CN103305635B - Method for molecularly detecting sorghum mosaic virus infecting sugarcane - Google Patents
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Abstract
The invention relates to a method for molecularly detecting a sorghum mosaic virus (SrMV) infecting sugarcane. The method comprises the following steps of: extracting the total RNA (Ribose Nucleic Acid) from leaves of the sugarcane; performing reverse transcription (RT) to obtain a template; performing polymerase chain reaction (PCR) through coat protein gene conserved sequence degenerate primers of different SrMV virus strains; carrying out double-digestion reaction on a PCR product through restriction enzymes NsiI and BsmI; and detecting restriction enzyme fragment length polymorphisms (RFLP) by 4% of low-melting-point agarose gel electrophoresis in order to identify whether the SrMV isinfected as well as identify the type of the SrMV strain. The method can detect different strains of SrMV isolates infecting the sugarcane quickly, simply and conveniently. The method for molecularly detecting the sorghum mosaic virus infecting sugarcane overcomes the shortcomings of biological assay, conventional RT-PCR and other methods and is a method capable of quickly, specifically and accurately detecting different SrMV strains.
Description
Technical field
The present invention relates to the detection method of a kind of plant virus, be specifically related to a kind of method that Molecular Detection infects the sorghum mosaic virus of sugarcane, belong to biology field.
Background technology
From Musschenbroek in 1892 since pawl low-lying area describes mosaic of sugarcane first, the country grown cane in the whole world at present generally occurs, and becomes one of important disease of sugarcane.Generally there is yellowish green alternate irregular decorative pattern, streak or mottled in disease plant, length, not of uniform size, is covered with whole blade, especially obvious with new leaf base symptom.Disease plant yellowing leaf, plant is downgraded, minimizing of tillering, shortened internodes, causes sugarcane yield and sugarcane stem sugar degree to reduce.Cause the cause of disease of mosaic of sugarcane to have 3 kinds, namely marmor upsilon section Potyvirus corn mosaic virus (
sugarcane mosaic virus, SCMV), sorghum mosaic virus (
sorghum mosaic virus, SrMV) and marmor upsilon section
poacevirusbelong to sugarcane stripe mosaic virus (
sugarcane stripe mosaic virus, SCSMV).Show according to our early-stage Study, SrMV is the main pathogen causing China's sugar material mosaic of sugarcane.
The diagnosis of mosaic of sugarcane can be identified by the Disease symptoms feature of blade, but Symptoms and sugarcane line, growth conditions, temperature is relevant with virus strain, and some disease plant does not show the symptom of floral leaf.Host range well can distinguish the different strain of Potyvirus corn mosaic virus subgroup with the biochemical characteristic of coat protein, but various biochemistry and serological technique are difficult to distinguish SCMV and SrMV strain.Host response can be used for distinguishing different virus strain, but this method expends time in and labour very much.Recently, Molecular Identification technology is usually used in studying the virus taxis of the marmor upsilon section of infecting sugarcane.Virus capsid protein (CP) gene order difference is one of criteria for classification that in marmor upsilon section, virus is planted.Identify that the different isolate of the SrMV infecting sugarcane has been reported both at home and abroad according to the CP sequence of SrMV, but, this common molecular detection method needs PCR primer to reclaim, be transformed into bacillus coli DH 5 alpha, then by order-checking and sequential analysis, experimental procedure is comparatively loaded down with trivial details, it is longer to expend time in.
Summary of the invention
The object of this invention is to provide a kind of method that Molecular Detection infects the sorghum mosaic virus of sugarcane, to overcome the weak point in common molecular detection.
The object of the invention is to realize by the following method.
A kind of Molecular Detection of the present invention infects the method for the sorghum mosaic virus of sugarcane, comprises Sugarcane Leaves Total RNAs extraction, degenerated primer design, RT-PCR amplification, restriction enzyme reaction, electrophoresis detection; It is characterized in that:
(1) degenerated primer design: degenerated primer sequence SrMV-CPF and the SrMV-CPR of amplification SrMV different strain coat protein gene conserved sequence are as follows:
SrMV-CPF：5'-ACADCAGADGCAACRGCACAAGC-3'
SrMV-CPR：5'-CTCRCCGACATTCCCATCYAAGCC-3'；
(2) RT-PCR amplification
Apply precious biotechnology (Dalian) company limited Reverse Transcription box and carry out RT reaction, reverse transcription reaction system cumulative volume is 10 μ L, include: 2 μ L 5 × PrimeScript damping fluids, 0.5 μ L PrimeScript ThermoScript II mixed solution, 0.5 μ L Oligo dT primer, 2 μ L Random 6 mers primers, 2 μ L Sugarcane Leaves total serum IgE templates, supply 10 μ L without RNase water; Reverse transcription reaction condition is 37 DEG C of 15 min, 85 DEG C of 5 s deactivation ThermoScript II;
Subsequently, apply precious biotechnology (Dalian) company limited PCR kit and carry out pcr amplification; PCR reaction system cumulative volume is 50 μ L, include 5.0 μ L 10 × PCR damping fluids, 4.0 μ L 2.5 mmol/L dNTPs, 10 μm of each 2.0 μ L of ol/L degenerated primer SrMV-CPF and SrMV-CPR, 0.25 U Taq DNA Polymerase, 1.0 μ L cDNA, sterilizing ultrapure water supplies 50 μ L; Described 10 × PCR damping fluid moiety is 500 mmol/L KCl, 100 mmol/L Tris-HCl pH 8.3,15 mmol/L MgCl
2; Pcr amplification program is as follows: 94 DEG C of denaturation 2 min, 94 DEG C of sex change 1 min, 55 DEG C of annealing 30 s, 72 DEG C of extensions 1 min, totally 35 circulations, and last 72 DEG C extend 10 min again;
(3) restriction enzyme reaction
Application Fermentas company
nsii and
bsmi restriction enzyme carries out double digestion reaction, and endonuclease reaction system cumulative volume is 20 μ L, includes 2.0 μ L 10 × enzyme cutting buffering liquids, PCR primer after 4.0 μ L purifying,
nsii and
bsmthe each 1.5 μ L of I restriction enzyme, sterilizing ultrapure water supplies 20 μ L; Endonuclease reaction condition: 37 DEG C of enzymes cut 12 hours; 65 DEG C of process 20 min, stop endonuclease reaction, are down to normal temperature;
(4) electrophoresis detection: get 5 μ L reaction solutions after endonuclease reaction terminates and carry out the low melting-point agarose gel electrophoresis that mass volume ratio is 4.0 %, in 0.5 × electrophoretic buffer environment, 80 V voltage stabilizing electrophoresis 2 h, then observe with gel imaging system and take pictures, in electrophoresis detection figure, there is PCR primer enzyme slitting band, represent that this sample is containing SrMV; There is not PCR primer enzyme slitting band, represent this sample not containing SrMV.
In electrophoresis detection figure, the position of PCR primer enzyme slitting band is different, represents that the strain of contained SrMV in sample is different.There is PCR primer enzyme slitting band at 608 bp and 241 bp positions simultaneously, represent in this sample containing SrMV-G1 strain; There is PCR primer enzyme slitting band in 849 bp positions, represent in this sample containing SrMV-G2 strain; There is PCR primer enzyme slitting band at 582 bp and 267 bp positions simultaneously, represent in this sample containing SrMV-G3 strain; There is PCR primer enzyme slitting band in 414 bp, 168 bp, 141 bp, 126 bp positions simultaneously, or there is PCR primer enzyme slitting band in 414 bp, 267 bp, 168 bp positions simultaneously, or there is PCR primer enzyme slitting band in 438 bp, 267 bp, 168 bp positions simultaneously, or there is PCR primer enzyme slitting band in 438 bp, 168 bp, 139 bp, 128 bp positions simultaneously, represent in these samples containing SrMV-G4 strain; There is PCR primer enzyme slitting band in 606 bp, 139 bp, 128 bp positions simultaneously, represent in this sample containing SrMV-G5 strain; There is PCR primer enzyme slitting band at 438 bp and 435 bp positions simultaneously, represent in this sample containing SrMV-G6 strain.If there is not PCR primer enzyme slitting band in electrophoresis detection figure, represent this sample not containing SrMV.Through repetition test, for not containing the sample of SrMV still containing other sugarcane disease, use detection method of the present invention, in electrophoresis detection figure, also do not occur PCR primer enzyme slitting band.
Advantage of the present invention is as follows: 1, with Sugarcane Leaves total serum IgE for template, according to specific degenerate primers SrMV-CPF and SrMV-CPR of SrMV different strain coat protein gene conserved sequence design, the method adopting RT-PCR and RFLP to combine, can detect whether sugarcane to be measured has infected sorghum mosaic virus easily and fast; 2, by restricted endoenzyme, RT-PCR product enzyme is cut, then electrophoresis detection restriction fragment length polymorphism (RFLP), just Rapid identification can go out different SrMV strains, the PCR primer in common molecular detection method that eliminates this authentication method reclaims until the step of order-checking and sequential analysis, can facilitate, special, detect and infect the different SrMV strains of sugarcane fast, comparatively ordinary method, substantially reduces the detection time of RT-PCR, and reduces testing cost.
Accompanying drawing explanation
Fig. 1 is the electrophoresis detection figure of the different strain of sorghum mosaic virus infecting sugarcane, wherein, 1 is DL 1000 bp DNA standard relative molecular mass, 2 ~ 10 is after testing containing the sample of SrMV, 11 is after testing not containing the sample of SrMV, 12 is sterilizing ultrapure water, and 13 is 20 bp DNA standard relative molecular masses.
Embodiment
In order to illustrate the present invention instead of restriction the present invention further, be illustrated below in conjunction with embodiment.Experimental technique described in following embodiment, if no special instructions, is ordinary method.Described reagent and biomaterial all can obtain if no special instructions from commercial channels.
Embodiment 1 one kinds of Molecular Detection infect the method for the sorghum mosaic virus of sugarcane, comprise the following steps:
1, Sugarcane Leaves Total RNAs extraction
1) have collected 104 parts of field Sugarcane Leaves samples from the sugarcane district of China Provinces (regions) ,-80 DEG C save backup.
2) take 0.1 g Sugarcane Leaves, and use liquid nitrogen grinding powdered.
3) in 1.5 mL centrifuge tubes, add powder, add 1 mL immediately when liquid nitrogen is just evaporated completely
reagent, lid upper tube cap, concussion mixing, in 4 DEG C, centrifugal 10 min of 12000 g.
4) Aspirate supernatant is in another new centrifuge tube, adds 0.2 mL chloroform, covers pipe lid, and vibration mixing, after room temperature leaves standstill 3 min, in 4 DEG C, centrifugal 15 min of 12000 g.
5) Aspirate supernatant is in another new centrifuge tube, add the Virahol (-20 DEG C) of 0.5 mL precooling, put upside down mixing, room temperature leaves standstill after 10 min, in 4 DEG C, the centrifugal 15 min(centrifuge tube openings of 12000 g keep placing towards centrifugal basket direction of principal axis).
6) pour out supernatant liquor in centrifuge tube, add pre-cooled ethanol (-20 DEG C) washing that 1 mL volume ratio is 75%, put upside down centrifuge tube 2 ~ 3 times, the centrifugal 5 min(centrifuge tube openings of 7500 g keep placing towards centrifugal basket direction of principal axis).Repeated washing precipitates 1 time.
7) pour out supernatant liquor in centrifuge tube, blotted with micro sample adding appliance, a suction nozzle used instead by a sample, and suction nozzle does not encounter the one side having precipitation, drying at room temperature 10 ~ 15 min.
8) add 50 μ L without RNas water, mixing, dissolve the RNA on tube wall, centrifugal 5 s of 2 000 g, save backup on ice.The RNA extracted should carry out RT-PCR amplification in 2 h; If need long-term preservation ,-80 DEG C of refrigerators should be placed.
9) on nucleic acid-protein analyser, measure the absorbance value of RNA, calculate the RNA concentration extracted.
2, degenerated primer design
According to sorghum mosaic virus (
sorghum mosaic virus) different strain coat protein gene conserved sequence, design degenerated primer sequence SrMV-CPF and SrMV-CPR, the object nucleic acid belt size of amplification is close to 900 bp, and degenerated primer sequence is as follows:
SrMV-CPF：5'-ACADCAGADGCAACRGCACAAGC-3'
SrMV-CPR：5'-CTCRCCGACATTCCCATCYAAGCC-3'。
3, RT-PCR amplification
Apply precious biotechnology (Dalian) company limited Reverse Transcription box and carry out RT reaction, in PCR pipe, add following reagent:
Mixing, 37 DEG C of denaturation 15 min, 85 DEG C of sex change 5 S after centrifugal 5 S of 8000 rpm.
Subsequently, take cDNA as template and SrMV-CPF and SrMV-CPR for primer carries out pcr amplification.Following reagent is added in PCR pipe:
Mixing, 94 DEG C of denaturation 2 min, 94 DEG C of sex change 1 min, 55 DEG C of annealing 30 s, 72 DEG C of extensions 1 min, totally 35 circulations after centrifugal 5 S of 8000 rpm, last 72 DEG C extend 10 min again.
4, PCR primer purifying
1) mixing of isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1) is added after being settled to 100 μ L with 50 μ L sterilizing ultrapure waters.
2) room temperature, 13,500 rpm 5 min are centrifugal, get supernatant.
3) mixing of isopyknic chloroform/primary isoamyl alcohol (24: 1) is added.
4) room temperature, 13,500 rpm 5 min are centrifugal, get supernatant.
5) add 10 μ L 3 mol/L sodium-acetates, the cold ethanol of 250 μ L, place 30 min for-20 DEG C.
6) 4 DEG C, 13,500 rpm 15 min are centrifugal, abandon supernatant.
7) add the cold ethanol of 500 μ L 70 % to clean, 4 DEG C, 13,500 rpm 5 min are centrifugal, abandon supernatant.
8) precipitation is dry.
9) add 100 μ L sterilizing ultrapure waters to dissolve.
5, endonuclease reaction
Application Fermentas company
nsii and
bsmi restriction enzyme carries out double digestion reaction, adds following reagent in PCR pipe:
Mixing, after centrifugal 5 S of 8000 rpm, 37 DEG C of enzymes cut 12 hours, and 65 DEG C of process 20 min, stop endonuclease reaction, be down to normal temperature.
6, electrophoresis detection
Getting 5 μ L digestion products through mass volume ratio is that the low melting-point agarose gel of 4 % is under 0.5 × TBE and 80 V voltages after electrophoresis 2 h, dye 15 min in the ethidium bromide staining liquid of 0.5 μ g/mL, observe with gel imaging system and take pictures, in electrophoresis detection figure, there is PCR primer enzyme slitting band, represent that this sample is containing SrMV; There is not PCR primer enzyme slitting band, represent this sample not containing SrMV.
The present embodiment have detected 104 samples collected from sugarcane district, Provinces (regions) of China, therefrom choose represent meaning electrophoresis detection result as shown in Figure 1, in Fig. 1,2-10 represents sample 1-9 after testing containing SrMV.Wherein, there is band at 608 bp and 241 bp positions in sample 1 simultaneously, represents that its sequence is the nucleotide sequence shown in SEQ ID NO:3 in sequence table containing SrMV-G1 strain in sample 1; There is band in 849 bp positions in sample 2, represents that its sequence is the nucleotide sequence shown in SEQ ID NO:4 in sequence table containing SrMV-G2 strain in sample 2; There is band at 582 bp and 267 bp positions in sample 3, represent that its sequence is the nucleotide sequence shown in SEQ ID NO:5 in sequence table containing SrMV-G3 strain in sample 3 simultaneously; There is band in 414 bp, 168 bp, 141 bp, 126 bp positions in sample 4, represent that its sequence is the nucleotide sequence shown in SEQ ID NO:6 in sequence table containing SrMV-G4 strain in sample 4 simultaneously; There is band in 414 bp, 267 bp, 168 bp positions in sample 5, represent that its sequence is the nucleotide sequence shown in SEQ ID NO:7 in sequence table containing SrMV-G4 strain in sample 5 simultaneously; There is band in 438 bp, 267 bp, 168 bp positions in sample 6, represent that its sequence is the nucleotide sequence shown in SEQ ID NO:8 in sequence table containing SrMV-G4 strain in sample 6 simultaneously; There is band in 438 bp, 168 bp, 139 bp, 128 bp positions in sample 7, represent that its sequence is the nucleotide sequence shown in SEQ ID NO:9 in sequence table containing SrMV-G4 strain in sample 7 simultaneously; There is band in 606 bp, 139 bp, 128 bp positions in sample 8, represent that its sequence is the nucleotide sequence shown in SEQ ID NO:10 in sequence table containing SrMV-G5 strain in sample 8 simultaneously; There is band at 438 bp and 435 bp positions in sample 9, represent that its sequence is the nucleotide sequence shown in SEQ ID NO:11 in sequence table containing SrMV-G6 strain in sample 9 simultaneously; There is not PCR primer enzyme slitting band in 11 in Fig. 1, represents this sample not containing SrMV.
The present invention checks order to the SrMV strain PCR primer that sample 1-9 infects.Sample 1 samples and samples sample and sample sample and sample sample and sample and sample from Yunnan (YN-ROC221) from Fujian (FJ-CI20031), sample 8 from Guangxi (GX-YG35), sample 9 from Yunnan (YN-FN21), sample 7 from Hainan (HN-YZ49), sample 6 from Guangxi (GX-YZ194), sample 5 from Guangdong (GD-YT861), sample 4 from Fujian (FJ-GT53), sample 3 from Guangxi (GX-LC1362), sample 2, and its sequence is respectively the nucleotide sequence shown in SEQ ID NO:3-11 in sequence table.By the nucleotide sequence shown in SEQ ID NO:3-11 respectively with U.S. SrMV-H(GenBank accession number U57358), SrMV-I(GenBank accession number U57358) and SrMV-M(GenBank accession number U57360) nucleotide sequence compare, consistence is 80.7% ~ 81.5%, 82.2% ~ 83.2%, 90.3% ~ 90.7%, 93.7% ~ 95.3%, 92.0% ~ 92.6%, 86.7% ~ 87.2%, demonstrates the reliability of detected result of the present invention further.
The main agents that the present invention adopts is as follows: (all chemical reagent are analytical pure)
1,
reagent purchased from American Invitrogen company.
2, sample-loading buffer: 0.1 % tetrabromophenol sulfonphthalein, 40 % sucrose;
3,0.5 × tbe buffer liquid: 44.5 mmol/L Tris, 50 mmol/L HBO
3, 1 mmol/L EDTA;
4, cDNA reverse transcription and pcr amplification reagent are purchased from the precious biotech firm in Dalian.
Instrument used in the present invention is mainly as follows:
1, PCR amplification instrument: German Eppendorf 5331 PCR amplification instrument;
2, electrophoresis apparatus: Liuyi Instruments Plant, Beijing DYCZ-20A DNA sequence analysis electrophoresis apparatus;
3, whizzer: German Eppendorf 5810/5810R multifunctional table-type whizzer;
4, gel imaging system: U.S. BIO-RAD Gel Doc 2000 gel imaging system;
5, spectrophotometer: U.S. GE NanoVue Plus ultramicrospectrophotometer.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
<110> University Of Agriculture and Forestry In Fujian
<120> Molecular Detection infects the method for the sorghum mosaic virus of sugarcane
<130>
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213> artificial sequence
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acadcagadg caacrgcaca agc 23
<210> 2
<211> 24
<212> DNA
<213> artificial sequence
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ctcrccgaca ttcccatcya agcc 24
<210> 3
<211> 849
<212> DNA
<213> sorghum mosaic virus (
sorghum mosaic virus)
<400> 3
acagcagatg caacagcaca agcgcagcga gaggctgcag caaaagctca acaggatgcc 60
gatgcgaaaa agagagcaga tgacgaagca gcagagaaac agagacaaga tgctgctgca 120
aagaagaaag ctgatgatga tgctaaagca aaagcagatg ccgatgcgaa aaagaaagca 180
gatgacgaag cagcgcaaag agcacaaaat cagaaagata aagacgttga tgctggaacg 240
tcaggcacag ttacagtacc aaaactcaaa gctatgtcca agaaaatgcg cttgccgcaa 300
gcaaaaggaa agaatatttt acatctcgat ttcttacttg gatataagcc acaacagcaa 360
gacatctcaa acacacgagc aacacgagca gagtttgata ggtggtatgc agccgtacaa 420
agagaatatg aacttgatga cacacaaatg acagttgtta tgagtggatt aatggtatgg 480
tgcatagaaa atggttgctc accgaacatc aatggtgttt ggaccatgat ggatggggaa 540
gaacaaagaa catttccttt aaagccaata attgaaaatg cttctccaac ttttaggcag 600
atcatgcatc actttagcga tgcagctgaa gcgtatatag aataccgtaa ttcaacggaa 660
cgctacatgc caagatacgg acttcagcga aacttgaccg actacagttt agcacgttat 720
gcttttgatt tttacgagat cacatcacgt acaactgccc gtgctaagga ggcccacatg 780
cagatgaaag ccgcagcagt tcgtggttca aacacccgaa tgttcggctt ggatgggaat 840
gtcggagag 849
<210> 4
<211> 849
<212> DNA
<213> sorghum mosaic virus (
sorghum mosaic virus)
<400> 4
acagcagatg caacagcaca agcgcagcga gaggctgcag caaaagctca acaggatgcc 60
gatgcgaaaa agagagcaga tgatgaagca gcagagaaac agagacaaga tgctgctgca 120
aagaagaaag ctgacgatga cgctaaagca aaagcagatg ctgatgcgaa gaagaaagca 180
gatgatgaag cagcgcaaag agcgcaaaat cagaaagaca aagacgttga tgctggaacg 240
tcagggacag tcacagtacc aaagcttaaa gctatgtcca agaagatgcg cttgccacaa 300
gcgaaaggaa agaacatctt acatcttgat ttcttacttg gatataaacc acagcaacaa 360
gacatttcaa acacacgagc aacacgagat gagtttgata gatggtatgc agccgtacaa 420
aaggaatatg aagttgatga cacacaaatg acagttgtca tgagtggact tatggtatgg 480
tgcatagaga atggttgctc accgaacatc aatggtgttt ggaccatgat ggatggagaa 540
gaacaaagga aatttccttt aaagccaata attgaaaatg cttctccaac ttttagacaa 600
ataatgcacc actttagtga tgcagctgaa gcgtatatag aataccgtaa ttcgacggaa 660
cgatacatgc caagatacgg acttcagcga aacttgaccg actacaattt agcacgatat 720
gcttttgatt tctatgaaat tacatcacgc acacctgctc gtgctaagga ggcccacatg 780
cagatgaaag ccgcagcagt tcgtggttca aacacccgaa tgttcggctt ggatgggaat 840
gtcggtgag 849
<210> 5
<211> 849
<212> DNA
<213> sorghum mosaic virus (
sorghum mosaic virus)
<400> 5
acagcagatg caacagcaca agcacagcgg gacgcagcgg caaaagcgca acgagatgct 60
gatgcgaaaa agaaggcaga tgatgaagca gcggaaaagc agagacaaga tgccgcagcg 120
aaaaagaaag ctgatgatga tgcaaaagcc aaggctgatg ctgatgctaa ggcaaaggcg 180
gatgctgatg cagctaatag agcgcaaaat cagaaagaca aagatgtgga tgccggcaca 240
tctggcacag tggcagcgcc taagctcaaa gcaatgtcaa agaaaatgaa attaccacaa 300
gcaaagggga aaaacatttt gcatttggat tttcttttgg ggtataagcc acaacaacaa 360
gacatttcaa acacaagagc aacaagagat gaatttgata gatggtatga agcactacag 420
agagagtatg aactagacga cacacagatg acagtagttg ctagtggact aatggtctgg 480
gccatagaga atggatgctc acctaatatc aatggtgttt ggacaatgat ggatggagat 540
gagcagagga aatttccgct caaacctgtt atagaatatg catctccaac attcagacag 600
ataatgcacc actttagtga tgcagctgaa gcgtacatag agtaccgaaa ctcaacagag 660
cgttacatgc caagatacgg acttcagcga aacttgaccg actataacct agcccggtac 720
gcgttcgatt tctatgaaat aacttcgcgt acaccggcga gagctagaga ggcccacatg 780
cagatgaaag cagcagcagt gcgtggttca aacacgcgca tgtttggctt ggatgggaat 840
gtcggagag 849
<210> 6
<211> 849
<212> DNA
<213> sorghum mosaic virus (
sorghum mosaic virus)
<400> 6
acagcagatg caacagcaca agcacagcgt gatgcagcag cgaaagctca acgagacgca 60
gaagcggcag agaagcagag acaagatgct gcagctaaga agaaagctga tgatgatgcg 120
aaagctaaag ctgatgcgga tgccaaagca aaatcagatg ctgacgcgaa aaagaaagca 180
gacgatgaag cagcaagtaa ggtacaaaat caaaaagata aggatgtgga tgccggcaca 240
tctggcacag tggcagtgcc taaactcaaa gcaatgtcca agaaaatgaa gctaccacaa 300
gcaaaaggga aaaacatttt acacttggat tttcttttgg gatataagcc acaacaacaa 360
gacatttcaa acaccagagc tacacgggat gagttcgata ggtggtatga tgcattgcag 420
aaagaatatg aactagatga cacgcagatg acagtggtcg caagcggact catggtctgg 480
gtcatagaaa acggatgctc acctaatatt aatggtgttt ggacaatgat ggatggagat 540
gagcaaagga aatttccact caagcccgtt attgagtatg catctccgac atttagacag 600
ataatgcacc actttagtga tgcagctgaa gcgtacatag agtacagaaa ctcgactgag 660
cgttacgtgc caagatacgg acttcagcga aacttaaccg actataacct agcccggtat 720
gcatttgatt tctatgaaat aacttcgcgt acaccggcga gagctagaga ggcccacatg 780
cagatgaaag cagcagcagt gcgtggttca aacacgcgca tgtttggctt ggatgggaat 840
gtcggagag 849
<210> 7
<211> 849
<212> DNA
<213> sorghum mosaic virus (
sorghum mosaic virus)
<400> 7
acagcagaag caacagcaca agcacagcgt gatgcagcag cgaaagctca acgagacgca 60
gaagcggcag agaagcagag acaagatgct gcagctaaga agaaagctga tgatgatgcg 120
aaagctaaag ctgacgcgga tgccaaagca aaatcagatg ctgacgcgaa aaagaaagca 180
gacgatgaag cagcaagtaa agtacaaaat caaaaagata aggatgtgga tgccggcaca 240
tctggcacag tggcagtgcc taaactcaaa gcaatgtcca agaaaatgaa gctaccgcaa 300
gcgaaaggga aaaacatttt acacttggat tttcttttgg gatataagcc acaacaacaa 360
gacatttcaa acaccagagc cacacgggat gagttcgata ggtggtatga tgcattgcag 420
aaggaatatg aactagatga cacacagatg acagtagtcg caagtgggct catggtttgg 480
gtcatagaga atgggtgctc gcctaatatc aatggtgttt ggacaatgat ggatggagat 540
gagcaaagga aatttccact caaaccagtt attgaatatg catctccaac atttagacag 600
ataatgcacc actttagtga tgcagctgaa gcgtacatag agtatcgaaa ctcaacggaa 660
cgttatatgc caagatacgg acttcagcga aacttaaccg actataacct agcccggtac 720
gcatttgatt tctatgaaat aacttcgcgt acaccggcga gagctagaga ggcccacatg 780
cagatgaaag cagcagcagt gcgtggttca aacacgcgca tgtttggctt ggatgggaat 840
gtcggagag 849
<210> 8
<211> 873
<212> DNA
<213> sorghum mosaic virus (
sorghum mosaic virus)
<400> 8
acagcagatg caacggcaca agcacagcgt gatgcagcag caaaagccca acgagatgct 60
gatgcaaaaa agaaggcaga tgacgaggcg gcggagaaac agagacaaga tgctgcagca 120
aagaagaaag ctgatgatga tgcaaaagct aaagctgatg cggatgccaa agcaaaatca 180
gatgctgatg cgaaaaagaa agcagatgat gcagcagcaa gtaaagcact aaatcaaaaa 240
gacaaggatg tggatgtcgg cacatctggc acagtggcag tgcctaaact caaagcaatg 300
tccaagaaaa tgaagctacc acaggcgaaa gggaaaaaca ttttacactt ggattttctt 360
ttgggatata agccacaaca acaagacatt tcaaacacta gagccacacg ggatgagttc 420
gataggtggt atgatgcatt gcagaaggaa aatgaactag atgacacaca gatgacagta 480
gtcgcaagtg ggctcatggt ttgggtcata gagaatgggt gctcgcctaa tatcaatggt 540
gtttggacaa tgatggatgg agatgagcaa aggaaatttc cactcaaacc agttattgaa 600
tatgcatctc caacattcag acagataatg caccacttta gtgatgcagc tgaagcgtac 660
atggagtatc gaaactcaac ggaacgttat atgccaagat acggacttca gcgaaactta 720
accgactata acctagcccg gtacgcattt gatttctatg aaataacttc gcgtacacca 780
gcgagagcta gagaggccca catgcagatg aaagcagcag cagtgcgtgg ttcaaacacg 840
cgcatgtttg gcttggatgg gaatgtcgga gag 873
<210> 9
<211> 873
<212> DNA
<213> sorghum mosaic virus (
sorghum mosaic virus)
<400> 9
acagcagaag caacagcaca agcacagcgt gatgcagcag caaaagctca acgagatgct 60
gatgcaaaga agaaggcaga tgatgaggcg gcagagaagc aaagacaaga tgctgcagca 120
aagaagaaag ctgatgatga tgcgaaggct aaagctgatg cagacgctaa agcaaaagca 180
gatgctgatg cgaaaaagaa agcagacaat gaagcagcaa gtaaagcact aaatcaaaaa 240
gacaaggatg tggatgtcgg cacatctggc acagtggcag tgcctaaact caaagcgatg 300
tccaagaaaa tgaagctacc acaagcaaaa gggaaaaaca ttttacactt ggattttcta 360
ttgggatata agccacaaca acaagacatt tcaaacacca gagccacgcg ggatgagttc 420
gataggtggt atgatgcatt gcagaaggaa tatgaacttg atgacacaca gatgacagta 480
gtcgcaagtg gacttatggt ttgggtcata gaaaacggat gctcacctaa tattaatggt 540
gtttggacaa tgatggatgg agatgaacaa aggaaatttc cactcaagcc tgttattgaa 600
tatgcatctc caacattcag acagataatg caccacttta gtgatgcagc tgaagcgtac 660
atagagtatc gaaattcgac agagcgatat atgccaagat atggacttca gcgaaactta 720
accgactata acctagctcg gtacgcattc gatttctatg aaataacttc gcgtacaccg 780
gcgagagcta gggaggccca catgcagatg aaagcagcag cagtgcgtgg ttcaaacacg 840
cgcatgtttg gcttggatgg gaatgtcgga gag 873
<210> 10
<211> 873
<212> DNA
<213> sorghum mosaic virus (
sorghum mosaic virus)
<400> 10
acagcagatg caacagcaca agcacagcgt gatgcagcag cgaaagctca acgagatgct 60
gatgcaaaga aaaaggcaga tgacgaagca gcagagaggc agagacaaga agctgcagca 120
aagaagaagg ctgatgatga tgcgaaagct aaagctgatg cggatgccaa agcaaaatca 180
gatgccgatg tgaaaaagaa agctgatgaa gaagcagcaa ataaagtaca aaatcaaaaa 240
gataaggatg tggatgccgg cacatctggc acagtggcag tgcctaaact caaggcaatg 300
tccaagaaaa tgaagctacc acaagcgaaa gggaaaaaca tcctacactt ggacttcctt 360
ttgggatata agccacaaca acaagatatt tcaaacacaa gagcaacaag agatgaattt 420
gatagatggt atgaagcatt acagagagag tatgaactag atgacacaca gatgacagta 480
gtcgctagtg gactcatggt ctgggccata gaaaatggat gctcacctaa tattaatggt 540
gtttggacaa tgatggatgg agatgaacaa aggaaatttc cactcaagcc cgttattgaa 600
tatgcatctc caacattcag acagataatg caccacttta gtgatgcagc tgaagcgtac 660
atagagtacc gaaactcaac agagcgttat atgccaagat atggacttca gcgaaactta 720
accgactata acctagcccg gtacgcattc gatttttacg aaataacttc gcgtacaccg 780
gcgagagcta gagaggccca catgcagatg aaagcagcag cagtgcgtgg ttcaaacacg 840
cgcatgtttg gcttggatgg gaatgtcgga gag 873
<210> 11
<211> 873
<212> DNA
<213> sorghum mosaic virus (
sorghum mosaic virus)
<400> 11
acagcagatg caacagcaca agcacagcgg gacgcagcag caaaagctca gcgggatgct 60
gatgcaaaga aaaaggctga tgatgaagca gcagaaaagc aaagacagga tgctgctgcg 120
aagaagaaag cggatgatga tgccaaggcc aaagctgatg ctgatgctaa ggctaaaact 180
gatgctgaag ctaaaaagaa agcagatgat gaagcagcaa agagaacaca aaaccagaag 240
gataaggatg ttgacgctgg aacatcaggc acagtggcag tgccaaagct caaagctatg 300
tctaagaaaa tgagattacc acaagctaaa ggaaagaaca ttttgcactt agactttctt 360
ttgggttaca agccacagca acaagatatt tcaaacacaa gatcaactag agacgaattc 420
gacagatggt acgatgcatt acagaaagag tatgagctgg atgatacaca gatgacagtc 480
gttgcaagcg ggctcatggt ttgggctatt gagaatggat gttcgcctaa tatcaatggt 540
gtttggacaa tgatggatgg agatgagcaa aggaaatttc ctttgaaacc tgtcatagaa 600
tatgcttctc caacgtttag acaaataatg caccacttta gtgatgcagc tgaagcgtat 660
attgagtata gaaactcaac agaacgttat atgccaagat atggacttca gcgaaactta 720
accgactata acctagcacg atacgcattt gatttttatg aaataacatc gcgtacacca 780
gcaagagcta gagaggccca catgcagatg aaagcagcag cagtgcgtgg ttcaaacacg 840
cgcatgtttg gcttggatgg gaatgtcgga gag 873
Claims (1)
1. Molecular Detection infects a method for the sorghum mosaic virus of sugarcane, comprises Sugarcane Leaves Total RNAs extraction, degenerated primer design, RT-PCR amplification, restriction enzyme reaction, electrophoresis detection; It is characterized in that:
(1) degenerated primer design: degenerated primer sequence SrMV-CPF and the SrMV-CPR of amplification SrMV different strain coat protein gene conserved sequence are as follows:
SrMV-CPF：5'-ACADCAGADGCAACRGCACAAGC-3'
SrMV-CPR：5'-CTCRCCGACATTCCCATCYAAGCC-3'；
(2) RT-PCR amplification:
RT reacts: reverse transcription reaction system cumulative volume is 10 μ L, include: 2 μ L 5 × PrimeScript damping fluids, 0.5 μ L PrimeScript ThermoScript II mixed solution, 0.5 μ L Oligo dT primer, 2 μ L Random 6 mers primers, 2 μ L Sugarcane Leaves total serum IgE templates, supply 10 μ L without RNase water; Reverse transcription reaction condition is 37 DEG C of 15 min, 85 DEG C of 5 s deactivation ThermoScript II;
Pcr amplification: PCR reaction system cumulative volume is 50 μ L, include 5.0 μ L 10 × PCR damping fluids, 4.0 μ L 2.5 mmol/L dNTPs, 10 μm of each 2.0 μ L of ol/L degenerated primer SrMV-CPF and SrMV-CPR, 0.25 U Taq DNA Polymerase, 1.0 μ L cDNA, sterilizing ultrapure water supplies 50 μ L; Described 10 × PCR damping fluid moiety is 500 mmol/L KCl, 100 mmol/L Tris-HCl pH 8.3,15 mmol/L MgCl
2; Pcr amplification program is as follows: 94 DEG C of denaturation 2 min, 94 DEG C of sex change 1 min, 55 DEG C of annealing 30 s, 72 DEG C of extensions 1 min, totally 35 circulations, and last 72 DEG C extend 10 min again;
(3) restriction enzyme reaction: application
nsii and
bsmi restriction enzyme carries out double digestion reaction, and endonuclease reaction system cumulative volume is 20 μ L, includes 2.0 μ L 10 × enzyme cutting buffering liquids, PCR primer after 4.0 μ L purifying,
nsii and
bsmthe each 1.5 μ L of I restriction enzyme, sterilizing ultrapure water supplies 20 μ L; Endonuclease reaction condition: 37 DEG C of enzymes cut 12 hours; 65 DEG C of process 20 min, stop endonuclease reaction, are down to normal temperature;
(4) electrophoresis detection: get 5 μ L reaction solutions after endonuclease reaction terminates and carry out the low melting-point agarose gel electrophoresis that mass volume ratio is 4.0 %, in 0.5 × electrophoretic buffer environment, 80 V voltage stabilizing electrophoresis 2 h, then observe with gel imaging system and take pictures, in electrophoresis detection figure, there is PCR primer enzyme slitting band, represent that this sample is containing SrMV; There is not PCR primer enzyme slitting band, represent this sample not containing SrMV.
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| CN108728581B (en) * | 2018-06-26 | 2021-02-05 | 广西大学 | Multiple RT-PCR method for simultaneously detecting 5 sugarcane viruses, primers and kit thereof |
| CN110951924A (en) * | 2020-01-05 | 2020-04-03 | 云南省农业科学院甘蔗研究所 | One-step multiple RT-PCR detection method for simultaneously detecting 3 pathogens of sugarcane mosaic disease |
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| WO2001007639A2 (en) * | 1999-07-27 | 2001-02-01 | Syngenta Limited | Selectable marker gene |
| CN101852807A (en) * | 2010-05-14 | 2010-10-06 | 云南省农业科学院生物技术与种质资源研究所 | Sorghum mosaic virus tri-anti sandwich enzyme-linked immuno sorbent assay kit |
| CN101974654A (en) * | 2010-10-26 | 2011-02-16 | 云南省烟草公司昭通市公司 | Method for quickly detecting different strains of potato virus Y tobacco isolates |
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| WO2001007639A2 (en) * | 1999-07-27 | 2001-02-01 | Syngenta Limited | Selectable marker gene |
| CN101852807A (en) * | 2010-05-14 | 2010-10-06 | 云南省农业科学院生物技术与种质资源研究所 | Sorghum mosaic virus tri-anti sandwich enzyme-linked immuno sorbent assay kit |
| CN101974654A (en) * | 2010-10-26 | 2011-02-16 | 云南省烟草公司昭通市公司 | Method for quickly detecting different strains of potato virus Y tobacco isolates |
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Effective date of registration: 20231226 Address after: 513055 Lin Wu, Xianjiao Keng Village, Qiaotou Town, Yingde City, Qingyuan City, Guangdong Province Patentee after: Yingde Fenglin Agricultural Professional Cooperative Address before: 350002, Jinshan District, Jinshan Town, Cangshan District, Fuzhou, Fujian Patentee before: FUJIAN AGRICULTURE AND FORESTRY University |