CN109943667A - Primer, probe, kit and application based on RPA technology detection soybean mosaic virus - Google Patents
Primer, probe, kit and application based on RPA technology detection soybean mosaic virus Download PDFInfo
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- CN109943667A CN109943667A CN201910393734.3A CN201910393734A CN109943667A CN 109943667 A CN109943667 A CN 109943667A CN 201910393734 A CN201910393734 A CN 201910393734A CN 109943667 A CN109943667 A CN 109943667A
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- mosaic virus
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Abstract
The invention discloses a kind of primer, probe, kit and applications based on RPA technology detection soybean mosaic virus.The base sequence of upstream primer is as shown in SEQ ID NO.1, and the base sequence of downstream primer is as shown in SEQ ID NO.2, and the base sequence of probe is as shown in SEQ ID NO.3.In addition to the primer, probe, the kit further includes flow measurement chromatograph test strip, flow measurement chromatograph test strip buffer solution, RPA enzyme, RPA buffer, magnesium acetate solution, ddH2O, one or more of reference substance.The invention also discloses the methods of detection soybean mosaic virus.The present invention is based on soybean mosaic virus CP genes to devise specific primer and probe, reapplies recombinase polymeric enzymatic amplification-flow measurement chromatography method and is prepared for kit, sensibility is high, high specificity, diagnosis is accurate, and entire detection process only needs 30 minutes, and judgement as a result is extremely simple.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of RPA primer, probe, examination for detecting soybean mosaic virus
Agent box and application.
Background technique
Soybean mosaic virus (Soybean mosaic virus, SMV), is the most common virus disease in world soybean producing region
One of.Under natural conditions, economic loss caused by SMV is 35%-50% (Arif and Hassan, 2002), is up to when serious
50%-100% (Liao et al., 2002).In addition to directly resulting in soybean Severe Reduction and germplasm decline (Hill, 1999) outside,
SMV's infects the immunity for being greatly reduced soybean to other pathogens.
Currently, the method for detection soybean mosaic virus mainly has indicator plant method, enzyme linked immunosorbent assay (ELISA), anti-
Transcriptional polymerase chain reaction (RT-PCR) and real-time fluorescence PCR method.Indicator plant detection method is fairly simple, but it detects speed
It is very slow, and can not determine the type for infecting virus, sensitivity is relatively low.It is existing both at home and abroad much to be detected using ELISA method
The report of soybean mosaic virus, this method high sensitivity is easy to operate, but required detection time is also at 1~2 day, and antibody
Long preparation period, costly, sensitivity is not as good as RT-PCR.RT-PCR, the immunocapture established using Protocols in Molecular Biology
The methods of RT-PCR, real-time fluorescence RT-PCR and probe hybridization check so that the testing result of soybean mosaic virus it is more quick,
It is sensitive, accurate.But the method for traditional based on PCR needs to be denaturalized, annealing, extends three steps, and the temperature of each step is different,
Special thermal cycler is needed to carry out, and limits its use scope.Therefore many isothermal amplification technique quilts for not depending on PCR instrument
It invents, is especially most widely used with LAMP.
RPA (Recombinasepolymeraseamplifcation) is a novel isothermal amplification technique, main
Principle is that microfilament is formed using recombinase and primer when searching the sequence of complete complementary pairing therewith on template DNA, in list
Make template DNA unwinding under the protein-bonded side group of chain DNA, primer starts to match in template DNA, and in the effect of archaeal dna polymerase
Lower duplication extends.There has been no the reports using RPA method detection soybean mosaic virus at present.
Summary of the invention
Goal of the invention: not there are problems that in the prior art RPA method detect soybean mosaic virus, the purpose of the present invention
One of there is provided based on RPA technology detection soybean mosaic virus primer, probe, kit, it is another object of the present invention to
The application of above-mentioned primer, probe, kit is provided, the third object of the present invention is to provide a kind of side for detecting soybean mosaic virus
Method.
Technical solution: the primer and probe combination of the present invention based on RPA technology detection soybean mosaic virus, packet
It includes:
Upstream primer: 5 '-AAGGAAGATGAATCTTCCAATGGTTGAAGGGAA-3 '；
Downstream primer: [5 ' BIOTIN] ATCAAGTTCATATTCATCTTTGACTGCATTGTA；
Probe: [5 ' FAM] GTTTAGACCACTTGCTTGAGTATAAACCTAAT [THF] AAGTTGATTTATTCAA [3 '
BLOCK]。
The present invention also provides a kind of kits based on RPA technology detection soybean mosaic virus, include the primer
And probe combinations.
The kit further includes flow measurement chromatograph test strip, flow measurement chromatograph test strip buffer solution, RPA enzyme, RPA buffering
Liquid, magnesium acetate solution, ddH2O, one or more of reference substance.These ingredients can be prepared voluntarily, can also use commercially available
Product, such as a kind of relatively good selection, flow measurement chromatograph test strip, flow measurement chromatograph test strip buffer solution use Germany Milenia
The Milenia GenLine HybriDetect kit of Biotec GmbH company；RPA enzyme, RPA buffer, magnesium acetate solution
Using the TwistAmp of TwistDX companyTMnfo kit。
The present invention also provides the primer and probe combinations based on RPA technology detection soybean mosaic virus to detect
Application in soybean mosaic virus.
The present invention also provides the kits based on RPA technology detection soybean mosaic virus in detection soybean mosaic
Application in virus.
The present invention finally provide it is a kind of detect soybean mosaic virus method, comprising: using the cDNA of sample to be tested as
Template carries out RPA amplification using the primer and probe combination, identifies amplified production.
Wherein, using identifying in Sidestream chromatography test strips amplified production, according to showing result judgement in test strips
Soybean mosaic virus is positive or negative.Method particularly includes: take 2 μ L amplified productions and 98 μ L flow measurement chromatograph test strip buffer solutions
Mixing forms mixed liquor, then draws the sample-adding end that 10 μ L mixed liquors are added drop-wise to flow measurement chromatograph test strip, and vertical by end is loaded
It immerses in the centrifuge tube equipped with 200 μ L flow measurement chromatograph test strip buffer solutions, observes show result in test strips after five minutes.
The overall reaction system of RPA amplification is 50 μ L, composition are as follows: 2.1 μ L of upstream primer that 10 μM of concentration, 10 μM of concentration
2.1 μ L of downstream primer, 10 μM of concentration of 0.6 μ L of probe, 29.5 μ L of RPA buffer, ddH212.2 μ L of O, 1 μ L sample to be tested
CDNA template, 2.5 μ L concentration be 280mM MgAc, RPA enzyme be originated from TwistDX company TwistAmpTMnfo kit。。
Amplification condition are as follows: 39 DEG C, 20 minutes.
The utility model has the advantages that
The present invention is directed to soybean mosaic virus CP gene, devises the primer and probe based on RPA technology, due to right at present
Design rule is not formed in the primer and probe of RPA technology, and the primer of Standard PCR design, probe and not applicable RPA are anti-
Answer, it is therefore desirable to technical staff continuously attempts to and explores, and inventor, which have passed through, largely to test and grope, obtain one group it is special
Property high, primer and probe combination that sensitivity is good.
The present invention establish on molecular biology mechanism, first based on soybean mosaic virus CP gene devise specific primer and
Probe, the gene is not only highly conserved, but also to other cause of disease high specials, reapplies recombinase polymeric enzymatic amplification-flow measurement layer
The method of analysis is prepared for quickly detecting the kit of soybean mosaic virus, and sensibility is high, high specificity, and diagnosis is accurate, entire to examine
Survey process only needs 30 minutes, and judgement as a result need to only observe the result that flow measurement chromatograph test strip is shown, operates extremely simple；
Obtaining entire reaction process after nucleic acid samples can carry out in a non-laboratory environment, be applicable not only to indoor identification and can be with
Applied to field on-site test.
Detailed description of the invention
Fig. 1 is the result figure of the foundation of embodiment 1RPA detection method, respectively is from left to right in figure: soybean mosaic
Virus, positive control, negative control；
Fig. 2 is RPA sensitivity experiment result figure, and the dilution of template cDNA respectively is 1,10 from left to right in figure-1、
10-2And 10-3, most right is positive control；
Fig. 3 is specificity experiments result figure, and respectively be from left to right in figure: soybean mosaic virus, Kidney bean are commonly spent
Mosaic virus, Brassica 2 et 4, negative control, positive control.
Specific embodiment
Combined with specific embodiments below, the present invention is furture elucidated, it should be understood that these embodiments are merely to illustrate the present invention
Rather than limit the scope of the invention, after the present invention has been read, those skilled in the art are to various equivalences of the invention
The modification of form falls within the application range as defined in the appended claims.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
RPA amplification kit TwistAmp in following embodimentsTMNfokits is the product of TwistDX company, product mesh
Record number is TANFO02KIT.
Soybean mosaic virus (Soybean mosaic virus) in following embodiments is in document " Cui Xiaoyan, Chen Xin, goldenrain tree
Crane Xiang, the structure of the soybean cDNA library applied to memebrane protein yeast two-hybrid of Zhi Haijian .2013. soybean mosaic virus induction
It builds Plant Pathology .43 (5): being disclosed in 556-560. ", the public can obtain from Jiangsu Province Agriculture Science Institute.But simultaneously not only
It is limited to this, other disclosed Strains of Soybean Mosaic Virus can also be used.
Bean common mosaic virus (Bean common mosaic virus) in following embodiments document " Shen Liang,
A kind of newfound Bean common mosaic virus Molecular Identification China for infecting mung bean of Cui Jin, Xia Yan, Cui Xiaoyan, Chen Xin .2014.
Northern agronomy reports .29 (4): being disclosed in 1-8. ", the public can obtain from Jiangsu Province Agriculture Science Institute.It is not limited to this, it can also
To use Bean common mosaic virus strain disclosed in other.
Brassica 2 et 4 (Turnip mosaic virus) in following embodiments document " Xiaoyan Cui,
HodaYaghmaiean,Guanwei Wu,Xiaoyun Wu,Xin Chen,Greg Thornc and Aiming
Wang.2017.The C-terminal region of the Turnip mosaic virus P3protein is
essential for viral infection via targeting P3to the viral replication
Complex.Virology.510:147-155. it is disclosed in ", the public can obtain from Jiangsu Province Agriculture Science Institute.But simultaneously not only
It is limited to this, other disclosed Turnip Mosaic Virus Strains On Oilseed can also be used.
Embodiment 1
RPA primer, probe and its RPA kit for detecting soybean mosaic virus include:
One, the design and synthesis of RPA primer, probe
With reference to the conserved sequence of the CP gene (genbank accession number be JF833015) of soybean mosaic virus in GenBank,
Selecting specific conservative region, (position is 8681-8840) in JF833015, designs RPA primer and probe.All primers and spy
Needle is synthesized by the handsome Bioisystech Co., Ltd in Shanghai, and the base sequence of primer and probe is as follows.
Upstream primer (SEQ ID NO.1):
5′-AAGGAAGATGAATCTTCCAATGGTTGAAGGGAA-3′；
Downstream primer (SEQ ID NO.2):
[5'BIOTIN]ATCAAGTTCATATTCATCTTTGACTGCATTGTA-3′；
Probe (SEQ ID NO.3): [5 ' FAM] GTTTAGACCACTTGCTTGAGTATAAAC
CTAAT[THF]AAGTTGATTTATTCAA[3'a phosphate]；
In probe, THF is tetrahydrofuran, and FAM is fluorescein, and phosphate is phosphate group；BIOTIN is biotin.
The fragment sequence (SEQ ID NO.4) of SMV detection:
AAGGAAGATGAATCTTCCAATGGTTGAAGGGAAGATTATCCTCAGTTTAGACCACTTGCTTGAGTATA
AACCTAATCAAGTTGATTTATTCAATACTCGGGCAACAAGAACACAGTTTGAGGCATGGTACAATGCAGTCAAAGA
TGAATATGAACTTGAT。
Two, kit forms:
(1) RPA primed probe mixed liquor: upstream primer shown in the NO.1 of ID containing SEQ, downstream shown in SEQ ID NO.2 are drawn
Probe shown in object, SEQ ID NO.3, each primer, probe concentration be 10 μM；
(2) (flow measurement chromatograph test strip is used cooperatively ingredient, Milenia to HybriDetect Assay Buffer solution
GenLine HybriDetect kit, German Milenia Biotec GmbH company, article No. 041)
(3) Sidestream chromatography test strips (Milenia GenLine HybriDetect, German Milenia Biotec GmbH
Company, article No. 041)
(4) RPA lyophozyme (containing three kinds of polymerase, recombinase and single strand binding protein) and RPA reaction premixed liquid, RPA are anti-
Answer premixed liquid mixed for 29.5 μ L Rehydration Buffer and 2.5 μ L 280mM Magnesium Acetate (magnesium acetate)
Close liquid (TwistDX company, TwistAmpTMNfo kit ingredient).
Mentioned reagent box specific is conventionally bought or is prepared the preparation method comprises the following steps: synthesize the primer and probe
Remaining each ingredient.
Embodiment 2
The method for quickly detecting soybean mosaic virus using mentioned reagent box, specifically includes the following steps:
(1) recombinase polymeric enzymatic amplification (RPA) is reacted:
Use TwistAmpTMNfo kit prepares the real-time RPA reaction system of 50 μ L, and 10 μM of concentration of upstream primer 2.1 is added
μ L, the 2.1 μ L of downstream primer that 10 μM of concentration, 0.6 μ L, Rehydration Buffer of probe, the 29.5 μ L that 10 μM of concentration, distillation
12.2 μ L of water, is transferred in the 0.2mL reaction tube of RPA lyophozyme after mixing, is 280mM's by 1 μ L cDNA template, 2.5 μ L concentration
MgAc is added in the reaction tube, and 39 DEG C, amplification obtains amplified production in 20 minutes.
(2) detection of amplified production:
2 μ L RPA amplified productions are drawn after amplified reaction and 98 μ L HybriDetect Assay Buffer solution mix
After form mixed liquor, then draw the sample-adding end that 10 μ L mixed liquors are added drop-wise to Sidestream chromatography test strips, it is finally that test strips are endways
In the centrifuge tube equipped with 200 μ L HybriDetect Assay Buffer solution of insertion, test strips are observed after five minutes and are shown
As a result, if test strips show two thick sticks, be then negative (Fig. 1) if it is a thick stick for the soybean mosaic virus positive.
The sensitivity experiments of the kit of the invention of embodiment 3
Experimentation are as follows: soybean mosaic virus cDNA is subjected to RPA amplified reaction, dilution is added in each amplified reaction
It is 1,10-1, 10-2, 10-3Soybean mosaic virus cDNA be template.It is quick according to soybean mosaic virus of the present invention after reaction
Detection method carry out the detection of Sidestream chromatography test strips, testing result as shown in fig. 2, it can be seen that this method can detecte it is anti-
Soybean mosaic virus cDNA in system (nucleic acid concentration 1.333ng/ μ L) is answered to dilute 100 times, detection sensitivity is very high.
Embodiment 4
The specificity experiments of kit of the invention:
Experimentation are as follows: with the cDNA of soybean mosaic virus, Bean common mosaic virus cDNA and Brassica 2 et 4
CDNA carries out specificity experiments, and experimental result is fig. 3, it is shown that only detect the test paper of soybean mosaic virus cDNA
There is Liang Tiaogang in item, and the test strips for detecting other viruses are Yi Tiaogang, illustrates kit of the present invention to soybean mosaic virus
CDNA has very strong specificity.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Jiangsu Province Agriculture Science Institute
<120>primer, probe, kit and application based on RPA technology detection soybean mosaic virus
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aaggaagatg aatcttccaa tggttgaagg gaa 33
<210> 2
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atcaagttca tattcatctt tgactgcatt gta 33
<210> 3
<211> 49
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (33)
<223>n=tetrahydrofuran
<400> 3
gtttagacca cttgcttgag tataaaccta atnaagttga tttattcaa 49
<210> 4
<211> 160
<212> DNA
<213>soybean mosaic virus (Soybean mosaic virus)
<400> 4
aaggaagatg aatcttccaa tggttgaagg gaagattatc ctcagtttag accacttgct 60
tgagtataaa cctaatcaag ttgatttatt caatactcgg gcaacaagaa cacagtttga 120
ggcatggtac aatgcagtca aagatgaata tgaacttgat 160
Claims (9)
1. a kind of primer and probe combination based on RPA technology detection soybean mosaic virus characterized by comprising
Upstream primer: 5 '-AAGGAAGATGAATCTTCCAATGGTTGAAGGGAA-3 '；
Downstream primer: [5 ' BIOTIN] ATCAAGTTCATATTCATCTTTGACTGCATTGTA-3 '；
Probe: [5 ' FAM] GTTTAGACCACTTGCTTGAGTATAAACCTAAT [THF] AAGTTGATTTATTCAA [3 '
BLOCK]。
2. a kind of kit based on RPA technology detection soybean mosaic virus, which is characterized in that comprising described in claim 1
Primer and probe combination.
3. the kit according to claim 2 based on RPA technology detection soybean mosaic virus, which is characterized in that also wrap
Include flow measurement chromatograph test strip, flow measurement chromatograph test strip buffer solution, RPA enzyme, RPA buffer, magnesium acetate solution, ddH2O, it compares
One or more of product.
4. the primer and probe combination according to claim 1 based on RPA technology detection soybean mosaic virus is big in detection
Application in bean mosaic virus.
5. the kit according to claim 2 or 3 based on RPA technology detection soybean mosaic virus is in detection soybean mosaic
Application in virus.
6. a kind of method for detecting soybean mosaic virus characterized by comprising using the cDNA of sample to be tested as template, benefit
RPA amplification is carried out with primer and probe described in claim 1 combination, identifies amplified production.
7. the method for detection soybean mosaic virus according to claim 6, which is characterized in that use Sidestream chromatography test strips
On amplified production is identified.
8. the method for detection soybean mosaic virus according to claim 6, which is characterized in that the overall reaction body of RPA amplification
System is 50 μ L, composition are as follows: 2.1 μ L of upstream primer that 10 μM of concentration, 10 μM of concentration of 2.1 μ L of downstream primer, 10 μM of concentration of spy
0.6 μ L of needle, 29.5 μ L of RPA buffer, ddH212.2 μ L of O, the cDNA template of 1 μ L sample to be tested, 2.5 μ L concentration are 280mM's
MgAc, RPA enzyme are originated from the TwistAmp of TwistDX companyTMnfo kit。。
9. the method for detection soybean mosaic virus according to claim 6, which is characterized in that amplification condition are as follows: 39 DEG C, 20
Minute.
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| CN201910393734.3A CN109943667A (en) | 2019-05-13 | 2019-05-13 | Primer, probe, kit and application based on RPA technology detection soybean mosaic virus |
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Cited By (1)
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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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| CN107034309A (en) * | 2016-02-03 | 2017-08-11 | 中国农业科学院兰州兽医研究所 | The real-time fluorescence RPA kits of quick detection PRV, test strips RPA kits and application thereof |
| CN107236734A (en) * | 2017-08-10 | 2017-10-10 | 中国检验检疫科学研究院 | Method, RPA IAC primers and kit based on RPA IAC technology examination soybean mosaic virus |
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2019
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| CN107034309A (en) * | 2016-02-03 | 2017-08-11 | 中国农业科学院兰州兽医研究所 | The real-time fluorescence RPA kits of quick detection PRV, test strips RPA kits and application thereof |
| CN107236734A (en) * | 2017-08-10 | 2017-10-10 | 中国检验检疫科学研究院 | Method, RPA IAC primers and kit based on RPA IAC technology examination soybean mosaic virus |
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| 袁俊杰等: "一步法逆转录重组酶聚合酶常温扩增技术(RT-RPA)检测大豆花叶病毒", 《检验检疫学刊》 * |
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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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