CN103966307A - Fluorescent quantitative PCR detection technology of brassicaceous vegetable plasmodiophoromycetes, and its application - Google Patents
Fluorescent quantitative PCR detection technology of brassicaceous vegetable plasmodiophoromycetes, and its application Download PDFInfo
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Abstract
The invention relates to a fluorescent quantitative PCR detection technology of plasmodiophoromycetes, and its application. The technology is used for rapidly and accurately quantitatively detecting brassicaceous vegetable plasmodiophoromycetes. The technology comprises the following steps: extracting DNA from a sample, designing a specific primer, carrying out real-time fluorescent quantitative PCR detection on the ITS region gene fragment of the plasmodiophoromycetes by applying an SYBRGreen I based dye process fluorescent quantitative PCR technique, constructing a standard curve, and calculating the copy number of the ITS region gene fragment in the sample and the concentration of the plasmodiophoromycetes. The detection technology has the advantages of simple and rapid operation, strong specificity and high sensitivity, realizes a highest detection sensitivity reaching 24 copy numbers per reaction, and can also realize the analysis of large batch samples. The technology provides a good basis for the early-stage prediction forecasting of diseases, so the technology is adapted to be widely applied in the plant disease diagnosis detection field.
Description
Technical field
The present invention is a kind of fluorescence quantitative PCR detection technique and application of Plasmodiophora brassicae Causing Cruciferae Clubroot, is exclusively used in detection Plasmodiophora brassicae Causing Cruciferae Clubroot, belongs to crops disease diagnosing and Prevention Technique field.
Background technology
Rape plasmodiophora brassicae (
plasmodiophora brassicae) causing the club root of brassicaceous vegetable, main parasitic is in the root of the multiple plant of Btassica.All have distribution in the whole world, its damaging range expands to some flower plants from brassicaceous vegetable in recent years, infect cultivation and more than 100 kinds of wild plant (mutation).The worldwide loss that club root causes every year reaches 10-15%.Disease plant is infected rear formation knee by plasmodiophora brassicae and organizes, short and small, the easy wilting of plant (Voorips, 1995).Pathogenic bacteria discharges formation statospore and can survive a lot of years (Wallenhammar, 1996) from rotten knee tissue.The adaptability of soil temperature that plasmodiophora brassicae statospore is sprouted and infected is 18～25 DEG C, and the suitableeest water capacity is 70%, and the suitableeest soil acidity or alkalinity is that 5.4～6.5(Lee is precious poly-, 2008).It is firm that plasmodiophora brassicae statospore germination rate in the solution of susceptible host's root exudates can reach 75%(Xiao Chong, and 2002).Rape belong to plant repeat plantation process in these statospores in soil gradually accumulation, degree of disease increases the weight of (Robert etc. 2009) year by year.
Obviously before performance, be not easy prediction and avoid infecting of pathogen plasmodiophora in disease symptom, even peasant think do not have club root to infect or the aseptic situation in field under, the statospore of plasmodiophora brassicae is quietly from a local soil and water transmission to another place, cause for all the foregoing reasons the control of crucifer club root very difficult, therefore, effectively and fast Cruciferae pathogen plasmodiophora detection technique of development, effectively avoid spreading and propagating of this disease, control tool for Cruciferae club root is of great significance.
Because pathogen plasmodiophora can not directly be cultivated, its conventional soil detects the raw survey experiment that depends on disease plant, or directly assesses by the symptom of root, or infects situation (Toxopeus etc. 1975) by microscopic examination plasmodiophora brassicae.Recently, fluorescent microscope, immunology and the detection method being based upon on DNA basis are applied to detection soil, the plasmodiophora brassicae in water or Plant samples gradually.These detection methods are quick not, and can not carry out quantitatively the plasmodiophora brassicae in plant, water and pedotheque, along with molecular diagnostic techniques is in the widespread use of medical science, Animal husbandry and plant pathology, and on the one hand from diagnosing single target to dual and multiplex PCR future development, (RTi-PCR) future development from qualitative PCR to real-time fluorescence quantitative PCR on the other hand, this is for we provide strong basis to the rapid detection of plasmodiophora brassicae in plant, soil and water.
RT-PCR is the amount of carrying out instant detection specificity product by the power of PCR continuous detecting fluorescent signal during index amplification, and the starting template amount of purpose of appraisals gene accordingly.Compared with conventional PCR, have advantages of easy and simple to handle, efficient fast, strong, the favorable repeatability of susceptibility and specificity high.The principle of work of SYBR Green I quantitative fluorescent PCR is in the system of conventional PCR, to add fluorescence dye SYBR Green I, and SYBR Green I is a kind of fluorescence dye being incorporated in double-stranded DNA ditch, and after being combined with double-stranded DNA, its fluorescence strengthens greatly.In PCR reaction system, add excessive SYBR Green I fluorescence dye, fluorescence dye can be incorporated into after DNA double chain specifically, emitting fluorescence signal, and the luminescent dye molecule not mixing in chain is not launched any fluorescent signal.Thereby along with the carrying out of PCR reaction, the enhancing and the PCR product that ensure fluorescent signal increase Complete Synchronization, the cycle index (Ct represents by cycle threshold) that signal is strengthened to a certain threshold value goes on record, in Ct value and PCR system, between the logarithm of starting template, there is strict linear relationship, utilize Ct value and the typical curve of positive plasmid standards for quantitation amplification, more just can accurately calculate existence and the quantity of certain nucleic acid in starting template according to the Ct value of testing sample.
Summary of the invention
The object of the invention is to overcome the deficiency of plasmodiophora brassicae traditional detection method, the method for the quantitative fluorescent PCR rapid detection of a kind of plasmodiophora brassicae is provided.
Another object of the present invention is to provide a kind of fluorescence quantitative PCR detection technique in the forecast of crucifer club root Epidemiology monitor, the application of fungus in soil and Seed-associated fungi aspect.
This detection technique, can large-scale promotion in Plant diseases diagnosis and treatment field.
Object of the present invention is achieved through the following technical solutions:
The experimental procedure of the method is: extract DNA from sample to be tested; To add machine on the fluorescent quantitation reaction solution of standard substance and testing sample, carry out PCR detection with fluorescent quantitative detector; By comparing the cycle threshold of testing sample and standard substance, calculate the initiate dna concentration of testing sample according to typical curve.
Technology of the present invention specifically comprises the steps:
1, get field natural occurrence soil, disease plant, brassicaceous vegetable seed, carry out the extraction purifying of total DNA, obtain DNA sample;
2, Auele Specific Primer design, for avoiding because primer specialization is lower, in PCR reaction, there is false positive results or non-target fungi is produced to non-specific amplification, the present invention is on systems analysis pathogen plasmodiophora ITS district, 18SrRNA and the phylogenetic basis of 5.8SrRNA nucleotide sequence, adopt CLUSTAL, compare with this region sequence of Plasmodiophoromycetes and soil-borne pathogen, find the specific sequence region of pathogen plasmodiophora; Select pathogen plasmodiophora specific sequence region, design real-time fluorescence PCR primer, by Blast pogram (
http:// blast.ncbi.nlm.nih.gov/Blast.cgi) more designed primer sequence and other biological species do not have remarkable homology.Primer pair 1, sense primer sequence is: (5
,-TCTTGCGTGTCGCTGTATTC-3
,), antisense primer sequence is: (5
,-ATAGGTTGGGGTAACTTGGC-3
,), amplified fragments size is 137bp(Fig. 1); Primer pair 2, sense primer sequence is: (CTCACAACGATGAAGAAC), antisense primer sequence is: (5
,-ACTCACAGCCAAAGACAAGC-3
,), amplified fragments size is 140bp(Fig. 2).
plasmodiophora brassicae18S rRNA, ITS1,5.8S rRNA and ITS2 partial sequence NCBI accession number are respectively (HM015883.1) and (EF195335.1).
3, real-time fluorescence quantitative PCR reaction system is: quantitative fluorescent PCR reaction system cumulative volume is 25 μ l, and wherein SYBR Premix Ex Taq (includes TaKaRa Ex Taq TM HS, dNTP Mixture, Mg
2+with SYBR Green I) 12.5 μ l, concentration is the sense primer PBF2(PBF3 of 10 μ mol/L) and antisense primer PBR2(PBR3) each 0.5 μ l, 10 times of gradient dilution liquid of standard substance or detected sample DNA solution 2 μ l, ddH
2o polishing to 25 μ l;
4, PCR response procedures is: PCR adopts two-step approach, and 95 DEG C of 3min then carry out 40 circulations, and each circulation comprises 95 DEG C of 10s, 60 DEG C of 30s; After PCR completes, by 0.1 DEG C of s
-1temperature rise rate rises to 95 DEG C of analysis verifications that carry out melt curve analysis from 72 DEG C;
5, insert the plasmon DNA extracting after the pMD-20 carrier conversion bacillus coli DH 5 alpha propagation in ITS district, DNA is quantitative and 10 times of gradient dilutions through ultraviolet spectrophotometer A260, be diluted to 6 concentration gradients, increase according to above-mentioned PCR reaction system and program; After reaction finishes, adopt computer automatic drafting quantitative fluorescent PCR typical curve according to the cycle threshold of each concentration gradient (Ct);
6, get the DNA sample obtaining in step 1) as template, increase, and carry out the amplification of negative control and positive control simultaneously by above-mentioned quantitative fluorescent PCR reaction system and response procedures on real-time fluorescence quantitative PCR instrument, wherein negative control adopts ddH
2o, positive control adopts plasmon DNA; After reaction finishes, the cycle threshold of DNA sample (Ct) is contrasted with typical curve, obtain DNA sample Zhong ITS district gene fragment copy number, and then obtain the concentration of pathogen plasmodiophora in original sample.
Plasmodiophora brassicae Causing Cruciferae Clubroot real-time fluorescence quantitative PCR detection technique can generate test kit and comprise standard positive DNA profiling, fluorescent quantitation reaction solution.
Standard positive DNA profiling is by containing the pGM-T(that inserts object fragment purchased from Tian Gen company) carrier is prepared from.The preparation process of standard substance is: adopt primer I TS1 and ITS4 amplification pathogen plasmodiophora ITS district, after PCR product electrophoresis, cut the gel that contains target fragment, target fragment is spent the night and is connected with 16 DEG C, pGM-T carrier after reclaiming purifying with gel reagents box, transforms bacillus coli DH 5 alpha competent cell.Converted product is coated with dull and stereotyped cultivation, chooses hickie, and adopting primer is that GZ10/GZ11 and PBF2/PBR2 and PBF3/PBF3 carry out PCR qualification.Positive colony is by the reliability of the further qualification result of sequential analysis.Select all positive clones of PCR and sequential analysis, be inoculated into the LB substratum incubated overnight that contains penbritin, adopt the little extraction reagent kit of plasmid (purchased from Tian Gen company) to prepare plasmid DNA.DNA is quantitative through ultraviolet spectrophotometer A260, is 104ng-104fg/ μ L and 216ng-216fg/ μ L through 10 times of gradient dilutions, is stored in-20 DEG C.
Fluorescent quantitation reaction solution includes TaKaRa Ex Taq TM HS, dNTP Mixture, Mg by sense primer PBF2, antisense primer PBR2, fluorescence dye SYBR Premix Ex Taq
2+form with SYBR Green I.
Fluorescent quantitation reaction solution includes TaKaRa Ex Taq TM HS, dNTP Mixture, Mg by sense primer PBF3, antisense primer PBR3, fluorescence dye SYBR Premix Ex Taq
2+form with SYBR Green I.
Pathogen plasmodiophora fluorescence quantitative PCR detection technique provided by the invention, for the special target sequence design primer in pathogen plasmodiophora genome ITS district, by the optimization of optimizing reaction system (primer concentration, amplification program etc.), adopt quantitative detection system (comprising ABI Prism system, PE Applied Bioasystems, Bio-Rad), can be used for the detection by quantitative of the pathogen plasmodiophora in various sources.The detection point of accumulation of primer pair 1 is that 1.04fg plasmid DNA/μ l is equivalent to 24 cells, and the detection point of accumulation of primer pair 2 is that 2.16fg plasmid DNA/μ l is equivalent to 45 cells.
The present invention compared with prior art has the following advantages and effect:
1) specificity is good, qualification is quick
Primer comprises the specific sequence of plasmodiophora brassicae, and detection specificity is high.In environment, particularly the rhizospheric microorganism of brassicaceous vegetable is of a great variety, and plasmodiophora brassicae is strict obligate parasite, can not isolated culture, therefore, can not adopt the method for traditional separation and Culture to detect, the present invention has overcome this weak point, and detects fast.
2) accurate quantitative analysis, highly sensitive
Compared with detecting with regular-PCR, the present invention can accurate quantitative analysis, and target dna, in 104ng-104fg/ μ L concentration range, has good linear relationship; Detect and there is reproducible feature simultaneously.
3) good, the applied range of practicality
Can detection by quantitative plant plant, plasmodiophora brassicae in soil, seed and water, in conjunction with the field morbidity threshold value of crucifer club root, can provide strong reference for the early prediction forecast of disease.
Brief description of the drawings
Fig. 1 is primer pair PBF2/PBR2 specific detection pcr amplification result.1,2 template is Chinese cabbage daetylorhiz genomic dna, the template of 3-10 is respectively spongospora subterranea DNA, many slime bacterias of cereal DNA, Fusarium oxysporum DNA, phytophthora blight of pepper DNA, melon and fruit pythium spp DNA, dry thread Pyrenomycetes DNA, Fusarium semitectum DNA, fusarium moniliforme DNA, and M is 150bp DNA ladder(Takara company).
Fig. 2 is primer pair PBF3/PBR3 specific detection pcr amplification result.1 template is Chinese cabbage daetylorhiz genomic dna, the template of 2-9 is respectively spongospora subterranea DNA, many slime bacterias of cereal DNA, Fusarium oxysporum DNA, phytophthora blight of pepper DNA, melon and fruit pythium spp DNA, dry thread Pyrenomycetes DNA, Fusarium semitectum DNA, fusarium moniliforme DNA, and M is 150bp DNA ladder(Takara company).
The typical curve of Fig. 3 embodiment 2 fluorescent quantitative PCR, the longitudinal axis is Ct, transverse axis is Log CO; Standard is mistaken for 0.995.
The amplification curve of Fig. 4 embodiment 2 quantitative fluorescent PCR positive criteria product, the longitudinal axis is Delta Rn; Transverse axis is Cycle Number, curve 1,2, and 3,4,5 and 6 the positive standard substance of template, in reaction system, DNA content is respectively 1.04ng, 104pg, 10.4pg, 1.04pg, 104fg, 10.4fg.
The amplification curve of Fig. 5 embodiment 3 quantitative fluorescent PCRs, the longitudinal axis is Delta Rn; Transverse axis is Cycle Number, curve 1,2, and 3,4,5 and 6 the positive standard substance of template, in reaction system, DNA content is respectively 1.04ng, 104pg, 10.4pg, 1.04pg, 104fg, 10.4fg; 7 and 8 is testing samples.
The typical curve of Fig. 6 embodiment 4 fluorescent quantitative PCR, the longitudinal axis is Ct, transverse axis is Log CO; Standard is mistaken for 0.999.
The amplification curve of Fig. 7 embodiment 4 quantitative fluorescent PCR positive criteria product, the longitudinal axis is Delta Rn; Transverse axis is Cycle Number, curve 1,2, and 3,4,5 and 6 the positive standard substance of template, in reaction system, DNA content is respectively 2.16ng, 216pg, 21.6pg, 2.16pg, 216fg, 21.6fg.
Embodiment
Below in conjunction with specific examples, further set forth invention.Should be appreciated that following example is only not used in the claimed scope of restriction the present invention for the present invention is described.What in application example, DNA extraction adopted is test kit, also can adopt the methods such as improvement CTAB in practical application.
Embodiment 1: the Auele Specific Primer of pathogen plasmodiophora detects
Adopting common soil-borne pathogen in Cruciferae vegetable rhizospheric microorganism is material, and pathogen plasmodiophora primer pair PBF2/PBR2 and PBF3/PBR3 are carried out to specific detection.Chinese cabbage daetylorhiz is preserved in-20 DEG C of refrigerators.Other soil-borne disease fungal pathogens bacterial strain is cultivated with PDA or PD liquid nutrient medium.All bacterial strains first activate on solid medium, then proceed to liquid nutrient medium and cultivate, and collect mycelia or thalline extraction DNA after 1 week.The extraction of DNA adopts the CTAB extraction method of improvement.
Taking the pathogenic bacteria fungal genomic DNA of rape plasmodiophora brassicae and other soil-borne disease as template, utilize primer PBF2/PBR2 to carry out regular-PCR amplification, to detect the specificity of this primer.Common PCR reaction system is as follows: total reaction volume 25 μ L, 10 × PCR buffer, 2.5 μ L, dNTP(10 μ mol/L) 0.5 μ L, the each 0.5 μ L of primer (10 μ mol/L), template DNA 2.0 μ L, TapDNA polysaccharase 0.5 μ L, finally with ddH
2o complements to 25 μ L.PCR response procedures is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 60 DEG C of annealing 1min, 72 DEG C are extended 1.5min, totally 38 circulations; 72 DEG C are extended 7 min.Pcr amplification product is electrophoresis detection (Fig. 1) on 1% sepharose.
Utilize primer PBF3/PBR3 to carry out regular-PCR amplification, to detect the specificity of this primer.Common PCR reaction system is as follows: total reaction volume 25 μ L, 10 × PCR buffer, 2.5 μ L, dNTP(10 μ mol/L) 0.5 μ L, the each 0.5 μ L of primer (10 μ mol/L), template DNA 2.0 μ L, TapDNA polysaccharase 0.5 μ L, finally with ddH
2o complements to 25 μ L.PCR response procedures is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 57 DEG C of annealing 1min, 72 DEG C are extended 1.5min, totally 35 circulations; 72 DEG C are extended 7 min.Pcr amplification product is electrophoresis detection (Fig. 2) on 1% sepharose.
Table 1 primer specificity detects bacterial strain
To PBF2/PBR2 and PBF3/PBR3, the pcr amplification result (Fig. 1,2) to pathogen plasmodiophora and sibling species, genus and other soil-borne pathogen shows that this primer pair has good specificity to specific detection primer.Under 60 DEG C and 58 DEG C of annealing conditions, only pathogen plasmodiophora there is is the amplified production of 137bp and 140bp, other bacterial strains are all without amplified production.
Embodiment 2: the detection of the inner pathogen plasmodiophora of Chinese cabbage interior plant in vegetative period
At the beginning of 2012 9 months, in the Experimental Base sampling of Chinese cabbage inherent Qingdao in vegetative period Shanxi Academy of Agricultural Sciences, sample is the Chinese cabbage root of field planting after 15 days in club root morbidity field.And what get is the plant root that does not show disease symptom, and sample is cleaned repeatedly, guarantee that sample surface is without plasmodiophora brassicae statospore, adopt (MP Biomedicals company of the U.S., FastDNA SPIN Kit for Soil) extraction, purify DNA.The DNA of each root is dissolved in 30 μ l TE damping fluids, gets 2 μ l DNA solutions as template, adopts primer pair GZ10/GZ11 to carry out PCR detection to each sample, and amplified production detects through 1.5% agarose electrophoresis, and meanwhile, each sample carries out fluorescence quantitative PCR detection.
Product after regular-PCR amplification produces without band on sepharose.Quantitative fluorescent PCR reaction system cumulative volume is 25 μ l, wherein SYBR Premix Ex Taq 12.5 μ l, the each 0.5 μ l of 10 μ mol sense primer PBF2 and antisense primer PBR2, standard substance 10 times of gradient dilution liquid (1.04ng, 104pg, 10.4pg, 1.04pg, 104fg, 10.4fg) and detected sample DNA solution 2 μ l, remainder ddH
2o polishing.PCR adopts two-step approach: 95 DEG C of 10min, then; 95 DEG C of 15s, 60 DEG C of 1min are 1 circulation, totally 40 circulations.
After loop ends, the analysis software that adopts instrument to carry, analysing amplified result production standard curve (Fig. 3) and amplification curve (Fig. 4), calculate testing sample pathogen plasmodiophora DNA content.Result shows that standard substance have fabulous linear relationship, and relation conefficient is 0.995; Testing sample has obvious fluorescent signal to increase, and there is good kinetic curve, the DNA amount that calculates pathogen plasmodiophora in testing sample reaction system according to typical curve is 42.8pg, show that fluorescence quantitative PCR detection system not only can be quantitative to the pathogen plasmodiophora in testing sample than regular-PCR detection system the method, and detection sensitivity detects higher than regular-PCR, in the time that plant is performance disease symptom, adopt regular-PCR to detect and pathogen plasmodiophora can not be detected, and quantitative fluorescent PCR reaches the object of an early stage rapid detection.
Embodiment 3: the soil sample of field natural occurrence detects
In our the national crucifer club root serious area of falling ill, during field disease survey is carried out year July in July, 2011-2012, gather Hubei, Yunnan, Guizhou, Qingdao, Sichuan, the soil sample of the field natural occurrence on the ground such as Chongqing, gather altogether 60 parts of soil samples, in these 60 parts of soil, there are 32 parts for grave illness field, and these fields were planted Cruciferae vegetable before 2011 always, 20 parts is hypopathia field, the time of these field plantation Cruciferae vegetables does not exceed 3 years, 8 parts for not showing the field of disease symptom, preceding crop is Chinese cabbage, the crop varieties of these regional cultures is in the majority with susceptible variety.The soil sample of getting from field is stored in-20 DEG C of refrigerators immediately, in the time extracting DNA, carries out frozen dried.Get freeze-drying sample 0.2-0.5g, adopt (MP Biomedicals company of the U.S., FastDNA SPIN Kit for Soil) extraction, purify DNA.DNA is dissolved in 40 μ l TE damping fluids, gets 2 μ l DNA solutions and makes template, carries out fluorescence quantitative PCR detection.
Quantitative fluorescent PCR reaction system cumulative volume is 25 μ l, wherein SYBR Premix Ex Taq 12.5 μ l, the each 0.5 μ l of 10 μ mol sense primer PBF2 and antisense primer PBR2, standard substance 10 times of gradient dilution liquid (1.04ng, 104pg, 10.4pg, 1.04pg, 104fg, 10.4fg) and detected sample DNA solution 2 μ l, remainder ddH
2o polishing.PCR adopts two-step approach: 95 DEG C of 10min, then; 95 DEG C of 15s, 60 DEG C of 1min are 1 circulation, totally 40 circulations.
After loop ends, the analysis software that adopts instrument to carry, analysing amplified result (Fig. 5), calculates testing sample pathogen plasmodiophora DNA content.Result shows that standard substance have fabulous linear relationship, and relation conefficient is 0.995; Testing sample has obvious fluorescent signal to increase, and has good kinetic curve, and in reaction system, in grave illness field and hypopathia field soil sample sample, pathogen plasmodiophora average dna content is respectively 55.61 and 17.06pg.
The CT value of table 2 quantitative fluorescent PCR and the DNA content of sample
The above results shows pathogen plasmodiophora in the soil long-term sequential cropping cultivation along with crop in cruciferae, can amount reproduction and accumulation in soil, cause the state of an illness to increase the weight of year by year, by the detection of the soil sample to hypopathia field and anosis field, can tentatively determine that pathogen plasmodiophora causes the general morbidity threshold value of pathogen plasmodiophora in field, and plasmodiophora brassicae also can be detected in anosis field, illustrate that the method also has very high susceptibility while application in field, the foundation of the method can to the plasmodiophora brassicae in field soil sample promptly and accurately carry out detection by quantitative, we are formulated to the aspects such as means of prevention such as cultivation disease-resistant variety and rotation of crops crop rotation and there is important directive significance.
Embodiment 4: artificial inoculation soil sample detects
In Vegetable & Flower Inst., Chinese Academy of Agriculture Science's field experiment, carry out the preparation of artificial inoculation soil sample in October, 2012.Get appropriate susceptible old complaint hollander and break into homogenate, leave standstill after 2 hours, proportionally add water and mix inoculation with sterilizing soil.Make the sick soil (1.0 × 10 of 9 kinds of different concns
8, 1.0 × 10
7, 1.0 × 10
6, 1.0 × 10
5, 1.0 × 10
4, 1.0 × 10
3, 5.0 × 10
2, 10,0 statospore number/g sterilizing soil), the pH value of soil sample is adjusted to 6.5 left and right, and-20 DEG C save backup.Get freeze-drying sample 0.2-0.5g, adopt (MP Biomedicals company of the U.S., FastDNA SPIN Kit for Soil) extraction, purify DNA.DNA is dissolved in 40 μ l TE damping fluids, gets 2 μ l DNA solutions and makes template, carries out fluorescence quantitative PCR detection.
Quantitative fluorescent PCR reaction system cumulative volume is 25 μ l, wherein SYBR Premix Ex Taq 12.5 μ l, the each 0.5 μ l of 10 μ mol sense primer PBF3 and antisense primer PBR3, standard substance 10 times of gradient dilution liquid (2.16ng, 216pg, 21.6pg, 2.16pg, 216fg, 21.6fg) and detected sample DNA solution 2 μ l, remainder ddH
2o polishing.PCR adopts two-step approach: 95 DEG C of 10min, then; 95 DEG C of 15s, 58 DEG C of 1min are 1 circulation, totally 40 circulations.
After loop ends, the analysis software that adopts instrument to carry, analysing amplified result production standard curve (Fig. 6) and amplification curve (Fig. 7), calculate testing sample pathogen plasmodiophora DNA content.
The CT value of table 3 quantitative fluorescent PCR and the DNA content of sample
Adopt the soil sample of artificial inoculation to verify the validity of quantitative fluorescent PCR system.The above results shows that the Plasmodiophora brassicae Causing Cruciferae Clubroot fluorescence quantitative PCR detection system that this research is set up has very high susceptibility and reliability, the detection point of accumulation of this system is 24fg plasmid DNA/μ l, and corresponding fungus in soil amount is 500 statospore/g soil.This detected result is combined with Fields detection result, can set up club root field prediction system accurately.
Claims (3)
1. a fluorescence quantitative PCR detection technique for Plasmodiophora brassicae Causing Cruciferae Clubroot, is characterized in that comprising the following steps:
Get field natural occurrence soil, disease plant, brassicaceous vegetable seed, carry out the extraction purifying of total DNA, obtain DNA sample;
Auele Specific Primer design, on systems analysis pathogen plasmodiophora ITS district, 18SrRNA and the phylogenetic basis of 5.8rRNA nucleotide sequence, adopt CLUSTAL, compare with this region sequence of Plasmodiophoromycetes and soil-borne pathogen, find the specific sequence region of pathogen plasmodiophora, design Auele Specific Primer is to 1(PBF2/PBR2), primer pair 2(PBF3/PBR3), described primer pair is:
Primer pair 1 forward primer 5
,-CTTGCGTGTCGCTGTATTC-3
,
reverse primer 5
,-ATAGGTTGGGGTAACTTGGC-3
,;
Primer pair 2 forward primers 5
,-CTCACAACGATGAAGAAC-3
,
Reverse primer 5
,-ACTCACAGCCAAAGACAAGC-3
,
3) real-time fluorescence quantitative PCR reaction system is: quantitative fluorescent PCR reaction system cumulative volume is 25 μ l, and wherein SYBR Premix Ex Taq (includes TaKaRa Ex Taq TM HS, dNTP Mixture, Mg
2+with SYBR Green I) 12.5 μ l, concentration is the sense primer PBF2(PBF3 of 10 μ mol/L) and antisense primer PBR2(PBR3) each 0.5 μ l, 10 times of gradient dilution liquid of standard substance or detected sample DNA solution 2 μ l, ddH
2o polishing to 25 μ l;
4) PCR response procedures is: PCR adopts two-step approach, and 95 DEG C of 3min then carry out 40 circulations, and each circulation comprises 95 DEG C of 10s, 60 DEG C of 30s; After PCR completes, by 0.1 DEG C of s
-1temperature rise rate rises to 95 DEG C of analysis verifications that carry out melt curve analysis from 72 DEG C;
5) insert the plasmon DNA extracting after the pMD-20 carrier conversion bacillus coli DH 5 alpha propagation in ITS district, DNA is quantitative and 10 times of gradient dilutions through ultraviolet spectrophotometer A260, be diluted to 6 concentration gradients, increase according to above-mentioned PCR reaction system and program; After reaction finishes, adopt computer automatic drafting quantitative fluorescent PCR typical curve according to the cycle threshold of each concentration gradient (Ct);
6) get the DNA sample obtaining in step 1) as template, increase, and carry out the amplification of negative control and positive control simultaneously by above-mentioned quantitative fluorescent PCR reaction system and response procedures on real-time fluorescence quantitative PCR instrument, wherein negative control adopts ddH
2o, positive control adopts plasmodiophora brassicae strain gene group DNA; After reaction finishes, the cycle threshold of DNA sample (Ct) is contrasted with typical curve, obtain DNA sample Zhong ITS district gene fragment copy number, and then obtain the concentration of pathogen plasmodiophora in original sample.
2. the fluorescence quantitative PCR detection technique of the detection crucifer club root described in claims 1 can change into commercial test kit, test kit comprises: sense primer PBF2, antisense primer PBR2(sense primer PBF3, antisense primer PBR3), fluorescence dye SYBR Premix Ex Taq includes TaKaRa Ex Taq TM HS, dNTP Mixture, Mg
2+transform with SYBR Green I and by the pMD-20 carrier in insertion ITS district the plasmid DNA of extracting after bacillus coli DH 5 alpha propagation, DNA is quantitative and 10 times of gradient dilutions through ultraviolet spectrophotometer A260.
3. this technology can be applied in farm environment, comprises pathogen plasmodiophora on survive the winter in soil bacterium amount and brassicaceous vegetable plant is carried out to Real-Time Monitoring, and detect the pathogen plasmodiophora in brassicaceous vegetable seed-borne fungi and field irrigation water.
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| CN104694658A (en) * | 2015-03-19 | 2015-06-10 | 中国农业大学 | Detection method for phytopathogen content in soil |
| CN107090449A (en) * | 2017-05-23 | 2017-08-25 | 中国科学院东北地理与农业生态研究所 | A kind of method for extracting plant root endogenetic bacteria DNA |
| CN107683338A (en) * | 2015-06-17 | 2018-02-09 | 东洋制罐集团控股株式会社 | The inspection method of microorganism, microorganism check kit, the microorganism checking with microarray, mould detection with carrier, the detection method of mould and mould detection kit |
| CN107937593A (en) * | 2017-12-21 | 2018-04-20 | 沈阳农业大学 | The Specific PCR primers and detection method of Plasmodiophora brassicae |
| CN110241242A (en) * | 2019-06-10 | 2019-09-17 | 沈阳农业大学 | The quantitative detecting method of plasmodiophora brassicae in different type sample |
| CN110669858A (en) * | 2019-08-05 | 2020-01-10 | 中国农业科学院蔬菜花卉研究所 | Triple fluorescence quantitative PCR detection method and kit for cruciferous vegetable rhizoctonia solani |
| CN113604590A (en) * | 2021-08-09 | 2021-11-05 | 上海沃吉基因科技有限公司 | Fluorescent quantitative PCR detection method and kit for plant pathogenic bacteria Pantoea ananatis |
| CN113621726A (en) * | 2020-05-09 | 2021-11-09 | 中国农业科学院蔬菜花卉研究所 | Establishment and application of potato ustilaginoidea virens rapid detection system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104694658A (en) * | 2015-03-19 | 2015-06-10 | 中国农业大学 | Detection method for phytopathogen content in soil |
| CN107683338A (en) * | 2015-06-17 | 2018-02-09 | 东洋制罐集团控股株式会社 | The inspection method of microorganism, microorganism check kit, the microorganism checking with microarray, mould detection with carrier, the detection method of mould and mould detection kit |
| CN107090449A (en) * | 2017-05-23 | 2017-08-25 | 中国科学院东北地理与农业生态研究所 | A kind of method for extracting plant root endogenetic bacteria DNA |
| CN107937593A (en) * | 2017-12-21 | 2018-04-20 | 沈阳农业大学 | The Specific PCR primers and detection method of Plasmodiophora brassicae |
| CN110241242A (en) * | 2019-06-10 | 2019-09-17 | 沈阳农业大学 | The quantitative detecting method of plasmodiophora brassicae in different type sample |
| CN110669858A (en) * | 2019-08-05 | 2020-01-10 | 中国农业科学院蔬菜花卉研究所 | Triple fluorescence quantitative PCR detection method and kit for cruciferous vegetable rhizoctonia solani |
| CN110669858B (en) * | 2019-08-05 | 2022-06-24 | 中国农业科学院蔬菜花卉研究所 | Triple fluorescence quantitative PCR detection method and kit for cruciferous vegetable rhizoctonia solani |
| CN113621726A (en) * | 2020-05-09 | 2021-11-09 | 中国农业科学院蔬菜花卉研究所 | Establishment and application of potato ustilaginoidea virens rapid detection system |
| CN113604590A (en) * | 2021-08-09 | 2021-11-05 | 上海沃吉基因科技有限公司 | Fluorescent quantitative PCR detection method and kit for plant pathogenic bacteria Pantoea ananatis |
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