CN104032003A - Method for quantitatively detecting ustilaginoidea virens from seed-borne and soil-borne media - Google Patents

Method for quantitatively detecting ustilaginoidea virens from seed-borne and soil-borne media Download PDF

Info

Publication number
CN104032003A
CN104032003A CN201410259131.1A CN201410259131A CN104032003A CN 104032003 A CN104032003 A CN 104032003A CN 201410259131 A CN201410259131 A CN 201410259131A CN 104032003 A CN104032003 A CN 104032003A
Authority
CN
China
Prior art keywords
reaction
concentration
sequence
realamp
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201410259131.1A
Other languages
Chinese (zh)
Other versions
CN104032003B (en
Inventor
孙文献
韩彦卿
彭军
张楠
方安菲
张亢
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
China Agricultural University
Original Assignee
China Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by China Agricultural University filed Critical China Agricultural University
Priority to CN201410259131.1A priority Critical patent/CN104032003B/en
Publication of CN104032003A publication Critical patent/CN104032003A/en
Application granted granted Critical
Publication of CN104032003B publication Critical patent/CN104032003B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a method for quantitatively detecting ustilaginoidea virens from seed-borne and soil-borne media. The method comprises the steps of (1) pre-treating a detected sample, and quickly extracting DNA of the detected sample; (2) constructing a standard curve; and (3) performing reaction by using a fluorescent nucleic acid thermostatic amplification detection technology. The method can be used for quantitatively detecting the ustilaginoidea virens, overcomes the defects of the conventional first generation LAMP (loop-mediated isothermal amplification) detection method, and has the advantages of high sensitivity, high specificity, low pollution, stable reaction and the like. By adopting the strategy of sensitively detecting the reaction product by adopting a closed-tube detection technique, the method disclosed by the invention is unlikely to be affected by pollutants and can be used for detecting a soil sample on site and analyzing and judging the reaction product, so that the method is extremely simple and suitable for being widely popularized and applied.

Description

A kind of from kind of biography and native medium are situated between the method for detection by quantitative ustilaginoidea virens
Technical field
The present invention relates to microorganism field, in particular a kind of from kind of biography and native medium are situated between the method for detection by quantitative ustilaginoidea virens.
Background technology
False smut is to infect by ustilaginoidea virens (Ustilaginoidea virens, having condition is Villosiclava virens) a kind of Rice Panicle fungal disease causing; Pathogen infection causes the little long-pending pathogenic bacteria bacterium colony that is several times as much as the grain of rice of organizer, i.e. the rice curve taken of paddy rice.In recent years, along with popularization and the establishing in large scale of high-quality hybrid rice, the application of a large amount of nitrogenous fertilizer, is the popular favourable condition of having created of generation of false smut, by one in history fragmentary, sporadic disease develop and become one of the Major Diseases in each paddy rice producing region of China.The generation of false smut not only makes a big impact to the output of paddy rice, and ustilaginoidea virens can produce a large amount of toxin, and the cell fission of people, animal and plant is had to strong restraining effect.Therefore, the generation of false smut not only makes the paddy rice underproduction, but also can pollute grain, has influence on grain security, controls false smut and shoulders heavy responsibilities.
The symptom of false smut only manifests at the late growth stage of paddy rice, appears at the fringe portion of paddy rice, and the position such as the blade of rice plant, stem stalk is without any disease symptom before this.Under the natural condition in field, generally within about 1 week after Rice Flowering, start to have the tender rice curve of children to occur, after blooming 3 weeks, rice curve volume reaches maximum and a large amount of chlamydospore of its periphery generation.Under the larger weather condition of day and night temperature, the surface of paddy rice rice curve in ripe late period can form one to several sclerotium in irregular shape, and sclerotium can be sprouted generation thecaspore under certain condition, may become the bacterium source of next time infecting.
At present more limited to the research of false smut, a lot of basic theory Study on Problems seldom, for the infection mechanism of pathogenic bacteria, there are a lot of disputes in Disease Cycle etc., and how the solution of these problems is for carrying out, rapid molecular detects and effectively the comprehensive prevention and control of disease are very important.Recently, the mechanism that infects site and infect of false smut has been had to important breakthrough.By the serial section of rice curve is shown, the first of false smut infected the filigree tissue that site is paddy rice, and the most easily infects 3 filigrees between ovary and lodicule.Ustilaginoidea virens never infects Rice Ovary and flower pesticide, but has once in a while the peripheral organization that secondary hyphae infects column cap and lodicule.Pathogen infection pattern belongs to extracellular and infects and expand, and pathogenic bacteria mycelia does not directly penetrate and enter host cell, only at iuntercellular, walk, and the cell walls of filigree tissue is degradable the most at last.In rice curve growth course, although producing toxin, ustilaginoidea virens directly do not kill host cell, so false smut Pseudomonas biotroph pathogenic bacteria.In addition,, by the bacterial strain of GFP mark, Hu et al. further confirms that to the result of study in the first dip-dye site of false smut false smut is that filigree by small ear inside carries out just infecting.Mycelia 24h after artificial inoculation starts to contaminate, and after inoculation, after 168h, mycelia accumulative total reaches highest level, and before Rice Heading, mycelia is wrapped up all floral organs and produces rice curve after extending upward along the filigree infecting the top that arrives flower pesticide.
Although obtain above-mentioned important breakthrough, for the Disease Cycle of false smut, do not get conforming result of study always.As far back as the sixties in 20th century, Ikegami etc. have just carried out Histological research to infecting the seedling stage of ustilaginoidea virens.Found that, ustilaginoidea virens can successfully infect the tender coleoptile of children at Seed Germination of Rice initial stage, but does not find that ustilaginoidea virens can arrive at the spike primordium of plants stems tip as ustilago.In addition, infect the seedling stage of follow-up ustilaginoidea virens and carry out cytological observation and find that ustilaginoidea virens can infect the tender radicle of paddy rice children, and PCR detected result shows that whole plant comprises all portability pathogenic bacterias of fringe and seed.
By rice curve is carried out to morphological observation and adopt GFP mark bacterial strain research false smut infect site and infection mechanism has all confirmed that false smut is that specific period (rice ear sprouting period), specific site (filigree of small ear inside) infect.In addition, although research shows that chlamydospore can survive in seed and soil, but chlamydospore can only survive the very short time after sprouting, may not having time enough to serve as primary source of infection at Rise's boot period completes and infects, also illustrate that ustilaginoidea virens is after infecting seedling stage, be difficult under field conditions (factors) or can not effectively expand to fringe portion and cause disease.At present whether rice paddy seed is carried disease germs and can cause rice pathogenesis to have a large amount of disputes, send out disease tassel yield lower, well below high sense of height morbidity time kind field natural occurrence rate.
Recent studies have shown that false smut spore can grow nonparasitically upon another plant field planting on biological and abiotic surface.Under the anhydrous state of high humidity, false smut spore can asymptomaticly infect also field planting at Gramineae, the plant leaf of Cruciferae and Solanaceae.And having under water condition, on plant leaf, preservative film or glassine paper, these spores just can sprout and form mycelia for several days afterwards, and part spore can also directly produce secondary spore.In addition, if reverse lack of water process, these spores or sprout and form new spore, or oppositely form chlamydospore.These results show that ustilaginoidea virens has the characteristic of growing nonparasitically upon another plant, and the sclerotium after surviving the winter or chlamydospore grow nonparasitically upon another plant on paddy rice or vector, such as barnyard grass, and cogongrass and duckweed.At high humidity or rainy day sprouting, become spore and grow nonparasitically upon another plant on the surface of different plants or non-biological material, thereby Rise's boot period causes morbidity along with the media such as rainwater and wind infect paddy rice small ear.These find that explanation seed-borne fungi is also may become the primary source of infection of false smut.In sum, it is former that the chlamydospore that the sclerotium scattering in soil and seed carry may be all that false smut first infected.Therefore, the ustilaginoidea virens that must have a kind of fast and stable, sensitive quantitative detecting method to carry soil and seed detects, and the morbidity of false smut is carried out to early warning, is one of important guarantee of producing of rice safety.
In recent years, the method that has a PCR-based is successfully for the report of the detection of false smut, conventional PCR method improves a lot compared with additive method on the operating time and aspect the specificity detecting and sensitivity, but, PCR detection technique needs special plant and instrument and testing cost higher, and its detection time still long (2～3 hours), be not suitable for the large-scale application fast in field.Therefore, strengthen rice paddy seed carry disease germs detect and soil in the monitoring of cause of disease amount, a kind of highly sensitive, easy and simple to handle, with low cost and result is swift with judgement in the urgent need to developing, can replace to a certain extent the detection method of PCR.
Summary of the invention
Technical problem to be solved by this invention is the deficiency existing in detecting ustilaginoidea virens for prior art, provide a kind of from kind of biography and native medium are situated between the method for detection by quantitative ustilaginoidea virens.
Technical scheme of the present invention is as follows:
A method for detection by quantitative ustilaginoidea virens from kind of biography and native medium Jie, its step is as follows:
(1) test sample is carried out to pre-treatment, the DNA of rapid extraction test sample;
(2) structure of typical curve
PCR reaction system is: 2.5 μ L10 * PCR buffer, and 2 μ L2.5mM dNTPs, 0.25 μ L5U/ μ L Taq polysaccharase, each 1 μ L of 10pM primer uvr1497F/UV-B3, the rice green smut genomic dna that 1 μ L extracts, mends sterile purified water to 25 μ L; Wherein, the sequence of uvr1497F is 5 '-CCGCTGCCTAAGATAAAGTCC-3 ', and the sequence of UV-B3 is 5 '-CAAGTGCGAGGATAACTGAAT-3 ';
The condition of PCR reaction: 94 ℃ of denaturation 3min, 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ extend 90s, carry out 35 circulations; 72 ℃ extend 7min; After reaction finishes, get 5 μ L reaction product, separated amplified production on 1% agarose gel electrophoresis, the EB observations that dyes; Pcr amplification product is cut and reclaimed with test kit from gel, reclaim after product and be connected to pMD-18-T carrier, after sequence verification sequence, by this plasmid called after pMD18-T-UV, its concentration is 10ng/ μ L; By this plasmid DNA as template, after the dilution of 10 times of gradient series for building the RealAmp typical curve that detects rice aspergillus; RealAmp typical curve is to build according to the relation between the plasmid concentration of pMD18-T-UV in seed and corresponding Tt value, and X-axis represents the logarithmic value of starting template concentration, and Y-axis represents that different concns template amplification reaches threshold value time T t used;
(3) fluorescence nucleic acid constant-temperature amplification detection technique reaction
RealAmp reaction system: cumulative volume is 25 μ L, sample DNA after 1 μ L extraction purifying is as template, 2.5 μ L10 * BstDNA polymerase buffer, 1.0 μ L concentration are that Bst DNA polymerase, the 1.0 μ L concentration that the outside primer UV-B3 of 0.2 μ M and inner side primer UV-FIP that UV-F3,0.5 μ L concentration are 1.6 μ M and UV-BIP, 1 μ L concentration are 8U/ μ L are 0.2 μ MSYTO-9 fluorescence dye, mend sterile purified water to 25 μ L, and then the paraffin oil of system such as add to cover whole reaction solution to prevent volatilization; Wherein the sequence of UV-F3 is 5 '-CTTGTGTTTTCCAATGCATGT-3 ', the sequence of UV-B3 is 5 '-CAAGTGCGAGGATAACTGAAT-3 ', the sequence of UV-FIP is 5 '-ATGCCCGCCAGAATACTGGCGCAGAATTCAGTGAATCATCG-3 ', and the sequence of UV-BIP is 5 '-CCTCAAGCTCTGTCTTGCGACGAGACCGCCGATTCATTT-3 '; RealAmp reaction is carried out on ESE-Quant Tube Scanner, and 63 ℃ of reaction 90min, hatch 10min with termination reaction at 80 ℃ subsequently; Reaction finishes rear instrument and automatically according to typical curve, shows quantitative result.
Described method, in step (3), 10 * Bst DNA polymerase buffer contains 1.4mM dNTPs, 0.8mM trimethyl-glycine and 8mM MgSO 4.
Described fluorescence nucleic acid constant-temperature amplification detection technique reaction also can be used for the Rapid identification of field sample; In described step (3), rice curve genome DNA is set as positive control, TE (100mM Tris-HCl, pH8.0,50mM EDTA) as negative control, in reaction tubes, cover and drip SYBR Green I, reaction finishes with hand, to get rid of or instantaneous centrifugal the SYBR Green I covering in reaction tubes is blended in reaction solution afterwards, adopt covered colour developing to carry out judged result, if reaction solution color is orange, represent that result is negative, if reaction solution color is green, represent that result is positive.
Method energy detection by quantitative ustilaginoidea virens of the present invention, overcome the deficiency of first-generation LAMP detection method in the past, there is the advantages such as highly sensitive, high specific, low pollution, stable reaction, adopt covered (closed-tube detection technique) just strategy of detection reaction product delicately, the method is impact and the energy Site Detection pedotheque of vulnerable to pollution thing not, the method of analyzing judgement reaction product is very simple, is suitable for applying widely.
Accompanying drawing explanation
Fig. 1 is the specificity analyses electrophorogram (agarose gel electrophoresis (2%) detects RealAmp constant-temperature amplification product) of ustilaginoidea virens real-time fluorescence nucleic acid constant-temperature amplification technology for detection; In figure, M represents DNA marker (molecular weight marker); 1-8 represents the amplified production that Different Kinds of Pathogens thing DNA is template, be respectively rice green smut (Ustilaginoidea virens), rice blast (Magnaporthe grisea), banded sclerotial blight (Thanatephorus cucumeris), rice bakanae disease (Gibberella moniliformis), bacterial leaf-blight (Xanthomonas oryzae pv.oryzae), bacterial stripe (Xanthomonas oryzae pv.oryzicola), green muscardine fungus (Metarhizium anisopliae) and water contrast.
Fig. 2 is the product that agarose gel electrophoresis detects ustilaginoidea virens nest-type PRC specific amplification; In figure, M represents DNA marker; 1-8 is respectively the amplified production that Different Kinds of Pathogens thing DNA is template, be respectively rice green smut (Ustilaginoidea virens), rice blast (Magnaporthe grisea), banded sclerotial blight (Thanatephorus cucumeris), rice bakanae disease (Gibberella moniliformis), bacterial leaf-blight (Xanthomonas oryzae pv.oryzae), bacterial stripe (Xanthomonas oryzae pv.oryzicola), green muscardine fungus (Metarhizium anisopliae) and water contrast.
Fig. 3 is the specific amplification graphic representation of ustilaginoidea virens real-time fluorescence nucleic acid constant-temperature amplification technology for detection; X-axis represents proliferation time (min), and Y-axis represents the fluorescence signal intensity (mV) of response.
Fig. 4 is the detection curve of the real-time fluorescence nucleic acid constant-temperature amplification technology of seed-borne fungi; X-axis represents proliferation time (min), and Y-axis represents the fluorescence signal intensity (mV) of response.
Fig. 5 is the examination criteria curve of the real-time fluorescence nucleic acid constant-temperature amplification technology of seed-borne fungi.
Fig. 6 is seed-borne fungi real-time fluorescence quantitative PCR detection curve.
Fig. 7 is seed-borne fungi real-time fluorescence quantitative PCR examination criteria curve.
Fig. 8 is the curve of real-time fluorescence nucleic acid constant-temperature amplification technology in artificial inoculation soil; X-axis represents proliferation time (min), and Y-axis represents the fluorescence signal intensity (mV) of response.
Fig. 9 is the typical curve of real-time fluorescence nucleic acid constant-temperature amplification technology in artificial inoculation soil.
Figure 10 is artificial inoculation soil real-time fluorescence quantitative PCR detection curve.
Figure 11 is the typical curve that artificial inoculation soil real-time fluorescence quantitative PCR detects.
Figure 12 is field seed sample segment real-time fluorescence nucleic acid constant-temperature amplification technology for detection curve.
Figure 13 is field seed sample segment real-time fluorescence quantitative PCR detection curve.
Figure 14 is pathogenic bacteria result comparison in RealAmp assay and two kinds of detection method detection by quantitative seeds of realtime PCR.
Figure 15 is field part pedotheque real-time fluorescence nucleic acid constant-temperature amplification technology for detection curve.
Figure 16 is field part pedotheque real-time fluorescence quantitative PCR detection curve.
Figure 17 is two kinds of detection method detection by quantitative result contrasts.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
1, test sample is carried out to pre-treatment, the DNA of rapid extraction test sample
(1) seed DNA extraction step: weigh rice paddy seed according to the standard of thousand seed weight, after threshing, kind of subshell is collected according to the operation instructions of Promega plant genome DNA extraction test kit (Pu Luomaige (Beijing) Bioisystech Co., Ltd) and operate.
(2) soil DNA extraction step: add 2ml SPCB damping fluid (100mmol/LTris-HCl, 100mmol/L EDTA, 120mM Na according to 1 gram of soil 3pO 4, the massfraction CTAB that is 2%, 1.5M NaCl, pH8.0, the mercaptoethanol that percent by volume is 2%), the ratio of 100 μ g/ml Proteinase Ks, processing 30-60min for 37 ℃, is then 65 ℃ of water-bath 1-2hr in the SDS solution of 1% (mass percentage concentration) at final concentration, and centrifugal (room temperature) gets supernatant, with isopyknic chloroform extracting, once remove albumen, if after extracting, the interface of water and chloroformic solution is also unintelligible, can consider that extracting is once again with chloroform.After centrifugal, get supernatant, the NaAc (pH5.2) that first adds 1/10th volume 0.3M, adds isopyknic Virahol again after mixing, place 1hr precipitation DNA after fully mixing in-20 ℃, centrifugal rear by percent by volume, be that 75% ethanol cleans 1-3 time, add the ddH of 100 μ l 2o.Subsequent purification.
Purification step: G-75 and PVPP are mixed with purifying pillar (or Micro Bio-Spin of employing Bio-rad), excessively after post, preserve DNA standby.
Pillar preparation: 0.8ml SephedexG-75slurry adds the about 13-14mm of centrifuge tube, adds the about 5-7mm of dry PVPP in the above, then adds after the water of 100ul the centrifugal 3min of 350 * g for twice.The last DNA that adds 100 μ l to extract from soil in the pillar preparing, the centrifugal 1min of 350g (the centrifugal DNA of allowing enters G-75), 350g recentrifuge 10min purify DNA.
2, the preparation of Auele Specific Primer
According to rice aspergillus U.virens16S rDNA conserved sequence, design 2 pairs of Auele Specific Primers, each is a pair of to comprise outside primer (UV-F3 and UV-B3) and inner side primer (UV-FIP and UV-BIP).Wherein UV-F3 (5 '-CTTGTGTTTTCCAATGCATGT-3 ') and UV-B3 (5 '-CAAGTGCGAGGATAACTGAAT-3 ') are outside primer, UV-FIP (5 '-ATGCCCGCCAGAATACTGGCGCAGAATTCAGTGAATCATCG-3 ') and UV-BIP (5 '-CCTCAAGCTCTGTCTTGCGACGAGACCGCCGATTCATTT-3 ') are inner side primer, and the concentration of above-mentioned outside primer UV-F3 and UV-B3 is respectively 5pmol/ μ l; The concentration of above-mentioned inner side primer UV-FIP and UV-BIP is 40pmol/ μ l.
3, fluorescence nucleic acid constant-temperature amplification detection technique (RealAmp) reaction
Sample through pre-treatment, primer, reaction buffer are mixed with Bst archaeal dna polymerase, at 63 ℃ of insulation 90min, carry out endless chain replacement(metathesis)reaction.RealAmp is at the enterprising line operate of ESE-Quant Tube Scanner, ESE-Quant Tube Scanner is that a thermal cycling and fluoroscopic examination are in the RealAmp of one amplification instrument, there are 8 bottoming holes, instrument self can directly show detected result with display screen, can also be connected on computer and be controlled by computer.After finishing, amplification has following numerical value at the amplification curve that shows screen display:
Tt value (threshold time, Tt) refers to the time that fluorescent signal in each reaction tubes experiences while arriving the thresholding of setting, with Ct value comparing class in fluorescent PCR seemingly.
Threshold value (threshold) refers to the fluorescent signal value of setting.
Fluorescent signal value refers to the fluorescence signal intensity that reaction tubes Instrumental is measured, and with milli-volts (mV), represents.
RealAmp reaction system: cumulative volume is 25 μ L, the sample DNA after 1 μ L extraction purifying is as template, and 2.5 μ L10 * Bst DNA polymerase buffer (contain 1.4mM dNTPs, 0.8mM trimethyl-glycine, 8mM MgSO 4), 1.0 μ L concentration are the outside primer UV-B3 of 0.2 μ M and inner side primer UV-FIP and UV-BIP, 1 μ L Bst DNA polymerase (8U/ μ L) that UV-F3,0.5 μ L concentration are 1.6 μ M, 1.0 μ L concentration are 0.2 μ MSYTO-9 fluorescence dye, mend sterile purified water to 25 μ L, and then the paraffin oil of system such as add to cover whole reaction solution to prevent volatilization.
RealAmp reaction is carried out on ESE-Quant Tube Scanner (ESE Gmbh, Stockach, Germany), and 63 ℃ of reaction 90min, hatch 10min with termination reaction at 80 ℃ subsequently.By rice curve genome DNA as positive control; With TE (100mM Tris-HCl, pH8.0,50mM EDTA) as negative control.The reaction tubes that contains the reaction soln preparing is placed in to 63 ℃ of reaction 90min.Reaction finishes rear demonstration screen display amplification curve, and X-axis represents proliferation time, and Y-axis shows fluorescent value.
4, the structure of RealAmp typical curve
(1) detection sensitivity for accurate assessment RealAmp also builds typical curve thus, we adopt specific PCR primer uvr1497F (5 '-CCGCTGCCTAAGATAAAGTCC-3 ') (Ashizawa et al., 2010) and the ITS fragment of UV-B3 (5 '-CAAGTGCGAGGATAACTGAAT-3 ') combination amplification 1240bp, this fragment has comprised between the amplification region of real-time PCR (Real-Time Fluorescent Quantitative PCR Technique) and RealAmp, subsequently, this PCR product cloning is arrived to pMD18-T carrier, after sequence verification is correct, called after pMD18-T-UV, detection sensitivity for assessment of seed-borne fungi, and build thus typical curve.
(2) PCR reaction system: 2.5 μ L10 * PCR buffer (Mg 2+), 2 μ L dNTPs (2.5mM each), 0.25 μ L Taq polysaccharase (5U/ μ L), each 1 μ L (10pM) of primer uvr1497F/UV-B3, rice green smut (Ustilaginoidea virens) genomic dna that 1 μ L extracts, mends sterile purified water to 25 μ L; The condition of PCR reaction: 94 ℃ of denaturation 3min, 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ extend 90s, carry out 35 circulations; 72 ℃ extend 7min.After reaction finishes, get 5 μ L reaction product, separated amplified production on 1% agarose gel electrophoresis, the EB observations that dyes.Pcr amplification product is cut and reclaimed with test kit from gel, reclaim after product and be connected to pMD-18-T carrier, after sequence verification sequence, by this plasmid called after pMD18-T-UV (10ng/ μ L).By this plasmid DNA, as template, the detection sensitivity for assessment of RealAmp after 10 times of gradient series dilutions also builds respectively and detects the RealAmp of rice aspergillus and the typical curve of real-time PCR thus.RealAmp typical curve is to build according to the relation between the plasmid concentration of pMD18-T-UV in seed and corresponding Tt value, X-axis represents the logarithmic value of starting template concentration, Y-axis represents that different concns template amplification reaches the threshold value time used (Tt), and the typical curve of resulting Seed-associated fungi is y=-3.1143+8.6866 (R 2=0.9935); The typical curve of real-timePCR builds according to the relation between the plasmid concentration of pMD18-T-UV in seed and corresponding Ct value, X-axis represents the logarithmic value of starting template concentration, Y-axis represents that different concns template amplification reaches the threshold value time used (Ct), and the typical curve of resulting Seed-associated fungi is y=-2.1+11.043 (R 2=0.9983).
5, result judgement
This experiment adopts two kinds of diverse ways judged results, and a kind of is detection by quantitative, and directly, after ESE-Quant Tube Scanner reaction finishes, instrument shows quantitative result according to typical curve automatically.In addition, in order to adapt to the Rapid identification of field sample, isothermal amplification reactions finishes with hand, to get rid of or instantaneous centrifugal the SYBR Green I covering in reaction tubes is blended in reaction solution afterwards, adopt covered colour developing to carry out judged result, if reaction solution color is orange, represent that result is negative, if reaction solution color is green, represent that result is positive.Covered detection can be avoided the crossed contamination between sample, and can be used as the auxiliary judgment mode outside detection by quantitative result.
The specificity analyses that embodiment 2RealAmp detects
Other rice pathogen thing for specificity analyses has rice blast (Magnaporthe grisea), banded sclerotial blight (Thanatephorus cucumeris), rice bakanae disease (Gibberella moniliformis), bacterial leaf-blight (Xanthomonas oryzae pv.oryzae), bacterial stripe (Xanthomonas oryzae pv.oryzicola), also has (specifically in Table 1) such as disinsection fungal green muscardine funguss (Metarhizium anisopliae).
Product electrophoresis detection result demonstration after constant-temperature amplification, only template is that false smut (Ustilaginoidea virens) genomic dna shows gradient band, all the other pathogen DNA are all without gradient band (being shown in Fig. 1); All bacterial strains adopt the nest-type PRC of ustilaginoidea virens to detect, and result demonstration, only has false smut genomic dna to amplify the specific band of 232bp, and other contrast without amplified production (seeing Fig. 2); The amplification curve that ESE-Quant Tube Scanner instrument carries out after RealAmp amplified reaction only has 1 (seeing Fig. 3), and corresponding numbering is corresponding is to take the PCR pipe that Ustilaginoidea virens genomic dna is template.Therefore, result shows that the designed primer of RealAmp is the Auele Specific Primer of false smut (Ustilaginoidea virens).
In this research of table 1 for testing real-time fluorescence nucleic acid constant-temperature amplification technology specificity bacterial strain used and numbering thereof
ATCC represents American Type Culture Collecti; ACCC represents Chinese agriculture microbial strains preservation center; CFCC represents China Forest microbial strains preservation center; CCCAU represents culture presevation head office of China Agricultural University."+" represents positive; "-" represents negative.
The analysis of embodiment 3 Seed-associated fungi susceptibilitys and typical curve thereof build
The pMD-18-T-UV plasmid DNA including between RealAmp amplification region of take is template, with the DNA of health kind skin, as 10 times of gradient series dilution methods of water, pMD-18-T-UV plasmid DNA is diluted to 3.8 * 10 successively from 38ng/ μ l -6ng/ μ l, with the negative contrast of sterilized water.Result shows, RealAmp can be respectively 38ng/ μ l～3.8 * 10 from pMD-18-T-UV plasmid DNA concentration -4in the template DNA of 6 orders of magnitude such as ng/ μ l, amplify product, after SYBR GreenI dyeing, be green, negative control colour developing, for orange, shows that the detection rice aspergillus DNA lower limit of RealAmp is about 3.8 * 10 -4ng/ μ l (seeing Fig. 4);
ESE-Quant Tube Scanner amplification curve shows the starting template DNA of different concns and between Tt value, has highly linear relation accordingly.The logarithmic value that represents starting template concentration with X-axis, Y-axis represents that different concns template amplification reaches the threshold value time used (Tt), the R of the typical curve building thus 2=0.9935, regression equation is Y=-3.1143X+8.6866 (seeing Fig. 5).If when quantitative by the method, only need to know that amplification reaches the threshold value time used (Tt), just can obtain the content that seed carries rice aspergillus.
Equally, employing pMD-18-T-UV plasmid DNA is diluted and is simulated as much as possible the detection sensitivity that field real detection case assessment real-timePCR detects seed pathogenic bacteria with the healthy DNA that plants skin, under the detection of result demonstration real-time PCR, is limited to 3.8 * 10 -6ng/ μ l (seeing Fig. 6), is 100 times of RealAmp, and its regression equation is Y=-2.1X+11.043, R 2=0.9983 (seeing Fig. 7).
In embodiment 4 artificial inoculation soil, detect the structure of chlamydospore susceptibility and typical curve
1, experimental technique
The chlamydospore artificial inoculation soil that the artificial inoculation of take morbidity produces is sample, from soil, extract total DNA, according to being used as the detection sensitivity of template assessment RealAmp and the amplification curve after ESE-Quant Tube Scanner amplified reaction after 1:10 series gradient dilution, determine the detection lower limit of RealAmp and realtime PCR.
2, experimental result
Result shows, the template DNA that RealAmp can dilute from five orders of magnitude, amplify product.Between the starting template DNA of different concns and corresponding Tt value, there is good linear relationship.The logarithmic value that represents starting template concentration with X-axis, Y-axis represents that different concns template amplification reaches the threshold value time used (Tt), take this, to build typical curve that RealAmp detects be y=-3.025x+31.051, R 2=0.9884 (seeing Fig. 8, Fig. 9).Equally, real-timePCR can amplify product from the template DNA of 6 order of magnitude dilutions, and amplification typical curve is y=-2.0629+30.63, R 2=0.9911 (seeing Figure 10, Figure 11).RealAmp can detect 4.5 * 10 3spore/gram pedotheque, and realtime PCR can detect 4.5 * 10 2spore/gram pedotheque.
The practical application that embodiment 5RealAmp detects
At Rice Cropping field random acquisition sample, on the spike of rice that has rice curve, gathering other grain of rice detects as a sample, in addition, the paddy rice cross breeding seed of each paddy fields of China is carried out random inspection and carries out RealAmp detection, positive control and negative control are set, result shows, in 85 samples, the positive that real-time PCR and RealAmp detect is respectively 70 (82.4%) and 68 (80%), at RealAmp, detect in 17 negative samples, real-time PCR only detects 2 positive more, the detected result of these two kinds of methods is basically identical, show that RealAmp detected result is reliable and stable, and the content of can quantitative analysis rice paddy seed organizing rice aspergillus, the results are shown in Figure 12, Figure 13, Figure 14.
136 parts of the pedotheques of field morbidity detect, it is 124 that the positive that real-time PCR and RealAmp detect is respectively, the detected result of these two kinds of methods is consistent, although illustrate that RealAmp is lower than real-time PCR in detection sensitivity, but being applied in the pedotheque with inhibitory substance is to have powerful advantage, the strand displacement activity of Bst archaeal dna polymerase has suitable tolerance, RealAmp detected result is reliable and stable, and content (Figure 15, Figure 16, Figure 17) that can quantitative analysis soil semilate rice aspergillus.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.

Claims (2)

1. a method for detection by quantitative ustilaginoidea virens from kind of biography and native medium Jie, is characterized in that, its step is as follows:
(1) test sample is carried out to pre-treatment, the DNA of rapid extraction test sample;
(2) structure of typical curve
PCR reaction system is: 2.5 μ L10 * PCR buffer, and 2 μ L2.5mM dNTPs, 0.25 μ L5U/ μ L Taq polysaccharase, each 1 μ L of 10pM primer uvr1497F/UV-B3, the rice green smut genomic dna that 1 μ L extracts, mends sterile purified water to 25 μ L; Wherein, the sequence of uvr1497F is 5 '-CCGCTGCCTAAGATAAAGTCC-3 ', and the sequence of UV-B3 is 5 '-CAAGTGCGAGGATAACTGAAT-3 ';
The condition of PCR reaction: 94 ℃ of denaturation 3min, 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ extend 90s, carry out 35 circulations; 72 ℃ extend 7min; After reaction finishes, get 5 μ L reaction product, separated amplified production on 1% agarose gel electrophoresis, the EB observations that dyes; Pcr amplification product is cut and reclaimed with test kit from gel, reclaim after product and be connected to pMD-18-T carrier, after sequence verification sequence, by this plasmid called after pMD18-T-UV, its concentration is 10ng/ μ L; By this plasmid DNA as template, after the dilution of 10 times of gradient series for building the RealAmp typical curve that detects rice aspergillus; RealAmp typical curve is to build according to the relation between the plasmid concentration of pMD18-T-UV in seed and corresponding Tt value, and X-axis represents the logarithmic value of starting template concentration, and Y-axis represents that different concns template amplification reaches threshold value time T t used;
(3) fluorescence nucleic acid constant-temperature amplification detection technique reaction
RealAmp reaction system: cumulative volume is 25 μ L, sample DNA after 1 μ L extraction purifying is as template, 2.5 μ L10 * Bst DNA polymerase buffer, 1.0 μ L concentration are that Bst DNA polymerase, the 1.0 μ L concentration that the outside primer UV-B3 of 0.2 μ M and inner side primer UV-FIP that UV-F3,0.5 μ L concentration are 1.6 μ M and UV-BIP, 1 μ L concentration are 8U/ μ L are 0.2 μ MSYTO-9 fluorescence dye, mend sterile purified water to 25 μ L, and then the paraffin oil of system such as add to cover whole reaction solution to prevent volatilization; Wherein the sequence of UV-F3 is 5 '-CTTGTGTTTTCCAATGCATGT-3 ', the sequence of UV-B3 is 5 '-CAAGTGCGAGGATAACTGAAT-3 ', the sequence of UV-FIP is 5 '-ATGCCCGCCAGAATACTGGCGCAGAATTCAGTGAATCATCG-3 ', and the sequence of UV-BIP is 5 '-CCTCAAGCTCTGTCTTGCGACGAGACCGCCGATTCATTT-3 '; RealAmp reaction is carried out on ESE-QuantTubeScanner, and 63 ℃ of reaction 90min, hatch 10min with termination reaction at 80 ℃ subsequently; Reaction finishes rear instrument and automatically according to typical curve, shows quantitative result.
2. method according to claim 1, is characterized in that, in step (3), 10 * BstDNA polymerasebuffer contains 1.4mMdNTPs, 0.8mM trimethyl-glycine and 8mM MgSO 4.
CN201410259131.1A 2014-06-11 2014-06-11 A kind of method of detection by quantitative ustilaginoidea virens from kind of biography and native medium are situated between Expired - Fee Related CN104032003B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410259131.1A CN104032003B (en) 2014-06-11 2014-06-11 A kind of method of detection by quantitative ustilaginoidea virens from kind of biography and native medium are situated between

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410259131.1A CN104032003B (en) 2014-06-11 2014-06-11 A kind of method of detection by quantitative ustilaginoidea virens from kind of biography and native medium are situated between

Publications (2)

Publication Number Publication Date
CN104032003A true CN104032003A (en) 2014-09-10
CN104032003B CN104032003B (en) 2016-01-20

Family

ID=51462997

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410259131.1A Expired - Fee Related CN104032003B (en) 2014-06-11 2014-06-11 A kind of method of detection by quantitative ustilaginoidea virens from kind of biography and native medium are situated between

Country Status (1)

Country Link
CN (1) CN104032003B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105969871A (en) * 2016-06-12 2016-09-28 华中农业大学 Ustilaginoidea virens PCR detection primers and application thereof
CN111621587A (en) * 2020-06-05 2020-09-04 南京农业大学 LAMP (loop-mediated isothermal amplification) rapid detection method for ustilaginoidea virens in rice seeds and application

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103589794A (en) * 2013-11-04 2014-02-19 中国热带农业科学院环境与植物保护研究所 Method for real-time fluorescence isothermal quantitative detection of citrus huanglongbing

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103589794A (en) * 2013-11-04 2014-02-19 中国热带农业科学院环境与植物保护研究所 Method for real-time fluorescence isothermal quantitative detection of citrus huanglongbing

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
郑静等: "稻曲病菌分生孢子实时PCR定量检测方法的建立和初步应用", 《中国水稻科学》, vol. 26, no. 4, 10 July 2012 (2012-07-10), pages 500 - 505 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105969871A (en) * 2016-06-12 2016-09-28 华中农业大学 Ustilaginoidea virens PCR detection primers and application thereof
CN105969871B (en) * 2016-06-12 2019-05-21 华中农业大学 Rice aspergillus PCR detection primer and application thereof
CN111621587A (en) * 2020-06-05 2020-09-04 南京农业大学 LAMP (loop-mediated isothermal amplification) rapid detection method for ustilaginoidea virens in rice seeds and application

Also Published As

Publication number Publication date
CN104032003B (en) 2016-01-20

Similar Documents

Publication Publication Date Title
Kumar et al. Rapid detection and quantification of Alternaria solani in tomato
Cohen et al. Monitoring physiological races of Podosphaera xanthii (syn. Sphaerotheca fuliginea), the causal agent of powdery mildew in cucurbits: factors affecting race identification and the importance for research and commerce
Yu et al. Organic fertilization alters the community composition of root associated fungi in Pisum sativum
Reddy et al. Treatment of millet crop plant (Sorghum bicolor) with the entomopathogenic fungus (Beauveria bassiana) to combat infestation by the stem borer, Chilo partellus Swinhoe (Lepidoptera: Pyralidae)
CN102220430B (en) Auxiliary screening method for stripe rust-resistance wheat and its special primers
Ferniah et al. Characterization and pathogenicity of Fusarium oxysporum as the Causal Agent of Fusarium wilt in chili (Capsicum annuum L.)
Barman et al. Identification and characterization of fungi associated with blister blight lesions of tea (Camellia sinensis L. Kuntze) isolated from Meghalaya, India
Campbell et al. Development of southern stem canker disease on soybean seedlings in the greenhouse using a modified toothpick inoculation assay
Qian et al. Funneliformis mosseae affects the root rot pathogen Fusarium oxysporum in soybeans
CN104232748B (en) Whether a kind of red bayberry nursery stock carries the rapid molecular detection method of wilting germ
Jamali et al. Species-specific ITS primers for the identification of Picoa juniperi and Picoa lefebvrei and using nested-PCR for detection of P. juniperi in planta
CN104032003B (en) A kind of method of detection by quantitative ustilaginoidea virens from kind of biography and native medium are situated between
CN103555850B (en) Specific primer, kit and method for detecting Lonsdalea quercina subsp.populi
CN104911262B (en) A kind of dalbergia odorifera tar spot bacterium molecule detection primer and quick determination method
Das et al. Detection of Phytophthora nicotianae in water used for irrigating citrus trees by Ypt1 gene based nested PCR
CN103882109B (en) The molecular detecting method of sugarcane toppers maize ear rot pathogenic bacteria microspecies gx1
Sousa et al. Detection and transmission of Fusarium verticillioides in corn seeds according to the plant stage
Sharma et al. PCR based assay for the detection of Alternaria brassicicola in crucifers
CN105602948A (en) Genes and method for identifying gossypium hirsutum linn. variety verticillium wilt resistance by fluorescence quantitative PCR technique
Li et al. Analysis of bacterial and fungal community structure in replant strawberry rhizosphere soil with denaturing gradient gel electrophoresis
Saad et al. Relationship between ganoderma ergosterol concentration and basal stem rot disease progress on Elaeis guineensis
Jamali et al. Nested-PCR for detecting Terfezia claveryi in roots of Helianthemum species in field and greenhouse conditions
CN104212896B (en) The Molecular Identification primer of a kind of Tobacco Angular Leaf Spot Disease bacterium and authentication method
CN113789405A (en) Method for detecting content of gibberella zeae in soil based on real-time fluorescent quantitative PCR
Dai et al. Establishment and evaluation of a TEF1-α based loop-mediated isothermal amplification assay for detection of Phomopsis longicolla

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20160120

Termination date: 20160611

CF01 Termination of patent right due to non-payment of annual fee