CN101985652A - High-throughput molecular detection of Fusarium graminearum on carbendazim medicament-resistant gene frequency - Google Patents
High-throughput molecular detection of Fusarium graminearum on carbendazim medicament-resistant gene frequency Download PDFInfo
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Abstract
The invention belongs to high-throughput molecular detection of Fusarium graminearum on carbendazim medicament-resistant gene frequency, which can be used for monitoring the medicament resistance of the Fusarium graminearum and early warning medicament-resistant epiphytotic disease of the Fusarium graminearum. The invention provides medicament-resistant high-throughput detection technology constructed based on the fact that over 97 percent of Fusarium graminearum medicament-resistant genetype to carbendazim results from mutation of the 167th locus of beta2-microtubulin gene. The detection method mainly comprises the following three steps of: (1) respectively extracting known sensitive and medicament-resistant strains and genome DNA of samples to be detected; (2) performing specific real-time quantitative PCR reaction and establishing a standard curve; and (3) contrasting the standard curve to solve the medicament-resistant gene frequency in the detected samples. The method has the characteristics of high throughput, rapidness and accuracy. The sensitivity of detecting the medicament-resistant gene frequency is millionth to one hundred thousandth, and the accuracy rate is over 96 percent.
Description
One, technical field
The invention belongs to the high-throughput molecular detecting method of Fusarium graminearum (Fusarium graminearum) to the carbendazim-resistance gene frequency, can be used for monitoring benzimidazole germicide resistance Fusarium graminearum colony development trend, carry out the fashion forecasting and the early warning of resistance wheat scab.
Two, technical background
Wheat scab is a kind of worldwide disease that is caused by Fusarium graminearum Fusarium graminearum Schwabe, has a strong impact on the yield and quality of wheat.Adopt benzimidazole germicide for a long time or carry out chemical prevention based on composite dose of this class medicament.Benzimidazole germicide is efficient as a class, the wide spectrum systemic fungicide is used on producing, and has solved the environmental toxicity problem of protective fungicide, has improved the ability of human this disease of control.Benzimidazole germicide comprises derosal, F-1991, thiabendazole, thiophanate etc.These sterilant have identical antimicrobial spectrum and antibacterial mechanisms, and they also have identical resistance mechanism, have the quadrature transreactance property of medicine each other.Because the height specialization of this class medicament, action site is single, adds the frequency of administration height, and resistance advantage physiological strain will appear in the use many plant pathogenic fungis in back colony for many years, makes chemical control lose effect fully.Through effort in recent years, Agricultural University Of Nanjing's sterilant laboratory study finds that fusarium graminearum mainly is because Fusarium graminearum β 2-microtubule protein gene (FGSG_06611.3) sudden change causes to the drug-fast strain of derosal, the codon mutation of these genes encoding 167 amino acids (sports tyrosine Tyr by phenylalanine Phe, base is by TTT → TAT, be single base mutation) cause the resistance of this bacterium to derosal, this genotype accounts for more than 97% in the gibberellic hypha colony of field drug-fastness.
Because pathogenic bacteria is in natural enormous amount, (1～5%) can cause that the resistance disease is popular when the ratio of resistance individuality in colony was very low, made the chemical control failure.Traditional detection method needs the separation and Culture pathogenic bacteria, cultivates on the pastille substratum then, according to medicament the effect of mycelial growth is differentiated resistance again.This sensitivity testing method to medicament needs several time-of-weeks usually, and workload is big, and it is limited to measure sample size; When the drug resistance gene frequency is difficult to when following find 1%.Therefore, whether traditional monitoring for resistance method can only examine the chemical control failure owing to resistance causes, can not early warning.The frequency of resistance physiological strain/drug resistance gene in the early detection cause of disease colony is implemented the resistance management strategy early and is implemented early warning, is the effective measures of resistance management.On resistance Mechanism Study basis, principle according to point mutation, use conventional nucleic acid technology such as ASO-PCR technology etc. and detect the resistance of pathogenic fungi benzimidazole germicides such as derosal, then can fast, accurately detect sample, but a PCR reaction system can only detect the susceptibility of a bacterial strain to Bavistin, and the single bacterial strain that can only obtain to be measured by electrophoresis detection after whole PCR reaction system finishes is to derosal susceptibility.Real-time quantitative PCR (Real-time quantitative PCR) method has then changed the reality that pathogenic bacteria can only single sample detects the detection of fungicide sensitivity, it has the high-throughput that (1) is detected, promptly in a reaction system, can be to the sensitivity testing of all samples to be checked to sterilant; (2) real-time of detected result can be understood the size of drug-fast strain subpopulation in the sample in PCR reaction is carried out; (3) high efficiency, the technical measurement drug-fast strains such as ASO-PCR of classical Plating (at least two weeks above could obtain the result), routine can only carry out the mensuration of single susceptibility to sterilant, this method is then measured a large amount of samples simultaneously, consuming time only is 6 hours, can accurately understand then the generation of drug-fast strain subpopulation, development, popular situation, effectively instruct medication then.(4) make and detect low-frequency drug resistance gene in early days and become possibility.
This invention adopts real time quantitative PCR method to detect the drug resistance gene frequency of Fusarium graminearum to derosal, comprises the pre-treatment of sample, the extraction of genomic dna, the process of real-time quantitative PCR amplification.
Three, summary of the invention
Technical problem the object of the present invention is to provide a kind of method for quick to Fusarium graminearum carbendazim-resistance gene frequency.This technology is utilized Cycling probe Real time PCR first, according to the resistant strain genotype, optimize reaction conditions, real-time quantitative PCR (Real-time quantitative PCR) can and carry out the diagnosis and the detection of a large amount of, quick and easy resistance subpopulation early stage in the wheat scab period of disease, detect more than the rate of accuracy reached to 96%, have a practical value the control this season resistance disease of in time adopting an effective measure is popular.
The gene test of technical scheme Fasarium graminearum for resisting carbendazim is based on β
2Its 167th amino acids codon origination point sudden change of encoding of-microtubule protein gene (FGSG_06611.3), TTT (Phe) → TAT (Tyr).
Fusarium graminearum was divided into for three steps to the high-throughput molecular detecting method of carbendazim-resistance gene frequency:
(1) adopts conventional phenol chloroform isoamyl alcohol method, extract the nuclear gene group DNA of known drug-resistant strains, susceptibility bacterial strain and testing sample respectively.
(2) the specific C ycling Probe of utilization primer and design carries out the real-time quantitative PCR reaction, sets up the typical curve that drug-fast strain and sensitive strain detect respectively.
The key of the real-time detection technique of high-throughput of drug resistance gene frequency is that the Cycling probe that is used for real-time quantitative PCR has specificity in the Fusarium graminearum colony, and the foundation of design is that drug-fast strain is at β
2The 167th of-tubulin is by Phe (TTT) → Tyr (TAT).
1. Fusarium graminearum β increases
2The pulsating primer of-microtubule protein gene is right
Codon167F?5′AAGCCATTGATGTTGTTCG?3′
Codon167R?5′CATGACGGTAGAAATCAGGTAG?3′
2. specific C ycling probe
P1?5’-(FAM)CATAACGGAA?
AGG(Eclipse)-3’
P2?5’-(HEX)CATAACGGAA?
AGG(Eclipse)-3’
Real-time quantitative PCR (Real-time quantitative PCR) reaction system and amplification program thereof:
Reaction system:
10×CycleavePCR?Buffer 2.5μL
DNTP Mixture (each 2.5mM) 3 μ L
Mg?Solution(25mM) 5μL
Codon167F(10μM) 0.5μL
Codon167R(10μM) 0.5μL
P1(5μM) 0.6μL
P2(5μM) 1μL
TliRNase?H?II(200U/μl) 0.5μL
TaKaRa?Ex?Taq
TM?HS(5U/μl) 0.25μL
DH
2O (sterile purified water) 9.15 μ L
Dna profiling * 2.0 μ L
* dna profiling: responsive and resistant strain respectively adds 1 μ L.
Amplification condition:
Pre-sex change: 95 ℃ of 30s
↓
Sex change: 95 ℃ of 5s
Annealing: 55 ℃ of 20s
Extend: 72 ℃ of 31s
40 circulations
(3) testing sample uses above-mentioned system and condition to carry out the real-time quantitative PCR reaction, contrasts two typical curves having set up, obtains the quantity and the ratio of drug-fast strain in the Fusarium graminearum total amount of corresponding drug-fast strain and susceptibility bacterial strain.
The detection method of beneficial effect Fasarium graminearum for resisting carbendazim subpopulation of the present invention, compared with prior art,
1. real-time quantitative PCR (Real-time quantitative PCR) method has changed pathogenic bacteria and can only carry out single pattern detection to the detection of fungicide sensitivity, compare with the ASO-PCR Molecular Detection with the plate detection of routine, have following advantage and positively effect.
(1) high-throughput of Jian Ceing, promptly in a reaction system, can be to the sensitivity testing of all samples to be checked to sterilant;
(2) real-time of detected result can be understood the size of drug-fast strain subpopulation in the sample in PCR reaction is carried out;
(3) detect high efficiency, classical Plating, conventional technical measurement drug-fast strains such as ASO-PCR can only carry out the mensuration of single susceptibility to sterilant, this method is then measured a large amount of samples simultaneously, consuming time only is 6 hours, can accurately understand then the generation of drug-fast strain subpopulation, development, popular situation, effectively instruct medication then.
(4) make and detect low-frequency drug resistance gene in early days and become possibility.
(5) the present invention is the resistance subpopulation that utilizes Cycling probe real-time quantitative PCR technology for detection Fasarium graminearum for resisting carbendazim sterilant in the world first.
(6) compare with common SYBR GREEN I dye method, this technology can detect resistance and sensitive strain simultaneously, need not be in charge of, and realizes multiple detection.
2, at present domestic and international research unit all adopts the mycelial growth method to detect the Fusarium graminearum resistance, takes 6 days at least but this method relates to sampling, separation and Culture needs 3～5 days and indoor a large amount of insecticide sensitivity determination experiment, and the cycle is longer.There is not sophisticated resisting carbendazim fusarium graminearum molecular detection technology to use at present as yet.Real-time quantitative PCR then can come out whole testing samples in a reaction system to the carbendazim-resistance level detection, promptly know the dynamic result of this reaction in the carrying out of PCR reaction.This method has high-throughput, quick, abridged edition.This in time, reasonably instructs the science medication to the development trend of timely understanding resistance pathogenic bacteria subpopulation, effectively administers resistance, and reduces cost and reduce environmental pollution and have realistic meaning.
3, have 4% bacterial strain that derosal is had resistance in the 100 strain Fusarium graminearums of being measured approximately, result's (4.02%) that this real-time quantitative PCR measurement result and traditional colony diameter method record matches.
Four, description of drawings
The amplification curve of Fig. 1 responsive probe (P2)
The amplification curve of Fig. 2 resistance probe (P1)
Fig. 3 quantitative PCR specific detection demonstration graph
Fig. 4 primer is to the pcr amplification condition of Codon167F/Codon167R
The typical curve of Fig. 5 responsive probe (P2)
The typical curve of Fig. 6 resistance probe (P1)
Five, embodiment
Embodiment 1Cycling Probe fluorescence dye quantitative PCR system optimization
1.1 stability and the reliability of the optimization of annealing temperature in order to guarantee to detect, the CycleavePCR of precious biotechnology (Dalian) company limited has been selected in experiment for use
TMCore Kit DCY501 test kit.
In the PCR reaction process, annealing temperature Tm value is the important assurance of atopic, will produce non-specific product if annealing temperature is crossed to hang down., lower among the present invention to the primer requirement because probe has stronger specificity, only need its assurance that sufficiently high reaction efficiency is arranged.With reference to primer Tm value, carry out grads PCR from 50～60 ℃, the binding reagents box is recommended annealing temperature, and the suitableeest annealing temperature of having determined quantitative PCR detection is 55 ℃.
1.2 it is to determine to obtain the suitableeest concentration and probe concentration that concentration and probe concentration is optimized the purpose of this step.The concentration of probe just can influence the height of fluorescent signal.When concentration and probe concentration is low excessively, the PCR product is excessive, then can't obtain correct typical curve.And, must adjust the concentration of two probes in order to prevent of the phase mutual interference of two fluorophors in same system.To add probe (5 μ M) in the experiment in the 25 μ l reaction systems and increase progressively with 0.1 μ l, to determine best concentration and probe concentration P1 (5 μ M) 0.6 μ l, P2 (5 μ M) 1 μ l from 0.1 μ l～2 μ l.
The sensitivity of embodiment 2Cycling Probe fluorescence dye quantitative PCR
Standard substance require purity height, homogeneous, stable.Choose in this experiment the PCR product fragment of purifying is cloned on the carrier as standard substance.Sensitive strain ZF43 standard substance copy number is 1.33 * 10
6～1.33 * 10
2Individual, resistant strain ZF52 standard substance copy number is 1.78 * 10
6～1.78 * 10
2Individual, be 10 times of gradient dilutions.Standard substance are increased, and (Fig. 1, Fig. 2), hence one can see that, and this technology lowest detection copy number is 10 to obtain two groups of amplification curves respectively
2The order of magnitude, sensitivity are 10～100 times of regular-PCR detection method at least, also than general SYBR GREEN I dye method sensitivity height.
The specificity checking of embodiment 3Cycling Probe fluorescence dye quantitative PCR
With sensitive strain ZF43, resistant strain ZF52 is that template is carried out quantitative PCR detection respectively, and reaction has excellent specificity.When template was ZF43, under the HEX passage, the P2 probe had signal, detect sensitive strain ZF43, and the P1 probe does not have signal (Fig. 3 A); This moment under the FAM passage two equal no signals of probe.When template is ZF52, only when the FAM passage, can detect signal (Fig. 3 B).
The typical curve of embodiment 4Cycling Probe fluorescence dye quantitative PCR is set up
Standard substance with sensitive strain ZF43, resistant strain ZF52 gradient dilution are masterplate, carry out the quantitative PCR reaction.Real-time quantitative PCR reaction system and condition:
Reaction system:
10×Cycleave?PCR?Buffer 2.5μL
DNTP Mixture (each 2.5mM) 3 μ L
Mg?Solution(25mM) 5μL
Codon167F(10μM) 0.5μL
Codon167R(10μM) 0.5μL
P1(5μM) 0.6μL
P2(5μM) 1μL
TliRNase?H?II(200U/μl) 0.5μL
TaKaRa?Ex?Taq
TM?HS(5U/μl) 0.25μL
DH
2O (sterile purified water) 9.15 μ L
Dna profiling * 2.0 μ L
* dna profiling: responsive and resistant strain respectively adds 1 μ L.
Amplification condition (three-step approach, Fig. 4):
Pre-sex change: 95 ℃ of 30s
↓
Sex change: 95 ℃ of 5s
Annealing: 55 ℃ of 20s
Extend: 72 ℃ of 31s
40 circulations
Can obtain five contacts, signal amplification curve clearly behind the quantitative pcr amplification respectively; Contrast as negative control (NTC) with clear water simultaneously, do not have fluorescent signal to detect.The typical curve equation that instrument draws after to interpretation of result is ZF43:y=-3.5198x+44.861 R
2=0.9965 (Fig. 1, Fig. 5); ZF52:y=-3.6133x+45.889 R
2=0.9991 (Fig. 2, Fig. 6).
The sensitive strain ZF43 and the resistant strain ZF52 that get concentration known mix (mass ratio ZF43: ZF52=1.39), carry out the quantitative PCR reaction as template, obtain the Ct value (table 1) of sample, by the typical curve Equation for Calculating draw ZF43, the ZF52 copy number is respectively 6371/μ l, 4400/μ l, copy number ZF43: ZF52=1.44: 1, result and known sample ratio basically identical.Show that it is believable that this technology is used for detecting quantitative fusarium graminearum and resisting/feel the ratio of bacterial strain.
Field, Tongzhou, embodiment Jiangsu in 62009 bacterial strain detects
The ripe thecaspore of collecting (100 field bacterial strains mix) is divided into two parts, a copy of it is diluted to 900 spore/ml, getting 100 μ l respectively is coated in and contains derosal (2ppm) and not on the pastille PDA flat board (add Streptomycin sulphate in the PDA flat board, quintozene suppresses bacterium and other fungi), each 5 repetition.Observe the sprouting situation in 1～2 day, calculate the resistance ratio.Extract another one's share of expenses for a joint undertaking cystospore genomic dna, detect with quantitative PCR technique.
Observed and recorded is sprouted situation, and thecaspore sprouting situation is respectively 70,61., 49,63,55 on pastille flat board not, and to sprout quantity be 4,3,2,1,2 to thecaspore on the pastille flat board.Calculating resistant strain, to account for bacterial strain total amount ratio be 4.03%.
Quantitative PCR detection the results are shown in Table 2.Draw resistant strain according to the typical curve Equation for Calculating and account for bacterial strain total amount ratio and be about 4%, with traditional biological measurement result basically identical.
Table 1 sample detection by quantitative result
Table?1?Results?of?test?by?Real?time?PCR
Table 2 field sample detection by quantitative result
Table?2?Results?of?test?of?field?samples?by?Real?time?PCR
Claims (2)
1. Fusarium graminearum (Fusarium graminearum) is characterized in that adopting the gene frequency that Fusarium graminearum colony develops immunity to drugs to benzimidazole germicide in the sick sample of real-time quantitative PCR (Real-time quantitative PCR) technology for detection to the high-throughput molecular detecting method of carbendazim-resistance gene frequency.
This detection method was divided into for three steps:
(1) adopts conventional phenol chloroform isoamyl alcohol method, extract the nuclear gene group DNA of known drug-resistant strains, susceptibility bacterial strain and testing sample respectively.
(2) the specific C ycling Probe of utilization primer and design carries out the real-time quantitative PCR reaction, sets up the typical curve that drug-fast strain and sensitive strain detect respectively.
Real-time quantitative PCR (Real-time quantitative PCR) reaction system and amplification program thereof:
Reaction system:
10×CycleavePCR?Buffer 2.5μL
DNTP Mixture (each 2.5mM) 3 μ L
Mg?Solution(25mM) 5μL
Codon167F(10μM) 0.5μL
Codon167R(10μM) 0.5μL
P1(5μM) 0.6μL
P2(5μM) 1μL
TliRNase?H?II(200U/μl) 0.5μL
TaKaRa?Ex?Taq
TM?HS(5U/μl) 0.25μL
Dna profiling (responsive and resistant strain respectively adds 1 μ L) 2.0 μ L
DH
2O (sterile purified water) complements to cumulative volume 25 μ L
Amplification condition:
Pre-sex change: 95 ℃ of 30s
↓
Sex change: 95 ℃ of 5s
Annealing: 55 ℃ of 20s
Extend: 72 ℃ of 31s
40 circulations
(3) testing sample uses above-mentioned system and condition to carry out the real-time quantitative PCR reaction, contrasts two typical curves having set up, obtains the quantity and the ratio of drug-fast strain in the Fusarium graminearum total amount of corresponding drug-fast strain and susceptibility bacterial strain.
2. according to claim 1, Fusarium graminearum is characterized in that the real-time detection technique of the high-throughput of carbendazim-resistance gene frequency: the Cycling probe that is used for real-time quantitative PCR has specificity.The foundation of design is drug-fast strain β
2The 167th amino acids codon of-microtubule protein gene is undergone mutation, by TTT (Phe) → TAT (Tyr).
1. Fusarium graminearum β increases
2The pulsating primer of-microtubule protein gene is right
Codon167F?5′AAGCCATTGATGTTGTTCG?3′
Codon167R?5′CATGACGGTAGAAATCAGGTAG?3′
2. specific C ycling probe
P1 5’-(FAM)CATAACGGAA
AGG(Eclipse)-3’
P2 5’-(HEX)CATAACGGAA
AGG(Eclipse)-3’
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436628A (en) * | 2013-09-23 | 2013-12-11 | 南京农业大学 | Method for rapidly detecting moderately-resistant strain in fusarium graminearum to carbendazim |
| CN105463133A (en) * | 2015-12-28 | 2016-04-06 | 深圳市生科源技术有限公司 | Swine fever virus DNA/RNA (deoxyribonucleic acid/ribonucleic acid) heterozygosis probe-process detection kit and detection method thereof |
| CN108841987A (en) * | 2018-07-05 | 2018-11-20 | 南京农业大学 | A kind of rapid detection method of Fusarium graminearum 2-cyano-3-amino-3-phenylancryic acetate resistant strain |
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|---|---|---|---|---|
| CN1657627A (en) * | 2005-01-31 | 2005-08-24 | 南京农业大学 | Detection gene of Fasarium graminearum for resisting carbendazim and its detection method |
| CN101475983A (en) * | 2008-11-17 | 2009-07-08 | 南京农业大学 | One-tube detection method for indetifying Gibberella zeae and confiming medium pesticide resistance level of Gibberella zeae to carbendazim |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1657627A (en) * | 2005-01-31 | 2005-08-24 | 南京农业大学 | Detection gene of Fasarium graminearum for resisting carbendazim and its detection method |
| CN101475983A (en) * | 2008-11-17 | 2009-07-08 | 南京农业大学 | One-tube detection method for indetifying Gibberella zeae and confiming medium pesticide resistance level of Gibberella zeae to carbendazim |
Non-Patent Citations (2)
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| 《中国植物病理学会2010年学术年会论文集》 20100703 仇剑波等 禾谷镰孢菌beta2-tubulin基因点突变在生物进化中的意义 第77页 1-2 , 2 * |
| 仇剑波等: "禾谷镰孢菌β2-tubulin基因点突变在生物进化中的意义", 《中国植物病理学会2010年学术年会论文集》, 3 July 2010 (2010-07-03), pages 77 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436628A (en) * | 2013-09-23 | 2013-12-11 | 南京农业大学 | Method for rapidly detecting moderately-resistant strain in fusarium graminearum to carbendazim |
| CN103436628B (en) * | 2013-09-23 | 2014-10-29 | 南京农业大学 | Method for rapidly detecting moderately-resistant strain in fusarium graminearum to carbendazim |
| CN105463133A (en) * | 2015-12-28 | 2016-04-06 | 深圳市生科源技术有限公司 | Swine fever virus DNA/RNA (deoxyribonucleic acid/ribonucleic acid) heterozygosis probe-process detection kit and detection method thereof |
| CN108841987A (en) * | 2018-07-05 | 2018-11-20 | 南京农业大学 | A kind of rapid detection method of Fusarium graminearum 2-cyano-3-amino-3-phenylancryic acetate resistant strain |
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