification.This mechanism will be made further in test from now on
Illustrate.
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 cucumber 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.3；
Upstream inner primer BIP sequence SEQ ID NO.4；
Downstream outer primer F3 sequence SEQ ID NO.1；
Downstream outer primer B3 sequence SEQ ID NO.2；
And ring primer LB sequence SEQ ID NO.5.
2. test kit as claimed in claim 1, also includes the rapid extraction liquid of the RNA of cucumber mosaic virus,
Described rapid extraction liquid by 10% the dimethyl methyl thiamine of ethyl acetate, 2～10% and 1M Tris-HClpH7.5 structure
Become.
3. test kit as claimed in claim 2, wherein, the concentration of described dimethyl methyl thiamine is 6%.
4. a cucumber mosaic virus RT-LAMP detection method, it is characterised in that comprise the following steps
It is quick that S1 uses that the dimethyl methyl thiamine of ethyl acetate, 2～10% by 10% and 1M Tris-HClpH7.5 constitute
Extracting solution, extracts total serum IgE from testing sample, 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 concentration of described dimethyl methyl thiamine is 6%.
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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| CN108624719A (en) * | 2018-06-21 | 2018-10-09 | 中国科学院寒区旱区环境与工程研究所 | A kind of kit and its detection method of specific detection lily cucumber mosaic virus |
| CN110117677A (en) * | 2019-06-05 | 2019-08-13 | 三明市农业科学研究院 | A kind of PCR primer combination and detection method of passion fruit cucumber mosaic virus |
| CN110295247A (en) * | 2019-04-19 | 2019-10-01 | 贵州出入境检验检疫局检验检疫综合技术中心（贵州国际旅行卫生保健中心） | The species-specific primer of the anti-CMV capsicum of Rapid identification is to, kit and method |
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| CN110117677A (en) * | 2019-06-05 | 2019-08-13 | 三明市农业科学研究院 | A kind of PCR primer combination and detection method of passion fruit cucumber mosaic virus |
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Application publication date: 20160928 |