CN101985652B - 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 Fusarium graminearum (Fusarium graminearum) to the high-throughput molecular detecting method of carbendazim-resistance gene frequency, can be used for monitoring benzimidazole germicide resistance Fusarium graminearum colony development trend, carry out 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 for a long time benzimidazole germicide or carry out chemical prevention take this class medicament as main built agent.Benzimidazole germicide is efficient as a class, the wide spectrum systemic fungicide is being produced application, 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 each other the quadrature transreactance property of medicine.Because the height specialization of this class medicament, action site is single, adds that frequency of administration is high, and the resistance dominant race will appear in many plant pathogenic fungis colony after using 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 this bacterium to the resistance of 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, then cultivates at the pastille substratum, 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 large, measures sample size limited; 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 early the resistance management strategy 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 such as ASO-PCR technology etc. and detect pathogenic fungi to the resistance of the benzimidazole germicides such as derosal, then can fast, accurately detect sample, but a PCR reaction system can only detect a bacterial strain to the susceptibility of Bavistin, and the single bacterial strain that can only obtain to measure 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, namely 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 drug-fast strain subpopulation generation, development, popular situation, effectively instruct then medication.
(4) make and detect in early days low-frequency drug resistance gene and become possibility.
This invention adopts real time quantitative PCR method to detect Fusarium graminearum to the drug resistance gene frequency of derosal, comprises the extraction of determination, 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 be at wheat scab period of disease and the diagnosis and detection that carries out a large amount of, quick and easy resistance subpopulation early stage, Detection accuracy reaches more than 96%, has a practical value to 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
2-microtubule protein gene (FGSG_06611.3), its 167th amino acids codon origination point mutation T TT (Phe) → TAT (Tyr) that encodes.
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 respectively the nuclear DNA of known drug-resistant strains, susceptibility bacterial strain and testing sample.
(2) use the specific C ycling probe of primer and design to carry out the real-time quantitative PCR reaction, set up respectively the typical curve that drug-fast strain and sensitive strain detect.
The key of the real-time detection technique of high-throughput of drug resistance gene frequency is that Cycling probe 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 primer pair of-microtubule protein gene segment
β
2-microtubule protein gene upstream primer 5 ' AAGCCATTGATGTTGTTCG 3 '
β
2-microtubule protein gene downstream primer 5 ' CATGACGGTAGAAATCAGGTAG 3 '
2. specific C ycling probe
P1 5’-(FAM)CATAACGGAA
GG(Eclipse)-3’
P2 5’-(HEX)CATAACGGAA
GG(Eclipse)-3’
Real-time quantitative PCR (Real-time quantitative PCR) reaction system and amplification program thereof:
Reaction system:
* dna profiling: responsive and resistant strain respectively adds 1 μ L.
Amplification condition:
Denaturation: 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 that established, and obtains 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 that detects, namely 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, the 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 drug-fast strain subpopulation generation, development, popular situation, effectively instruct then medication.
(4) make and detect in early days low-frequency drug resistance gene and become possibility.
(5) the present invention utilizes Cycling probe Real-time quantitative PCR to detect the resistance subpopulation of 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 at least 6 days but the 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.Not yet there is at present ripe resisting carbendazim fusarium graminearum molecular detection technology to use.Real-time quantitative PCR then can in a reaction system with whole testing samples to the carbendazim-resistance level detection out, namely be known 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 Scientific Usage of Drugs to the development trend of timely understanding resistance pathogenic bacteria subpopulation, effectively administers resistance, and reduces cost and environmental contamination reduction has realistic meaning.
3, approximately have 4% bacterial strain that derosal is had resistance in the 100 strain Fusarium graminearums of measuring, 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 pair β
2-microtubule protein gene upstream primer/β
2The pcr amplification condition of-microtubule protein gene downstream primer
The typical curve of Fig. 5 responsive probe (P2)
The typical curve of Fig. 6 resistance probe (P1)
Five, embodiment
Embodiment 1Cycling fluorescence probe dyestuff 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
TMCore Kit DCY501 test kit.
In the PCR reaction process, annealing temperature Tm value is the important guarantee of atopic, will produce non-specific product if annealing temperature is crossed to hang down.Because probe has stronger specificity, lower to the primer requirement among the present invention, only need it to ensure sufficiently high reaction efficiency.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 affect the height of fluorescent signal.When concentration and probe concentration is excessively low, the PCR product is excessive, then can't obtain correct typical curve.And in the phase mutual interference of same system, must adjust the concentration of two probes in order to prevent two fluorophors.To add probe (5 μ M) in the experiment in the 25 μ l reaction systems and increase progressively with 0.1 μ l from 0.1 μ l～2 μ l, to determine best concentration and probe concentration P1 (5 μ M) 0.6 μ l, P2 (5 μ M) 1 μ l.
The sensitivity of embodiment 2Cycling fluorescence probe dyestuff quantitative PCR
Standard substance requirement purity is high, homogeneous, stable.The PCR product fragment of choosing in this experiment 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, obtain respectively two groups of amplification curves (Fig. 1, Fig. 2), hence one can see that, and this technology lowest detection copy number is 10
2The order of magnitude, sensitivity are 10～100 times of regular-PCR detection method at least, and be also high than general SYBR GREEN I dye method sensitivity.
The specificity checking of embodiment 3Cycling fluorescence probe dyestuff quantitative PCR
Carry out respectively quantitative PCR detection take sensitive strain ZF43, resistant strain ZF52 as template, reaction has good 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 fluorescence probe dyestuff quantitative PCR is set up
Take the standard substance of sensitive strain ZF43, resistant strain ZF52 gradient dilution as masterplate, carry out the quantitative PCR reaction.Real-time quantitative PCR reaction system and condition:
Reaction system:
* dna profiling: responsive and resistant strain respectively adds 1 μ L.
Amplification condition (three-step approach, Fig. 4):
Denaturation: 95 ℃ of 30s
↓
Sex change: 95 ℃ of 5s
Annealing: 55 ℃ of 20s
Extend: 72 ℃ of 31s
40 circulations
Can obtain respectively clearly amplification curve of five contacts, signal behind the quantitative pcr amplification; 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+45.889R
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 are 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 (100 field bacterial strains mix) of collecting is divided into two parts, a copy of it is diluted to 900 spore/ml, getting respectively 100 μ l is coated in and contains derosal (2ppm) and not (adding Streptomycin sulphate, quintozene anti-bacteria and other fungi in the PDA flat board) on the pastille PDA flat board, 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%, basically identical with the traditional biological measurement result.
Table 1 sample detection by quantitative result
Table1 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
Fusarium graminearum is to the high-throughput Molecular Detection .txt of carbendazim-resistance gene frequency
SEQUENCE LISTING
＜110〉Agricultural University Of Nanjing
＜120〉Fusarium graminearum is to the high-throughput Molecular Detection of carbendazim-resistance gene frequency
<140>201010247003.7
<141>2010-08-06
<160>4
<210>1
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the upstream primer Codon167F of amplification β 2-microtubule protein gene
<400>1
aagccattga tgttgttgg
<210>2
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the upstream primer Codon167R of amplification β 2-microtubule protein gene
<400>2
catgacggta gaaatcaggt ag
<210>3
<211>14
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the specific C ycling probe P1 of amplification sensitive strain β 2-microtubule protein gene
<400>3
cataacggaa tagg
<210>4
<211>14
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the specific C ycling probe P2 of amplification sensitive strain β 2-microtubule protein gene
<400>4
cataacggaa aagg
Claims (1)
1. Fusarium graminearum (Fusarium graminearum) produces the real-time detection method of the drug resistance gene frequency of medium resistance level (in anti-) to derosal, it is characterized in that adopting the Cycling probe specificity of real-time quantitative PCR (Real-time quantitative PCR) technology, according to the horizontal bacterial strain β of medium resistance
2The 167th amino acids codon of-microtubule protein gene has been designed by TTT (Phe) → TAT (Tyr)
1. Fusarium graminearum β increases
2The primer pair of-microtubule protein gene segment
β
2-microtubule protein gene upstream primer 5 ' AAGCCATTGATGTTGTTCG 3 '
β
2-microtubule protein gene downstream primer 5 ' CATGACGGTAGAAATCAGGTAG 3 '
2. specific C ycling probe
P1 5’-(FAM)CATAACGGAA
GG(Eclipse)-3’
P2 5’-(HEX)CATAACGGAA
GG(Eclipse)-3，
The flat drug resistance gene frequency of water resistant during this flora body produces benzimidazole germicide in can the high throughput testing sick sample.
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| 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 |
Citations (2)
| 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 |
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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)
| Title |
|---|
| 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》.2010,第77页. |
| 禾谷镰孢菌β2-tubulin基因点突变在生物进化中的意义;仇剑波等;《中国植物病理学会2010年学术年会论文集》;20100703;第77页 * |
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