CN102382876A - Method for detecting avermectin pesticide resistance produced by diamondback moth - Google Patents
Method for detecting avermectin pesticide resistance produced by diamondback moth Download PDFInfo
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Abstract
The invention discloses a method for detecting avermectin pesticide resistance produced by diamondback moth. The method comprises the following steps of 1, carrying out single-end genome DNA extraction of a diamondback moth sample, 2, carrying out specific gene fragment PCR amplification of a DNA template extracted by the step 1; 3, recovering a PCR amplified product DNA obtained by the step 2, 4, according to a sequencing result, detecting if an N-end of the recovered PCR amplified product DNA has a mutational site, and 5, according to a sequencing result obtained by the step 4, carrying out determination of avermectin pesticide resistance produced by the diamondback moth sample. The method does not need a mass of captive-bred experimental insects and saves labor forces and resources. Through the method, accurate resistance detection results are obtained and occurrence frequency of an insect individual with resistance is shown.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of detection small cabbage moth the drug-fast method of Avermectins agricultural chemicals.
Background technology
At present small cabbage moth the resistance of Avermectins agricultural chemicals is detected mainly is to carry out through traditional bioassay-blade medicine embrane method: got in the Avermectins soup that fresh free of contamination cabbage leaves is immersed in series concentration 10 seconds; Compare with zero(ppm) water (containing 0.01%Triton-X100); Connect 10 to 20 of 3 instar larvaes of the same size after indoor the drying, each concentration repeats for 3 times.Check result after 48 hours.With POLO or SAS software data processing, calculate the LC50 value, then according to the resistance level of relatively judging small cabbage moth of LC50 value.
But above-mentioned traditional monitoring small cabbage moth exists significantly not enough to Avermectins pesticide resistance method: (1) needs artificial breeding examination worm in enormous quantities.The length of time that traditional bioassay method requires worm to be tested is consistent with size, and this just need be after the examination worm of work is adopted back in the field carries out artificial breeding indoor, and the standard that obtains capacity is tried worm.(2) need the long test duration.Traditional bioassay time needs more than two days; (3) traditional survey result that gives birth to is to chemical sproof overall judge of tested insect colony, can not react the frequency of the individual appearance of insect-resistant.
For this reason, be badly in need of a kind of novel, detection method fast of invention.
Summary of the invention
In order to realize the foregoing invention purpose, the technical scheme that the present invention adopts is following:
A kind of detection small cabbage moth comprises that to the drug-fast method of Avermectins agricultural chemicals step is following:
The first step is carried out the extraction of single head genomic dna to the small cabbage moth sample, and step comprises,
(1) said small cabbage moth sample is placed the homogenizer of precooling, adds the homogenate of TE damping fluid,
(2) add cell pyrolysis liquid, mixing changes in the centrifuge tube, adds Proteinase K again, place, during shake up several times, be translucent thick liquid up to sample,
(3) add chloroform, mixing gently,
(4) add precipitated liquid, room temperature is placed, centrifugal treating then,
(5) sop up supernatant, add sodium chloride solution immediately, vibration causes deposition and dissolves fully gently, adds RNA enzyme A, and mixing is placed,
(6) absolute ethyl alcohol of adding precooling is placed, and centrifugal treating siphons away ethanol then, washes once with ethanol, is inverted on the clean filter paper, and drying at room temperature with TE damping fluid dissolving DNA, obtains dna profiling, and cryopreservation is subsequent use;
Second step, the dna profiling that extracts in the said the first step is carried out the segmental pcr amplification of specific gene, step comprises,
(1) in the PCR pipe, set up the PCR reaction system: the PCR damping fluid, deoxyribonucleotide, upstream primer, downstream primer, dna profiling, archaeal dna polymerase, deionized water,
(2) above-mentioned reaction system is carried out the PCR reaction, reaction conditions is: after the sex change, sex change is annealed in advance, extends, and repeatedly circulation is extended, then the cryopreservation sample at last;
The 3rd step, the product behind the said pcr amplification is reclaimed, step comprises,
(1) product of getting behind the said pcr amplification carries out electrophoresis with sepharose,
(2) cut off 100～300 milligrams of purpose bands from said sepharose rapidly under the uv lamp, place micro-centrifuge tube, uv lamp operation does not down surpass 30 seconds,
(3) the about 100-300 μ of the binding buffer liquid L that adds in the centrifuge tube, 55～60 ℃ of heating dissolved glue in 5～10 minutes fully, every at a distance from 2-3 minute mixing once,
(4) homomixture that obtains in said the 3rd step (3) is forwarded in the recovery post,, removes supernatant in the room temperature centrifugal treating,
(5) in said recovery post, add binding buffer liquid again and wash said recovery post,, remove supernatant in the room temperature centrifugal treating,
(6) add the SWP damping fluid in said recovery post, centrifugal treating is removed supernatant,
(7) repeat in said the 3rd step (5), (6) once,
(8) Ex-all supernatant after the centrifugal treating adds the DNA lysate, centrifugal treating,
(9) agarose electrophoresis detects and reclaims the result;
In the 4th step, the DNA low tempertaure storage that said recovery is obtained checks order then, or the DNA that said recovery is obtained checks order immediately;
The 5th step, according to said sequencing result, check whether the N-end of the DNA that said recovery obtains the mutational site occurs, promptly the 78th the leucine Methionin that becomes Xie Ansuan or the 82nd becomes leucine;
The 6th step; According to the sequencing result in the 5th step; Said small cabbage moth sample is differentiated the resistance of Avermectins agricultural chemicals; Become the arbitrary mutational site among the leucine if said the 78th leucine becomes Xie Ansuan or the 82nd 's Methionin, just explain that said small cabbage moth sample resistance occurred to Avrmectin.
Also comprised for the 7th step, step comprises,
Repeatedly repeat six steps of the first step to the, according to the number of the said small cabbage moth sample that detects with the number of the sample in said mutational site wherein occurred, calculate the fastness frequency of said small cabbage moth sample population.
Also comprised for the 8th step; Whether detection need change medicament kind is prevented and treated said small cabbage moth sample population; Step comprises; According to the fastness frequency of the said small cabbage moth sample population that calculates,, prevents and treats the fastness frequency of a said small cabbage moth sample population if, just should changing medicament kind greater than 5%.
Said small cabbage moth sample is the small cabbage moth worm of adopting back from the field alive, and perhaps said small cabbage moth sample is the alcohol-pickled sample of adopting back from the field of small cabbage moth process.
Said small cabbage moth sample is a diamondback moth larvae.
In said the 3rd step, what the product behind the said pcr amplification was reclaimed employing is to reclaim test kit fast; In said the 4th step, the DNA that said recovery is obtained is stored in-20 ℃ of refrigerators and checks order then or check order immediately.
The said the first step, step comprise,
(1) said small cabbage moth sample is placed the homogenizer of-4 ℃ of precoolings, adds 200 μ L TE damping fluid homogenate,
(2) add 200 μ L cell pyrolysis liquids, mixing changes in the 1.5mL centrifuge tube, adds 3 μ L Proteinase Ks again, placed 10 minutes for 55 ℃, during shake up 3 times, be translucent thick liquid up to sample,
(3) add 300 μ L chloroforms, mixing gently,
(4) add 250 μ L precipitated liquid, room temperature was placed 2 minutes, 10000rpm centrifugal treating 2 minutes,
(5) sop up supernatant, add the sodium chloride solution of 1.2 moles every liter of 50 μ L immediately, vibration causes deposition and dissolves fully gently, adds 1 μ L RNA enzyme A, and mixing was placed 10 minutes for 55 ℃,
(6) absolute ethyl alcohol of adding 150 μ L precoolings was placed 1 hour for-20 ℃, and 10000rpm centrifugal treating 10 minutes siphons away ethanol; Wash once with 75% ethanol, be inverted on the clean filter paper, drying at room temperature 10 minutes; With 25 μ L TE damping fluid dissolving DNAs, obtain dna profiling, subsequent use in-20 ℃ of preservations;
In said second step, step comprises,
(1) in 0.25mL PCR pipe, sets up the PCR reaction system that TV is 25 μ L: 2.5 μ L, 10 * PCR damping fluid; Every liter of concentration deoxyribonucleotide of 10 mmoles of 2 μ L, 1 μ L upstream primer, 1 μ L downstream primer; 1 μ L dna profiling; 0.5 μ L archaeal dna polymerase, 17.0 μ L deionized waters
(2) above-mentioned reaction system is carried out PCR reaction, reaction conditions is: 94 ℃ of preparatory sex change are after 5 minutes, 94 ℃ of sex change 45 seconds, and 52 ℃ of annealing 45 seconds, 72 ℃ were extended 1 minute, 35 circulations of coreaction, last 72 ℃ were extended 10 minutes, and preserved samples in 4 ℃ then.
In said the 3rd step, step comprises,
(1) product of getting behind the said pcr amplification of 5 μ L carries out electrophoresis with 1.0% sepharose, deposition condition: 80 millivolts of constant voltages, and electrophoresis 40 minutes,
(2) cut off 100～300 milligrams of purpose bands from said sepharose rapidly under the uv lamp, place the 1.5mL micro-centrifuge tube, uv lamp operation does not down surpass 30 seconds,
(3) the about 100-300 μ of the binding buffer liquid L that adds in the centrifuge tube, 55～60 ℃ of heating dissolved glue in 5～10 minutes fully, every at a distance from 2-3 minute mixing once,
(4) homomixture that obtains in said the 3rd step (3) is forwarded in the recovery post of 2mL, in room temperature 10,000rpm centrifugal treating 1 minute is removed supernatant,
(5) in said recovery post, add 300 μ L binding buffer liquid again and wash said recovery post, in room temperature 10,000rpm centrifugal treating 1 minute is removed supernatant,
(6) add 700 μ L SWP damping fluids in said recovery post, 10,000rpm centrifugal treating 1 minute is removed supernatant,
(7) repeat in said the 3rd step (5), (6) once,
(8) Ex-all supernatant after the centrifugal treating adds 10 μ L DNA lysates, and 10,000rpm centrifugal treating 1 minute,
(9) 1% agarose electrophoresiss detect and reclaim the result, and the pcr amplification product size should be 131bp.
Said the 3rd the step (8) afterwards, repeat said the 3rd the step (5) at least once after, carry out again said the 3rd the step (9).
So-called TE damping fluid among the present invention is meant the edta solution of Tutofusin tris hydrochloric acid soln+1 mmole of 10 mmoles.
So-called upstream primer among the present invention is meant 10 micro-molar concentrations, and the deoxyribonucleotide sequence is: TAAGGCCGAACTATGGAG; So-called downstream primer is meant 10 micro-molar concentrations, and the deoxyribonucleotide sequence is: GGGGATCTGTCCAAAACT; So-called archaeal dna polymerase is meant 5 units/μ L.
So-called the 78th leucine among the present invention, nucleotide sequence is: TGT; So-called Xie Ansuan, nucleotide sequence is: TCT; So-called the 82nd Methionin, nucleotide sequence is: AAA; So-called leucine, nucleotide sequence is: TCA.
Detection small cabbage moth of the present invention is at first extracted single head small cabbage moth genomic dna to the drug-fast method of Avermectins agricultural chemicals, carries out the segmental pcr amplification of specific gene with designed primer, and the PCR product carries out the dna direct order-checking through after the purifying and recovering.If N-brings out existing any one mutational site: the 78th leucine (nucleotide sequence is: TGT) become Xie Ansuan (nucleotide sequence is: TCT) or the 82nd Methionin (nucleotide sequence is: (nucleotide sequence is: TCA) AAA) to become leucine; Just explain that this small cabbage moth resistance occurred to Avrmectin; According to the percentage of resistance individuality, judge the size of small cabbage moth population resistance again in detection worm amount.
Like 30 of one-time detection small cabbage moths, wherein there are 3 the mutational site to occur, then the fastness frequency of this small cabbage moth population is 10% (3/30*100%).If the fastness frequency of a population, just should consider that changing medicament kind prevents and treats greater than 5%.
Among the present invention, said the 3rd the step (8) afterwards, repeat said the 3rd the step (5) one or many after, carry out again said the 3rd the step (9), can improve organic efficiency.
Beneficial effect of the present invention is following:
1, the present invention does not need artificial breeding examination worm in enormous quantities, saves labor force and resource.
2, the present invention is directly test of sampling from the field, reduces pilot process, can be alcohol-pickled sample, conveniently posts and stores.
3, gained resistance result of the present invention is accurate, and can react the individual frequency that occurs of insect-resistant.
Embodiment
Specify optimized technical scheme of the present invention below.
A kind of detection small cabbage moth comprises that to the drug-fast method of Avermectins agricultural chemicals step is following:
The first step, at first adopt back small cabbage moth from the field after (can be alcohol-pickled sample or live in worm), carry out the extraction of single head genomic dna, step comprises,
(1) said small cabbage moth sample is placed the homogenizer of-4 ℃ of precoolings, adds 200 μ L TE damping fluid homogenate,
(2) add 200 μ L cell pyrolysis liquids, mixing changes in the 1.5mL centrifuge tube, adds 3 μ L Proteinase Ks again, placed 10 minutes for 55 ℃, during shake up 3 times, be translucent thick liquid up to sample,
(3) add 300 μ L chloroforms, mixing gently,
(4) add 250 μ L precipitated liquid, room temperature was placed 2 minutes, 10000rpm centrifugal treating 2 minutes,
(5) sop up supernatant, add the sodium chloride solution of 1.2 moles every liter of 50 μ L immediately, vibration causes deposition and dissolves fully gently, adds 1 μ L RNA enzyme A, and mixing was placed 10 minutes for 55 ℃,
(6) absolute ethyl alcohol of adding 150 μ L precoolings was placed 1 hour for-20 ℃, and 10000rpm centrifugal treating 10 minutes siphons away ethanol; Wash once with 75% ethanol, be inverted on the clean filter paper, drying at room temperature 10 minutes; With 25 μ L TE damping fluid dissolving DNAs, obtain dna profiling, subsequent use in-20 ℃ of preservations;
Second step, the dna profiling that extracts in the said the first step is carried out the segmental pcr amplification of specific gene, step comprises,
(1) in 0.25mL PCR pipe, sets up the PCR reaction system that TV is 25 μ L: 2.5 μ L, 10 * PCR damping fluid; Every liter of concentration deoxyribonucleotide of 10 mmoles of 2 μ L, 1 μ L upstream primer, 1 μ L downstream primer; 1 μ L dna profiling; 0.5 μ L archaeal dna polymerase, 17.0 μ L deionized waters
(2) above-mentioned reaction system is carried out PCR reaction, reaction conditions is: 94 ℃ of preparatory sex change are after 5 minutes, 94 ℃ of sex change 45 seconds, and 52 ℃ of annealing 45 seconds, 72 ℃ were extended 1 minute, 35 circulations of coreaction, last 72 ℃ were extended 10 minutes, and preserved samples in 4 ℃ then;
The 3rd step, adopt and to reclaim the product of test kit after fast and reclaim said pcr amplification, step comprises,
(1) product of getting behind the said pcr amplification of 5 μ L carries out electrophoresis with 1.0% sepharose, deposition condition: 80 millivolts of constant voltages, and electrophoresis 40 minutes,
(2) cut off 100～300 milligrams of purpose bands from said sepharose rapidly under the uv lamp, place the 1.5mL micro-centrifuge tube, uv lamp operation does not down surpass 30 seconds,
(3) the about 100-300 μ of the binding buffer liquid L that adds in the centrifuge tube, 55～60 ℃ of heating dissolved glue in 5～10 minutes fully, every at a distance from 2-3 minute mixing once,
(4) homomixture that obtains in said the 3rd step (3) is forwarded in the recovery post of 2mL, in room temperature 10,000rpm centrifugal treating 1 minute is removed supernatant,
(5) in said recovery post, add 300 μ L binding buffer liquid again and wash said recovery post, in room temperature 10,000rpm centrifugal treating 1 minute is removed supernatant
(6) add 700 μ L SWP damping fluids in said recovery post, 10,000rpm centrifugal treating 1 minute is removed supernatant,
(7) repeat in said the 3rd step (5), (6) once,
(8) Ex-all supernatant after the centrifugal treating adds 10 μ L DNA lysates, and 10,000rpm centrifugal treating 1 minute,
(9) repeat said the 3rd step (5) once, can improve organic efficiency,
(10) 1% agarose electrophoresiss detect and reclaim the result, and the pcr amplification product size should be 131bp;
In the 4th step, the DNA that said recovery is obtained checks order immediately;
The 5th step, according to said sequencing result, check whether the N-end of the DNA that said recovery obtains the mutational site occurs, promptly the 78th the leucine Methionin that becomes Xie Ansuan or the 82nd becomes leucine;
The 6th step; According to the sequencing result in the 5th step; Said small cabbage moth sample is differentiated the resistance of Avermectins agricultural chemicals; Become the arbitrary mutational site among the leucine if said the 78th leucine becomes Xie Ansuan or the 82nd 's Methionin, just explain that said small cabbage moth sample resistance occurred to Avrmectin;
The 7th step repeatedly repeated six steps of the first step to the, according to the number of the said small cabbage moth sample that detects with the number of the sample in said mutational site wherein occurred, calculated the fastness frequency of said small cabbage moth sample population;
The 8th step; Whether detection need change medicament kind is prevented and treated said small cabbage moth sample population; According to the fastness frequency of the said small cabbage moth sample population that calculates,, prevents and treats the fastness frequency of a said small cabbage moth sample population if, just should changing medicament kind greater than 5%.
Like 30 of one-time detection small cabbage moths, wherein there are 3 the mutational site to occur, then the fastness frequency of this small cabbage moth population is 10% (3/30*100%).If the fastness frequency of a population, just should consider that changing medicament kind prevents and treats greater than 5%.
More than through the detailed description of concrete and preferred embodiment the present invention; But those skilled in the art should be understood that; The present invention is not limited to the above embodiment; All within spirit of the present invention and principle, any modification of being done, be equal to replacement etc., all should be included within protection scope of the present invention.
Claims (10)
1. one kind is detected small cabbage moth to the drug-fast method of Avermectins agricultural chemicals, it is characterized in that, comprises that step is following:
The first step is carried out the extraction of single head genomic dna to the small cabbage moth sample, and step comprises,
(1) said small cabbage moth sample is placed the homogenizer of precooling, adds the homogenate of TE damping fluid,
(2) add cell pyrolysis liquid, mixing changes in the centrifuge tube, adds Proteinase K again, place, during shake up several times, be translucent thick liquid up to sample,
(3) add chloroform, mixing gently,
(4) add precipitated liquid, room temperature is placed, centrifugal treating then,
(5) sop up supernatant, add sodium chloride solution immediately, vibration causes deposition and dissolves fully gently, adds RNA enzyme A, and mixing is placed,
(6) absolute ethyl alcohol of adding precooling is placed, and centrifugal treating siphons away ethanol then, washes once with ethanol, is inverted on the clean filter paper, and drying at room temperature with TE damping fluid dissolving DNA, obtains dna profiling, and cryopreservation is subsequent use;
Second step, the dna profiling that extracts in the said the first step is carried out the segmental pcr amplification of specific gene, step comprises,
(1) in the PCR pipe, set up the PCR reaction system: the PCR damping fluid, deoxyribonucleotide, upstream primer, downstream primer, dna profiling, archaeal dna polymerase, deionized water,
(2) above-mentioned reaction system is carried out the PCR reaction, reaction conditions is: after the sex change, sex change is annealed in advance, extends, and repeatedly circulation is extended, then the cryopreservation sample at last;
The 3rd step, the product behind the said pcr amplification is reclaimed, step comprises,
(1) product of getting behind the said pcr amplification carries out electrophoresis with sepharose,
(2) cut off 100～300 milligrams of purpose bands from said sepharose rapidly under the uv lamp, place micro-centrifuge tube, uv lamp operation does not down surpass 30 seconds,
(3) the about 100-300 μ of the binding buffer liquid L that adds in the centrifuge tube, 55～60 ℃ of heating dissolved glue in 5～10 minutes fully, every at a distance from 2-3 minute mixing once,
(4) homomixture that obtains in said the 3rd step (3) is forwarded in the recovery post,, removes supernatant in the room temperature centrifugal treating,
(5) in said recovery post, add binding buffer liquid again and wash said recovery post,, remove supernatant in the room temperature centrifugal treating,
(6) add the SWP damping fluid in said recovery post, centrifugal treating is removed supernatant,
(7) repeat in said the 3rd step (5), (6) once,
(8) Ex-all supernatant after the centrifugal treating adds the DNA lysate, centrifugal treating,
(9) agarose electrophoresis detects and reclaims the result;
In the 4th step, the DNA low tempertaure storage that said recovery is obtained checks order then, or the DNA that said recovery is obtained checks order immediately;
The 5th step, according to said sequencing result, check whether the N-end of the DNA that said recovery obtains the mutational site occurs, promptly the 78th the leucine Methionin that becomes Xie Ansuan or the 82nd becomes leucine;
The 6th step; According to the sequencing result in the 5th step; Said small cabbage moth sample is differentiated the resistance of Avermectins agricultural chemicals; Become the arbitrary mutational site among the leucine if said the 78th leucine becomes Xie Ansuan or the 82nd 's Methionin, just explain that said small cabbage moth sample resistance occurred to Avrmectin.
2. detection small cabbage moth according to claim 1 is characterized in that the drug-fast method of Avermectins agricultural chemicals, also comprises for the 7th step, and step comprises,
Repeatedly repeat six steps of the first step to the, according to the number of the said small cabbage moth sample that detects with the number of the sample in said mutational site wherein occurred, calculate the fastness frequency of said small cabbage moth sample population.
3. detection small cabbage moth according to claim 2 is to the drug-fast method of Avermectins agricultural chemicals; It is characterized in that, also comprised for the 8th step, whether detection need change medicament kind is prevented and treated said small cabbage moth sample population; Step comprises; According to the fastness frequency of the said small cabbage moth sample population that calculates,, prevents and treats the fastness frequency of a said small cabbage moth sample population if, just should changing medicament kind greater than 5%.
According to claim 1 or 2 or 3 described detection small cabbage moths to the drug-fast method of Avermectins agricultural chemicals; It is characterized in that: said small cabbage moth sample is the small cabbage moth worm of adopting back from the field alive, and perhaps said small cabbage moth sample is the alcohol-pickled sample of adopting back from the field of small cabbage moth process.
According to claim 1 or 2 or 3 described detection small cabbage moths to the drug-fast method of Avermectins agricultural chemicals, it is characterized in that: said small cabbage moth sample is a diamondback moth larvae.
According to claim 1 or 2 or 3 described detection small cabbage moths to the drug-fast method of Avermectins agricultural chemicals, it is characterized in that in said the 3rd step, what the product behind the said pcr amplification was reclaimed employing is to reclaim test kit fast; In said the 4th step, the DNA that said recovery is obtained is stored in-20 ℃ of refrigerators and checks order then or check order immediately.
According to claim 1 or 2 or 3 described detection small cabbage moths to the drug-fast method of Avermectins agricultural chemicals, it is characterized in that the said the first step, step comprise,
(1) said small cabbage moth sample is placed the homogenizer of-4 ℃ of precoolings, adds 200 μ L TE damping fluid homogenate,
(2) add 200 μ L cell pyrolysis liquids, mixing changes in the 1.5mL centrifuge tube, adds 3 μ L Proteinase Ks again, placed 10 minutes for 55 ℃, during shake up 3 times, be translucent thick liquid up to sample,
(3) add 300 μ L chloroforms, mixing gently,
(4) add 250 μ L precipitated liquid, room temperature was placed 2 minutes, 10000rpm centrifugal treating 2 minutes,
(5) sop up supernatant, add the sodium chloride solution of 1.2 moles every liter of 50 μ L immediately, vibration causes deposition and dissolves fully gently, adds 1 μ L RNA enzyme A, and mixing was placed 10 minutes for 55 ℃,
(6) absolute ethyl alcohol of adding 150 μ L precoolings was placed 10000rpm centrifugal treating 10 minutes 1 hour for-20 ℃; Siphon away ethanol, wash once, be inverted on the clean filter paper with 75% ethanol; Drying at room temperature 10 minutes; With 25 μ L TE damping fluid dissolving DNAs, obtain dna profiling, subsequent use in-20 ℃ of preservations.
According to claim 1 or 2 or 3 described detection small cabbage moths to the drug-fast method of Avermectins agricultural chemicals, it is characterized in that in said second step, step comprises,
(1) in 0.25mL PCR pipe, sets up the PCR reaction system that TV is 25 μ L: 2.5 μ L, 10 * PCR damping fluid; Every liter of concentration deoxyribonucleotide of 10 mmoles of 2 μ L, 1 μ L upstream primer, 1 μ L downstream primer; 1 μ L dna profiling; 0.5 μ L archaeal dna polymerase, 17.0 μ L deionized waters
(2) above-mentioned reaction system is carried out PCR reaction, reaction conditions is: 94 ℃ of preparatory sex change are after 5 minutes, 94 ℃ of sex change 45 seconds, and 52 ℃ of annealing 45 seconds, 72 ℃ were extended 1 minute, 35 circulations of coreaction, last 72 ℃ were extended 10 minutes, and preserved samples in 4 ℃ then.
According to claim 1 or 2 or 3 described detection small cabbage moths to the drug-fast method of Avermectins agricultural chemicals, it is characterized in that in said the 3rd step, step comprises,
(1) product of getting behind the said pcr amplification of 5 μ L carries out electrophoresis with 1.0% sepharose, deposition condition: 80 millivolts of constant voltages, and electrophoresis 40 minutes,
(2) cut off 100～300 milligrams of purpose bands from said sepharose rapidly under the uv lamp, place the 1.5mL micro-centrifuge tube, uv lamp operation does not down surpass 30 seconds,
(3) the about 100-300 μ of the binding buffer liquid L that adds in the centrifuge tube, 55～60 ℃ of heating dissolved glue in 5～10 minutes fully, every at a distance from 2-3 minute mixing once,
(4) homomixture that obtains in said the 3rd step (3) is forwarded in the recovery post of 2mL, in room temperature 10,000rpm centrifugal treating 1 minute is removed supernatant,
(5) in said recovery post, add 300 μ L binding buffer liquid again and wash said recovery post, in room temperature 10,000rpm centrifugal treating 1 minute is removed supernatant,
(6) add 700 μ L SWP damping fluids in said recovery post, 10,000rpm centrifugal treating 1 minute is removed supernatant,
(7) repeat in said the 3rd step (5), (6) once,
(8) Ex-all supernatant after the centrifugal treating adds 10 μ L DNA lysates, and 10,000rpm centrifugal treating 1 minute,
(9) 1% agarose electrophoresiss detect and reclaim the result, and the pcr amplification product size should be 131bp.
According to claim 1 or 2 or 3 described detection small cabbage moths to the drug-fast method of Avermectins agricultural chemicals, it is characterized in that: said the 3rd step (8) afterwards, repeat said the 3rd step (5) at least once after, carry out said the 3rd step (9) again.
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| CN105713959A (en) * | 2014-12-01 | 2016-06-29 | 南京农业大学 | Molecular detection method of resistance of diamond back moth against avermectin target |
| CN106222128A (en) * | 2016-08-16 | 2016-12-14 | 浙江大学 | The cultural method of a kind of diamondback moth embryo cell line and application thereof |
| CN107841539A (en) * | 2017-12-13 | 2018-03-27 | 中国农业科学院蔬菜花卉研究所 | A kind of CAPS labeling methods for detecting tetranychid and being mutated to AVM resistance related gene |
| CN110607378A (en) * | 2019-10-31 | 2019-12-24 | 贵州省烟草科学研究院 | Primer pair for detecting drug resistance of myzus persicae to imidacloprid and detection method |
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| CN105713959B (en) * | 2014-12-01 | 2019-04-19 | 南京农业大学 | Molecular detecting method of the diamondback moth to avermectin target resistance |
| CN106222128A (en) * | 2016-08-16 | 2016-12-14 | 浙江大学 | The cultural method of a kind of diamondback moth embryo cell line and application thereof |
| CN107841539A (en) * | 2017-12-13 | 2018-03-27 | 中国农业科学院蔬菜花卉研究所 | A kind of CAPS labeling methods for detecting tetranychid and being mutated to AVM resistance related gene |
| CN110607378A (en) * | 2019-10-31 | 2019-12-24 | 贵州省烟草科学研究院 | Primer pair for detecting drug resistance of myzus persicae to imidacloprid and detection method |
| CN110607378B (en) * | 2019-10-31 | 2022-11-29 | 贵州省烟草科学研究院 | Primer pair for detecting drug resistance of myzus persicae to imidacloprid and detection method |
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