CN102952901B - Method for synchronously detecting TBTV (Tobacco Bushy Top Virus), TVDV (Tobacco Vein Distorting Virus), Sat-TBTV (Tobacco Bushy Top Virus Satellite-Like Ribose Nucleic Acid) and TVDVaRNA (TVDA-associated Ribonucleic Acid) - Google Patents
Method for synchronously detecting TBTV (Tobacco Bushy Top Virus), TVDV (Tobacco Vein Distorting Virus), Sat-TBTV (Tobacco Bushy Top Virus Satellite-Like Ribose Nucleic Acid) and TVDVaRNA (TVDA-associated Ribonucleic Acid) Download PDFInfo
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Abstract
The invention relates to a method for synchronously detecting TBTV (Tobacco Bushy Top Virus), TVDV (Tobacco Vein Distorting Virus), Sat-TBTV (Tobacco Bushy Top Virus Satellite-Like Ribose Nucleic Acid) and TVDVaRNA (TVDA-associated Ribonucleic Acid), which belongs to the field of plant protection. The synchronous and quick detection of four pathogens can be achieved by respectively designing and synthetizing forward and reverse primers of the TBTV, the TVDV, the Sat-TBTV and the TVDVaRNA, extracting total RNA of infected plant tissues or virus transmission vector insects by a CTAB (Cetyl Trimethyl Ammonium Bromide) method and adopting a one-step method quadruple RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The specificity of designed primers is strong, and the four pathogens can be amplified at the same time; the total RNA of the infected plant tissues and the virus transmission vector insects are extracted by using the CTAB method, thus not only can the quality of extracted RNA be ensured, but also the detection cost can be also reduced; and the reverse transcription and the multiplex-PCR are completed in one step, and thus the detection time is greatly saved. The method disclosed by the invention has the advantages of fast speed, high efficiency, strong specificity, high sensitivity, low cost and the like, the synchronous detection of the four pathogens related to the TBTV can be realized in one step, and the method has a wide application prospect.
Description
technical field
The present invention relates to the method for synchronous detection TBTV, TVDV, Sat-TBTV and TVDVaRNA a kind of, platymiscium protection field.
background technology
Tobacco bushy top disease (tobacco bushy top disease) occurred in the Chun Kewangli in Zimbabwe the north cigarette district first in nineteen fifty-seven, after this eruption and prevalence cause very large loss.Tobacco bushy top disease early occurs in the Yunnan of China, but for a long time because this disease is only fragmentary generation, tobacco is not produced and causes heavy losses and out in the cold, until just receive publicity gradually after the ground big area eruption and prevalences such as Baoshan, Yunnan, Dali for 1993.After this this disease is popular on a large scale in cigarette district, Yunnan Province of China Midwest, cause crushing loss to part cigarette district, diseased region also has tobacco bushy top disease sporadicly to occur except relating to the Baoshan, Dali, 3 Wai， Kunming, Ge Zhou city, Chuxiong, Yuxi, Red River, Simao, mountain of papers, Xishuangbanna and Deng Zhou city, Nujiang.End calendar year 2001, Yunnan Province's accumulative total onset area reaches 51300 hm
2, 8700 hm wherein
2total crop failure, 1400 hm
2replant, direct economic loss is up to 2.1 hundred million yuan, and tobacco bushy top disease has become Yunnan Province's unique tobacco diseases that causes big area total crop failure since the nineties in 20th century.
Tobacco bushy top disease by the tobacco bushy top virus of Umbravi-rus (
tobacco bushy top virus, TBTV) and the tobacco of Lutoevirus section Polerovirus turn round arteries and veins virus (
tobacco vein distorting virus, TVDV) Combined Infection causes.A microRNA of following TBTV appearance and its molecular weight to be about 1000 bp in the susceptible cigarette strain of Yunnan Tobacco thick grass top sickness, often detected in addition, according to current research, infer that this microRNA is the satellite RNA of TBTV, be called the similar satellite RNA of tobacco bushy top virus (Tobacco bushy top virus satellite-like RNA, Sat-TBTV).2010 have detected size again from the plant of infection tobacco bushy top disease is the RNA that 3.0 kbp are relevant to tobacco bushy top disease, in the international virus taxis report of Jiu Ci by its called after TVDVaRNA(Tobacco vein distorting virus-associated RNA, TVDVaRNA).TBTV genome is not containing the gene of coded housing albumen, and it can be propagated by frictional inoculation, under the help of helper virus TVDV, can pass through aphis propagation.Current result of study shows, under natural condition, show the cigarette strain of typical tobacco bushy top disease symptom, all by above-mentioned 4 kinds of pathogen Combined Infections, caused, and a lot of not cigarette strains of reveal any symptoms in Yunnan Tobacco thick grass top sickness region of disease also often can detect TVDV and TVDVaRNA.So far, there is no especially effectively Control of Plant Virus Disease medicament both at home and abroad, therefore except being strictly on guard against the propagation of aphid, strengthen detection and the monitoring of 4 kinds of pathogens of tobacco bushy top disease in cigarette strain and aphid, for Accurate Diagnosis disease and prevent that the spread and epidemic of virus disease from having important practical significance, be to produce upper one of important measures of the comprehensive regulation that tobacco bushy top disease is carried out.
Conventionally the method that detects plant identification virus has biology, electron microscopy, serological technique and Protocols in Molecular Biology etc.Biological method is simple to operate, but the method sense cycle is long, sensitivity is low, is also subject to the impact of the aspects such as season, environment and viral species, is therefore difficult to the Accurate Diagnosis of 4 kinds of pathogens of tobacco bushy top disease.Due to TBTV, Sat-TBTV and TVDVaRNA coded housing albumen not, there is no plastochondria structure, electron microscopy and conventional serological technique cannot be identified for this 3 detection that grows tobacco clump top sickness pathogen; Electron microscopy only can be observed viral plastochondria form, and very difficult precise Identification goes out viral species, and apparatus expensive, is not suitable as the usual way that TVDV detects.The genome of TVDV can coded housing albumen, virus has complete plastochondria structure, can be used as antigen and prepare antiserum(antisera), but because the distribution of TVDV is confined to phloem, and viral level is lower, be difficult for directly from diseased plant, being purified to virus particle, therefore there is no at present the report of relevant TVDV antiserum(antisera) aspect both at home and abroad.Protocols in Molecular Biology, especially RT-PCR technology is not only simple to operate, and high specificity, highly sensitive, but conventional RT-PCR method can only detect a kind of virus at every turn, for the situation of multiple pathogen Combined Infection, need to detect separately for every kind of virus, workload is large, cost is high, and efficiency is low.
The present invention adopts single stage method RT-PCR, 4 kinds of pathogens for tobacco bushy top disease, design respectively the Auele Specific Primer of 4 pairs of tobacco bushy top disease pathogens, can detect 4 pathogens that grow tobacco clump top sickness by a PCR, have efficient, quick, special, sensitive and save time and reduce the advantages such as cost.
summary of the invention
The present invention has overcome prior art in the deficiency to the multiple pathogen Combined Infection of tobacco bushy top disease tobacco and when passing virus mediator insect and detecting, and provides a kind of efficient, sensitive, special and detect cheaply tobacco and pass the method for TBTV, TVDV, Sat-TBTV and 4 kinds of pathogens of TVDVaRNA in virus mediator insect.
Technical scheme of the present invention is: the method for synchronous detection TBTV, TVDV, Sat-TBTV and TVDVaRNA, according to step below, carry out:
(1) design of primers is synthetic:
(a) forward primer TBTVdF, TVDVdF, SatTBTVdF and the TVDVaRNAdF that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdF：5’-TACCACACCTAAACAGCGTTG-3’，
TVDVdF：5’-GCAACAGCGAGACTTTCATCT-3’，
SatTBTVdF：5’-TGTTGGCCGTGGGCAGCAA-3’，
TVDVaRNAdF：5’-GCGTACTGCGGAAGTCTCAC-3’，
(b) reverse primer TBTVdR, TVDVdR, SatTBTVdR and the TVDVaRNAdR that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdR：5’-CTCATCTCCCGCTAAGTCAG-3’，
TVDVdR:5 '-CRTTGCCTTTATAGAGCAGCC-3 ', wherein, the R=A in sequence or G,
SatTBTVdR：5’-TTCGTATTTGGCTCCGTCGC-3’，
TVDVaRNAdR：5’-TGTACTGGAGTGCTTCAACCG-3’；
(2) CTAB method is extracted the total nucleic acid of catch an illness plant tissue or biography virus mediator insect, as RT-PCR amplification template;
(3) single stage method RT-PCR amplification;
(4) electrophoresis detection;
(5) Analysis of test results: according to every pair of primer amplification clip size and viral relation, observation is infected the tobacco material of TBTV, TVDVaRNA, Sat-TBTV and TVDV or is passed the different nucleic acid belts that detect in virus mediator insect, the kind of concrete TBTV, TVDVaRNA, Sat-TBTV and the TVDV pathogen infecting in judgement morbidity tobacco or biography virus mediator insect.
Described CTAB method is extracted diseased plant tissue or is passed the total nucleic acid of virus mediator insect, and step is as follows:
The first step, preparation of samples
(1) diseased plant tissue is prepared: 3 ~ 5 sick leaves are superimposed, take 50 ~ 100 mg to mortar;
(2) passing virus mediator insect prepares: get 30 ~ 50 aphids to mortar;
Second step, add the CTAB damping fluid of 1.2 mL, the EDTA of the Tris-HCl of 100 mM of the CTAB that this damping fluid is 2% by mass percent, 2% PVP-40 and pH 8.0, the NaCl of 1.4 M and 20 mM forms mixed solution, finally according to the volume of mixing solutions, adds 0.2% mercaptoethanol;
The 3rd step, constant temperature are processed: after fully grinding, proceed in the centrifuge tube of 1.5 mL, under 65 ℃ of temperature are bathed, process 15 ~ 60 min;
The 4th step, centrifugal treating: first, centrifuge tube is put into whizzer 12000 ~ 13000 r/min centrifugal treating 15 min, get 750 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, add the chloroform that 750 μ L volume ratios are 24: 1: primary isoamyl alcohol, vortex concussion mixes 30 s; Then, again the centrifuge tube that supernatant liquor is housed is put into whizzer 12000 ~ 13000 r/min centrifugal treating 10 min, then, with micropipet, draw 600 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, and add 600 μ L Virahols, and put upside down centrifuge tube and fully mix liquid, under room temperature, place 10 min, finally the centrifuge tube that supernatant liquor and Virahol are housed is put into whizzer 12000 ~ 13000 r/min centrifugal treating 15 min, with micropipet sucking-off supernatant liquor; Precipitation part adds 500 μ L 70 % ethanol, and puts into whizzer 12000 ~ 13000 r/min centrifugal treating 10 min;
The 5th step, dry: use micropipet sucking liquid, precipitation part seasoning 10 ~ 15 min under 20 ℃ ~ 25 ℃ conditions;
The 6th step, dissolving: add the Tris-HCl of 100 μ L 20 mmol/L pH8.0 and be placed in 10 min on ice, with micropipet piping and druming, make for 5 ~ 10 times precipitation part fully dissolve;
The 7th step, preservation: nucleic acid precipitation lysate-20 ℃ save backup; Or obtained total nucleic acid is put into-80 ℃ with dry powder, and to carry out prolonged preservation standby.
The PrimeScript that described single stage method RT-PCR adopts precious biotechnology (Dalian) company limited to produce
?one Step RT-PCR Kit Ver.2 test kit, reaction system is 15 μ L, by 2 * 1 Step Buffer of PrimeScript 1 Step Enzyme Mix, the 7.5 μ L of 0.6 μ L, the forward and reverse primer SatTBTVdF of 10 μ M of the forward and reverse primer TVDVaRNAdF of 10 μ M of the forward and reverse primer TBTVdF of 10 μ M of each 0.6 μ L and TBTVdR, each 0.5 μ L and TVDVaRNAdR, each 0.4 μ L and SatTBTVdR, the forward and reverse primer TVDVdF of 10 μ M of each 0.3 μ L and the ddH of TVDVdR, 2.8 μ L
2the RNA template of O and 0.5 μ L mixes.
The reaction conditions of described single stage method RT-PCR is: in the RT-PCR reaction by the synthetic cDNA of RNA, controlling temperature is 50 ℃, reaction times 30 min; Then when temperature is 94 ℃, keep 2 min, make RTase inactivation; Temperature is controlled at 94 ℃ and carries out sex change 30 sec, carries out anneal 30 sec, then between 55 ℃ ~ 60 ℃ of temperature, temperature is controlled at 72 ℃, extend 1 min, from sex change, to extending, carry out altogether 30 circulations, last 72 ℃ are extended PCR product after 10 min again and ℃ preserve in temperature-4.
In electrophoresis detection, adopt agarose gel electrophoresis to detect amplification, prepare the sepharose of 1.0 %, in the gel preparing, every 100 mL add 5 μ L Gold View staining agents to mix.In 0.5 * tbe buffer pendular ring border, 90 V voltage stabilizing electrophoresis 60 min, then observe and take pictures in gel imaging instrument.
Described detection primer amplification clip size is as shown in table 1 with the corresponding relation of the pathogen that detects:
Beneficial effect of the present invention:
(1) TBTV, TVDV, Sat-TBTV and TVDVaRNA are 4 kinds of pathogens finding in current tobacco bushy top disease, and these 4 kinds of pathogens often Combined Infection tobacco cause serious harm.The present invention is directed to these 4 kinds of pathogens and designed respectively multipair Auele Specific Primer, for reduce the interaction between primer as far as possible, through testing sieve repeatedly, select TBTVdF and TBTVdR, TVDVaRNAdF and TVDVaRNAdR, SatTBTVdF and SatTBTVdR, TVDVdF and these 4 pairs of high specificities of TVDVdR and mutual glitch-free detection primer, and these 4 pairs of primers can amplify target product respectively equably.
accompanying drawing explanation
Fig. 1 is specificity and the compatibility of multiple RT-PCR;
Fig. 2 is the multiple RT-PCR expanding effect of different extension rate RNA templates;
Fig. 3 is the detected result of Partial Tobacco clump top sickness field sample multiple RT-PCR;
In figure: swimming lane 1 is TBTV; Swimming lane 2 is TVDVaRNA; Swimming lane 3 is Sat-TBTV; Swimming lane 4 is TVDV; Swimming lane 5 is TBTV+Sat-TBTV; Swimming lane 6 is TVDVaRNA+TVDV; Swimming lane 7 is tobacco sample TBTV+TVDVaRNA+Sat-TBTV+TVDV detected result; Swimming lane 8 is viruliferous aphid worm TBTV+TVDVaRNA+Sat-TBTV+TVDV detected result; Swimming lane 9 is health tobacco blade amplification; Swimming lane 10 is template 10
0the expanding effect of * dilution; Swimming lane 11 is template 10
1the expanding effect of * dilution; Swimming lane 12 is template 10
2the expanding effect of * dilution; Swimming lane 13 is template 10
3the expanding effect of * dilution; Swimming lane 14 is 10
4the expanding effect of * dilution; Swimming lane 15 is the detected result of tobacco sample TBTV+Sat-TBTV; Swimming lane 16 is the detected result of tobacco sample TVDV; Swimming lane 17 is the detected result of health tobacco blade; Swimming lane 18 is the detected result of tobacco sample TBTV+TVDVaRNA+Sat-TBTV+TVDV; Swimming lane 19 is the detected result of viruliferous aphid worm TBTV+TVDVaRNA+Sat-TBTV+TVDV; Swimming lane 20 is the detected result of tobacco sample TVDVaRNA+TVDV; Swimming lane 21 is the detected result of tobacco sample TVDVaRNA+Sat-TBTV+TVDV; Swimming lane M is 2 Kb Plus DNA Ladder.
embodiment
Below in conjunction with drawings and Examples, the present invention is described in more detail, to facilitate the technical staff to understand.
embodiment 1:
The susceptible tobacco plant in field of take is material, and the method for synchronous detection TBTV, TVDV, Sat-TBTV and TVDVaRNA is carried out according to step below:
(1) design of primers is synthetic:
(a) forward primer TBTVdF, TVDVdF, SatTBTVdF and the TVDVaRNAdF that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdF：5’-TACCACACCTAAACAGCGTTG-3’，
TVDVdF：5’-GCAACAGCGAGACTTTCATCT-3’，
SatTBTVdF：5’-TGTTGGCCGTGGGCAGCAA-3’，
TVDVaRNAdF：5’-GCGTACTGCGGAAGTCTCAC-3’，
(b) reverse primer TBTVdR, TVDVdR, SatTBTVdR and the TVDVaRNAdR that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdR：5’-CTCATCTCCCGCTAAGTCAG-3’，
TVDVdR:5 '-CRTTGCCTTTATAGAGCAGCC-3 ', the wherein R=A in sequence or G,
SatTBTVdR：5’-TTCGTATTTGGCTCCGTCGC-3’，
TVDVaRNAdR：5’-TGTACTGGAGTGCTTCAACCG-3’；
Above-mentioned primer is synthetic by Beijing six directions Hua Da genome company;
(2) CTAB method is extracted the total nucleic acid of the plant tissue of catching an illness, and as RT-PCR amplification template, specifically according to step below, carries out:
The first step, diseased plant tissue are prepared: 3 sick leaves are superimposed, take 50 mg to mortar;
Second step, add the CTAB damping fluid of 1.2 mL, the EDTA of the Tris-HCl of 100 mM of the CTAB that this damping fluid is 2% by mass percent, 2% PVP-40 and pH 8.0, the NaCl of 1.4 M and 20 mM forms mixed solution, finally according to the volume of mixing solutions, adds 0.2% mercaptoethanol;
The 3rd step, constant temperature are processed: after fully grinding, proceed in the centrifuge tube of 1.5 mL, under 65 ℃ of temperature are bathed, process 15 min;
The 4th step, centrifugal treating: first, centrifuge tube is put into whizzer 12000 r/min centrifugal treating 15 min, get 750 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, add the chloroform that 750 μ L volume ratios are 24: 1: primary isoamyl alcohol, vortex concussion mixes 30 s; Then, again the centrifuge tube that supernatant liquor is housed is put into whizzer 12000 r/min centrifugal treating 10 min, then, with micropipet, draw 600 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, and add 600 μ L Virahols, and put upside down centrifuge tube and fully mix liquid, under room temperature, place 10 min, finally the centrifuge tube that supernatant liquor and Virahol are housed is put into whizzer 12000 r/min centrifugal treating 15 min, with micropipet sucking-off supernatant liquor; Precipitation part adds 500 μ L 70 % ethanol, and puts into whizzer 12000 r/min centrifugal treating 10 min;
The 5th step, dry: use micropipet sucking liquid, precipitation part seasoning 10 min under 20 ℃ of conditions;
The 6th step, dissolving: add the Tris-HCl of 100 μ L 20 mmol/L pH8.0 and be placed in 10 min on ice, with micropipet piping and druming, make for 5 times precipitation part fully dissolve;
The 7th step, preservation: nucleic acid precipitation lysate-20 ℃ save backup.
(3) single stage method RT-PCR amplification: the PrimeScript that described single stage method RT-PCR adopts precious biotechnology (Dalian) company limited to produce
?one Step RT-PCR Kit Ver.2 test kit, reaction system is 15 μ L, by 2 * 1 Step Buffer of PrimeScript 1 Step Enzyme Mix, the 7.5 μ L of 0.6 μ L, the forward and reverse primer SatTBTVdF of 10 μ M of the forward and reverse primer TVDVaRNAdF of 10 μ M of the forward and reverse primer TBTVdF of 10 μ M of each 0.6 μ L and TBTVdR, each 0.5 μ L and TVDVaRNAdR, each 0.4 μ L and SatTBTVdR, the forward and reverse primer TVDVdF of 10 μ M of each 0.3 μ L and the ddH of TVDVdR, 2.8 μ L
2the RNA template of O and 0.5 μ L mixes.
The reaction conditions of described single stage method RT-PCR is: in the RT-PCR reaction by the synthetic cDNA of RNA, controlling temperature is 50 ℃, reaction times 30 min; Then when temperature is 94 ℃, keep 2 min, make RTase inactivation; Temperature is controlled at 94 ℃ and carries out sex change 30 sec, at 55 ℃ of anneal 30 sec, then, temperature is controlled at 72 ℃, extend 1 min, from sex change, to extending, carry out altogether 30 circulations, last 72 ℃ are extended PCR product after 10 min again and ℃ preserve in temperature-4.
(4) electrophoresis detection: adopt agarose gel electrophoresis to detect amplification in electrophoresis detection, prepare 1.0% sepharose, every 100 mL add 5 μ L Gold View staining agents to mix in the gel preparing.In 0.5 * tbe buffer pendular ring border, 90 V voltage stabilizing electrophoresis 60 min, then observe and take pictures in gel imaging instrument.
(5) Analysis of test results: observe and find, 1049 bp, 792 bp, 598 bp and 4 nucleic acid belts of 357 bp from the susceptible tobacco plant in field, have been detected, and any band in normal healthy controls, do not detected, according to the every pair of primer amplification clip size in table 1 and viral relation, band referring to 17 and No. 18 swimming lanes of Fig. 3, in the tobacco bushy top disease tobacco leaf of judgement field morbidity, infected TBTV, TVDVaRNA, Sat-TBTV and 4 kinds of pathogens of TVDV, primer specificity is strong, compatible high.
proof analysis: amplified production authenticity verification
Select 1049 bp, 792 bp, 598 bp and 4 nucleic acid belts of 357 bp in above-mentioned amplified production, under ultraviolet lamp, from gel, cut respectively respective strap, after the AxyPrepTM DNA Gel Extraction Kit 50-prp purifying of biotechnology (Hangzhou) company limited of adopt liking to pursue progress reclaims, to reclaim the pMD18-T Vector that product connects most valuable treasure biotechnology (Dalian) company limited, molecular cloning method is converted into routinely
e. coliin DH5 α competent cell, adopt the plasmid of Beijing hundred Tyke Bioisystech Co., Ltd to prepare in a small amount test kit (centrifugal column type) extraction recombinant plasmid, each fragment at least selects 3 positive colonies to send Beijing Liuhe Huada Genomics Technology Co., Ltd to carry out two-way order-checking.Sequential analysis shows, the Nucleotide consistence of the TBTV logining in the cDNA of 1049 bp and GenBank reaches 99%, the Nucleotide consistence of the TVDVaRNA logining in the cDNA of 792 bp and GenBank reaches 96%, the Nucleotide consistence of the Sat-TBTV logining in the cDNA of 598 bp and GenBank reaches 99%, the Nucleotide consistence of the TVDV logining in the cDNA of 357 bp and GenBank reaches 99%, 1049 bp that proof obtains from the susceptible tobacco in field by the present invention, 792 bp, 598 bp and 4 nucleic acid belts of 357 bp are exactly TBTV, TVDVaRNA, the corresponding cDNA amplified production of Sat-TBTV and TVDV, amplified production of the present invention is genuine and believable.
embodiment 2:
The susceptible tobacco plant of greenhouse artificial inoculation of take is material, and the method for synchronous detection TBTV, TVDV, Sat-TBTV and TVDVaRNA is carried out according to step below:
(1) design of primers is synthetic:
(a) forward primer TBTVdF, TVDVdF, SatTBTVdF and the TVDVaRNAdF that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdF：5’-TACCACACCTAAACAGCGTTG-3’，
TVDVdF：5’-GCAACAGCGAGACTTTCATCT-3’，
SatTBTVdF：5’-TGTTGGCCGTGGGCAGCAA-3’，
TVDVaRNAdF：5’-GCGTACTGCGGAAGTCTCAC-3’，
(b) reverse primer TBTVdR, TVDVdR, SatTBTVdR and the TVDVaRNAdR that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdR：5’-CTCATCTCCCGCTAAGTCAG-3’，
TVDVdR:5 '-CRTTGCCTTTATAGAGCAGCC-3 ', the wherein R=A in sequence or G,
SatTBTVdR：5’-TTCGTATTTGGCTCCGTCGC-3’，
TVDVaRNAdR：5’-TGTACTGGAGTGCTTCAACCG-3’，
Above-mentioned primer is synthetic by Beijing six directions Hua Da genome company;
(2) CTAB method is extracted the total nucleic acid of the plant tissue of catching an illness, and as RT-PCR amplification template, specifically according to step below, carries out:
The first step, diseased plant tissue are prepared: 4 sick leaves are superimposed, take 70 mg to mortar;
Second step, add the CTAB damping fluid of 1.2 mL, the EDTA of the Tris-HCl of 100 mM of the CTAB that this damping fluid is 2% by mass percent, 2% PVP-40 and pH 8.0, the NaCl of 1.4 M and 20 mM forms mixed solution, finally according to the volume of mixing solutions, adds 0.2% mercaptoethanol;
The 3rd step, constant temperature are processed: after fully grinding, proceed in the centrifuge tube of 1.5 mL, under 65 ℃ of temperature are bathed, process 35 min;
The 4th step, centrifugal treating: first, centrifuge tube is put into whizzer 12500 r/min centrifugal treating 15 min, get 750 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, add the chloroform that 750 μ L volume ratios are 24: 1: primary isoamyl alcohol, vortex concussion mixes 30 s; Then, again the centrifuge tube that supernatant liquor is housed is put into whizzer 12500 r/min centrifugal treating 10 min, then, with micropipet, draw 600 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, and add 600 μ L Virahols, and put upside down centrifuge tube and fully mix liquid, under room temperature, place 10 min, finally the centrifuge tube that supernatant liquor and Virahol are housed is put into whizzer 12500 r/min centrifugal treating 15 min, with micropipet sucking-off supernatant liquor; Precipitation part adds 500 μ L 70% ethanol, and puts into whizzer 12500 r/min centrifugal treating 10 min;
The 5th step, dry: use micropipet sucking liquid, precipitation part seasoning 13 min under 23 ℃ of conditions;
The 6th step, dissolving: add the Tris-HCl of 100 μ L 20 mmol/L pH8.0 and be placed in 10 min on ice, with micropipet piping and druming, make for 7 times precipitation part fully dissolve;
The 7th step, preservation: nucleic acid precipitation lysate-20 ℃ save backup.
(3) single stage method RT-PCR amplification: the PrimeScript that described single stage method RT-PCR adopts precious biotechnology (Dalian) company limited to produce
?one Step RT-PCR Kit Ver.2 test kit, reaction system is 15 μ L, by 2 * 1 Step Buffer of PrimeScript 1 Step Enzyme Mix, the 7.5 μ L of 0.6 μ L, the forward and reverse primer SatTBTVdF of 10 μ M of the forward and reverse primer TVDVaRNAdF of 10 μ M of the forward and reverse primer TBTVdF of 10 μ M of each 0.6 μ L and TBTVdR, each 0.5 μ L and TVDVaRNAdR, each 0.4 μ L and SatTBTVdR, the forward and reverse primer TVDVdF of 10 μ M of each 0.3 μ L and the ddH of TVDVdR, 2.8 μ L
2the RNA template of O and 0.5 μ L mixes.
The reaction conditions of described single stage method RT-PCR is: in the RT-PCR reaction by the synthetic cDNA of RNA, controlling temperature is 50 ℃, reaction times 30 min; Then when temperature is 94 ℃, keep 2 min, make RTase inactivation; Temperature is controlled at 94 ℃ and carries out sex change 30 sec, at 60 ℃ of anneal 30 sec, then, temperature is controlled at 72 ℃, extend 1 min, from sex change, to extending, carry out altogether 30 circulations, last 72 ℃ are extended PCR product after 10 min again and ℃ preserve in temperature-4.
(4) electrophoresis detection: adopt agarose gel electrophoresis to detect amplification in electrophoresis detection, prepare the sepharose of 1.0 %, every 100 mL add 5 μ L Gold View staining agents to mix in the gel preparing.In 0.5 * tbe buffer pendular ring border, 90 V voltage stabilizing electrophoresis 60 min, then observe and take pictures in gel imaging instrument.
(5) Analysis of test results: observe and find, 1049 bp, 792 bp, 598bp and 4 nucleic acid belts of 357 bp in tobacco plant, have been detected, and any band in normal healthy controls, do not detected, according to the every pair of primer amplification clip size in table 1 and viral relation, 7 and No. 9 swimming lanes referring to Fig. 1, in the tobacco bushy top disease tobacco leaf of judgement greenhouse artificial inoculation morbidity, infected TBTV, TVDVaRNA, Sat-TBTV and 4 kinds of pathogens of TVDV, reproducible.
proof analysis: amplified production authenticity verification
1049 bp in the above-mentioned amplified production of random choose, 792 bp, 598bp and 4 nucleic acid belts of 357 bp, under ultraviolet lamp, from gel, cut respectively respective strap, after the AxyPrepTM DNA Gel Extraction Kit 50-prp purifying of biotechnology (Hangzhou) company limited of adopt liking to pursue progress reclaims, to reclaim the pMD18-T Vector that product connects most valuable treasure biotechnology (Dalian) company limited, molecular cloning method is converted into routinely
e. colidH5 α competent cell, adopt the plasmid of Beijing hundred Tyke Bioisystech Co., Ltd to prepare in a small amount test kit (centrifugal column type) extraction recombinant plasmid, each fragment at least selects 3 positive colonies to send Beijing Liuhe Huada Genomics Technology Co., Ltd to carry out two-way order-checking.Sequential analysis shows, the Nucleotide consistence of the TBTV logining in the cDNA of 1049 bp and GenBank reaches 98%, the Nucleotide consistence of the TVDVaRNA logining in the cDNA of 792 bp and GenBank reaches 96%, the Nucleotide consistence of the Sat-TBTV logining in the cDNA of 598 bp and GenBank reaches 96%, the Nucleotide consistence of the TVDV logining in the cDNA of 357 bp and GenBank reaches 99%, 1049 bp that obtain in the susceptible tobacco of proof by greenhouse of the present invention artificial inoculation, 792 bp, 598 bp and 4 nucleic acid belts of 357 bp are exactly TBTV, TVDVaRNA, the corresponding cDNA amplified production of Sat-TBTV and TVDV, proving again amplified production of the present invention is genuine and believable.
embodiment 3:
The susceptible tobacco of a kind of greenhouse artificial inoculation of take is material, and the method for synchronous detection TBTV, TVDV, Sat-TBTV and TVDVaRNA is carried out according to step below:
(1) design of primers is synthetic:
(a) forward primer TBTVdF, TVDVdF, SatTBTVdF and the TVDVaRNAdF that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdF：5’-TACCACACCTAAACAGCGTTG-3’，
TVDVdF：5’-GCAACAGCGAGACTTTCATCT-3’，
SatTBTVdF：5’-TGTTGGCCGTGGGCAGCAA-3’，
TVDVaRNAdF：5’-GCGTACTGCGGAAGTCTCAC-3’，
(b) reverse primer TBTVdR, TVDVdR, SatTBTVdR and the TVDVaRNAdR that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdR：5’-CTCATCTCCCGCTAAGTCAG-3’，
TVDVdR:5 '-CRTTGCCTTTATAGAGCAGCC-3 ', the wherein R=A in sequence or G,
SatTBTVdR：5’-TTCGTATTTGGCTCCGTCGC-3’，
TVDVaRNAdR：5’-TGTACTGGAGTGCTTCAACCG-3’，
Above-mentioned primer is synthetic by Beijing six directions Hua Da genome company;
(2) CTAB method is extracted the total nucleic acid of the plant tissue of catching an illness, and as RT-PCR amplification template, specifically according to step below, carries out:
The first step, diseased plant tissue are prepared: 5 sick leaves are superimposed, take 100 mg to mortar;
Second step, add the CTAB damping fluid of 1.2 mL, the EDTA of the Tris-HCl of 100 mM of the CTAB that this damping fluid is 2% by mass percent, 2% PVP-40 and pH 8.0, the NaCl of 1.4 M and 20 mM forms mixed solution, finally according to the volume of mixing solutions, adds 0.2% mercaptoethanol;
The 3rd step, constant temperature are processed: after fully grinding, proceed in the centrifuge tube of 1.5 mL, under 65 ℃ of temperature are bathed, process 60 min;
The 4th step, centrifugal treating: first, centrifuge tube is put into whizzer 13000 r/min centrifugal treating 15 min, get 750 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, add the chloroform that 750 μ L volume ratios are 24: 1: primary isoamyl alcohol, vortex concussion mixes 30 s; Then, again the centrifuge tube that supernatant liquor is housed is put into whizzer 12500 r/min centrifugal treating 10 min, then, with micropipet, draw 600 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, and add 600 μ L Virahols, and put upside down centrifuge tube and fully mix liquid, under room temperature, place 10 min, finally the centrifuge tube that supernatant liquor and Virahol are housed is put into whizzer 13000 r/min centrifugal treating 15 min, with micropipet sucking-off supernatant liquor; Precipitation part adds 500 μ L 70% ethanol, and puts into whizzer 13000 r/min centrifugal treating 10 min;
The 5th step, dry: use micropipet sucking liquid, precipitation part seasoning 15 min under 25 ℃ of conditions;
The 6th step, dissolving: add the Tris-HCl of 100 μ L 20 mmol/L pH8.0 and be placed in 10 min on ice, with micropipet piping and druming, make for 10 times precipitation part fully dissolve;
The 7th step, preservation: obtained total nucleic acid is put into-80 ℃ with dry powder, and to carry out prolonged preservation standby.
(3) single stage method RT-PCR amplification: the PrimeScript that described single stage method RT-PCR adopts precious biotechnology (Dalian) company limited to produce
?one Step RT-PCR Kit Ver.2 test kit, reaction system is 15 μ L, by 2 * 1 Step Buffer of PrimeScript 1 Step Enzyme Mix, the 7.5 μ L of 0.6 μ L, the forward and reverse primer SatTBTVdF of 10 μ M of the forward and reverse primer TVDVaRNAdF of 10 μ M of the forward and reverse primer TBTVdF of 10 μ M of each 0.6 μ L and TBTVdR, each 0.5 μ L and TVDVaRNAdR, each 0.4 μ L and SatTBTVdR, the forward and reverse primer TVDVdF of 10 μ M of each 0.3 μ L and the ddH of TVDVdR, 2.8 μ L
2the RNA template of O and 0.5 μ L mixes.
The reaction conditions of described single stage method RT-PCR is: in the RT-PCR reaction by the synthetic cDNA of RNA, controlling temperature is 50 ℃, reaction times 30 min; Then when temperature is 94 ℃, keep 2 min, make RTase inactivation; Temperature is controlled at 94 ℃ and carries out sex change 30 sec, at 57 ℃ of anneal 30 sec, then, temperature is controlled at 72 ℃, extend 1 min, from sex change, to extending, carry out altogether 30 circulations, last 72 ℃ are extended PCR product after 10 min again and ℃ preserve in temperature-4.
(4) electrophoresis detection: adopt agarose gel electrophoresis to detect amplification in electrophoresis detection, prepare the sepharose of 1.0 %, every 100 mL add 5 μ L Gold View staining agents to mix in the gel preparing.In 0.5 * tbe buffer pendular ring border, 90 V voltage stabilizing electrophoresis 60 min, then observe and take pictures in gel imaging instrument.
(5) Analysis of test results: observe and find, 1049 bp and 2 nucleic acid belts of 598 bp in susceptible tobacco plant, have been detected, and any band in normal healthy controls, do not detected, according to the every pair of primer amplification clip size in table 1 and viral relation, swimming lane 5 and 9 referring to Fig. 1, in the tobacco bushy top disease tobacco leaf of judgement greenhouse artificial inoculation morbidity, infected TBTV and 2 kinds of pathogens of Sat-TBTV, reproducible.
proof analysis: amplified production authenticity verification
1049 bp in the above-mentioned amplified production of random choose and 2 nucleic acid belts of 598 bp, under ultraviolet lamp, from gel, cut respectively respective strap, after the AxyPrepTM DNA Gel Extraction Kit 50-prp purifying of biotechnology (Hangzhou) company limited of adopt liking to pursue progress reclaims, to reclaim the pMD18-T Vector that product connects most valuable treasure biotechnology (Dalian) company limited, molecular cloning method is converted into routinely
e. coliin DH5 α competent cell, adopt the plasmid of Beijing hundred Tyke Bioisystech Co., Ltd to prepare in a small amount test kit (centrifugal column type) extraction recombinant plasmid, each fragment at least selects 3 positive colonies to send Beijing Liuhe Huada Genomics Technology Co., Ltd to carry out two-way order-checking.Sequential analysis shows, the Nucleotide consistence of the TBTV logining in the cDNA of 1049 bp and GenBank reaches 98%, the Nucleotide consistence of the Sat-TBTV logining in the cDNA of 598 bp and GenBank reaches 96%, 1049 bp that obtain in the susceptible tobacco of proof by greenhouse of the present invention artificial inoculation and 2 nucleic acid belts of 598 bp are exactly the corresponding cDNA amplified production of TBTV and Sat-TBTV, and proving again amplified production of the present invention is genuine and believable.
embodiment 4:
Take field, to pass virulent aphis worm be material, and the method for synchronous detection TBTV, TVDV, Sat-TBTV and TVDVaRNA is carried out according to step below:
(1) design of primers is synthetic:
(a) forward primer TBTVdF, TVDVdF, SatTBTVdF and the TVDVaRNAdF that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdF：5’-TACCACACCTAAACAGCGTTG-3’，
TVDVdF：5’-GCAACAGCGAGACTTTCATCT-3’，
SatTBTVdF：5’-TGTTGGCCGTGGGCAGCAA-3’，
TVDVaRNAdF：5’-GCGTACTGCGGAAGTCTCAC-3’，
(b) reverse primer TBTVdR, TVDVdR, SatTBTVdR and the TVDVaRNAdR that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdR：5’-CTCATCTCCCGCTAAGTCAG-3’，
TVDVdR:5 '-CRTTGCCTTTATAGAGCAGCC-3 ', the wherein R=A in sequence or G,
SatTBTVdR：5’-TTCGTATTTGGCTCCGTCGC-3’，
TVDVaRNAdR：5’-TGTACTGGAGTGCTTCAACCG-3’，
Above-mentioned primer is synthetic by Beijing six directions Hua Da genome company;
(2) CTAB method is extracted the total nucleic acid that passes virus mediator insect, as RT-PCR amplification template; Specifically according to step below, carry out:
The first step, biography virus mediator insect are prepared: get 30 aphids to mortar;
Second step, add the CTAB damping fluid of 1.2 mL, the EDTA of the Tris-HCl of 100 mM of the CTAB that this damping fluid is 2% by mass percent, 2% PVP-40 and pH 8.0, the NaCl of 1.4 M and 20 mM forms mixed solution, finally according to the volume of mixing solutions, adds 0.2% mercaptoethanol;
The 3rd step, constant temperature are processed: after fully grinding, proceed in the centrifuge tube of 1.5 mL, under 65 ℃ of temperature are bathed, process 15 min;
The 4th step, centrifugal treating: first, centrifuge tube is put into whizzer 12000 r/min centrifugal treating 15 min, get 750 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, add the chloroform that 750 μ L volume ratios are 24: 1: primary isoamyl alcohol, vortex concussion mixes 30 s; Then, again the centrifuge tube that supernatant liquor is housed is put into whizzer 12000 r/min centrifugal treating 10 min, then, with micropipet, draw 600 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, and add 600 μ L Virahols, and put upside down centrifuge tube and fully mix liquid, under room temperature, place 10 min, finally the centrifuge tube that supernatant liquor and Virahol are housed is put into whizzer 12000 r/min centrifugal treating 15 min, with micropipet sucking-off supernatant liquor; Precipitation part adds 500 μ L 70% ethanol, and puts into whizzer 12000 r/min centrifugal treating 10 min;
The 5th step, dry: use micropipet sucking liquid, precipitation part seasoning 10 min under 20 ℃ of conditions;
The 6th step, dissolving: add the Tris-HCl of 100 μ L 20 mmol/L pH8.0 and be placed in 10 min on ice, with micropipet piping and druming, make for 5 times precipitation part fully dissolve;
The 7th step, preservation: nucleic acid precipitation lysate-20 ℃ save backup.
(3) single stage method RT-PCR amplification: the PrimeScript that described single stage method RT-PCR adopts precious biotechnology (Dalian) company limited to produce
?one Step RT-PCR Kit Ver.2 test kit, reaction system is 15 μ L, by 2 * 1 Step Buffer of PrimeScript 1 Step Enzyme Mix, the 7.5 μ L of 0.6 μ L, the forward and reverse primer SatTBTVdF of 10 μ M of the forward and reverse primer TVDVaRNAdF of 10 μ M of the forward and reverse primer TBTVdF of 10 μ M of each 0.6 μ L and TBTVdR, each 0.5 μ L and TVDVaRNAdR, each 0.4 μ L and SatTBTVdR, the forward and reverse primer TVDVdF of 10 μ M of each 0.3 μ L and the ddH of TVDVdR, 2.8 μ L
2the RNA template of O and 0.5 μ L mixes.
The reaction conditions of described single stage method RT-PCR is: in the RT-PCR reaction by the synthetic cDNA of RNA, controlling temperature is 50 ℃, reaction times 30 min; Then when temperature is 94 ℃, keep 2 min, make RTase inactivation; Temperature is controlled at 94 ℃ and carries out sex change 30 sec, at 55 ℃ of anneal 30 sec, then, temperature is controlled at 72 ℃, extend 1 min, from sex change, to extending, carry out altogether 30 circulations, last 72 ℃ are extended PCR product after 10 min again and ℃ preserve in temperature-4.
(4) electrophoresis detection: adopt agarose gel electrophoresis to detect amplification in electrophoresis detection, prepare 1.0% sepharose, every 100 mL add 5 μ L Gold View staining agents to mix in the gel preparing.In 0.5 * tbe buffer pendular ring border, 90 V voltage stabilizing electrophoresis 60 min, then observe and take pictures in gel imaging instrument.
(5) Analysis of test results: observe and find, 1049 bp, 792 bp, 598bp and 4 nucleic acid belts of 357 bp in aphid material, have been detected, according to the every pair of primer amplification clip size in table 1 and viral relation, No. 19 swimming lanes referring to Fig. 3, judgement field passes TBTV, TVDVaRNA, Sat-TBTV and the 4 kinds of pathogens of TVDV that infect in virulent aphis worm, reproducible.
proof analysis: amplified production authenticity verification
1049 bp in the above-mentioned amplified production of random choose, 792 bp, 598bp and 4 nucleic acid belts of 357 bp, under ultraviolet lamp, from gel, cut respectively respective strap, after the AxyPrepTM DNA Gel Extraction Kit 50-prp purifying of biotechnology (Hangzhou) company limited of adopt liking to pursue progress reclaims, to reclaim the pMD18-T Vector that product connects most valuable treasure biotechnology (Dalian) company limited, molecular cloning method is converted into routinely
e. colidH5 α competent cell, adopt the plasmid of Beijing hundred Tyke Bioisystech Co., Ltd to prepare in a small amount test kit (centrifugal column type) extraction recombinant plasmid, each fragment at least selects 3 positive colonies to send Beijing Liuhe Huada Genomics Technology Co., Ltd to carry out two-way order-checking.Sequential analysis shows, the Nucleotide consistence of the TBTV logining in the cDNA of 1049 bp and GenBank reaches 98%, the Nucleotide consistence of the TVDVaRNA logining in the cDNA of 792bp and GenBank reaches 96%, the Nucleotide consistence of the Sat-TBTV logining in the cDNA of 598 bp and GenBank reaches 96%, the Nucleotide consistence of the TVDV logining in the cDNA of 357 bp and GenBank reaches 99%, proof passes 1049 bp that obtain malicious aphid from field by the present invention, 792bp, 598 bp and 4 nucleic acid belts of 357 bp are exactly TBTV, TVDVaRNA, the corresponding cDNA amplified production of Sat-TBTV and TVDV, proving again amplified production of the present invention is genuine and believable.
embodiment 5:
Take greenhouse manually raises virulent aphis worm as material, and the method for synchronous detection TBTV, TVDV, Sat-TBTV and TVDVaRNA is carried out according to step below:
(1) design of primers is synthetic:
(a) forward primer TBTVdF, TVDVdF, SatTBTVdF and the TVDVaRNAdF that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdF：5’-TACCACACCTAAACAGCGTTG-3’，
TVDVdF：5’-GCAACAGCGAGACTTTCATCT-3’，
SatTBTVdF：5’-TGTTGGCCGTGGGCAGCAA-3’，
TVDVaRNAdF：5’-GCGTACTGCGGAAGTCTCAC-3’，
(b) reverse primer TBTVdR, TVDVdR, SatTBTVdR and the TVDVaRNAdR that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdR:5 '-CTCATCTCCCGCTAAGTCAG-3 ', wherein, the R=A in sequence or G,
TVDVdR：5’-CRTTGCCTTTATAGAGCAGCC-3’，
SatTBTVdR：5’-TTCGTATTTGGCTCCGTCGC-3’，
TVDVaRNAdR：5’-TGTACTGGAGTGCTTCAACCG-3’，
Above-mentioned primer is synthetic by Beijing six directions Hua Da genome company;
(2) CTAB method is extracted the total nucleic acid that passes virus mediator insect, as RT-PCR amplification template; Specifically according to step below, carry out:
The preparation of the first step, biography virus mediator insect: get 40 aphids to mortar;
Second step, add the CTAB damping fluid of 1.2 mL, the EDTA of the Tris-HCl of 100 mM of the CTAB that this damping fluid is 2% by mass percent, 2% PVP-40 and pH 8.0, the NaCl of 1.4 M and 20 mM forms mixed solution, finally according to the volume of mixing solutions, adds 0.2% mercaptoethanol;
The 3rd step, constant temperature are processed: after fully grinding, proceed in the centrifuge tube of 1.5 mL, under 65 ℃ of temperature are bathed, process 35 min;
The 4th step, centrifugal treating: first, centrifuge tube is put into whizzer 12500 r/min centrifugal treating 15 min, get 750 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, add the chloroform that 750 μ L volume ratios are 24: 1: primary isoamyl alcohol, vortex concussion mixes 30 s; Then, again the centrifuge tube that supernatant liquor is housed is put into whizzer 12500 r/min centrifugal treating 10 min, then, with micropipet, draw 600 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, and add 600 μ L Virahols, and put upside down centrifuge tube and fully mix liquid, under room temperature, place 10 min, finally the centrifuge tube that supernatant liquor and Virahol are housed is put into whizzer 12500 r/min centrifugal treating 15 min, with micropipet sucking-off supernatant liquor; Precipitation part adds 500 μ L 70 % ethanol, and puts into whizzer 12500 r/min centrifugal treating 10 min;
The 5th step, dry: use micropipet sucking liquid, precipitation part seasoning 10 min under 23 ℃ of conditions;
The 6th step, dissolving: add the Tris-HCl of 100 μ L 20 mmol/L pH8.0 and be placed in 10 min on ice, with micropipet piping and druming, make for 7 times precipitation part fully dissolve;
The 7th step, preservation: nucleic acid precipitation lysate-20 ℃ save backup.
(3) single stage method RT-PCR amplification: the PrimeScript that described single stage method RT-PCR adopts precious biotechnology (Dalian) company limited to produce
?one Step RT-PCR Kit Ver.2 test kit, reaction system is 15 μ L, by 2 * 1 Step Buffer of PrimeScript 1 Step Enzyme Mix, the 7.5 μ L of 0.6 μ L, the forward and reverse primer SatTBTVdF of 10 μ M of the forward and reverse primer TVDVaRNAdF of 10 μ M of the forward and reverse primer TBTVdF of 10 μ M of each 0.6 μ L and TBTVdR, each 0.5 μ L and TVDVaRNAdR, each 0.4 μ L and SatTBTVdR, the forward and reverse primer TVDVdF of 10 μ M of each 0.3 μ L and the ddH of TVDVdR, 2.8 μ L
2the RNA template of O and 0.5 μ L mixes.
The reaction conditions of described single stage method RT-PCR is: in the RT-PCR reaction by the synthetic cDNA of RNA, controlling temperature is 50 ℃, reaction times 30 min; Then when temperature is 94 ℃, keep 2min, make RTase inactivation; Temperature is controlled at 94 ℃ and carries out sex change 30 sec, at 57 ℃ of anneal 30 sec, then, temperature is controlled at 72 ℃, extend 1 min, from sex change, to extending, carry out altogether 30 circulations, last 72 ℃ are extended PCR product after 10 min again and ℃ preserve in temperature-4.
(4) electrophoresis detection: adopt agarose gel electrophoresis to detect amplification in electrophoresis detection, prepare 1.0% sepharose, every 100 mL add 5 μ L Gold View staining agents to mix in the gel preparing.In 0.5 * tbe buffer pendular ring border, 90 V voltage stabilizing electrophoresis 60 min, then observe and take pictures in gel imaging instrument.
(5) Analysis of test results: observe and find, 1049 bp, 792 bp, 598bp and 4 nucleic acid belts of 357 bp in aphid material, have been detected, and any band in normal healthy controls, do not detected, according to the every pair of primer amplification clip size in table one and viral relation, 8 and No. 9 swimming lanes referring to Fig. 1, judgement greenhouse manually raised virulent aphis insect infection TBTV, TVDVaRNA, Sat-TBTV and 4 kinds of pathogens of TVDV, reproducible.
proof analysis: amplified production authenticity verification
1049 bp in the above-mentioned amplified production of random choose, 792 bp, 598bp and 4 nucleic acid belts of 357 bp, under ultraviolet lamp, from gel, cut respectively respective strap, after the AxyPrepTM DNA Gel Extraction Kit 50-prp purifying of biotechnology (Hangzhou) company limited of adopt liking to pursue progress reclaims, to reclaim the pMD18-T Vector that product connects most valuable treasure biotechnology (Dalian) company limited, molecular cloning method is converted into routinely
e. colidH5 α competent cell, adopt the plasmid of Beijing hundred Tyke Bioisystech Co., Ltd to prepare in a small amount test kit (centrifugal column type) extraction recombinant plasmid, each fragment at least selects 3 positive colonies to send Beijing Liuhe Huada Genomics Technology Co., Ltd to carry out two-way order-checking.Sequential analysis shows, the Nucleotide consistence of the TBTV logining in the cDNA of 1049 bp and GenBank reaches 98%, the Nucleotide consistence of the TVDVaRNA logining in the cDNA of 792bp and GenBank reaches 96%, the Nucleotide consistence of the Sat-TBTV logining in the cDNA of 598 bp and GenBank reaches 96%, the Nucleotide consistence of the TVDV logining in the cDNA of 357 bp and GenBank reaches 99%, proof is manually raised 1049 bp that obtain virulent aphis worm from greenhouse by the present invention, 792bp, 598 bp and 4 nucleic acid belts of 357 bp are exactly TBTV, TVDVaRNA, the corresponding cDNA amplified production of Sat-TBTV and TVDV, proving again amplified production of the present invention is genuine and believable.
embodiment 6:
1, experiment material
The susceptible tobacco plant in field of take is material, and the method for synchronous detection TBTV, TVDV, Sat-TBTV and TVDVaRNA is carried out according to step below:
(1) design of primers is synthetic:
(a) forward primer TBTVdF, TVDVdF, SatTBTVdF and the TVDVaRNAdF that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdF：5’-TACCACACCTAAACAGCGTTG-3’，
TVDVdF：5’-GCAACAGCGAGACTTTCATCT-3’，
SatTBTVdF：5’-TGTTGGCCGTGGGCAGCAA-3’，
TVDVaRNAdF：5’-GCGTACTGCGGAAGTCTCAC-3’，
(b) reverse primer TBTVdR, TVDVdR, SatTBTVdR and the TVDVaRNAdR that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdR：5’-CTCATCTCCCGCTAAGTCAG-3’，
TVDVdR:5 '-CRTTGCCTTTATAGAGCAGCC-3 ', wherein, the R=A in sequence or G,
SatTBTVdR：5’-TTCGTATTTGGCTCCGTCGC-3’，
TVDVaRNAdR：5’-TGTACTGGAGTGCTTCAACCG-3’，
Above-mentioned primer is synthetic by Beijing six directions Hua Da genome company;
(2) CTAB method is extracted the total nucleic acid of the plant tissue of catching an illness, and as RT-PCR amplification template, specifically according to step below, carries out:
The first step, diseased plant tissue are prepared: 5 sick leaves are superimposed, take 80 mg to mortar;
Second step, add the CTAB damping fluid of 1.2 mL, the EDTA of the Tris-HCl of 100 mM of the CTAB that this damping fluid is 2% by mass percent, 2% PVP-40 and pH 8.0, the NaCl of 1.4 M and 20 mM forms mixed solution, finally according to the volume of mixing solutions, adds 0.2% mercaptoethanol;
The 3rd step, constant temperature are processed: after fully grinding, proceed in the centrifuge tube of 1.5 mL, under 65 ℃ of temperature are bathed, process 60 min;
The 4th step, centrifugal treating: first, centrifuge tube is put into whizzer 13000 r/min centrifugal treating 15 min, get 750 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, the chloroform that the volume ratio that adds 750 μ L is 24: 1: primary isoamyl alcohol, vortex concussion mixes 30 s; Then, again the centrifuge tube that supernatant liquor is housed is put into whizzer 13000 r/min centrifugal treating 10 min, then, with micropipet, draw 600 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, and add 600 μ L Virahols, and put upside down centrifuge tube and fully mix liquid, under room temperature, place 10 min, finally the centrifuge tube that supernatant liquor and Virahol are housed is put into whizzer 13000 r/min centrifugal treating 15 min, with micropipet sucking-off supernatant liquor; Precipitation part adds 500 μ L 70 % ethanol, and puts into whizzer 13000 r/min centrifugal treating 10 min;
The 5th step, dry: use micropipet sucking liquid, precipitation part seasoning 10min under 25 ℃ of conditions;
The 6th step, dissolving: add 100 μ L 20 mmol/L
pthe Tris-HCl of H8.0 is also placed in 10 min on ice, with micropipet piping and druming, makes for 10 times precipitation part fully dissolve;
The 7th step, preservation: nucleic acid precipitation lysate-20 ℃ save backup.
(3) single stage method RT-PCR amplification: the PrimeScript that described single stage method RT-PCR adopts precious biotechnology (Dalian) company limited to produce
?one Step RT-PCR Kit Ver.2 test kit, reaction system is 15 μ L, by 2 * 1 Step Buffer of PrimeScript 1 Step Enzyme Mix, the 7.5 μ L of 0.6 μ L, the forward and reverse primer SatTBTVdF of 10 μ M of the forward and reverse primer TVDVaRNAdF of 10 μ M of the forward and reverse primer TBTVdF of 10 μ M of each 0.6 μ L and TBTVdR, each 0.5 μ L and TVDVaRNAdR, each 0.4 μ L and SatTBTVdR, the forward and reverse primer TVDVdF of 10 μ M of each 0.3 μ L and the ddH of TVDVdR, 2.8 μ L
2the RNA template of O and 0.5 μ L mixes.
The reaction conditions of described single stage method RT-PCR is: in the RT-PCR reaction by the synthetic cDNA of RNA, controlling temperature is 50 ℃, reaction times 30min; Then when temperature is 94 ℃, keep 2min, make RTase inactivation; Temperature is controlled at 94 ℃ and carries out sex change 30 sec, at 60 ℃ of anneal 30 sec, then, temperature is controlled at 72 ℃, extend 1 min, from sex change, to extending, carry out altogether 30 circulations, last 72 ℃ are extended PCR product after 10 min again and ℃ preserve in temperature-4.
(4) electrophoresis detection: adopt agarose gel electrophoresis to detect amplification in electrophoresis detection, prepare 1.0% sepharose, every 100 mL add 5 μ L Gold View staining agents to mix in the gel preparing.In 0.5 * tbe buffer pendular ring border, 90 V voltage stabilizing electrophoresis 60 min, then observe and take pictures in gel imaging instrument.
(5) Analysis of test results: observe and find, 792 bp and 2 nucleic acid belts of 357 bp in the susceptible tobacco plant blade in field, have been detected, and any band in normal healthy controls, do not detected, according to the every pair of primer amplification clip size in table 1 and viral relation, referring to 17 and No. 20 swimming lanes of Fig. 3, judgement has detected TVDVaRNA and this 2 kinds of pathogens of TVDV from the susceptible tobacco leaf in field.
proof analysis: amplified production authenticity verification
792 bp in the above-mentioned amplified production of random choose and 2 nucleic acid belts of 357 bp, under ultraviolet lamp, from gel, cut respectively respective strap, after the AxyPrepTM DNA Gel Extraction Kit 50-prp purifying of biotechnology (Hangzhou) company limited of adopt liking to pursue progress reclaims, to reclaim the pMD18-T Vector that product connects most valuable treasure biotechnology (Dalian) company limited, molecular cloning method is converted into routinely
e. colidH5 α competent cell, adopt the plasmid of Beijing hundred Tyke Bioisystech Co., Ltd to prepare in a small amount test kit (centrifugal column type) extraction recombinant plasmid, each fragment at least selects 3 positive colonies to send Beijing Liuhe Huada Genomics Technology Co., Ltd to carry out two-way order-checking.Sequential analysis shows, the Nucleotide consistence of the TVDVaRNA logining in the cDNA of 792 bp and GenBank reaches 99%, the Nucleotide consistence of the TVDV logining in the cDNA of 357 bp and GenBank reaches 99%, these 2 nucleic acid belts of 792 bp that proof obtains from the susceptible tobacco plant in field by the present invention and 357 bp are exactly the corresponding cDNA amplified production of TVDVaRNA and TVDV, and proving again amplified production of the present invention is genuine and believable.
By the verity that the target stripe that the tobacco in greenhouse and field and aphid sample are increased taps rubber that purifying reclaims, institute's amplified production has all been verified in cDNA clone and sequential analysis, and the working concentration of each primer, annealing temperature etc. are optimized, finally determined and detected the reaction system of these 4 kinds of pathogens and the loop parameter of pcr amplification etc.
For saving as far as possible testing cost, when carrying out RT-PCR amplification, reaction system can be optimized and is reduced to 15 μ L by common 50 μ L, in 15 μ L reaction systems, the best effort concentration of each primer is: the forward and reverse primer TBTVdF of 10 μ M and TBTVdR each 0.6 μ L, the forward and reverse primer TVDVaRNAdF of 10 μ M and TVDVaRNAdR each 0.5 μ L, the forward and reverse primer SatTBTVdF of 10 μ M and SatTBTVdR each 0.4 μ L, the forward and reverse primer TVDVdF of 10 μ M and each 0.3 μ L of TVDVdR.The optimum annealing temperature of pcr amplification is 60 ℃.In addition, the present invention is to being diluted to 10
-3sample template still can obtain 4 kinds of pathogens amplification (referring to Fig. 2) clearly simultaneously.Therefore, the primer specificity of the present invention's design is strong, has avoided as much as possible the phase mutual interference between primer, can really accomplish the accurate detection of TBTV, TVDV, Sat-TBTV and TVDVaRNA to identify, reproducible, highly sensitive.
Tradition RT-PCR will detect one by one to TBTV, TVDV, Sat-TBTV and 4 kinds of pathogens of TVDVaRNA while detecting tobacco bushy top disease pathogen, and amount of reagent used is larger, consuming time longer.Present method completes reverse transcription and multiplex PCR one step, primary first-order equation can detect 4 kinds of pathogens simultaneously, and reaction system is only 1/3rd of common system, compares with traditional RT-PCR, when being detected, batch samples can save at most 2/3rds detection time and reagent cost.In addition, in the extraction of total RNA, by CTAB method, extract total RNA of catch an illness plant tissue and biography virus mediator insect, can guarantee the quality of put forward RNA, also can significantly reduce testing cost.Therefore, the present invention has high-level efficiency, feature cheaply, is especially applicable to compared with the rapid detection of large sample amount.
Result shows: no matter be to infect separately in a kind of virus, or the in the situation that of two kinds or 4 kinds of pathogen Combined Infections, method of the present invention all can detect corresponding pathogen under same experimental conditions from the susceptible tobacco sample in greenhouse and field and vector aphid, and negative control occurs without non-specific result, detected result high specificity, true and reliable.Because reverse transcription and multiplex PCR one step complete, once amplification just can detect TBTV, TVDVaRNA, Sat-TBTV and 4 kinds of pathogens of TVDV simultaneously, has both simplified detection formality, has saved again detection time and testing cost.In addition, the total nucleic acid that CTAB method is extracted catch an illness plant tissue or vector aphid can guarantee the quality of put forward total RNA, also can effectively reduce testing cost.Therefore the present invention have that efficiency is high, high specificity, highly sensitive and low cost and other advantages, can be applicable to tobacco or other corresponding plants and pass the synchronous detection of the 4 kinds of pathogens such as TBTV, TVDVaRNA, Sat-TBTV and TVDV that occur on virus mediator insect, have broad application prospects.
The present invention describes by accompanying drawing, without departing from the present invention, can also carry out various conversion and be equal to replacement patent of the present invention, therefore, patent of the present invention is not limited to disclosed specific implementation process, and should comprise the whole embodiments that fall within the scope of Patent right requirement of the present invention.
Claims (5)
1. the method for synchronous detection TBTV, TVDV, Sat-TBTV and TVDVaRNA, is characterized in that, synchronous detection is carried out according to step below:
(1) design of primers is synthetic:
(a) forward primer TBTVdF, TVDVdF, SatTBTVdF and the TVDVaRNAdF that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdF：5’-TACCACACCTAAACAGCGTTG-3’，
TVDVdF：5’-GCAACAGCGAGACTTTCATCT-3’，
SatTBTVdF：5’-TGTTGGCCGTGGGCAGCAA-3’，
TVDVaRNAdF：5’-GCGTACTGCGGAAGTCTCAC-3’，
(b) reverse primer TBTVdR, TVDVdR, SatTBTVdR and the TVDVaRNAdR that detects TBTV, TVDV, Sat-TBTV and TVDVaRNA synthesized in design respectively, and primer sequence is as follows:
TBTVdR：5’-CTCATCTCCCGCTAAGTCAG-3’，
TVDVdR:5 '-CRTTGCCTTTATAGAGCAGCC-3 ', wherein, the R=A in sequence or G,
SatTBTVdR：5’-TTCGTATTTGGCTCCGTCGC-3’，
TVDVaRNAdR：5’-TGTACTGGAGTGCTTCAACCG-3’；
(2) CTAB method is extracted the total nucleic acid of catch an illness plant tissue or biography virus mediator insect, as RT-PCR amplification template;
(3) single stage method RT-PCR amplification;
(4) electrophoresis detection;
(5) Analysis of test results: according to every pair of primer amplification clip size and viral relation, observation is infected the tobacco material of TBTV, TVDVaRNA, Sat-TBTV and TVDV or is passed the different nucleic acid belts that detect in virus mediator insect, the kind of concrete TBTV, TVDVaRNA, Sat-TBTV and the TVDV pathogen infecting in judgement morbidity tobacco or biography virus mediator insect.
2. the method for synchronous detection TBTV according to claim 1, TVDV, Sat-TBTV and TVDVaRNA, is characterized in that, described CTAB method is extracted diseased plant tissue or passed the total nucleic acid of virus mediator insect, and step is as follows:
The first step, preparation of samples
(1) diseased plant tissue is prepared: 3 ~ 5 sick leaves are superimposed, take 50 ~ 100 mg to mortar;
(2) passing virus mediator insect prepares: get 30 ~ 50 aphids to mortar;
Second step, add the CTAB damping fluid of 1.2 mL, the EDTA of the Tris-HCl of 100 mM of the CTAB that this damping fluid is 2% by mass percent, 2% PVP-40 and pH 8.0, the NaCl of 1.4 M and 20 mM forms mixed solution, finally according to the volume of mixing solutions, adds 0.2% mercaptoethanol;
The 3rd step, constant temperature are processed: after fully grinding, proceed in the centrifuge tube of 1.5 mL, under 65 ℃ of temperature are bathed, process 15 ~ 60 min;
The 4th step, centrifugal treating: first, centrifuge tube is put into whizzer 12000 ~ 13000 r/min centrifugal treating 15 min, get 750 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, add the chloroform that 750 μ L volume ratios are 24: 1: primary isoamyl alcohol, vortex concussion mixes 30 s; Then, again the centrifuge tube that supernatant liquor is housed is put into whizzer 12000 ~ 13000 r/min centrifugal treating 10 min, then, with micropipet, draw 600 μ L supernatant liquors to the centrifuge tube of another 1.5 mL, and add 600 μ L Virahols, and put upside down centrifuge tube and fully mix liquid, under room temperature, place 10 min, finally the centrifuge tube that supernatant liquor and Virahol are housed is put into whizzer 12000 ~ 13000 r/min centrifugal treating 15 min, with micropipet sucking-off supernatant liquor; Precipitation part adds the ethanol of 500 μ L 70%, and puts into whizzer 12000 ~ 13000 r/min centrifugal treating 10 min;
The 5th step, dry: use micropipet sucking liquid, precipitation part seasoning 10 ~ 15 min under 20 ℃ ~ 25 ℃ conditions;
The 6th step, dissolving: add the Tris-HCl of 100 μ L 20 mmol/L pH 8.0 and be placed in 10 min on ice, with micropipet piping and druming, make for 5 ~ 10 times precipitation part fully dissolve;
The 7th step, preservation: nucleic acid precipitation lysate-20 ℃ save backup; Or obtained total nucleic acid is put into-80 ℃ with dry powder, and to carry out prolonged preservation standby.
3. the method for synchronous detection TBTV according to claim 1, TVDV, Sat-TBTV and TVDVaRNA, is characterized in that, the PrimeScript that described single stage method RT-PCR adopts precious biotechnology Dalian company limited to produce
?one Step RT-PCR Kit Ver.2 test kit, reaction system is 15 μ L, by 2 * 1 Step Buffer of PrimeScript 1 Step Enzyme Mix, the 7.5 μ L of 0.6 μ L, the forward and reverse primer SatTBTVdF of 10 μ M of the forward and reverse primer TVDVaRNAdF of 10 μ M of the forward and reverse primer TBTVdF of 10 μ M of each 0.6 μ L and TBTVdR, each 0.5 μ L and TVDVaRNAdR, each 0.4 μ L and SatTBTVdR, the forward and reverse primer TVDVdF of 10 μ M of each 0.3 μ L and the ddH of TVDVdR, 2.8 μ L
2the RNA template of O and 0.5 μ L mixes.
4. the method for synchronous detection TBTV according to claim 1, TVDV, Sat-TBTV and TVDVaRNA, it is characterized in that, the reaction conditions of described single stage method RT-PCR is: in the RT-PCR reaction by the synthetic cDNA of RNA, controlling temperature is 50 ℃, reaction times 30 min; Then when temperature is 94 ℃, keep 2 min, make RTase inactivation; Temperature is controlled at 94 ℃ and carries out sex change 30 sec, carries out anneal 30 sec, then between 55 ℃ ~ 60 ℃ of temperature, temperature is controlled at 72 ℃, extend 1 min, from sex change, to extending, carry out altogether 30 circulations, last 72 ℃ are extended PCR product after 10 min again and ℃ preserve in temperature-4.
5. the method for synchronous detection TBTV according to claim 1, TVDV, Sat-TBTV and TVDVaRNA, it is characterized in that, in electrophoresis detection, adopt agarose gel electrophoresis to detect amplification, prepare the sepharose of 1.0 %, in the gel preparing, every 100 mL add 5 μ L Gold View staining agents to mix, in 0.5 * tbe buffer pendular ring border, 90 V voltage stabilizing electrophoresis 60 min, then observe and take pictures in gel imaging instrument.
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| CN1793859A (en) * | 2005-12-22 | 2006-06-28 | 云南农业大学 | Method of synchronously detecting tobacco congting virus and tobacco niumai virus |
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| Title |
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| AJ315135;Li.F;《Genebank database》;20041101;全文 * |
| CTAB法用于染病烟草植株中烟草丛顶病毒RNA的提取;左瑞娟 等;《云南农业大学学报》;20111231;第26卷(第1期);27、28页 * |
| EF529624、EF529625;Mo X. et al.;《Genebank database》;20080401;全文 * |
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