CN103667528A - Reverse transcription loop-mediated isothermal detection method of bean common mosaic virus - Google Patents
Reverse transcription loop-mediated isothermal detection method of bean common mosaic virus Download PDFInfo
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Abstract
The invention discloses a reverse transcription loop-mediated isothermal detection method of a bean common mosaic virus. A group of primers disclosed in the invention is composed of a DNA molecule (1) represented by SEQ ID No.1, a DNA molecule DNA molecule (2) represented by SEQ ID No.2, a DNA molecule (3) represented by SEQ ID No.3 and a DNA molecule (4) represented by SEQ ID No.4. The bean common mosaic virus detection method has the advantages of rapidness, sensitivity and high specificity, and provides a new means for the rapid detection of the virus.
Description
Technical field
The present invention relates to a kind of reverse transcription loop-mediated isothermal detection method of Bean common mosaic virus.
Background technology
Bean common mosaic virus (Bean common mosaic virus, BCMV) is one of member of Potyvirus Potyvirus, in the whole world, extensively distributes, and be one of topmost virus disease on Kidney bean.BCMV is a kind of typical Seed-borne Virus, almost can infect all edible bean crops, plants biography rate up to 93% on Kidney bean.BCMV infect cause that blade narrows down after Kidney bean, curling, shrinkage, floral leaf, beanpod is mottled or lopsided etc., causes serious production loss.
Loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) the earliest by Notomi T etc. invention in 2000, it is a kind of isothermal nucleic acid amplification method based on highly sensitive strand displacement technology, its principle of work is: for 6 zone design of target gene 4 special primers, utilize a kind of strand displacement archaeal dna polymerase-Bst (Bacillus stearothermophilus) DNA polymerase to react and can complete nucleic acid amplification reaction in 1 hour under the condition of constant temperature (65 ℃), reaction product both can be observed by electrophoresis ultraviolet, also can directly estimate by fluorescent dye.The method is easy and simple to handle, and without specific apparatus (PCR instrument), and detected result is accurate, is a kind of fast and effectively detection method.Disease detection and quick diagnosis that the mankind and the various virus of animals and plants, bacterium, parasite etc. cause have been widely used at present.The method has wide range of applications, and is suitable for laboratories and carries out rapid detection.
At present in the detection of Bean common mosaic virus, more conventional method has two kinds: a kind of is molecular biology for detection, wherein common with PCR, but PCR needs 30-35 circulating reaction, instrument (PCR instrument), schedule of operation very complicated, time more time-consuming and need to be special be long, the technical requirements higher to testing staff, has limited using and promoting as fast diagnosis method greatly; Another kind is Serology test, common are ELISA, and Serology test is except Shortcomings in sensitivity, and the specificity of its detection is also restricted.And LAMP method has well overcome the deficiency of current these two class methods, it is short that LAMP detection method has the reaction times, without specific apparatus (constant-temperature metal bath), cost is low, detection sensitivity is high, specificity is good, has not yet to see the report that utilizes LAMP technology for detection Bean common mosaic virus.
Summary of the invention
The reverse transcription loop-mediated isothermal detection method that the object of this invention is to provide a kind of Bean common mosaic virus.
One group of primer provided by the invention, is comprised of the DNA molecular shown in following (1)-(4):
(1) DNA molecular shown in SEQ ID No.1;
(2) DNA molecular shown in SEQ ID No.2;
(3) DNA molecular shown in SEQ ID No.3;
(4) DNA molecular shown in SEQ ID No.4.
The test kit that detects Bean common mosaic virus also belongs to a protection scope of the present invention, and this test kit comprises described primer, ThermoScript II and Bst archaeal dna polymerase;
Described ThermoScript II is M-MLV ThermoScript II or AMV ThermoScript II.
A kind of method that detects Bean common mosaic virus also belongs to protection scope of the present invention; the method is that to take total RNA of testing sample be template; the described primer of take carries out reverse transcription loop-mediated isothermal nucleic acid amplification as primer; if obtain reverse transcription loop-mediated isothermal nucleic acid amplification product; in described testing sample, candidate is contained Bean common mosaic virus; if can not get reverse transcription loop-mediated isothermal nucleic acid amplification product, in described testing sample, candidate is not contained Bean common mosaic virus.
In aforesaid method, the determination methods whether described pcr amplification product obtains is following (1) and/or (2):
(1) reverse transcription loop-mediated isothermal nucleic acid amplification reaction product is carried out to agarose gel electrophoresis, the testing sample with disperse shape nucleic acid electrophoresis band is the sample that contains Bean common mosaic virus, does not have the testing sample of disperse shape nucleic acid electrophoresis band for not containing the sample of Bean common mosaic virus;
(2) reverse transcription loop-mediated isothermal nucleic acid amplification reaction product is reacted to obtain to reaction solution with SYBR Green I, it is the sample that contains Bean common mosaic virus that reaction solution is emerald testing sample candidate, and it is the sample that does not contain Bean common mosaic virus that reaction solution is orange testing sample candidate.
In above-mentioned arbitrary described method, in described primer, the DNA molecular shown in the DNA molecular shown in SEQ ID No.1 and SEQ ID No.2 is outer primer, and the DNA molecular shown in the DNA molecular shown in SEQ ID No.3 and SEQ ID No.4 is inner primer;
In described outer primer, the molar concentration rate of the DNA molecular shown in the DNA molecular shown in SEQ ID No.1 and SEQ ID No.2 in described reverse transcription loop-mediated isothermal nucleic acid amplification is 1:1;
In described inner primer, the molar concentration rate of the DNA molecular shown in the DNA molecular shown in SEQ ID No.3 and SEQ ID No.4 in described reverse transcription loop-mediated isothermal nucleic acid amplification is 1:1.
Described outer primer and the described inner primer molar concentration rate in described reverse transcription loop-mediated isothermal nucleic acid amplification is specially 1:5.
In above-mentioned arbitrary described method, the temperature of reaction in described reverse transcription loop-mediated isothermal nucleic acid amplification is 62 ℃.
In above-mentioned arbitrary described method, the magnesium ion concentration in described reverse transcription loop-mediated isothermal nucleic acid amplification is 5mM.
In above-mentioned arbitrary described method, the system of described reverse transcription loop-mediated isothermal nucleic acid amplification is as follows:
10 * Thermopol buffer2.5 μ L, DNA molecular 0.2 μ M shown in SEQ ID No.1, the DNA molecular 0.2 μ M shown in SEQ ID No.2, the DNA molecular 1.0 μ M shown in SEQ ID No.3, DNA molecular 1.0 μ M shown in SEQ ID No.4, dNTPs1.0mM, MgCl25mM, ThermoScript II 200U, Bst archaeal dna polymerase 12U, total RNA40ng of described testing sample, surplus is ddH2O, system 25 μ L;
Described 10 * Thermopol buffer is purchased from Niu Yinglun biotechnology (Beijing) company limited, and catalog number is M0275;
Described ThermoScript II is M-MLV ThermoScript II or AMV ThermoScript II;
The trade name of described Bst archaeal dna polymerase is Bst DNApolymerase, and purchased from Niu Yinglun biotechnology (Beijing) company limited, catalog number is M0275;
The reaction conditions of described reverse transcription loop-mediated isothermal nucleic acid amplification is: 62 ℃ of isothermal 1h; 80 ℃ of 5min.
In above-mentioned arbitrary described method, described M-MLV ThermoScript II is purchased from TaKaRa company, and catalog number is 2641A;
Described sample is specially the blade of testing sample.
The application of above-mentioned primer in detecting Bean common mosaic virus also belongs to protection scope of the present invention;
And/or,
The application of mentioned reagent box in detecting Bean common mosaic virus also belongs to protection scope of the present invention.
The present invention has set up a kind of method of detection Bean common mosaic virus of quick, sensitive, high special, for this viral rapid detection provides new means.
Accompanying drawing explanation
Fig. 1 is the primer screening electrophoresis result that RT-LAMP method detects Bean common mosaic virus.
Fig. 2 is the primer screening fluorescent dye result that RT-LAMP method detects Bean common mosaic virus.
Fig. 3 is RT-LAMP reaction system optimization result electrophoresis detection.
Fig. 4 is that the fluorescent dye of RT-LAMP reaction system optimization result detects.
Fig. 5 is specificity and the stability that electrophoretic analysis RT-LAMP method detects Bean common mosaic virus.
Fig. 6 is that specificity and the stability that RT-LAMP method detects Bean common mosaic virus is analyzed in fluorescent dye.
Fig. 7 is the susceptibility that electrophoretic analysis RT-LAMP method detects Bean common mosaic virus.
Fig. 8 is that the susceptibility that RT-LAMP method detects Bean common mosaic virus is analyzed in fluorescent dye.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Do not infect the soybean of any virus and infect Bean common mosaic virus soybean, Kidney bean and French beans, do not infect any virus Kidney bean, infect CMV(cucumber mosaic virus) Kidney bean and infect TMV(tobacco mosaic virus (TMV)) tomato all pick up from Chinese Tai'an, collected specimens is stored in-80 ℃ of refrigerators.
Bst DNApolymerase is purchased from Niu Yinglun biotechnology (Beijing) company limited, and catalog number is M0275.
M-MLV ThermoScript II is purchased from TaKaRa company, and catalog number is 2641A.
DNTPs is purchased from TaKaRa company, and catalog number is 4019.
SYBR Green I is purchased from Invitrogen company.
10 * Thermopol buffer is purchased from Niu Yinglun biotechnology (Beijing) company limited, and catalog number is M0275.
The reverse transcription loop-mediated isothermal amplification detection method of embodiment 1, Bean common mosaic virus
One, design of primers:
According to the Bean common mosaic virus nucleotide sequence of having reported on NCBI, utilize LAMP primer-design software Primer Explorer V4 design 7 cover special primers, primer sequence is as follows:
First group of primer:
F3：5’-ACGGATTGCTTCGGAATT-3’；
B3：5’-GCCTCTCAGTATTTTCGCTG-3’；
FIP:5’-TCCGATGTTTTGGATGTCACC-GGGATAAAAATCTAGCTCGCT-3’；
BIP:5’-TCGAGCCAGAGAAGCAGTAG-TACCATCAAGTCCAAACAACT-3’。
Second group of primer:
F3：5’-GCATACATTGAGATGAGAAATTCC-3’；
B3：5’-CATTACCATCAAGTCCAAACA-3’；
FIP：5’-GTAGCGAGCTAGATTTTTATCCCTC-AGAAACCGTACATGCCTAG-3’；
BIP：5’-GAGGTGACATCCAAAACATCGGAT-CTTGCTGCTAACGTTGCT-3’。
The 3rd group of primer:
F3：5’-ACAAGGATGTGAACGCAG-3’；
B3：5’-CATTGTACCACATTTCAAACTG-3’；
FIP：5’-CACCATGGGCAAGTTCATCC-GCTCCAAAGGGAAGGTTG-3’；
BIP：5’-TAGACCATCTATTGGATTACAAGCC-CATCTTTGTTGCTCTTGTGTT-3’。
The 4th group of primer:
F3：5’-TCATGCACCATTTCTCAGA-3’；（SEQ?ID?No.1）
B3：5’-ACAACTTGCTGCTAACGT-3’；（SEQ?ID?No.2）
FIP:5’-CCTCAAATTCCGAAGCAATCCG-CATTGAGATGAGAAATTCCGAG-3’；（SEQ?IDNo.3）
BIP:5’-TCGCTACGCTTTTGATTTCTATGA-CTGCTGCCTTCATCTGTG-3’。（SEQ?ID?No.4）
The 5th group of primer:
F3：5’-CCAAAGGGAAGGTTGTCC-3’；
B3：5’-CATTACAATTGACATTTGTGCA-3’；
FIP:5’-GGCTTGTAATCCAATAGATGGTCTA-TCAAAAGATCACAAAAAGGATGA-3’；
BIP:5’-TTAACACAAGAGCAACAAAGATGC-CTCATACTCGCCCTTCAC-3’。
The 6th group of primer:
F3：5’-CACGGCTTCAAAAGATCAC-3’；
B3：5’-GCCATTCATTACAATTGACATT-3’；
FIP：5’-GGCTTGTAATCCAATAGATGGTCTA-AAAAAGGATGAACTTGCCCA-3’；
BIP：5’-TTAACACAAGAGCAACAAAGATGC-CTCATACTCGCCCTTCAC-3’。
The 7th group of primer:
F3：5’-ATTTGAGGGATAAAAATCTAGCT-3’；
B3：5’-TGTGCATGTTTTGATTGACG-3’；
FIP：5’-ACTGCTTCTCTGGCTCGATC-CTACGCTTTTGATTTCTATGAGG-3’；
BIP：5’-TCAGCAACGTTAGCAGCAAG-AGTATTTTCGCTGGTTGTTG-3’。
Two, total RNA of the soybean leaves of extraction infection Bean common mosaic virus is experimental group, extracts the negative control group of total RNA of the soybean leaves that does not infect any virus simultaneously.
Three, reverse transcription loop-mediated isothermal nucleic acid amplification (RT-LAMP) reaction
Reaction system is as follows:
10 * Thermopol buffer2.5 μ L(20mM Tris-HCl, 10mM KCl, 2mM MgSO
4, 10mM (NH
4)
2sO
4, the Triton X-100 of volumn concentration 0.1%), concentration is primers F 3 and each 0.5 μ L of B3 of 10 μ M, concentration is primers F IP and each 2.0 μ L of BIP of 10 μ M, the dNTPs2.5 μ L that concentration is 10mM, the MgCl that concentration is 25mM
23 μ L, 200U/ μ L M-MLV ThermoScript II 1 μ L, the Bst DNA polymerase1.5 μ L of 8000U/mL, the template ribonucleic acid 2.0 μ L of 20ng/ μ L, ddH
2o supplies system to 25 μ L.
Reaction conditions: 62 ℃ of constant temperature 1h; 80 ℃ of 5min.
Template ribonucleic acid is respectively the experimental group of step 2 extraction or total RNA of negative control group.
Through above-mentioned reaction, obtain the RT-LAMP reaction product of experimental group and negative control group.
Four, the detection of RT-LAMP reaction product
(1) electrophoresis detection: get respectively the RT-LAMP reaction product of 5 μ L experimental group and negative control group through 1.5% sepharose electrophoresis 25min under 0.5 * tbe buffer liquid and 155V voltage conditions, gel is dyeed in the ethidium bromide (EB) of 0.5 μ g/mL 10min, dyeing is placed in gel imaging system to be observed.
Result as shown in Figure 1.
In Fig. 1, M:Trans2K Plus DNA Marker, 1-7 is followed successively by the RT-LAMP reaction product that 7 groups of primers detect the soybean that infects Bean common mosaic virus, and 8-14 is followed successively by the RT-LAMP reaction product that 7 groups of primers detect the soybean (negative control group) that does not infect any virus.
Fig. 1 shows, take in 7 groups of primers that total RNA of the soybean leaves that infects Bean common mosaic virus is template, the RT-LAMP reaction product that can observe the 4th group of primer is the nucleic acid swimming band of disperse shape, and the RT-LAMP reaction product of all the other 6 groups of primers does not have disperse shape nucleic acid electrophoresis band, take in the RT-LAMP reaction product of 7 groups of primers that total RNA of the soybean leaves that do not infect any virus is template all free nucleic acid electrophoretic bands.
(2) fluorescent dye detects: to adding 2.5 μ l SYBR Green I (1: 1000) in the RT-LAMP reaction product of 20 μ L volume experimental group and negative control group, (1:1000 refers to by 1 μ L SYBR Green I and adds 999uL ddH respectively
2the ratio of O is to SYBR Green I dilution), observations after dyeing 5min.
Result as shown in Figure 2.
In Fig. 2,1-7 is followed successively by the fluorescent dye result that 7 groups of primers detect the RT-LAMP reaction product of the soybean that infects Bean common mosaic virus, and 8-14 is followed successively by the fluorescent dye result that 7 groups of primers detect the RT-LAMP reaction product (negative control group) of the soybean that does not infect any virus.
Fig. 2 shows, take in 7 groups of primers that total RNA of the soybean leaves that infects Bean common mosaic virus is template, after can observing the RT-LAMP reaction product dyeing of the 4th group of primer, be emerald green, react positive, and the RT-LAMP reaction product of 7 groups of primers that the RT-LAMP reaction product of all the other 6 groups of primers and the total RNA of the soybean leaves that do not infect any virus of take are template keeps the orange of dyestuff, react negative.
The optimization of embodiment 2, RT-LAMP detection system
By example 1, can filter out optimum primer is the 4th group of primer, but the nucleic acid electrophoresis band of the 4th group of primer is not fairly obvious, so by two of the major effect of constructive system factor Mg2+ concentration and the screening of inside and outside primer concentration ratio, determine optimal detection system.
(25 μ L) is as follows for reaction system:
A:10 * Thermopol buffer2.5 μ L(20mM Tris-HCl, 10mM KCl, 2mM MgSO
4, 10mM (NH
4)
2sO
4, the Triton X-100 of volumn concentration 0.1%), in the 4th group of primer, primer (F3 and B3) is 0.2 μ M, and in the 4th group of primer, primer (FIP and BIP) is 1.0 μ M, dNTPs1.0mM, MgCl
25mM, 200U/ μ L M-MLV ThermoScript II 1.0 μ L, the Bst DNApolymerase1.5 μ L of 8000U/mL, the template ribonucleic acid 2.0 μ L of 20ng/ μ L, ddH
2o supplies system to 25 μ L;
B:10 * Thermopol buffer2.5 μ L(20mM Tris-HCl, 10mM KCl, 2mM MgSO
4, 10mM (NH
4)
2sO
4, the Triton X-100 of volumn concentration 0.1%), in the 4th group of primer, primer (F3 and B3) is 0.2 μ M, and in the 4th group of primer, primer (FIP and BIP) is 0.8 μ M, dNTPs1.0mM, MgCl
23mM, 200U/ μ L M-MLV1.0 μ L, the Bst DNApolymerase1.5 μ L of 8000U/mL, the template ribonucleic acid 2.0 μ L of 20ng/ μ L, ddH
2o supplies system to 25 μ L;
C:10 * Thermopol buffer2.5 μ L(20mM Tris-HCl, 10mM KCl, 2mM MgSO
4, 10mM (NH
4)
2sO
4, the Triton X-100 of volumn concentration 0.1%), in the 4th group of primer, primer (F3 and B3) is 0.2 μ M, and in the 4th group of primer, primer (FIP and BIP) is 0.8 μ M, dNTPs1.0mM, MgCl
24mM, 200U/ μ L M-MLV1.0 μ L, the Bst DNApolymerase1.5 μ L of 8000U/mL, the template ribonucleic acid 2.0 μ L of 20ng/ μ L, ddH
2o supplies system to 25 μ L;
D:10 * Thermopol buffer2.5 μ L(20mM Tris-HCl, 10mM KCl, 2mM MgSO
4, 10mM (NH
4)
2sO
4, the Triton X-100 of volumn concentration 0.1%), in the 4th group of primer, primer (F3 and B3) is 0.2 μ M, and in the 4th group of primer, primer (FIP and BIP) is 1.2 μ M, dNTPs1.0mM, MgCl
24mM, 200U/ μ L M-MLV1.0 μ L, the Bst DNApolymerase1.5 μ L of 8000U/mL, the template ribonucleic acid 2.0 μ L of 20ng/ μ L, ddH
2o supplies system to 25 μ L.
Reaction conditions: 62 ℃ of constant temperature 1h; 80 ℃ of 5min.
The total RNA that extract to infect the soybean leaves of Bean common mosaic virus is experimental group, extracts the negative control group of total RNA of the soybean leaves that does not infect any virus simultaneously, and the RNA of each sample of take carries out respectively above-mentioned reaction as template, obtains RT-LAMP reaction product.
The detection of RT-LAMP reaction product is as step 4 in embodiment 1.
Electrophoresis detection result as shown in Figure 3.
In Fig. 3, M:Trans2K Plus DNA Marker, total RNA that 1-4 is followed successively by infect the soybean of Bean common mosaic virus is template, the product that carries out RT-LAMP reaction in the system of A, B, C and D; Total RNA that 5-8 is followed successively by not infect the soybean (negative control group) of any virus is template, the product that carries out RT-LAMP reaction in the system of A, B, C and D.
Fig. 3 shows, the RT-LAMP reaction product that the A system that the total RNA of the soybean that infects Bean common mosaic virus of take is template obtains is bright disperse shape nucleic acid electrophoresis band, the nucleic acid electrophoresis band disperse shape of the RT-LAMP reaction product of B, D system is not obvious, the RT-LAMP reaction product of C system is bright disperse shape nucleic acid electrophoresis band but is slightly weaker than A system, equal free nucleic acid electrophoresis band in negative control group.
Fluorescent dye detected result as shown in Figure 4.
In Fig. 4, total RNA that 1-4 is followed successively by infect the soybean of Bean common mosaic virus is template, in the system of A, B, C and D, carry out the fluorescent dye result of the product of RT-LAMP reaction, total RNA that 5-8 is followed successively by not infect the soybean (negative control group) of any virus is template, carries out the fluorescent dye result of the product of RT-LAMP reaction in the system of A, B, C and D.
Fig. 4 shows, the total RNA of the soybean that infects Bean common mosaic virus of take is template, the fluorescent dye result of carrying out the product of RT-LAMP reaction in the system of A, B, C and D all presents emerald green, react positive, the total RNA of the soybean (negative control group) that do not infect any virus of take is template, the fluorescent dye result of carrying out RT-LAMP reaction product in the system of A, B, C and D is all orange, reacts negative.
These results suggest that A system is optimum system.
Extract total RNA of the blade of the Kidney bean that infects the soybean of Bean common mosaic virus, the French beans that infect Bean common mosaic virus, infection Bean common mosaic virus, extract to infect CMV(cucumber mosaic virus simultaneously) Kidney bean, infect TMV(tobacco mosaic virus (TMV)) tomato and do not infect total RNA of Kidney bean (negative control group) blade of any virus, by the method for embodiment 2, adopt A system to organize sample to each and carry out RT-LAMP reaction.
Electrophoresis detection result as shown in Figure 5.
In Fig. 5, M:Trans2K Plus DNA Marker; Swimming lane 1 is for infecting the RT-LAMP reaction product of the soybean group of Bean common mosaic virus; Swimming lane 2 is for infecting the RT-LAMP reaction product of the French beans group of Bean common mosaic virus; Swimming lane 3 is for infecting the RT-LAMP reaction product of the Kidney bean group of Bean common mosaic virus; Swimming lane 4 is for infecting CMV(cucumber mosaic virus) the RT-LAMP reaction product of Kidney bean group; Swimming lane 5 is for infecting TMV(tobacco mosaic virus (TMV)) the RT-LAMP reaction product of tomato group; The RT-LAMP reaction product of swimming lane 6 negative control groups.
Fig. 5 shows, infect the RT-LAMP reaction product of the sample of Bean common mosaic virus and all can observe the bright nucleic acid swimming band that is disperse shape, and the RT-LAMP reaction product that does not infect the sample of Bean common mosaic virus all do not have nucleic acid electrophoresis band.
Fluorescent dye detected result as shown in Figure 6.
In Fig. 6,1 for infecting the RT-LAMP reaction product fluorescent dye result of the soybean group of Bean common mosaic virus; 2 for infecting the RT-LAMP reaction product fluorescent dye result of the French beans group of Bean common mosaic virus; 3 for infecting the RT-LAMP reaction product fluorescent dye result of the Kidney bean group of Bean common mosaic virus; 4 for infecting CMV(cucumber mosaic virus) the RT-LAMP reaction product fluorescent dye result of Kidney bean group; 5 for infecting TMV(tobacco mosaic virus (TMV)) the RT-LAMP reaction product fluorescent dye result of tomato group; The RT-LAMP reaction product fluorescent dye result of 6 negative control groups.
Fig. 6 shows, the RT-LAMP reaction product fluorescent dye result that infects the sample of Bean common mosaic virus all presents emerald green, react positive, other RT-LAMP reaction product fluorescent dye results that do not infect the sample of Bean common mosaic virus are all orange, react negative.
Result shows, adopts A system can special, stably detect Bean common mosaic virus, with other viral no cross reaction according to the method for embodiment 2.
The total RNA that extracts the soybean leaves that infects Bean common mosaic virus, RNA concentration is 20ng/ μ L, uses ddH
2o solution does 10 by the RNA extracting
1-10
8doubling dilution, make the RNA of different concns.According to the method for embodiment 2, adopt A system to the RNA of each concentration and ddH
2o(is as blank) carry out RT-LAMP reaction.
Electrophoresis detection result as shown in Figure 7.
In Fig. 7, M:Trans2K Plus DNA Marker; Swimming lane 1 is 10 times of dilution groups; Swimming lane 2 is 10
2times dilution group; Swimming lane 3 is 10
3times dilution group; Swimming lane 4 is 10
4times dilution group; Swimming lane 5 is 10
5times dilution group; Swimming lane 6 is 10
6times dilution group; Swimming lane 7 is 10
7times dilution group; Swimming lane 8 is 10
8times dilution group; Swimming lane 9 is blank group.
Fig. 7 shows, the product that the RNA of soybean of each dilution infection Bean common mosaic virus of take carries out RT-LAMP reaction as template all can be observed the bright nucleic acid swimming band that is disperse shape, and blank group does not have nucleic acid electrophoresis band.
Fluorescent dye detected result as shown in Figure 8.
In Fig. 8,1 is 10 times of dilution groups; 2 is 10
2times dilution group; 3 is 103 times of dilution groups; 4 is 10
4times dilution group; 5 is 10
5times dilution group; 6 is 10
6times dilution group; 7 is 10
7times dilution group; 8 is 10
8times dilution group; 9 is blank group.
Fig. 8 shows, when total RNA of the soybean leaves of the infection Bean common mosaic virus of 20ng/ μ L is in dilution 10
8times time, according to the method for embodiment 2, adopt A system to carry out after the product S YBR Green I dyeing of RT-LAMP reaction, can also present special emerald green fluorescence, illustrate that to adopt the RT-LAMP method of embodiment 2 to adopt A system to detect Bean common mosaic virus susceptibility very high.
Claims (10)
1. one group of primer, is comprised of the DNA molecular shown in following (1)-(4):
(1) DNA molecular shown in SEQ ID No.1;
(2) DNA molecular shown in SEQ ID No.2;
(3) DNA molecular shown in SEQ ID No.3;
(4) DNA molecular shown in SEQ ID No.4.
2. detect a test kit for Bean common mosaic virus, this test kit comprises primer claimed in claim 1, ThermoScript II and Bst archaeal dna polymerase;
Described ThermoScript II is M-MLV ThermoScript II or AMV ThermoScript II.
3. a method that detects Bean common mosaic virus, the method is that to take total RNA of testing sample be template, the primer claimed in claim 1 of take carries out reverse transcription loop-mediated isothermal nucleic acid amplification as primer, if obtain reverse transcription loop-mediated isothermal nucleic acid amplification product, in described testing sample, candidate is contained Bean common mosaic virus, if can not get reverse transcription loop-mediated isothermal nucleic acid amplification product, in described testing sample, candidate is not contained Bean common mosaic virus.
4. method according to claim 3, is characterized in that: the determination methods whether described reverse transcription loop-mediated isothermal nucleic acid amplification product obtains is following (1) and/or (2):
(1) reverse transcription loop-mediated isothermal nucleic acid amplification reaction product is carried out to agarose gel electrophoresis, the testing sample with disperse shape nucleic acid electrophoresis band is the sample that contains Bean common mosaic virus, does not have the testing sample of disperse shape nucleic acid electrophoresis band for not containing the sample of Bean common mosaic virus;
(2) reverse transcription loop-mediated isothermal nucleic acid amplification reaction product is reacted to obtain to reaction solution with SYBR Green I, it is the sample that contains Bean common mosaic virus that reaction solution is emerald testing sample candidate, and it is the sample that does not contain Bean common mosaic virus that reaction solution is orange testing sample candidate.
5. according to the method described in claim 3 or 4, it is characterized in that: in described primer claimed in claim 1, the DNA molecular shown in the DNA molecular shown in SEQ ID No.1 and SEQ ID No.2 is outer primer, the DNA molecular shown in the DNA molecular shown in SEQ ID No.3 and SEQ ID No.4 is inner primer;
In described outer primer, the molar concentration rate of the DNA molecular shown in the DNA molecular shown in SEQ ID No.1 and SEQ ID No.2 in described reverse transcription loop-mediated isothermal nucleic acid amplification is 1:1;
In described inner primer, the molar concentration rate of the DNA molecular shown in the DNA molecular shown in SEQ ID No.3 and SEQ ID No.4 in described reverse transcription loop-mediated isothermal nucleic acid amplification is 1:1.
Described outer primer and the described inner primer molar concentration rate in described reverse transcription loop-mediated isothermal nucleic acid amplification is specially 1:5.
6. according to the arbitrary described method of claim 3-5, it is characterized in that: the temperature of reaction in described reverse transcription loop-mediated isothermal nucleic acid amplification is 62 ℃.
7. according to the arbitrary described method of claim 3-6, it is characterized in that: the magnesium ion concentration in described reverse transcription loop-mediated isothermal nucleic acid amplification is 5mM.
8. according to the arbitrary described method of claim 3-7, it is characterized in that: the system of described reverse transcription loop-mediated isothermal nucleic acid amplification is as follows:
10 * Thermopol buffer2.5 μ L, DNA molecular 0.2 μ M shown in SEQ ID No.1, the DNA molecular 0.2 μ M shown in SEQ ID No.2, the DNA molecular 1.0 μ M shown in SEQ ID No.3, DNA molecular 1.0 μ M shown in SEQ ID No.4, dNTPs1.0mM, MgCl25mM, ThermoScript II 200U, Bst archaeal dna polymerase 12U, total RNA40ng of described testing sample, surplus is ddH2O, system 25 μ L;
Described 10 * Thermopol buffer is purchased from Niu Yinglun biotechnology (Beijing) company limited, and catalog number is M0275;
Described ThermoScript II is M-MLV ThermoScript II or AMV ThermoScript II;
The trade name of described Bst archaeal dna polymerase is Bst DNApolymerase, and purchased from Niu Yinglun biotechnology (Beijing) company limited, catalog number is M0275;
The reaction conditions of described reverse transcription loop-mediated isothermal nucleic acid amplification is: 62 ℃ of isothermal 1h; 80 ℃ of 5min.
9. according to the arbitrary described method of claim 3-8, it is characterized in that: described M-MLV ThermoScript II is purchased from TaKaRa company, and catalog number is 2641A;
Described sample is specially the blade of testing sample.
10. the application of primer claimed in claim 1 in detecting Bean common mosaic virus;
And/or,
The application of test kit claimed in claim 2 in detecting Bean common mosaic virus.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1600865A (en) * | 2003-09-23 | 2005-03-30 | 北京金长河科技发展有限公司 | Gene chip for detecting plant virus |
| CN1952648A (en) * | 2005-10-19 | 2007-04-25 | 中华人民共和国北京出入境检验检疫局 | A gene chip, nucleotide sequence and reagent kit for detecting virus of leguminous plant |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1600865A (en) * | 2003-09-23 | 2005-03-30 | 北京金长河科技发展有限公司 | Gene chip for detecting plant virus |
| CN1952648A (en) * | 2005-10-19 | 2007-04-25 | 中华人民共和国北京出入境检验检疫局 | A gene chip, nucleotide sequence and reagent kit for detecting virus of leguminous plant |
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| 王杰 等: "菜豆种子中菜豆普通花叶病毒的RT-PCR检测", 《植物保护学报》, vol. 33, no. 4, 31 December 2006 (2006-12-31), pages 345 - 350 * |
| 秦文韬 等: "环介导恒温扩增技术(LAMP)及其在植物病毒检测中的研究进展", 《中国农学通报》, vol. 29, no. 21, 31 July 2013 (2013-07-31), pages 170 - 174 * |
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