CN104313188A - Kit for detecting and judging genotype of sugarcane yellow leaf virus - Google Patents
Kit for detecting and judging genotype of sugarcane yellow leaf virus Download PDFInfo
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Abstract
The invention relates to a kit for detecting and judging genotype of a sugarcane yellow leaf virus. The kit at least comprises an SCYLV-P0/F3 and SCYLV-P0/R2 degenerate primer pair, and also comprises an RT reaction liquid, a PCR reaction liquid, restriction enzymes and a buffer solution of the restriction enzymes, a positive control RNA template, a negative control RNA template, and sterilized water containing no nuclease. Primer has specificity, strip length multiformity of the restriction enzyme has uniqueness, and a simple, rapid and accurate method is provided for sugarcane yellow leaf virus detection and judgment. By adoption of the kit, a susceptible plant total RNA sample to be detected is subjected to RT-PCR amplification, and enzyme digestion reaction of four restriction enzymes which are BamHI, BsaI, PvuII and KpnI, enzyme-digested products are detected through 4% low melting-point agarose gel electrophoresis, whether the products are infected by the sugarcane yellow leaf virus or not can be judged according to the strip length multiformity of the restriction enzyme, and the genotype of the virus can also be judged.
Description
Technical field
The present invention relates to a kind of test kit detecting plant virus, be specifically related to a kind of detection and the genotypic test kit of qualification sugarcane yellow leaf virus, belong to plant virus detection technique field.
Background technology
Sugarcane yellow leaf virus (
sugarcane yellow leaf virus,sCYLV) belong to Lutoevirus section (
luteoviridae) Polerovirus (
polerovirus) member, by protein enclosure and the strand be wrapped in, just RNA(ssRNA) form, being mainly distributed in plant body in phloem tissue screen casing companion cell tenuigenin, is the cause of disease causing sugarcane yellow leaf (Yellow leaf).Sugarcane yellow leaf found at Hawaii, America early than 1989, and this disease causes sugarcane yield and sugar degree to decline, and is a kind of global virus disease of serious harm sugarcane production.In recent years, sugarcane yellow leaf generally occurs in Chinese main cane district, and has the trend increased the weight of year by year, forms potential threat to Sugar Industry development.
What current SCYLV reported has 10 kinds of genotype (Lin etc., 2014) at least: Brazilian genotype (BRA), Cuba's genotype (CUB), Peru's genotype (PER), French R é union island genotype (REU), Hawaii genotype (HAW), Colombia's type (COL), India's genotype (IND) and Chinese genotype (CHN1, CHN2, CHN3).Because BRA and PER, HAW and PER have the sequence identity of height respectively, they are called BRA-PER and HAW-PER genotype by the scholar therefore had.In China, at least there are 7 kinds of genotype, i.e. BRA, HAW, PER, CUB, CHN1, CHN2 and CHN3(Xu etc. in SCYLV, and 2010; Wang etc., 2012; Lin etc., 2014).SCYLV genome contains 6 open reading frame (Open reading frame), wherein the P0 albumen of ORF0 open reading frame coding has the activity suppressing RNA silence, pathogenic relevant with virus, and P0 gene exists genetic diversity, be desirable detection and the target sequence distinguishing different genotype.
The symptom characteristic of sugarcane yellow leaf is arteries and veins lower epidermis yellow in Sugarcane Leaves, and then blade tip starts withered downright bad.The diagnosis of this disease can tentatively be identified by the Disease symptoms feature of blade, but, Symptoms is relevant with sugarcane line, growth conditions, temperature and virus strain, and Disease symptoms performance needs certain latent period, growth early metaphase does not have symptom, some disease plant does not show obvious symptom, and this brings difficulty to the diagnosis of sugarcane yellow leaf field.The technology of sugarcane yellow leaf Pathogen identification and detection has electron microscopic diagnosis, tissue blot to hybridize immunoassay (TBIA), enzyme-linked immunoassay technology (ELISA), conventional RT-PCR and real-time fluorescence RT-PCR method etc.But above-mentioned Serology test can determine whether to infect SCYLV, but is difficult to distinguish the concrete genotype of SCYLV.RT-PCR molecular detecting method needs PCR primer to reclaim, be transformed into bacillus coli DH 5 alpha, and then by order-checking and sequential analysis, experimental procedure is comparatively loaded down with trivial details, it is longer to expend time in.According to the reagent that test kit of the present invention provides, disease plant total serum IgE sample to be detected is through RT-PCR amplification, use
bamhI,
bsai,
pvuiI and
kpni digestion with restriction enzyme reacts, and with the low melting-point agarose detected through gel electrophoresis digestion products of 4%, can determine whether to infect sugarcane yellow leaf virus and genotype type thereof according to restriction enzyme band length polymorphism.Primer of the present invention has specificity, and restriction enzyme band length polymorphism has uniqueness, detects and qualification provides a kind of easy, method fast and accurately for sugarcane yellow leaf virus.
Summary of the invention
The object of this invention is to provide a kind of detection and the genotypic test kit of qualification sugarcane yellow leaf virus.
Object of the present invention realizes by the following method:
One of the present invention detects and the genotypic test kit of qualification sugarcane yellow leaf virus, and it is characterized in that at least comprising degenerated primer in test kit to SCYLV-P0/F3 and SCYLV-P0/R2, its sequence is as follows:
SCYLV-P0/F3:5 '-TGGACCMAGCCTCTGRYT-3 ', wherein, M=A or M=C, R=A or R=G, Y=C or Y=T;
SCYLV-P0/R2:5 '-CAATCGWACACAGGATGC-3 ', wherein, W=A or W=T.
One of the present invention detects and the genotypic test kit of qualification sugarcane yellow leaf virus, is made up of following reagent:
(1) RT reaction solution: each inverse transcription reaction liquid comprises 5 × RT damping fluid 2.0 μ L, AMV ThermoScript II liquid 0.5 μ L, 10 μm of ol/L SCYLV-P0/R2 primer 2 .5 μ L, totally 5.0 μ L, 50 reaction systems amount to 250 μ L, and-20 DEG C save backup;
(2) PCR reaction solution: each PCR reaction solution comprises 10 × PCR damping fluid 5.0 μ L, 2.5 mmol/L dNTPs 4.0 μ L, and concentration is each 2.0 μ L of degenerated primer SCYLV-P0/F3 and SCYLV-P0/R2 of 10 μm of ol/L, totally 11.0 μ L, 50 reaction systems amount to 550 μ L, and-20 DEG C save backup;
(3) restriction enzyme damping fluid: each endonuclease reaction liquid comprises 10 × enzyme cutting buffering liquid 4.0 μ L, 50 reaction systems amount to 200 μ L, and-20 DEG C save backup;
(4) Ex Taq enzyme: each PCR reaction system comprises 5 U/ μ L Ex Taq enzyme 0.25 μ L, and 50 reaction systems amount to 12.5 μ L, and-20 DEG C save backup;
(5)
bamhI restriction endonuclease: each restriction enzyme reaction system comprises 10 U/ μ L
bamhI restriction endonuclease 1.0 μ L, 50 reaction systems amount to 50 μ L, and-20 DEG C save backup;
(6)
bsai restriction endonuclease: each restriction enzyme reaction system comprises 10 U/ μ L
bsai restriction endonuclease 1.0 μ L, 50 reaction systems amount to 50 μ L, and-20 DEG C save backup;
(7)
pvuiI restriction endonuclease: each restriction enzyme reaction system comprises 10 U/ μ L
pvuiI restriction endonuclease 1.0 μ L, 50 reaction systems amount to 50 μ L, and-20 DEG C save backup;
(8)
kpni restriction endonuclease: each restriction enzyme reaction system comprises 10 U/ μ L
kpni restriction endonuclease 1.0 μ L, 50 reaction systems amount to 50 μ L, and-20 DEG C save backup;
(9) positive RNA: adopt the RNA of T7 RNA polymerase in-vitro transcription synthesis containing different SCYLV genotype target fragment as standard model, 1 × 10
10copy/μ L, 50 μ L/ bottles ,-20 DEG C save backup;
(10) negative sample RNA: adopt the Sugarcane Leaves tissue infected without sugarcane yellow leaf virus, extracting blade total serum IgE is negative control, 100 ng/ μ L, 50 μ L/ bottles, and-20 DEG C save backup;
(11) nuclease free sterilized water: 2 × 10 mL/ bottles.
Advantage of the present invention: advantage of the present invention is cut by RT-PCR product enzyme by restricted endoenzyme, then electrophoresis detection restriction fragment length polymorphism, just Rapid identification can go out the different genotype of sugarcane yellow leaf virus, this authentication method overcomes the weak point of common molecular detection and authentication method, eliminating PCR primer in common molecular detection method reclaims until the step of order-checking and sequential analysis, substantially reduce the detection time of RT-PCR, and reduce testing cost, more realistic demand, has a extensive future.Primer of the present invention has specificity, and restriction enzyme band length polymorphism has uniqueness, detects and qualification provides a kind of easy, method fast and accurately for sugarcane yellow leaf virus.
Accompanying drawing explanation
Fig. 1 is the RT-PCR product restriction enzyme digestion and electrophoresis detection figure of sugarcane yellow leaf virus different genotype, and wherein, 1 is DL 100 bp DNA standard relative molecular mass, 2 is BRA genotype, and 3 is PER genotype, and 4 is HAW genotype, 5 and 6 is CHN3 genotype, and 7 is CHN1 genotype.
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.
1, Sugarcane Leaves Total RNAs extraction
1) have collected 332 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 TRIZOL reagent immediately, lid upper tube cap when liquid nitrogen is just evaporated completely, concussion mixing, in 4 DEG C, centrifugal 10 min of 12000 rpm.
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 rpm.
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 rpm 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 rpm 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 nuclease free sterilized waters, 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
The primer-design software that amplimer application of the present invention is relevant designs according to sugarcane yellow leaf virus genome 5 ' terminal sequence, from the tens pairs of primers obtained, select the higher primer pair of scoring again, then the primer sequence designed is sent to Sangon Biotech (Shanghai) Co., Ltd. and carries out synthesis and obtain.Finally determine degenerated primer sequence SCYLV-P0/F3 and SCYLV-P0/R2, the object nucleic acid belt size of amplification is 968 bp, and degenerated primer sequence is as follows:
SCYLV-P0/F3:5 '-TGGACCMAGCCTCTGRYT-3 ', wherein, M=A or M=C, R=A or R=G, Y=C or Y=T;
SCYLV-P0/R2:5 '-CAATCGWACACAGGATGC-3 ', wherein, W=A or W=T.
3, RT-PCR amplification
The RT reaction solution that applying this test kit provides carries out reverse transcription reaction, adds following reagent in PCR pipe:
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 SCYLV-P0/F3 and SCYLV-P0/R2 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 30 s, 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 nuclease free sterilized 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 nuclease free sterilized waters to dissolve.
5, endonuclease reaction
Apply that this test kit provides
bamhI,
bsai,
pvuiI and
kpni restriction enzyme and damping fluid thereof carry out endonuclease reaction, add following reagent in PCR pipe:
Mixing, after centrifugal 5 s of 8000 rpm, 37 DEG C of enzymes cut 2 hours, and 65 DEG C of process 5 min, stop endonuclease reaction, be down to normal temperature.
6, 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, 100 V voltage stabilizing electrophoresis 1.5 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 SCYLV; There is not PCR primer enzyme slitting band, represent this sample not containing SCYLV.Banding pattern according to occurring distinguishes different SCYLV genotype.
Sugarcane yellow leaf virus different genotype restriction enzyme digestion sites and band length polymorphism as shown in table 1, as seen from Table 1, the position of RT-PCR product enzyme slitting band is different, represents that the genotype of contained SCYLV in sample is different.There is RT-PCR product enzyme slitting band in 968 bp positions, represent in this sample containing BPH genotype (containing BRA, PER and HAW genotype); There is RT-PCR product enzyme slitting band at 325 bp and 674 bp positions simultaneously, represent in this sample containing CHN3 genotype; There is RT-PCR product enzyme slitting band at 274 bp and 727 bp positions simultaneously, represent in this sample containing REU genotype; At 101 bp, there is RT-PCR product enzyme slitting band in 204 bp, 325 bp and 431 bp positions simultaneously, represents in this sample containing CUB genotype; There is RT-PCR product enzyme slitting band at 395 bp and 604 bp positions simultaneously, represent in this sample containing CHN1 genotype; At 101 bp, there is RT-PCR product enzyme slitting band in 325 bp and 604 bp positions simultaneously, represents in this sample containing IND genotype.If there is not RT-PCR product enzyme slitting band in electrophoresis detection figure, represent this sample not containing SCYLV.Through repetition test, for not containing the sample of SCYLV still containing other sugarcane disease, use detection method of the present invention, in electrophoresis detection figure, also do not occur RT-PCR product enzyme slitting band.
Table 1 sugarcane yellow leaf virus different genotype restriction enzyme digestion sites and band length polymorphism
Remarks: BPH refers to containing BRA, PER and HAW genotype.
The present embodiment have detected 332 samples collected from sugarcane district, Provinces (regions) of China, therefrom chooses representational electrophoresis detection result as shown in Figure 1, and in Fig. 1,2-7 represents sample 1-6 after testing containing SCYLV.Wherein, all only there is band in 968 bp positions in sample 1,2 and 3, represents in these three samples containing BPH genotype (containing BRA, PER and HAW genotype); There is PCR primer enzyme slitting band in 325bp and 643bp position simultaneously, represent in this sample containing CHN3 genotype; There is PCR primer enzyme slitting band in 395bp and 573bp position simultaneously, represent in this sample containing CHN1 genotype.Sample 1 is from Laibin, Guangxi Province sugar cane breed cloud sugarcane 06-407; Sample 2 is from Sanya, Hainan sugarcane line precipice city 84-125; Sample 3 is from Wengyuan, Guangdong sugar material sugarcane GD-WY19; Sample 4 is from Guizhou Wangmo County sugarcane line Ganzhe 18; Sample 5 is from Sanya, Hainan sugarcane line CP49-50; Sample 6 is from Guangzhou Guangdong sugarcane line CP93-1309.
The main agents that the present invention adopts is as follows: (all chemical reagent are analytical pure)
1, TRIZOL 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;
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.
SEQUENCE LISTING
<110> University Of Agriculture and Forestry In Fujian
<120> mono-kind detects and the genotypic test kit of qualification sugarcane yellow leaf virus
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> artificial sequence
<400> 1
tggaccmagc ctctgryt 18
<210> 2
<211> 18
<212> DNA
<213> artificial sequence
<400> 2
caatcgwaca caggatgc 18
Claims (2)
1. one kind is detected and the genotypic test kit of qualification sugarcane yellow leaf virus, it is characterized in that at least comprising degenerated primer in test kit to SCYLV-P0/F3 and SCYLV-P0/R2, the sequence of SCYLV-P0/F3 is 5 '-TGGACCMAGCCTCTGRYT-3 ', wherein, M=A or M=C, R=A or R=G, Y=C or Y=T; The sequence of SCYLV-P0/R2 is 5 '-CAATCGWACACAGGATGC-3 ', wherein, W=A or W=T.
2. one according to claim 1 detects and the genotypic test kit of qualification sugarcane yellow leaf virus, it is characterized in that this test kit is made up of following reagent:
(1) RT reaction solution: each inverse transcription reaction liquid comprises 5 × RT damping fluid 2.0 μ L, AMV ThermoScript II liquid 0.5 μ L, 10 μm of ol/L SCYLV-P0/R2 primer 2 .5 μ L, totally 5.0 μ L, 50 reaction systems amount to 250 μ L, and-20 DEG C save backup;
(2) PCR reaction solution: each PCR reaction solution comprises 10 × PCR damping fluid 5.0 μ L, 2.5 mmol/L dNTPs 4.0 μ L, and concentration is each 2.0 μ L of degenerated primer SCYLV-P0/F3 and SCYLV-P0/R2 of 10 μm of ol/L, totally 11.0 μ L, 50 reaction systems amount to 550 μ L, and-20 DEG C save backup;
(3) restriction enzyme damping fluid: each endonuclease reaction liquid comprises 10 × enzyme cutting buffering liquid 4.0 μ L, 50 reaction systems amount to 200 μ L, and-20 DEG C save backup;
(4) Ex Taq enzyme: each PCR reaction system comprises 5 U/ μ L Ex Taq enzyme 0.25 μ L, and 50 reaction systems amount to 12.5 μ L, and-20 DEG C save backup;
(5)
bamhI restriction endonuclease: each restriction enzyme reaction system comprises 10 U/ μ L
bamhI restriction endonuclease 1.0 μ L, 50 reaction systems amount to 50 μ L, and-20 DEG C save backup;
(6)
bsai restriction endonuclease: each restriction enzyme reaction system comprises 10 U/ μ L
bsai restriction endonuclease 1.0 μ L, 50 reaction systems amount to 50 μ L, and-20 DEG C save backup;
(7)
pvuiI restriction endonuclease: each restriction enzyme reaction system comprises 10 U/ μ L
pvuiI restriction endonuclease 1.0 μ L, 50 reaction systems amount to 50 μ L, and-20 DEG C save backup;
(8)
kpni restriction endonuclease: each restriction enzyme reaction system comprises 10 U/ μ L
kpni restriction endonuclease 1.0 μ L, 50 reaction systems amount to 50 μ L, and-20 DEG C save backup;
(9) positive RNA: adopt the RNA of T7 RNA polymerase in-vitro transcription synthesis containing different SCYLV genotype target fragment as standard model, 1 × 10
10copy/μ L, 50 μ L/ bottles ,-20 DEG C save backup;
(10) negative sample RNA: adopt the Sugarcane Leaves tissue infected without sugarcane yellow leaf virus, extracting blade total serum IgE is negative control, 100 ng/ μ L, 50 μ L/ bottles, and-20 DEG C save backup;
(11) nuclease free sterilized water: 2 × 10 mL/ bottles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201410639123.XA CN104313188A (en) | 2014-11-13 | 2014-11-13 | Kit for detecting and judging genotype of sugarcane yellow leaf virus |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222291A (en) * | 2016-08-19 | 2016-12-14 | 福建农林大学 | A kind of Molecular Detection and the test kit identifying the Caulis Sacchari sinensis different strain of red streak bacterium |
-
2014
- 2014-11-13 CN CN201410639123.XA patent/CN104313188A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| WANG M Q等: "Genotype identification and genetic diversity of Sugarcane yellow leaf virus in China", 《PLANT PATHOLOGY》 * |
| 张荣跃等: "甘蔗黄叶病毒基因型研究进展", 《中国糖料》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222291A (en) * | 2016-08-19 | 2016-12-14 | 福建农林大学 | A kind of Molecular Detection and the test kit identifying the Caulis Sacchari sinensis different strain of red streak bacterium |
| CN106222291B (en) * | 2016-08-19 | 2019-06-28 | 福建农林大学 | A kind of kit of Molecular Detection strain different with identification sugarcane red streak bacterium |
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Application publication date: 20150128 |