CN105274241A - Molecular detection primer and quick detection method for thielaviopsis basicola - Google Patents
Molecular detection primer and quick detection method for thielaviopsis basicola Download PDFInfo
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Abstract
The invention discloses a molecular detection primer and a quick detection method for thielaviopsis basicola. The molecular detection primer and the quick detection method have the advantages that the specific primer which is high in detection sensitivity and specificity and can diagnose black roots of tobaccos in fields at early stages and monitor and identify germs is designed according to multiple comparative analysis on beta-tubulin gene sequences of the thielaviopsis basicola and other species, the detection sensitivity of the molecular primer can reach 10 pg, the tobacco infection thielaviopsis basicola can be detected within 24 hours, and the lengths of fragments of amplification products are 254 bp; the timely and accurate detection method and scientific evidence can be provided for identifying the thielaviopsis basicola, monitoring the thielaviopsis basicola at early stages and timely preventing and treating the thielaviopsis basicola.
Description
Technical field
The invention belongs to agricultural biological technical field.Be specifically related to the Molecular Detection to black root of tobacco bacterium rapid sensitive, can be used for the early diagnosis of black root of tobacco and the Testing and appraisal of germ simultaneously.
Background technology
Black root of tobacco is the important soil-borne disease of one on the tobacco leaf production that caused by thielaviopsis sp [Thielaviopsisbasicola (Berk.etBr.)], and the main Chan Yan district of its throughout world, makes tobacco leaf production suffer heavy losses.This pathogenic bacteria can infect the various plants that causes harm, as tobacco, cotton, beans, peanut, citrus and multiple ornamental plant.Black root of tobacco is global disease, economizes in Yunnan Province of China, Guizhou, Hubei etc. to occur heavier, and serious plot sickness rate can reach more than 30%.Shandong, Henan, Anhui, Jilin, Fujian etc. are economized also generation, and this disease has the trend increasing the weight of to cause harm in China in recent years.Black root of tobacco mainly betides on cigarette strain root system, makes root be that specific black rots, all can fall ill from seedling to Adult plant.Seedling is caught an illness and is caused " dampinging off ", but roots blacking, not in samping off.Germ invades from rhizome portion, and scab, around stem one week, is upwards invaded as leaf, under invade to side root, make whole strain seedling withered.Comparatively opium strain is caught an illness side root, tip of a root blackening, and diseased plant growth retardation, plant is short and small, and leaf look yellowish-brown.It is brown, downright bad that grave illness strain pulls up the blackening of visible whole strain root system, diseased plant blade turn yellow, thinning, have a strong impact on tobacco production and quality.Invalid body and band soil bacteria are the primary source of infection of this disease.When condition is suitable for, conidium or chlamydospore sprouting produce intrusion silk and invade epidermal cells of host by wound, and after intrusion, mycelia branch between Tobacco Epidermis spreads, and produces a large amount of conidium and chlamydospore, then infects.This disease morbidity thermophilic 17 ~ 23 DEG C, sends out lighter for less than 15 DEG C or more than 26 DEG C.When relative humidity is more than 80%, morbidity is heavy.Rainy or the cloudy weather for several days running sky of low temperature easily causes popular.This germ can infect 30 section 120 various plants such as pulse family, Curcurbitaceae, falls ill heavy with these section's plant continuous croppings.Low-lying lower wetland, barren saltings susceptible disease.
Because root Black Rotten is soil-borne disease, usually mix with the disease of other Tobacco Root stem and occur, cause difficulty to the correct diagnosis of disease.Root black rot harm tobacco was called harm commitment before naked eyes can observe illness, and at the commitment that root Black Rotten disease occurs, can not show roots blacking, scab is around black root of tobacco illnesss such as stem, side root, tip of a root blackening.Usual root black rot harm tobacco just can observe illness in 5-6 days, therefore, effectively prevents and treats pointedly, cause root black rot to endanger serious, cause larger financial loss in being difficult in early days of occurring of black root of tobacco evil.Therefore, set up the specific rapid molecular detection system of black root of tobacco bacterium, early the pathogenic bacteria that carries disease germs in disease plant or soil is detected rapidly and accurately, for the generation, popular taking scientific and reasonable prophylactico-therapeutic measures to control disease in time, reduce the financial loss that disease causes and there is important directive significance.
Being conventionally used to the Testing and appraisal method of phytopathogen is the separation and Culture first utilizing selective medium to carry out pathogenic bacteria, after obtaining pure culture bacterial strain, identify according to the morphological features such as colony characteristics, spore shape size, Pathogenicity, physio-biochemical characteristics etc.These method time and effort consumings, program is loaded down with trivial details, and the isolation identification of pathogenic bacteria is empirical very strong.There are some researches show, the morphological differences between black root of tobacco bacterium different strains is obvious, and some bacterial strain can produce a large amount of fan-shaped albefaction bacterial strains, and the monospore bacterium colony of some albefaction bacterial strain can be returned to wild-type, is subject to the interference of the factors such as man-made environment during qualification.In addition, due to directly pathogenic bacteria can not be detected from the diseased tissues of vegetation zone, be difficult to quick, accurate, the sensitive testing requirement met needed for disease control, easily miss the best period of disease control.
In recent years, along with the fast development of Protocols in Molecular Biology, the molecular detecting method being representative with polymerase chain reaction (PCR) technology obtains and develops rapidly and apply.Fast development based on the round pcr of genomic dna compensate for the deficiency of traditional classification authentication method effectively, and the features such as its specificity had, susceptibility and rapidity become phytopathogen classification and identify one of most widely used technology.Rapid detection and diagnosis can be carried out to Diseased Plant Tissues because round pcr does not need to carry out separation and purification to pathogenic bacteria, therefore can be used for black root of tobacco show disease before early diagnosis, for the precise Identification of disease and in time control provide scientific basis.
The PCR detection technique overwhelming majority at present for phytopathogen is the Auele Specific Primer developed based on rrna the Internal Transcribed Spacer (rDNA-ITS) sequence.Domestic and international researchist also develops the specificity amplification primer for round pcr to the rDNA-ITS sequence of black root of tobacco bacterium.But more and more study discovery, in the pathogenic bacteria of some sibling specieses, rDNA-ITS sequence difference is very little, be that the primer of drone design is difficult to them to distinguish with ITS, therefore need to develop some new genes and detect.Tubulin (Tubulin) is the protein families that a class contains multiple member.The modal member of eukaryote tubulin family is alpha-tubulin and 'beta '-tubulin, and they are main components of composition microtubule.β-tubulin gene includes multiple exon and intron, and multiple exon has conservative property, and these introns have larger variation between different plant species, is suitable for design primer and carries out Molecular Detection and distinguish different species.At home and abroad be not reported based on the detection technique of β-tubulin Data mining to black root of tobacco bacterium at present.
Summary of the invention
For solving the existing early diagnosis to black root of tobacco bacterium difficulty, sensitivity is low, cause and be difficult to propose to prevent and treat scientific basis in time, accurately, and rDNA-ITS sequence difference is very little in existing Molecular Detection, take ITS as the technical problem that the primer of drone design is difficult to the pathogenic bacteria of some sibling specieses to distinguish, the invention provides a kind of highly sensitive, early diagnosis can go out Tobacco Root black rot, the molecular detection primer of high specificity and result accurately, the black root of tobacco bacterium rapid molecular detection method of easy handling.
Technical scheme of the present invention is as follows:
1. a black root of tobacco bacterium molecule detects primer, it is characterized in that: described black root of tobacco bacterium molecule detects primer and is made up of upstream primer Tbas-tubF and downstream primer Tbas-tubR, the base sequence of described upstream primer Tbas-tubF is as shown in SEQIDNO:1, the base sequence of described downstream primer Tbas-tubR is as shown in SEQIDNO:2, and target product fragment length is 254bp.
2. a method for quick for black root of tobacco bacterium, is characterized in that comprising the following steps:
(1) from tobacco plant tissue to be checked, genomic dna is extracted;
(2) genomic dna extracted with step (1), for template, detects primer with the black root of tobacco bacterium molecule described in technical scheme 1 and carries out pcr amplification;
(3) pcr amplification product getting 5 ~ 8 μ l steps (2) carries out agarose gel electrophoresis, to take pictures under gel imaging system after biological nucleic acid dyeing observation, if the fragment of a 254bp can be amplified specifically, then there is black root of tobacco bacterium in tobacco plant tissue to be checked.
3. the method for quick of black root of tobacco bacterium according to technical scheme 2, it is characterized in that: the reaction system of step (2) described pcr amplification is: PCR reaction system cumulative volume 20 μ l, wherein 10 × reactionBuffer2 μ l, 100mmol/LMgCl
2buffer1.2 μ l, 2.5mmol/LdNTPMixture1.6 μ l, 10 μm of ol/L upstream primer Tbas-tubF0.4 μ l, 10 μm of ol/L downstream primer Tbas-tubR0.4 μ l, 5U/ μ lrTaqPolymerase0.1 μ l, template DNA 1 μ l, sterilizing ultrapure water supplies cumulative volume; Pcr amplification program is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 40s, 60 DEG C of annealing 30s, and 72 DEG C extend 40s, totally 35 circulations, and last 72 DEG C extend 10min.
Compared with prior art, beneficial effect of the present invention is as follows:
1, high specificity: the present invention is after carrying out multiple compare of analysis according to black root of tobacco bacterium β-tubulin gene order and other species, designs PCR detection primer black root of tobacco bacterium to specific amplified effect.The present invention is by black root of tobacco bacterium, band root Black Rotten Tissues of Tobacco, common 18 kind of plant pathogenic bacterias: Phytophthora nicotianae, phytophthora infestans, Phytophthora capsici, soybean phytophthora, cotton epidemic disease is mould, hidden ground epidemic disease is mould, palm mould, Pythium ultimum, melon and fruit corruption are mould, woods dwells rotten mould, Pyricularia oryzae, Fusarium oxysporum, Fusarium graminearum, peanut black rot, dry thread Pyrenomycetes, Penicillum glaucum, bread mould, trichoderma harziarum, carried out detection validation, the high specificity of sufficient proof primer of the present invention, the reliability of detected result from the fungi of soil separation at random.
2, highly sensitive, can detect the black root of tobacco bacterium of trace: the present invention design Auele Specific Primer can reach 10pg to the detection sensitivity of black root of tobacco bacterium on DNA level, can detect the black root of tobacco bacterium of trace.
3, early detection can go out Tobacco Root black rot: Auele Specific Primer of the present invention and detection method thereof can to before infecting tobacco 24h and 4 day at root black rot namely the commitment of the not yet aobvious illness of plant the former bacterium of root Black Rotten all can be detected, root black rot is diagnosed accurately and identifies.
4. practicality is good: the Auele Specific Primer of the present invention's design can be used for the early diagnosis of black root of tobacco cingula diseased tissues and highly sensitive micro-rapid detection, therefore, present method practical, can meet the black root of tobacco bacterium existed in band hyphostroma is carried out early, fast and reliable detection and qualification.
5. simple and efficient to handle: application detection method, from the sick sample tissue wash of tobacco Black Rotten, pathogenic bacteria DNA extraction, pcr amplification, agarose gel electrophoresis to obtaining detected result, whole process only needs 3 ~ 4 hours, compared with standard biologic detection method, simple and efficient to handle, substantially reduce the time needed for qualification.
In sum, the inventive method is easy and simple to handle, quick, can carry out early diagnosis, detection sensitivity up to 10pg to black root of tobacco, for qualification and the early monitoring of black root of tobacco evil, Instructing manufacture carries out preventing and treating in time the method and scientific basis that provide promptly and accurately to this disease.
Shown in SEQ ID NO:1 is the base sequence of upstream primer Tbas-tubF.
Shown in SEQ ID NO:2 is the base sequence of downstream primer Tbas-tubR.
Accompanying drawing explanation
Fig. 1 is the specific PCR amplification figure of the present invention to black root of tobacco bacterium.In figure: M is DL2000DNAmarker; Swimming lane 1 ~ 4 is black root of tobacco bacterium; Swimming lane 5 is Phytophthora nicotianae; Swimming lane 6 is phytophthora infestans; Swimming lane 7 is Phytophthora capsici; Swimming lane 8 is soybean phytophthora; Swimming lane 9 is that cotton epidemic disease is mould; Swimming lane 10 is that hidden ground epidemic disease is mould; Swimming lane 11 is palm mould; Swimming lane 12 is Pythium ultimum; Swimming lane 13 is that melon and fruit corruption is mould; Swimming lane 14 is rotten mould for woods dwells; Swimming lane 15 is Pyricularia oryzae; Swimming lane 16 is Fusarium oxysporum; Swimming lane 17 is Fusarium graminearum; Swimming lane 18 is peanut black rot; Swimming lane 19 is dry thread Pyrenomycetes; Swimming lane 20 is Penicillum glaucum; Swimming lane 21 is bread mould; Swimming lane 22 is trichoderma harziarum; Swimming lane 23 is soil separation fungi; Swimming lane 24 is negative control.
The sensitive amplification figure that Fig. 2 the present invention detects black root of tobacco bacterium.In figure: M is DL2000DNAmarker; Swimming lane 1 is 100ng; Swimming lane 2 is 10ng; Swimming lane 3 is 1ng; Swimming lane 4 is 100pg; Swimming lane 5 is 10pg; Swimming lane 6 is 1pg; Swimming lane 7 is 100fg; Swimming lane 8 is 10fg; Swimming lane 9 is negative control.
The result figure that Fig. 3 the present invention detects the early stage infected tissue of black root of tobacco bacterium.In figure: M is DL2000DNAmarker; Swimming lane 1 is health tobacco tissue (negative control); Swimming lane 2 infects the Tissues of Tobacco of 6 hours for root black rot; Swimming lane 3 infects the Tissues of Tobacco of 12 hours for root black rot; Swimming lane 4 infects the Tissues of Tobacco of 24 hours for root black rot; Swimming lane 5 infects the Tissues of Tobacco of 48 hours for root black rot; Swimming lane 6 infects the Tissues of Tobacco of 72 hours for root black rot; Swimming lane 7 infects the Tissues of Tobacco of 5 days for root black rot; Swimming lane 8 is root black rot DNA (positive control).
Embodiment
Detailed description below by embodiment sets forth the present invention further, but is not limitation of the present invention, only does example explanation.The reagent used in following embodiment, the large gold dollar of tobacco bred safflower all can be bought from commercial channel.Without specified otherwise in following embodiment is ordinary method.
The specific amplification of embodiment 1 PCR primer design of the present invention and primer pair black root of tobacco bacterium
One, design of primers and synthesis
Download the β-tubulin gene order of black root of tobacco bacterium and other species from GenBank, carry out multiple Multiple Sequence Alignment, analysis with ClustalW software, find the specific sequence of black root of tobacco bacterium β-tubulin gene; Primers3 software is utilized to carry out design of primers, designed black root of tobacco bacterium molecule detects primer and is made up of upstream primer Tbas-tubF and downstream primer Tbas-tubR, the base sequence of described upstream primer Tbas-tubF is as shown in SEQIDNO:1, the base sequence of described downstream primer Tbas-tubR, as shown in SEQIDNO:2, is 254bp to the expection clip size of black root of tobacco bacterium specific amplification.The primer designed is synthesized by Shanghai invitrogen company.
Two, for examination material: black root of tobacco bacterium (Thielaviopsisbasicola), Phytophthora nicotianae (Phytophthoranicotianae), phytophthora infestans (Phytophthorainfestans), Phytophthora capsici (Phytophthoracapsici), soybean phytophthora (Phytophthorasojae), cotton epidemic disease mould (Phytophthoraboehmeriae), hidden ground epidemic disease mould (Phytophthoracryptogea), palm mould (Phytophthorapalmivora), Pythium ultimum (Pythiumultimum), melon and fruit corruption mould (Pythiumaphanidermatum), woods dwells rotten mould (Pythiumsylvaticum), Pyricularia oryzae (Magnaportheoryzae), Fusarium oxysporum (Fusariumoxysporum), Fusarium graminearum (Fusariumgraminearum), peanut black rot (Cylindrocladiumparasiticum), Penicillum glaucum (Penicilliumglaucum), bread mould (Rhizopusnigricans), trichoderma harziarum (Trichodermaharzianum), dry thread Pyrenomycetes (Rhizoctoniasolani) is separated fungi with the soil be separated from soil at random.Except the random soil be separated from soil is separated except fungi, all the other are all for open in examination material non-patent literature listed by table 1, applicant Yunnan Prov Academy Of Agricultural Sciences, Agricultural Research Institute Of Environmental Resources from the applying date in Two decades years to germ listed by public's granting table 1, address, Yunnan Prov Academy Of Agricultural Sciences, Agricultural Research Institute Of Environmental Resources: No. 2238, Panlong District Beijing Road, Kunming, Yunnan Province, postcode: 650205.
It is the soil got at random in the vega of Dali Prefecture, Yunnan Province Midu County that soil is separated fungi, obtains by dilution-plate method culture of isolated.
Three, rapid molecular detects
(1) extraction of strains tested genomic dna
The extraction of each strains tested genomic dna all adopts CTAB method to extract the genomic dna of strains tested, and concrete grammar is:
1. in the EP centrifuge tube of 1.5,900 μ l2%CTAB extracting solutions and 90 μ l10%SDS are added; 2%CTAB extracting solution consists of: 2%CTAB, 100mmol/LTris-HCl (PH8.0), 20mmol/LEDTA (PH8.0) and 0.5mol/LNaCl.
2. the mycelium of strains tested is collected, moisture is pressed dry with filter paper, hypohostroma is used liquid nitrogen grinding powdered, levigate powder joins in the EP pipe installing 900 μ l2%CTAB extracting solutions and 90 μ l10%SDS, vortex oscillation 1min fully mixes, be placed in 65 DEG C of water-bath 1h, turn upside down 2-3 mixing every 10min; The centrifugal 15min of 12000rpm after water-bath 1h, get supernatant liquor add isopyknic mixed liquor A vibration mixing, described mixed liquor A be phenol, chloroform and primary isoamyl alcohol by phenol: chloroform: the volume ratio of primary isoamyl alcohol is that 25:24:1 mixes.
3. the centrifugal 5min of 12000rpm, gets supernatant liquor, adds isopyknic chloroform, vibration mixing, and the centrifugal 5min of 12000rpm, gets supernatant, adds isopyknic ice Virahol and fully mixes, and places the centrifugal 10min of 1h, 12000rpm for-20 DEG C; Abandon supernatant liquor, the volume fraction adding 700 μ l ice is 70% washing with alcohol 2 times, and the centrifugal 1min of 12000rpm, abandons supernatant; Dried up by white precipitate in EP pipe in Bechtop, the sterilizing ultrapure water added containing RNase dissolves, and namely obtains Genomic DNA solution; Measure the concentration of DNA sample with Nanodrop, be diluted to 100ng/ μ l, be placed in-20 DEG C for subsequent use.
(2) PCR reaction
With the genomic dna of said extracted for template, carry out pcr amplification with upstream primer Tbas-tubF and downstream primer Tbas-tubR.
The reaction system of pcr amplification is: PCR reaction system cumulative volume 20 μ l, wherein 10 × reactionBuffer2 μ l, 100mmol/LMgCl
2buffer1.2 μ l, 2.5mmol/LdNTPMixture1.6 μ l, 10 μm of ol/L upstream primer Tbas-tubF0.4 μ l, 10 μm of ol/L downstream primer Tbas-tubR0.4 μ l, 5U/ μ lrTaqPolymerase0.1 μ l, template DNA 1 μ l, sterilizing ultrapure water supplies cumulative volume.
Pcr amplification program is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 40s, 60 DEG C of annealing 30s, and 72 DEG C extend 40s, totally 35 circulations, and last 72 DEG C extend 10min.
(3), the detected result of pcr amplification product
6 × the sample-loading buffer getting 5 μ l amplified productions and 2 μ l after PCR reaction terminates mixes, and be separated through 1.5% agarose gel electrophoresis, observation of taking pictures under gel imaging system after biological nucleic acid dyeing, result as shown in Figure 1.Except black root of tobacco bacterium genomic dna amplifies the fragment that size is 254bp specifically, other Phytophthora nicotianae detected, phytophthora infestans, Phytophthora capsici, soybean phytophthora, cotton epidemic disease is mould, hidden ground epidemic disease is mould, palm mould, Pythium ultimum, melon and fruit corruption is mould, woods dwells rotten mould, Pyricularia oryzae, Fusarium oxysporum, Fusarium graminearum, peanut black rot, dry thread Pyrenomycetes, Penicillum glaucum, bread mould, trichoderma harziarum, soil is separated fungi and all fails to amplify spawn with the DNA compared, show that the upstream primer Tbas-tubF that the present invention designs and downstream primer Tbas-tubR has very strong specificity, the existence of black root of tobacco bacterium can only be detected, may be used in production practice the quick diagnosis of black root of tobacco and qualification.
The susceptibility of embodiment 2 primer pair black root of tobacco of the present invention bacterium detects
1.DNA concentration dilution: the black root of tobacco bacterium genomic dna of extraction, after ThermoNanoDrop spectrophotometric determination concentration, adopts 10 times of serial dilution genomic dna concentration to be that 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg are for subsequent use.
2. with upstream primer Tbas-tubF of the present invention and downstream primer Tbas-tubR, the susceptibility of black root of tobacco bacterium is detected
Utilize upstream primer Tbas-tubF and downstream primer Tbas-tubR to carry out pcr amplification to black root of tobacco bacterium 10 times of serial dilution genomic dnas respectively, the reaction system of pcr amplification is identical with embodiment 1 with pcr amplification program.
3. carry out 1.5% agarose gel electrophoresis after detected result: PCR terminates and detect amplified production.As Fig. 2 display, in 20 μ lPCR reaction systems, with black root of tobacco bacterium genomic dna for template, all can amplify a size from 100ng to 10pg be the specific band of 254bp, and DNA concentration lower than 1pg time, fail to amplify corresponding band, show: the detection sensitivity of primer of the present invention can reach 10pg, detection sensitivity is high, can detect the black root of tobacco bacterium of trace.
The early diagnosis of root black rot in embodiment 3 tobacco pathogenesis tissue
1. the preparation of the sick sample tissue of black root of tobacco
Concentration is about 1 × 10
5the black root of tobacco bacterium conidial suspension of cfu/ml or cut PDA with scalpel and cultivate the black root of tobacco bacterium inoculated by hypha block of 1 week in the growth tobacco bred safflower large gold dollar rhizome portion of 6 weeks, in 22 ~ 23 DEG C of growth cabinets, moisturizing is cultivated.Get the Tobacco Root stem of inoculation rear 6h, 12h, 24h, 48h, 72h and 5d respectively as the extraction of sick sample tissue for DNA.
2. tobacco tissue DNA rapid extraction and pcr amplification in spite of illness
The tobacco of respectively black root of tobacco bacterium being infected 6h, 12h, 24h, 48h, 72h and 5d in spite of illness sick stem or old complaint tissue extracts DNA according to NaOH rapid cleavage method, specific as follows:
The tobacco of infecting black root of tobacco bacterium in spite of illness sick stem or old complaint tissue tap water clean up, thieving paper suck dry moisture, cut incidence tissue with scalpel and be placed in mortar; Add 10 μ l lysate (0.5mol/LNaOH, 0.5%PVP) meterings by 1mg diseased tissues, be transferred in the centrifuge tube of 1.5ml after fully diseased tissues being ground in mortar, the centrifugal 5min of 12000rpm; The 0.1mol/LTris getting 100 μ l supernatant liquors and isopyknic pH8.0 mixes; Get 1 μ l mixed solution and be directly used in pcr amplification reaction as template, reaction system and the pcr amplification program of described pcr amplification are identical with embodiment 1, and each sick sample tissue DNA sample repeats for 3 times.
3. detected result
Carry out 1.5% agarose gel electrophoresis after PCR terminates and detect amplified production, as shown in Figure 3, the sick sample tissue infecting 6h, 12h as the health tobacco plant of negative control, black root of tobacco bacterium does not have amplified production; And the sick sample tissue that black root of tobacco bacterium infects 24h, 48h, 72h and 5d and the root black rot DNA being used as positive control all amplified one clearly size be the band of 254bp, show: detected root black rot in Tissues of Tobacco, incidence tissue has infected black root of tobacco bacterium.Result also shows further: adopt the rapid molecular detection method of upstream primer Tbas-tubF provided by the invention and downstream primer Tbas-tubR and black root of tobacco bacterium when the initial stage that root black rot infects and 6h and 12h by tobacco band diseased tissues, pathogenic bacteria can not be detected from Tissues of Tobacco, but root black rot infect tobacco 24h and after, namely at the commitment of the not yet aobvious illness of tobacco plant, pathogenic bacteria can be detected from Tissues of Tobacco, can diagnose accurately root black rot and identify, can to the generation taking scientific and reasonable prophylactico-therapeutic measures to control black root of tobacco disease in time, popular, the financial loss that minimizing disease causes provides scientific basis.
Table 1 embodiment 1 detects the relevant information of pathogenic bacteria material used
<110> Yunnan Tobacco Co., Ltd., Dali Branch
Yunnan Prov Academy Of Agricultural Sciences, Agricultural Research Institute Of Environmental Resources
<120> black root of tobacco bacterium molecule detects primer and method for quick thereof
<130>/
<160>2
<170>PatentInversion3.3
<210>1
<211>22
<212>DNA
<213> black root of tobacco bacterium (
thielaviopsisbasicola)
<400>1
cgccctatcaatccacttatcc22
<210>2
<211>20
<212>DNA
<213> black root of tobacco bacterium (
thielaviopsisbasicola)
<400>2
atgcgcttgaacagctcctg20
Claims (3)
1. a black root of tobacco bacterium molecule detects primer, it is characterized in that: described black root of tobacco bacterium molecule detects primer and is made up of upstream primer Tbas-tubF and downstream primer Tbas-tubR, the base sequence of described upstream primer Tbas-tubF is as shown in SEQIDNO:1, the base sequence of described downstream primer Tbas-tubR is as shown in SEQIDNO:2, and target product fragment length is 254bp.
2. a method for quick for black root of tobacco bacterium, is characterized in that comprising the following steps:
(1) from tobacco plant tissue to be checked, genomic dna is extracted;
(2) genomic dna extracted with step (1), for template, detects primer with black root of tobacco bacterium molecule according to claim 1 and carries out pcr amplification;
(3) pcr amplification product getting 5 ~ 8 μ l steps (2) carries out agarose gel electrophoresis, to take pictures under gel imaging system after biological nucleic acid dyeing observation, if the fragment of a 254bp can be amplified specifically, then there is black root of tobacco bacterium in tobacco plant tissue to be checked.
3. the method for quick of black root of tobacco bacterium according to claim 2, it is characterized in that: the reaction system of step (2) described pcr amplification is: PCR reaction system cumulative volume 20 μ l, wherein 10 × reactionBuffer2 μ l, 100mmol/LMgCl
2buffer1.2 μ l, 2.5mmol/LdNTPMixture1.6 μ l, 10 μm of ol/L upstream primer Tbas-tubF0.4 μ l, 10 μm of ol/L downstream primer Tbas-tubR0.4 μ l, 5U/ μ lrTaqPolymerase0.1 μ l, template DNA 1 μ l, sterilizing ultrapure water supplies cumulative volume; Pcr amplification program is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 40s, 60 DEG C of annealing 30s, and 72 DEG C extend 40s, totally 35 circulations, and last 72 DEG C extend 10min.
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| CN109280715A (en) * | 2018-07-31 | 2019-01-29 | 仲恺农业工程学院 | LAMP primer group for detecting peanut black rot, and rapid detection method and kit thereof |
| CN109280715B (en) * | 2018-07-31 | 2022-02-08 | 仲恺农业工程学院 | LAMP primer group for detecting peanut black rot, and rapid detection method and kit thereof |
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