CN101519697A - Phytophthora sojae molecule detecting primer and detection reagent kit thereof - Google Patents
Phytophthora sojae molecule detecting primer and detection reagent kit thereof Download PDFInfo
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Abstract
The invention provides a phytophthora sojae molecule detecting primer and a detection reagent kit thereof. The primer comprises a forward primer PSD11 and a backward primer PSD12; and the detection reagent kit comprises PCR buffer solution, MgCl2, dNTPs, Taq polymerase, positive control DNA, DMSO, PSD11/PSD12 and ultrapure water. The detection reagent kit based on the primer specifically amplifies specific amplifying products with the fragment length of 382bp in phytophthora sojae pure DNA, bacteria-bearing pathological tissues and soil. The specific molecule detecting primer and the detection reagent kit thereof can be applied to the rapid, sensitive and specific detection for plant tissues infected with phytophthora sojae and the phytophthora sojae in soil and can be also applied to the early diagnosis of field diseases and the high-sensitive and rapid molecule detection and identification for the phytophthora sojae carried by soybean exporting or importing from customs.
Description
Technical field
The present invention more specifically relates to a kind of phytophthora sojae molecule and detects primer and detection kit thereof in the field of corps diseases detection evaluation and Plant Quarantine.
Background technology
The soybean phytophthora root rot that is caused by pathogen of soybean root rot (Phytophthora sojae) is a kind of destructive soil-borne disease, outside being listed in the I class, China examines object, this disease was found in China Heilongjiang Province first in 1991, after this be the trend that enlarges year by year, Fujian Province 1997 finds soybean blight in area, ZhangZhou, be separated to the soybean blight bacterium in area, ZhangZhou in 2002, this disease has constituted threat to soybean in China at present.Soybean phytophthora root rot is a kind of soil-borne disease, pathogenic bacteria can be propagated from the region of disease to region of disease not through rainwater, soil, invalid body etc., and its infection ability is strong, belongs to polycyclic disease by its disease that causes more, can cause plant death in the short period of time.Therefore, carry out pathogen of soybean root rot and detect, prevent that it from propagating from the region of disease to region of disease not, and carry out diagnostic detection at its their early stage, significant to the propagation of soybean phytophthora root rot with control.
Since finding pathogen of soybean root rot, the various countries researchist just studies its detection technique, traditional detection method is to adopt selective medium isolated strains from soil, plant tissue or water body, again form of these bacterial strains etc. is identified, thereby determine whether to exist pathogen of soybean root rot, perhaps with the naked eye and by microscopy disease symptom is judged the disease that whether causes by pathogen of soybean root rot.Traditional detection method is not only time-consuming, accuracy is low, and require the testing staff that rich experience will be arranged, more most important is that it can not satisfy in the disease control and to formulate the pass in and out demand of rapid detection and evaluation of best control period and customs, and therefore traditional etiology detection method can not satisfy the needs of modern plants pathological research.
Part pathogenic bacteria immunoserology authentication method is set up now, but specificity is relatively poor, usually is subjected to the interference of sibling species, also is subjected to the influence of antiserum(antisera) quality, and the accuracy of serology detected result depends on sero-fast quality.The specialization of monoclonal antibody is too strong, is mainly used in and detects fungus strain in the epidemiology, usually multiple monoclonal antibody is mixed and uses, to eliminate the excessively problem of specialization.Think at present and detecting application facet, polyclonal antibody is better than monoclonal antibody, and cross reaction takes place easily close with the sibship bacterium of polyclonal antibody.Therefore, the application of conventional serological method has been subjected to certain restriction.Along with the development of Protocols in Molecular Biology, it is more and more to the successful example that pathogenic bacteria carries out special, sensitive rapid molecular detection to use round pcr.Having the scholar to utilize based on the Molecular Detection of ITS base sequence design primer to pathogen of soybean root rot both at home and abroad conducts a research, but, from ITS sequence design primer two high kinds of similarity are distinguished and to have difficulty greatly because that ITS changes between the sibling species of pathogenic bacteria is very little.Chinese scholars utilization at present is relatively poor to the molecular detection specific of pathogen of soybean root rot based on ITS base sequence design primer, false-positive phenomenon often occurring, mainly is owing to do not take into full account the difference of primer sequence between sibling species during the design primer.Therefore to carry out high specific to pathogen of soybean root rot and detect, design high special primer from phytophthora ITS base sequence and be very important.
Summary of the invention
The objective of the invention is to provide a kind of phytophthora sojae molecule and detect primer and detection kit thereof, long at the required cycle of the biological detection method of pathogen of soybean root rot in the prior art, present phytophthora sojae molecule detection method poor specificity, the problem that sensitivity is low, by measuring the ITS base sequence of pathogen of soybean root rot and other phytophthora, and carry out multiple ratio to analyzing, ITS base distinguished sequence at pathogen of soybean root rot has been designed a pair of high special primer, be used to detect with the plant tissue of pathogen of soybean root rot and the highly sensitive rapid molecular of soil, and provide a kind of reliable results, easy handling, high specificity, the rapid molecular detection kit of highly sensitive pathogen of soybean root rot, the highly sensitive rapid molecular that this test kit can be used for germ-carrying plant tissue and soil detects, and causes that for pathogen of soybean root rot disease shows early monitoring before the disease and customs and passes in and out that the entrained pathogen of soybean root rot highly sensitive of soybean rapid molecular detects and evaluation has crucial meaning.
The one couple of PCR primers that beans phytophthora root rot bacterium molecule of the present invention detects, i.e. the sequence of special molecular detection primer is:
Upstream primer PSD11 (18bp): 5 '～TGAGGTGTCTTGCGGCGT～3 '
Downstream primer PSD12 (19bp): 5 '～AATTGAGATGCCACCTCCG～3 '.
The rapid molecular detection kit that this phytophthora sojae molecule detects primer comprises:
| The pipe | Title | Concentration | |
| 1 | Ultrapure water d.d.H 2O | 〉=99% (weight concentration) | |
| 2 | The PCR damping fluid | 10× | |
| 3 | MgCl 2 | 5mM | |
| 4 | | 10mM | |
| 5 | The Taq polysaccharase | 5U/ |
|
| 6 | Positive control dna | 100ng/ |
|
| 7 | Dimethyl sulfoxide (DMSO) (DMSO) | 〉=99.5% (weight concentration) | |
| 8 | Upstream primer PSD11 | 20pmol/ |
|
| 9 | Downstream primer PSD12 | 20pmol/μl |
Pcr amplification reaction adopts 25 μ l systems, wherein:
10×PCR buffer 2.5μl；
MgCl
2(5mM) 2.5μl；
dNTPs(10mM) 2.0μl；
PSD11(20pmol/μl) 0.2μl；
PSD12(20pmol/μl) 0.2μl；
Taq polysaccharase 0.2 μ l;
Template DNA (5ng/ μ l) 1.0 μ l;
DMSO 0.5μl
d.d.H
2O 15.90μl
The pcr amplification program is: 94 ℃ of sex change 5min; 94 ℃ of sex change 1min, 68 ℃ of annealing 30sec, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min.
The foundation of above-mentioned pathogen of soybean root rot special molecular detection kit system:
(1) from the plant tissue that is infected by described pathogen of soybean root rot or have a DNA isolation in the pedotheque of pathogen of soybean root rot;
(2) pcr amplification: PCR reaction system 25 μ l comprise 2.5 μ l, 10 * PCR reaction buffer, 5mM MgCl
22.5 μ l, 10mM dNTPs 2.0 μ l, 1.0U Taq archaeal dna polymerase, each 0.2 μ l of 20pmol/ μ l primer PSD11/PSD12, weight concentration 99.5% DMSO, 0.5 μ l and 10ng template DNA,, d.d.H
2O supplies 25 μ l.The pcr amplification program is: 94 ℃ of sex change 5min; 94 ℃ of sex change 1min, 68 ℃ of annealing 30sec, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 10min.
1. when being used for plant tissue and having pathogen of soybean root rot, adopt the CTAB method to extract the DNA of pathogen of soybean root rot, carry out pcr amplification by following PCR reaction system and reaction conditions with designed primer: PCR reaction system 25 μ l, comprise 2.5 μ l, 10 * PCR reaction buffer, 5mM MgCl
22.5 μ l, 10mM dNTPs 2.0 μ l, 1.0U Taq archaeal dna polymerase, each 0.2 μ l of 20pmol/ μ l primer PSD11/PSD12, weight concentration 99.5% DMSO, 0.5 μ l and 10ng template DNA, d.d.H
2O supplies 25 μ l.The pcr amplification program is: 94 ℃ of sex change 5min; 94 ℃ of sex change 1min, 68 ℃ of annealing 30sec, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 10min.
2. when in pedotheque, existing under the pathogen of soybean root rot condition, adopt soil DNA extraction method (embodiment 3 is seen in concrete operations) to extract the DNA of pathogen of soybean root rot in the soil, carry out pcr amplification by following PCR reaction system and reaction conditions with designed primer: PCR reaction system 25 μ l, comprise 2.5 μ l, 10 * PCR reaction buffer, 5mM MgCl
22.5 μ l, 10mM dNTPs 2.0 μ l, 1.0U Taq archaeal dna polymerase, each 0.2 μ l of 20pmol/ μ l primer PSD11/PSD12, weight concentration 99.5% DMSO, 0.5 μ l and 10ng template DNA,, d.d.H
2O supplies 25 μ l.The pcr amplification program is: 94 ℃ of sex change 5min; 94 ℃ of sex change 1min, 68 ℃ of annealing 30sec, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 10min.
(3) then 8 μ l PCR products are separated with 1.5% agarose electrophoresis, through behind the ethidium bromide staining under ultraviolet lamp according to the big or small result of determination of amplified production.
(4) if can amplify the 382bp product specifically the time, can judge in described plant tissue or the pedotheque to have pathogen of soybean root rot; Otherwise there is not pathogen of soybean root rot in described plant tissue or the pedotheque.
Gordian technique of the present invention is the efficient specific amplification primer sequence and the amplification method thereof of pathogen of soybean root rot.In order to obtain the special primer sequence of pathogen of soybean root rot, the present invention serves as for the examination material with province such as China Fujian, Heilungkiang and external pathogen of soybean root rot and a plurality of phytophthora sibling species and common several pathogenic fungies, adopt the CTAB method to extract the strains tested genomic dna, concrete grammar is as follows: get hypha powder after the 50mg lyophilize in the 1.5ml centrifuge tube, add 900 μ l weight concentration 2% CTAB (cetyl trimethylammonium bromide) extracting solution (weight concentration weight concentration 2% CTAB; 100mmol/L Tris-HCl, PH 8.0; 20mmol/L EDTA, pH8.0; 1.4mol/L NaCl) with 90 μ l weight concentration 10% SDS (Sodium dodecylbenzene sulfonate) back vibration mixing, in 55～60 ℃ of water-bath 1.5h, every 10min puts upside down mixing once, behind the water-bath 1.5h centrifugal (12,000rpm) 15min, get supernatant liquor and add isopyknic phenol, chloroform and primary isoamyl alcohol mixing solutions, phenol in the described mixing solutions: chloroform: the volume ratio of primary isoamyl alcohol is: 25: 24: 1, centrifugal (12,000rpm) 5min, get supernatant liquor (water), add equal-volume chloroform extracting once (12, the centrifugal 5min of 000rpm), suct clear liquid, add the 3mol/LNaAC solution of 0.1 volume and-20 ℃ of dehydrated alcohols of 2 volumes,-20 ℃ down behind the precipitation 30min 12, the centrifugal 5min of 000rpm, remove supernatant liquor lightly, add 700 μ l-20 ℃ volumetric concentrations, 70% ethanol and wash (centrifugal slightly, as to incline and fall supernatant), it is back with 1 * TE (10mmol/LTris-HCL to dry alcohol-free flavor on Bechtop naturally, 0.1mmol/LEDTA, pH8.0) solution dissolves (containing 5 μ g/ μ L RNase among the TE), obtains dna solution, with UV spectrophotometer measuring DNA concentration and to be diluted to 50ng/ μ l stand-by.On the basis of pathogen of soybean root rot specific DNA fragment sequence, use ClustalX software comparison design special primer, through the specificity of strains tested and pathogen of soybean root rot being carried out PCR checking (PCR reaction system 25 μ l, comprise 2.5 μ l, 10 * PCR reaction buffer, 5mM MgCl
22.5 μ l, 10mM dNTPs 2.0 μ l, 1.0U Taq archaeal dna polymerase, each 0.2 μ l of 20pmol/ μ l primer PSD11/PSD12, weight concentration 99.5% DMSO, 0.5 μ l and 10ng template DNA,, d.d.H
2O supplies 25 μ l, and the pcr amplification program is: 94 ℃ of sex change 5min; 94 ℃ of sex change 1min, 68 ℃ of annealing 30sec, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 10min, and this special primer amplifies the product of 382bp specifically on pathogen of soybean root rot.This illustrates that this primer can be used in the production practice rapid and reliable detection of pathogen of soybean root rot and evaluation in morbidity plant tissue and the soil sample.
Beneficial effect of the present invention: the inventive method is applicable to the fast and reliable detection and the evaluation of pathogen of soybean root rot in soil sample and the plant tissue, and disease control that causes for pathogen of soybean root rot in the agriculture production and customs pass in and out, and the entrained pathogen of soybean root rot highly sensitive of soybean rapid molecular detects and evaluation has important practical value.The present invention compared with prior art has following technical superiority and positively effect:
1, high specificity: detection method of the present invention be utilize the rDNA gene of pathogen of soybean root rot conservative in having kind, plant between the characteristics of variation, detect according to polytropy distinguished sequence design primer.Economize and the pathogen of soybean root rot of external different areas and the checking of phytophthora sibling species and other oomycetes coming from from China Fujian, Heilungkiang etc., the result has very strong specificity;
2, practicality is good: the designed a pair of Auele Specific Primer that goes out of the present invention, can be used for detecting with the plant tissue of pathogen of soybean root rot and the highly sensitive rapid molecular of soil, therefore present method is practical, can satisfy the needs that the pathogen of soybean root rot that exists in carry disease germs soil and the morbidity plant tissue is carried out rapid and reliable detection and evaluation;
3, easy and simple to handle quick: as to use the inventive method, the band soil sample of pathogen of soybean root rot and plant tissue are carried out getting final product result of determination after germ DNA extraction, pcr amplification and the conventional agarose electrophoresis, need not amplified production is carried out digestion with restriction enzyme.General whole testing process can be finished in 8 hours.
Description of drawings
The specific PCR amplification figure of the pathogen of soybean root rot that Fig. 1 will detect for the present invention.
Among the figure: swimming lane M is 100bp ladder molecular weight marker, swimming lane 1 positive contrast, swimming lane 2 negative controls, swimming lane 3-5 is the isolating pathogen of soybean root rot in Fujian Province, swimming lane 6,7 is the isolating pathogen of soybean root rot in Heilongjiang Province, and swimming lane 8,9 is external pathogen of soybean root rot.
Fig. 2 is the detected result figure of the present invention to the incidence tissue and the soil that carries disease germs.
Among the figure: swimming lane M is 2000bp molecular weight marker, swimming lane 1 positive contrast, and swimming lane 2 negative controls, swimming lane 3,4,5 is the plant tissue of morbidity, swimming lane 6,7,8 is the soil that carries disease germs.
Embodiment
Technology contents of the present invention comprises the special detection primer of pathogen of soybean root rot, and designed primer and sequence thereof are:
Upstream primer PSD11 (18bp): 5 '～TGAGGTGTCTTGCGGCGT～3 '
Downstream primer PSD12 (19bp): 5 '～AATTGAGATGCCACCTCCG～3 '
Utilize this primer can be from pathogen of soybean root rot specific amplified go out the product of 382bp.
Specifically set forth the present invention below in conjunction with embodiment
Embodiment 1: primer is to the specific amplification of pathogen of soybean root rot
1. the special detection of pathogen of soybean root rot
PCR reaction system 25 μ l comprise 2.5 μ l, 10 * PCR reaction buffer, 5mM MgCl
22.5 μ l, 10mM dNTPs2.0 μ l, 1.0U Taq archaeal dna polymerase, each 0.2 μ l of 20pmol/ μ l primer PSD11/PSD12, weight concentration 99.5% DMSO0.5 μ l and 10ng template DNA,, d.d.H
2O supplies 25 μ l.The pcr amplification program is: 94 ℃ of sex change 5min; 94 ℃ of sex change 1min, 68 ℃ of annealing 30sec, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 10min.
2. detected result
The specificity that detects: except amplifying the product of 382bp specifically from province such as China Fujian, Heilungkiang and external pathogen of soybean root rot DNA, other 13 kinds of mould kinds of epidemic disease, downy mildew, pythium spp and bacterial strain DNA such as other fungi and bacterium that detected oomycetes all fail to amplify spawn, have very strong specificity.
Embodiment 2: the detection of pathogen of soybean root rot in the morbidity plant tissue.
1. sample collecting: the plant tissue sample picks up from soybean vegetables base, list mountain, Longhai City, Fujian Province town
2.DNA extract and detect
The morbidity plant tissue adopts the CTAB method to extract DNA, carries out pcr amplification by the method that the mentioned reagent box is implemented, and PCR reaction system 25 μ l comprise 2.5 μ l, 10 * PCR reaction buffer, 5mM MgCl
22.5 μ l, 10mM dNTPs 2.0 μ l, the 1.0UTaq archaeal dna polymerase, each 0.2 μ l of 20pmol/ μ l primer PSD11/PSD12, weight concentration 99.5% DMSO, 0.5 μ l and 10ng template DNA,, d.d.H2O supplies 25 μ l.The pcr amplification program is: 94 ℃ of sex change 5min; 94 ℃ of sex change 1min, 68 ℃ of annealing 30sec, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 10min.The electrophoresis detection amplified production.
3. detected result
The results are shown in Figure 2, see-bar clearly molecular weight be the specific band of 382bp, thereby judge that incidence tissue infects pathogen of soybean root rot.
Embodiment 3: the detection of pathogen of soybean root rot in the pedotheque.
1. sample collecting: pedotheque picks up from soybean vegetables base, Fu Gong town, Longhai City, Fujian Province
2.DNA extract and detect
(1) from the morbidity pedotheque, extract DNA:
Add a small amount of quartz sand after getting the freezing 24-48h of draining of the soil that sieves, pour liquid nitrogen into and fully grind, the soil fine powder branch after grinding is filled in the 1.5ml centrifuge tube, every pipe adds 500 μ l weight concentrations, 0.4% skim-milk solution, vortex mixing.The centrifugal 15min of 12000rpm.Get supernatant and add equal-volume Proteinase K damping fluid, adding final concentration is 10 μ g/ml Proteinase Ks, 55 ℃ of water-bath 1-3h.After water-bath finishes, add the 7.5M NH of 1/2 volume
4AC solution, mixing turns upside down.The centrifugal 15min of 12000rpm.Suct and reset and add 2 times of volume dehydrated alcohols-20 ℃ precipitation (sedimentation time 1.5h).After precipitation finishes, the centrifugal 15min of 12000rpm.Go with the hypsokinesis of volumetric concentration 70% washing with alcohol precipitation, room temperature is dried.DNA that every duplicate samples is carried dissolves with 10 μ l1 * TE (or aseptic ultrapure water), and-20 ℃ of preservations are standby.
(2) PCR of pathogen of soybean root rot detects
Carry out pcr amplification as stated above, PCR reaction system 25 μ l comprise 2.5 μ l, 10 * PCR reaction buffer, 5mMMgCl
22.5 μ l, 10mM dNTPs 2.0 μ l, 1.0U Taq archaeal dna polymerase, each 0.2 μ l of 20pmol/ μ l primer PSD11/PSD12, weight concentration 99.5% DMSO, 0.5 μ l and 10ng template DNA,, d.d.H
2O supplies 25 μ l.The pcr amplification program is: 94 ℃ of sex change 5min; 94 ℃ of sex change 1min, 68 ℃ of annealing 30sec, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 10min.The electrophoresis detection amplified production.
3. detected result
The results are shown in Figure 2, see one clearly molecular weight be the specific band of 382bp, judge that there is pathogen of soybean root rot in pedotheque.
Sequence table
＜110〉Inst. of Plant Protection, fujian Academy of Agricultural Science
＜120〉a kind of phytophthora sojae molecule detects primer and detection kit thereof
<160>2
<210>1
<211>18
<212>DNA
＜213〉pathogen of soybean root rot (Phytophthora sojae)
<220>
<400>1
<210>2
<211>19
<212>DNA
＜213〉pathogen of soybean root rot (Phytophthora sojae)
<220>
<400>2
Claims (10)
1. a phytophthora sojae molecule detects primer, it is characterized in that: the one couple of PCR primers that described phytophthora sojae molecule detects, i.e. and the sequence of special molecular detection primer is:
Upstream primer PSD11 (18bp): 5 '～TGAGGTGTCTTGCGGCGT～3 '
Downstream primer PSD12 (19bp): 5 '～AATTGAGATGCCACCTCCG～3 '.
2. phytophthora sojae molecule according to claim 1 detects primer, it is characterized in that: described phytophthora sojae molecule detection primer PSD11 and PSD12 amplify the product of 382bp specifically on pathogen of soybean root rot.
3. phytophthora sojae molecule according to claim 1 detects primer, it is characterized in that: the sequence preparation method that described phytophthora sojae molecule detects primer is: pathogen of soybean root rot and a plurality of phytophthora sibling species and common several pathogenic fungies are for the examination material, adopt the CTAB method to extract the strains tested genomic dna, concrete grammar is as follows: get hypha powder after the 50mg lyophilize in the 1.5ml centrifuge tube, add the mixing that vibrates behind 900 μ l weight concentrations, 2% cetyl trimethylammonium bromide CTAB extracting solution and 90 μ l weight concentrations, the 10% Sodium dodecylbenzene sulfonate SDS, in 55～60 ℃ of water-bath 1.5h, every 10min puts upside down mixing once, centrifugal 15min behind the water-bath 1.5h, centrifugal speed is 12,000rpm, get supernatant liquor and add isopyknic phenol, chloroform and primary isoamyl alcohol mixing solutions, phenol in the described mixing solutions: chloroform: the volume ratio of primary isoamyl alcohol is: 25: 24: 1, centrifugal 5min, centrifugal speed is 12,000rpm, get supernatant liquor, it is water, add the extracting of equal-volume chloroform once, centrifugal 5min, centrifugal speed is 12,000rpm, suct clear liquid, add the 3mol/LNaAC solution of 0.1 volume and-20 ℃ of dehydrated alcohols of 2 volumes,-20 ℃ down behind the precipitation 30min 12, the centrifugal 5min of 000rpm, remove supernatant liquor lightly,-20 ℃ of ethanol that add 700 μ l volumetric concentrations 70% wash, naturally (solution dissolves with 1 * TE to dry alcohol-free flavor back on Bechtop, obtain dna solution, with UV spectrophotometer measuring DNA concentration and to be diluted to 50ng/ μ l stand-by, on the basis of pathogen of soybean root rot specific DNA fragment sequence, use ClustalX software comparison design special primer, obtain phytophthora sojae molecule and detect primer sequence, through the specificity of strains tested and pathogen of soybean root rot being carried out the PCR checking, described phytophthora sojae molecule detects primer amplifies 382bp specifically on pathogen of soybean root rot product.
4. phytophthora sojae molecule according to claim 3 detects primer, and it is characterized in that: described cetyl trimethylammonium bromide CTAB extracting solution is weight concentration 2%CTAB; 100mmol/L Tris-HCl, PH8.0; 20mmol/LEDTA, pH8.0; 1.4mol/L NaC.
5. phytophthora sojae molecule according to claim 3 detects primer, and it is characterized in that: described 1 * TE solution is 10mmol/L Tris-HCL, 0.1mmol/LEDTA, and pH8.0 wherein contains 5 μ g/ μ L RNase among the TE.
6. phytophthora sojae molecule according to claim 3 detects primer, it is characterized in that: the concrete reaction system that described warp carries out the PCR checking to the specificity of strains tested and pathogen of soybean root rot is: PCR reaction system 25 μ l, comprise 2.5 μ l, 10 * PCR reaction buffer, 5mM MgCl
22.5 μ l, 10mM dNTPs 2.0 μ l, 1.0U Taq archaeal dna polymerase, each 0.2 μ l of 20pmol/ μ l primer PSD11/PSD12, weight concentration 99.5%DMSO 0.5 μ l and 10ng template DNA, d.d.H
2O supplies 25 μ l; The pcr amplification program is: 94 ℃ of sex change 5min; 94 ℃ of sex change 1min, 68 ℃ of annealing 30sec, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 10min.
7. employing such as claim 1,2,3,4,5 or 6 described phytophthora sojae molecules detect the rapid molecular detection kit of primers, and it is characterized in that: described rapid molecular detection kit comprises: the ultrapure water d.d.H that weight concentration 〉=99% is housed
2The ultrapure water pipe of O; The pipe of concentration 10 * PCR damping fluid is housed; 5mM MgCl is housed
2Pipe; The pipe of 10mM dNTPs is housed; The pipe of 5U/ μ l Taq polysaccharase is housed; The pipe of 100ng/ μ l positive control dna is housed; The pipe of weight concentration 〉=99.5% dimethyl sulfoxide (DMSO) DMSO is housed; The pipe of 20pmol/ μ l upstream primer PSD11 is housed; The pipe of 20pmol/ μ l downstream primer PSD12 is housed; Adopt this test kit to carry out pcr amplification reaction to be: pcr amplification reaction adopts 25 μ l systems, wherein:
10×PCR buffer 2.5μl；
5mM MgCl
2 2.5μl；
10mM dNTPs 2.0μl；
20pmol/μl PSD11 0.2μl；
20pmol/μl PSD12 0.2μl；
Taq polysaccharase 0.2 μ l;
5ng/ μ l template DNA 1.0 μ l;
DMSO 0.5μl；
d.d.H
2O 15.90μl；
The pcr amplification program is: 94 ℃ of sex change 5min; 94 ℃ of sex change 1min, 68 ℃ of annealing 30sec, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 10min.
8. foundation of adopting phytophthora sojae molecule as claimed in claim 7 to detect primer rapid molecular detection kit system is characterized in that: set up according to following steps:
(1) from the plant tissue that is infected by described pathogen of soybean root rot or have a DNA isolation in the pedotheque of pathogen of soybean root rot;
(2) adopt described molecular detection kit to carry out pcr amplification: PCR reaction system 25 μ l comprise 2.5 μ l, 10 * PCR reaction buffer, 5mM MgCl
22.5 μ l, 10mM dNTPs 2.0 μ l, 1.0U Taq archaeal dna polymerase, each 0.2 μ l of 20pmol/ μ l primer PSD11/PSD12, weight concentration 99.5% DMSO, 0.5 μ l and 10ng template DNA, d.d.H
2O supplies 25 μ l, and the pcr amplification program is: 94 ℃ of sex change 5min; 94 ℃ of sex change 1min, 68 ℃ of annealing 30sec, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 10min;
(3) the PCR product with 8 μ l steps (2) separates with 1.5% agarose electrophoresis, through behind the ethidium bromide staining under ultraviolet lamp according to the big or small result of determination of amplified production; If in the time of amplifying the 382bp product specifically, can judge in described plant tissue or the pedotheque to have pathogen of soybean root rot; Otherwise there is not pathogen of soybean root rot in described plant tissue or the pedotheque.
9. phytophthora sojae molecule according to claim 8 detects the foundation of primer rapid molecular detection kit system, it is characterized in that: when being used for plant tissue and having pathogen of soybean root rot, adopt the CTAB method to extract the DNA of pathogen of soybean root rot.
10. phytophthora sojae molecule according to claim 8 detects the foundation of primer rapid molecular detection kit system, it is characterized in that: when in pedotheque, existing under the pathogen of soybean root rot condition, adopt the soil DNA extraction method to extract the DNA of pathogen of soybean root rot in the soil, described soil DNA extraction method is: add a small amount of quartz sand after getting the freezing 24-48h of draining of the soil that sieves, pouring liquid nitrogen into fully grinds, soil fine powder branch after grinding is filled in the 1.5ml centrifuge tube, every pipe adds 500 μ l weight concentrations, 0.4% skim-milk solution, the vortex mixing, the centrifugal 15min of 12000rpm, get supernatant and add equal-volume Proteinase K damping fluid, adding final concentration is 10 μ g/ml Proteinase Ks, 55 ℃ of water-bath 1-3h after water-bath finishes, add the 7.5M NH of 1/2 volume
4AC solution, mixing turns upside down, the centrifugal 15min of 12000rpm sucts and resets and add 2 times of volume dehydrated alcohol-20 ℃ precipitations, sedimentation time 1.5h, after precipitation finishes, the centrifugal 15min of 12000rpm goes with the hypsokinesis of volumetric concentration 70% washing with alcohol precipitation, and room temperature is dried, DNA that every duplicate samples is carried dissolves with 10 μ l 1 * TE or aseptic ultrapure water, and-20 ℃ of preservations are standby.
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| CN200910111424A Pending CN101519697A (en) | 2009-04-07 | 2009-04-07 | Phytophthora sojae molecule detecting primer and detection reagent kit thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101805796A (en) * | 2010-04-13 | 2010-08-18 | 南京农业大学 | Primer for detecting the soybean phytophthora and kit and method thereof |
| CN101942520A (en) * | 2010-10-14 | 2011-01-12 | 南京农业大学 | Marker of avirulence gene PsAvr3b of phytophthora sojae |
| CN102424851A (en) * | 2011-12-27 | 2012-04-25 | 中华人民共和国昆山出入境检验检疫局 | Molecular detection method for Edrus deodara (Roxb) Lobd blight and primers therefor |
| CN102643925A (en) * | 2012-05-16 | 2012-08-22 | 南京农业大学 | Loop-mediated isothermal amplification (LAMP) primer composition for detecting phytophthora rot and application thereof |
| CN104946622A (en) * | 2014-03-31 | 2015-09-30 | 华中农业大学 | Method for rapidly extracting fungal genome DNA |
| CN106754872A (en) * | 2016-12-02 | 2017-05-31 | 华中农业大学 | A kind of method that microbe genome DNA is extracted from poplar wood |
| CN108949749A (en) * | 2018-08-14 | 2018-12-07 | 中国热带农业科学院环境与植物保护研究所 | A method of the rapidly extracting DNA long fragment from fresh fungal mycelium |
| CN109943624A (en) * | 2019-03-14 | 2019-06-28 | 南京林业大学 | A kind of method and its primer special and probe combinations based on RPA- Sidestream chromatography technology detection soybean phytophthora |
| CN113755625A (en) * | 2021-09-06 | 2021-12-07 | 宁波海关技术中心 | Primer, probe, kit and detection method for detecting cotton root rot |
| CN116516058A (en) * | 2023-06-28 | 2023-08-01 | 海南大学三亚南繁研究院 | Method and kit for visually detecting phytophthora sojae |
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2009
- 2009-04-07 CN CN200910111424A patent/CN101519697A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101805796A (en) * | 2010-04-13 | 2010-08-18 | 南京农业大学 | Primer for detecting the soybean phytophthora and kit and method thereof |
| CN101805796B (en) * | 2010-04-13 | 2012-09-05 | 南京农业大学 | Primer for detecting the soybean phytophthora and kit and method thereof |
| CN101942520A (en) * | 2010-10-14 | 2011-01-12 | 南京农业大学 | Marker of avirulence gene PsAvr3b of phytophthora sojae |
| CN101942520B (en) * | 2010-10-14 | 2012-10-17 | 南京农业大学 | Marker of avirulence gene PsAvr3b of phytophthora sojae |
| CN102424851B (en) * | 2011-12-27 | 2013-05-08 | 中华人民共和国昆山出入境检验检疫局 | Molecular detection method for Edrus deodara (Roxb) Lobd blight and primers therefor |
| CN102424851A (en) * | 2011-12-27 | 2012-04-25 | 中华人民共和国昆山出入境检验检疫局 | Molecular detection method for Edrus deodara (Roxb) Lobd blight and primers therefor |
| CN102643925A (en) * | 2012-05-16 | 2012-08-22 | 南京农业大学 | Loop-mediated isothermal amplification (LAMP) primer composition for detecting phytophthora rot and application thereof |
| CN104946622A (en) * | 2014-03-31 | 2015-09-30 | 华中农业大学 | Method for rapidly extracting fungal genome DNA |
| CN106754872A (en) * | 2016-12-02 | 2017-05-31 | 华中农业大学 | A kind of method that microbe genome DNA is extracted from poplar wood |
| CN106754872B (en) * | 2016-12-02 | 2019-07-05 | 华中农业大学 | A method of extracting microbe genome DNA from poplar wood |
| CN108949749A (en) * | 2018-08-14 | 2018-12-07 | 中国热带农业科学院环境与植物保护研究所 | A method of the rapidly extracting DNA long fragment from fresh fungal mycelium |
| CN109943624A (en) * | 2019-03-14 | 2019-06-28 | 南京林业大学 | A kind of method and its primer special and probe combinations based on RPA- Sidestream chromatography technology detection soybean phytophthora |
| CN113755625A (en) * | 2021-09-06 | 2021-12-07 | 宁波海关技术中心 | Primer, probe, kit and detection method for detecting cotton root rot |
| CN116516058A (en) * | 2023-06-28 | 2023-08-01 | 海南大学三亚南繁研究院 | Method and kit for visually detecting phytophthora sojae |
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