CN102634608B - Method for detecting apple viruses by adopting reverse transcription loop-mediated isothermal amplification technology - Google Patents
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Abstract
The invention relates to a method for detecting apple viruses by adopting a reverse transcription loop-mediated isothermal amplification technology, wherein the method comprises the following steps of total RNA (Ribose Nucleic Acid) extraction of apple viruses, RT-LAMP (reverse transcription loop-mediated isothermal amplification) reaction and electrophoresis detection and virus type determination, wherein the apple viruses are selected from an ASGV (Apple Stem Grooving Virus), an ACLSV (Apple Chlorotic Leaf Spot Virus) and an ASPV (Apple Stem Pitting Virus). According to the invention, an RT-LAMP primer is designed according to the gene conserved region of the apple viruses, and a rapid, flexible and highly specific method for detecting the apple viruses by adopting the one-step method reverse transcription loop-mediated isothermal amplification technology, and a new means for rapid detection of the apple viruses is provided.
Description
Technical field
The invention belongs to plant virology technical field, particularly relate to a kind of method adopting reverse transcription loop-mediated isothermal amplification technique to detect Apple virus.
Background technology
China is the major production areas of apple, and its cultivated area and output account for global 1/4th.The apple production in Shaanxi Province ranks first in the whole country position especially.Apple is bred mainly through grafting, also easily causes the propagation of virus in the process of grafting.Investigation finds, the sickness rate of apple latent virus is very high, and apple stem grooving virus (ASGV) is with malicious rate up to 90%, can bring serious harm to the production of apple.Apple tree, once can be with poison throughout one's life after infecting virus, endangers lastingly, and main manifestations is for weakening growth, malformation, reduction resistance, what have causes the symptoms such as tree body sharply fails, cause Growth status bad, fruit quality and production declining, and bring serious financial loss.Virus disease is large with its area, damage losses seriously becomes serious disease in apple production.
The harm of Apple virus disease is serious and control is difficult, is apple production and a theoretic major issue always.America and Europe, day etc., country just carried out the research of Apple virus since twentieth century forties, proposed that effective virus removes, detection technique and qualification program.The detection method generally adopted at present mainly contains plant indicator method, electron microscopy, immunological technique and molecular Biological Detection etc.But aforesaid method all exists different shortcomings, such as, longer, complex operation of required test period, sensitivity is not high, specificity is strong, accuracy is lower, be subject to test material restriction etc.
Ring mediated isothermal nucleic acid amplification (Loop-mediated isothermalmplification, LAMP) technology is a kind of novel nucleic acids amplification technique invented by T.Notomi, this technology relies on four special primers and a kind of archaeal dna polymerase with strand displacement characteristic, under isothermal conditions fast, efficient, high special, amplified target sequence (Notomietal, 2000) with sensitivity.Establish the LAMP detection method for multiple pathogenic bacteria, virus, fungi, parasite etc. so far, in drug resistance gene detection, the judgement of domestic animal early embryo sex etc., also have relevant report.The method has wide range of applications, and is suitable for laboratories and carries out rapid detection.The present invention is LAMP primer according to Apple virus gene conserved regions design, adopts single stage method reverse transcription loop-mediated isothermal amplification technique (RT-LAMP) to establish the method for the main virus of a kind of detection apple that is quick, sensitive, high special.Rapid detection for Apple virus provides new means.
Summary of the invention
The invention provides a kind of method adopting reverse transcription loop-mediated isothermal amplification technique to detect Apple virus, wherein the method comprises Apple virus Total RNAs extraction, RT-LAMP reaction and electrophoresis detection, determine viral species, wherein said Apple virus is selected from apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV) and apple stem pitting virus (ASPV), preferably, described primer sets comprises the primer being selected from one of following 3 groups:
1st group of primer, the RT-LAMP for ASGV virus reacts:
Outer primer (F3 and B3):
SEQ ID NO.1：5’-CAAGAAATGGCCCAAAGC-3’；
SEQ ID NO.2：5’-CTAACCCTCCAGTTCCAG-3’；
Inner primer (FIP and BIP):
SEQ ID NO.3：5’-AGGTGTTAGACGATTCATTTTGAGATTCGAAAAAAGCCCGTGG-3’；
SEQ ID NO.4：5’-ACAGGTGATTGATAGGATGACCAAATTACTCTCTGAACCTGCC-3’；
2nd group of primer, the RT-LAMP for ACLSV virus reacts:
Outer primer (F3 and B3):
SEQ ID NO.5：5’-ACTACCCCAGTCTCAACA-3’；
SEQ ID NO.6：5’-CCAGAGTTTTGCAACCAG-3’；
Inner primer (FIP and BIP):
SEQ ID NO.7：5’-AGGTTTTCCACCTTCTTCTCCTGTTATAAGTGGATTCCAATATGAAC-3’；
SEQ ID NO.8：5’-TTACCTACCAGAGAAAACTAGGGAAGAGTGATTAACATAAGCGAATG-3’；
3rd group of primer, the RT-LAMP for ASPV virus reacts:
Outer primer (F3 and B3):
SEQ ID NO.9：5’-TGCGGTGAATCAGGTGAATG-3’；
SEQ ID NO.10：5’-TGCAACCCCAACTGCTCTA-3’；
Inner primer (FIP and BIP):
SEQ ID NO.11：5’-GTTGGAGGCAGCACTTCTTGGACGTAGTGGTGGTGGAACAC-3’；
SEQ ID NO.12：5’-GAGACGCCTAGATTCTGTGGGCCGGTTGCTTGCGACAACT-3’。
In a specific embodiment, employing reverse transcription loop-mediated isothermal amplification technique of the present invention detects the method for Apple virus, and it comprises the steps:
1) extraction of virus total RNA:
Extract the virus total RNA in Folium Mali pumilae to be measured;
2) RT-LAMP reaction:
Use outer primer (F3 and B3) and inner primer (FIP and BIP) to carry out RT-LAMP reaction to virus total RNA, obtain amplified production;
3) electrophoresis detection:
Agarose gel electrophoresis is carried out to amplified production, obtains virogene amplified production band, determine whether apple infects described virus.
In a specific embodiment, employing reverse transcription loop-mediated isothermal amplification technique of the present invention detects the method for Apple virus, and it comprises the steps:
1) extraction of virus total RNA:
Extract the virus total RNA in Folium Mali pumilae to be measured;
2) RT-LAMP reaction:
Use outer primer (F3 and B3) and inner primer (FIP and BIP) to carry out RT-LAMP reaction to virus total RNA, obtain amplified production;
3) SYBR Green I green fluorescence method detects amplified production:
Directly detect by an unaided eye after adding SYBR Green I, determine whether apple infects described virus.
In a specific embodiment, in the method stated on the invention, the primer concentration ratio used in RT-LAMP reaction is: outer primer (F3 and B3): inner primer (FIP and BIP)=0.2-0.4: 1.2-1.6; Preferably, outer primer (F3 and B3): inner primer (FIP and BIP)=1: 4.
In a specific embodiment, in the method stated on the invention, the temperature of reaction in RT-LAMP reaction is preferably 50-65 DEG C; For ACLSV primer, the temperature of reaction of use is more preferably 60 DEG C, and for ASGV and ASPV primer, the temperature of reaction of use is more preferably 50 DEG C.
In a specific embodiment, in the method stated on the invention, the Mg in RT-LAMP reaction
2+concentration is preferably greater than or equal to 5mM, is more preferably 5mM.
Apple virus disease is often Combined Infection, and the present invention establishes through optimizing and groping experiment condition the method that the main cryptovirus of apple infects in three kinds, detection China, greatly improves detection efficiency, reduces testing cost.Method of the present invention has the features such as quick, sensitive, high degree of specificity.Method of the present invention has successfully been applied to synchronous detection that is indoor and field Apple virus disease Combined Infection.Present invention demonstrates that RT-LAMP is a kind of effective means detecting plant virus.
Accompanying drawing explanation
Figure 1A: RT-LAMP result electrophorogram;
Figure 1B: RT-LAMP fluorescence dye result figure, M1:MarkerIV ,+: susceptible sample ,-: negative control, M2:Marker I;
Fig. 2 A:RT-PCR result electrophorogram;
Fig. 2 B:RT-LAMP result electrophorogram M1:MarkerIV, 1-9:pMD18-T-ASGV plasmid dilution 10
1-10
9doubly do template, 10: negative control, M2:Marker I;
Embodiment
In order to understand the present invention, further illustrate the present invention with embodiment below, but following embodiment does not limit the present invention.
material and reagent
Apple healthy leaves, diseased plant blade picks up from the ground such as Shaanxi, China Yang Ling, Qian County respectively, gathers blade and is stored in-80 DEG C of refrigerators.
Main agents:
Bst DNApolymerase is purchased from NEB company;
Betaine, MgSO
4available from Sigma;
RNA extracts test kit purchased from TaKaRa company;
M-MLV ThermoScript II is purchased from Promega company;
Taq archaeal dna polymerase is purchased from TaKaRa company;
SYBR Green I is purchased from Invitrogen company;
Intestinal bacteria (Escherichia.coli) JM109, DNA standard Marker 1 is purchased from Voson company;
Glue reclaims test kit purchased from Bioteke company.
embodiment 1
1. design of primers
First the present inventor has downloaded the sequence of the full-length genome of the main virus of apple three kinds from NCBI, use Primer4.0 (http://primerexplorer.jp/e/v4_manual/index.html) to devise and organize primer more, then have selected often kind of viral primer according to composite factors such as the hairpin structure of the conservative property of primer place sequence area, primer, dimer GC content and Tm values.Finally have selected 4 primers for often kind of Apple virus, comprise 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP).Described primer is see SEQ ID NO.1-12.The primer of RT-PCR, the present invention uses the primer of model Xu Dong etc., see SEQ ID NO.13-18.The information of all primers is in table 1.
The information of table 1 primer sequence SEQ ID NO.1-18:
2, RNA extracting
Use polysaccharide polyphenol plant total serum IgE rapid extraction test kit (centrifugal cylindricality) (Bioteke), according to operation instructions, extract the total serum IgE of diseased plant Apple Leaves and healthy Apple Leaves respectively, be finally dissolved in 30 μ l elution buffers, be stored in-80 DEG C of refrigerators.
3, RT-LAMP reaction system
Reaction system (25 μ l) is as follows: 10 × ThermoPol damping fluid 2.5 μ l (20mM Tris-HCl, 10mMKCl, 2mMMgSO
4, 10mM (NH
4)
2sO
4, 0.1%Triton X-100), primer (F3 and B3) 0.2 μM, primer (FIP and BIP) 1.6 μMs, dNTPs 1mM, MgSO
45mM, betaine 1M, M-MLV 10u, Bst DNApolymerase (NEB) 10u, template ribonucleic acid 2 μ l.Reaction conditions is: be 50 DEG C of constant temperature 1h to ASGV and ASPV, is 60 DEG C of constant temperature 1h to ACLSV.
Reaction result is analyzed by the agarose gel electrophoresis of 2% and the two kinds of methods that directly detect by an unaided eye after adding SYBR Green I.
4, RT-LAMP and RT-PCR susceptibility compares with specificity
In order to compare RT-LAMP and RT-PCR susceptibility, the present invention uses a pair of model Xu Dong etc. the special primer comprising ASGV part CP gene.
RT-PCR process is as follows:
RT-PCR reaction system is 12.5 μ l, comprise the RNA of 3.5 μ lDEPC process water, 0.5 μ l downstream primer, 1 μ lASGV, then 70 DEG C of water-baths 5 minutes, then ice puts 5 minutes, then adds 1.75 μ lDEPC process water, 2.5 μ l dNTPs (10mM), 2.5 μ l 5 × M-MLV damping fluid (50mM Tri s-HCL, 75mMKCL, 3mM MgCl
2, 10mM DTT), 0.5 μ l M-MLV ThermoScript II, 0.25 μ lRNA enzyme inhibitors, then 42 DEG C reaction 1h, then 95 DEG C reaction 5 minutes.
Then RT-PCR amplified production Bioteke gel reclaims after test kit reclaims and is connected on pMD18-T carrier with DNA Ligation Kit (TaKaRa), obtains pMD18-T-ASGV.PMD18-T-ASGV is proceeded in intestinal bacteria (Escherichia.coli) JM109, after cultivation, extract plasmid, obtain pMD18-T-ASGV plasmid.Recording pMD18-T-ASGV plasmid starting point concentration is 150ng/ μ l.
PMD18-T-ASGV plasmid is diluted 10 respectively
1, 10
2, 10
3, 10
4, 10
5, 10
6, 10
7, 10
8, 10
9doubly be used for doing the template of RT-PCR and RT-LAMP.
RT-PCR result with 2% agarose gel electrophoresis analysis, RT-LAMP result with 2% agarose gel electrophoresis and the two kinds of methods that directly detect by an unaided eye after adding SYBR Green I analyze.
The specificity of RT-PCR is detected.The primer of the present invention ACLSV detects the sample of ASGV, ASPV, result only have ACLSV for positive, other be all negative, two-strain uses the same method in addition, obtains same result, shows as specificity good.
Carrying out detecting the present invention to the specificity of RT-LAMP also adopts the LAMP primer of ACLSV to detect the sample of ASGV, ASPV respectively, result be also only have ACLSV for positive, other be negative, two-strain uses the same method in addition, obtain same result, show as specificity good, namely one group of special LAMP primer can only detect corresponding one virus.
embodiment 2, adopt agarose gel electrophoresis method, SYBR Green I green fluorescence method to detect RT-LAMP amplified production
RT-LAMP amplified production is detected respectively by agarose gel electrophoresis method, SYBR Green I green fluorescence method.
RT-LAMP amplified production gel electrophoresis spectrum positive in scalariform (Fig. 1, A+).
In SYBR Green I fluoroscopic examination, the stoste of SYBR Green I is diluted 10 times, get 1 μ l and add in reaction tubes, the positive is emerald green (Fig. 1, B+), and feminine gender is orange (Fig. 1, B-).
Take two kinds of methods to detect the result of RT-LAMP in this experiment, one adds SYBR Green I, directly detects by an unaided eye, and another kind is the agarose gel electrophoresis analysis with 2%.Generally direct first method just clearly can distinguish the result of yin and yang attribute, can verify when uncertain by second method.
embodiment 3, RT-LAMP and RT-PCR susceptibility compares:
PMD18-T-ASGV plasmid is diluted 10 respectively
1, 10
2, 10
3, 10
4, 10
5, 10
6, 10
7, 10
8, 10
9doubly be used for doing the template of RT-PCR and RT-LAMP.Result is in dilution 10
8times time RT-PCR can't detect (Fig. 2 A), and RT-LAMP is in dilution 10
9times time can also detect (Fig. 2 B).The susceptibility that this also illustrates RT-LAMP is higher than RT-PCR more than 100 times.
embodiment 4, RT-PCR and RT-LAMP detect field sample:
Have detected multiple fields sample by RT-PCR and RT-LAMP two kinds of methods respectively, the detected result of result two kinds of methods is consistent.
RT-LAMP detection method does not need expensive plant and instrument, schedule of operation simple, and have rapidly and efficiently, the feature of high specific, high sensitivity, its range of application is more and more extensive, in the pathogenic microorganism examination, give play to great advantage, and be successfully applied in the correlative study of oncogene.Have detected multiple apple diseased plant sample by the RT-LAMP method described in this experiment and RT-PCR method, its result is consistent simultaneously, it is obvious that a large amount of time can be saved by RT-LAMP method, and also simple to operate.
In a word, the present invention establishes the method for five kinds of main viruses on a set of quick, easy, special, sensitive detection apple.Therefore, have reason to believe that RT-LAMP is as the molecular biological method for quick of one, will have more wide application prospect.
Method of the present invention is described by specific embodiment.Those skilled in the art can use for reference the links such as content appropriate change raw material of the present invention, processing condition and realize other object corresponding, its relevant change does not all depart from content of the present invention, all similar replacements and change will become apparent to those skilled in the art that and be all deemed to be included within scope of the present invention.
Reference:
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11 Cameron，H.R.，1989.Pear vein yellows.In：Fridlund，P.R.(Ed.)，Virus and Viruslike Diseases of Pome Fruits and Simulating Noninfectious Disorders.Cooperative Extension College of Agriculture and Home Economics Washington State Uni-versity，Pullman，WA，pp.175-181.
12 Hassan，M.，Myrta，A.，Polak，J.，2006.Simultaneous detection and identification of four pome fruit viruses by one-tube pentaplex RT-PCR.J.Virol.Methods 133，124-129.
13 Li Gangs, Fan Xiaojuan, Li Wei. the foundation of rapid detection PPR virus RT-LAMP method, Chinese Preventive Veterinary Medicine report, 2009, Vol.31, No.5;
14 Li Jian, Chen Qin, Xiong Wei. the foundation virus journal 2009, Vol.25No.2 of foot and mouth disease virus RT-LAMP detection method;
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Claims (1)
1. the method adopting reverse transcription loop-mediated isothermal amplification technique to detect apple stem grooving virus, wherein the method comprises Apple virus Total RNAs extraction, RT-LAMP reaction and electrophoresis detection, determine viral species, wherein said Apple virus is selected from apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV) and apple stem pitting virus (ASPV), and the method comprises the steps:
1) extraction of virus total RNA:
Extract the virus total RNA in Folium Mali pumilae to be measured;
2) RT-LAMP reaction:
Use outer primer (F3 and B3) and inner primer (FIP and BIP) to carry out RT-LAMP reaction to virus total RNA, obtain amplified production;
3) electrophoresis detection:
Agarose gel electrophoresis is carried out to amplified production, obtains virogene amplified production band, determine whether apple infects described virus;
Described outer primer (F3 and B3) is as follows:
SEQ ID NO.1：5’-CAAGAAATGGCCCAAAGC-3’；
SEQ ID NO.2：5’-CTAACCCTCCAGTTCCAG-3’；
Described inner primer (FIP and BIP) is as follows:
SEQ ID NO.3：5’-AGGTGTTAGACGATTCATTTTGAGATTCGAAAAAAGCCCGTGG-3’；
SEQ ID NO.4：5’-ACAGGTGATTGATAGGATGACCAAATTACTCTCTGAACCTGCC-3’；
Primer concentration in described RT-LAMP reaction is outer primer (F3 and B3): inner primer (FIP and BIP)=1:4; The temperature of reaction that ACLSV primer uses is 60 DEG C, and the temperature of reaction that ASGV and ASPV primer uses is 50 DEG C; Mg
2+concentration is 5mM.
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| CN104831000B (en) * | 2015-05-27 | 2017-08-11 | 中国农业科学院植物保护研究所 | Multiple RT-PCR kit for detecting cherry virus and detection method |
| CN106119414A (en) * | 2016-03-07 | 2016-11-16 | 魏芳 | A kind of apple chlorotic leaf spot virus detection kit and detection method |
| CN105648117A (en) * | 2016-03-07 | 2016-06-08 | 魏芳 | Apple stem grooving virus detection kit and method |
| CN107267548A (en) * | 2016-04-08 | 2017-10-20 | 西北农林科技大学 | The construction method of apple stem grooving virus infectious clone carrier |
| CN109423527A (en) * | 2017-08-23 | 2019-03-05 | 河南省农业科学院植物保护研究所 | Using the method for reverse transcription loop-mediated isothermal amplification technique detection Prunus necrotic ring spot virus (PNRSV) |
| CN113913558B (en) * | 2021-11-09 | 2022-07-19 | 中国科学院西北生态环境资源研究院 | Detection kit and detection method for apple latent spherical viruses |
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