CN1546689A - Molecular detecting method of resistance of Nilaparvata lugens to imidacloprid target - Google Patents
Molecular detecting method of resistance of Nilaparvata lugens to imidacloprid target Download PDFInfo
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- CN1546689A CN1546689A CNA2003101185238A CN200310118523A CN1546689A CN 1546689 A CN1546689 A CN 1546689A CN A2003101185238 A CNA2003101185238 A CN A2003101185238A CN 200310118523 A CN200310118523 A CN 200310118523A CN 1546689 A CN1546689 A CN 1546689A
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Abstract
The invention relates to a molecular detecting method of resistance of Nilaparvata lugens to imidacloprid target, wherein the employed specific primers being P: 5'-ACA CGT CCC CAG TGA GCA-3', Q: 5'-GTC GGT GGA ATG ATC TCT GC-3', A: 5'-ggggggggcc GTT TGG ATC CTG TAC ATC-3', B: 5'-ggggggggcg CAT GAT TGC CGT CGT- 3'. The 25 uL reaction system being, 2.5uL 10Xbuffer, 1.25 U Ex-Taq DNA polymerase, 2.5 uL single-head Nilaparvata lugens genome DNA, dNTPs each 0.2mmol/L, MgcL2 1mmol/L, primers each 1umol/L, adding double steaming water to total reaction volume of 25 uL. The PCR expansion product can be directly detected through gel electrophoresis.
Description
(1) technical field
Brown paddy plant hopper of the present invention (Nilaparvata lugens St l) to the molecular detecting method of Provado (Imidacloprid) target resistance, belongs to biological technical field, is exclusively used in the highly sensitive rapid detection of brown paddy plant hopper to the Provado target resistance.
(2) background technology
The rice in China plantation is with a long history, and the distribution region is very extensive, is topmost food crop, and rice yield accounts for more than 40% of total output of grain.China is maximum in the world Rice Production and country of consumption, paddy rice year about 2,860 ten thousand hectares of cultivated area, produce 1.85 hundred million tons in rice per year, account for the water globe seed rice and plant 1/5 of area, account for 1/3 of world's rice ultimate production.Therefore, the rich apology of paddy is very big to the influence of national economy and people's lives.In the rice cropping process of growth, often suffer multiple natural disasteies such as water, drought, yet great insect one brown paddy plant hopper of the paddy rice of causing harm also is that present many rice district influences one of major reason of the rich apology of paddy.Since nineteen eighty, brown paddy plant hopper accounts for 50% of national rice area in annual area 1300～2,000 ten thousand hectare of taking place of China.Though since the eighties in 20th century, " based on pest-resistant cultivar; chemical prevention, biological control are coordinated tentatively to have set up for the integrated control system of " center "; and in constantly improving; but chemical prevention is still accounting for mainly status with its instant effect, superiority and characteristics such as effective, easy to use in the control of brown paddy plant hopper.From the forties to the end of the seventies in 20th century (beginning of the eighties), the chemical prevention of brown paddy plant hopper mainly is organochlorine, organophosphorus and carbamate insecticides.Since 20th century the mid-80s, chitin synthesis inhibitor-buprofezin is demonstrated and is promoted in China.But because there is some defective in this sterilant, as take effect slow, to shortcomings such as brown paddy plant hopper ovum poor efficiencys, since the mid-90 in 20th century, new and effective systemic insecticide-Provado is widely used, and progressively becomes the main pesticide species of control planthopper.
Provado, Imidacloprid belongs to anabasine insecticide.To the research of this insecticides, originate from the discovery that Soloway in 1979 etc. have insecticidal activity to the heterocycle Nitromethylene compounds.Nineteen ninety, synthesized this compound abroad first.Beyer Co., Ltd begins to develop this insecticides in 1991, puts on market in 1992.1992, domestic (Jiangsu Prov. Inst. of Agricultural Chemicals) also synthesized this compound first.Provado is that (Nicotinic Acetylcholine Receptor, effector nAChR) are acetylcholine receptor bonded imperfect competition agonist to the insect acetylcholine receptor, and the mechanism of action uniqueness is very low to mammalian toxicity.Provado has extremely good systemic activity, and seed treatment, soil treatment and foliar spray have efficiently preventing and treating a lot of Agricultural pests, and sucking pest particularly is as plant hopper, leafhopper, thrips, aphid etc.Yet, the extra high preventive effect of Provado just, make this medicament on producing, exist undesirable conditions such as single use, abuse, and monitored the resistance of several insects at present, indicating the possibility that brown paddy plant hopper produces this sterilant resistance Provado.Though also do not monitor of the remarkable resistance generation of brown paddy plant hopper field population at present to Provado, but existing monitoring shows fastness frequency in the population of field the trend of increase is arranged, explanation is used under the situation of Provado on a large scale continuously, and the possibility that brown paddy plant hopper field population produces remarkable resistance is very big.[Zhao Jianzhou. insect is to the progress of Provado resistance. plant protection, 1998,24 (6): 40～41. Liu Ze literary compositions, Wang Yinchang, Han Zhaojun, etc. two kinds of planthoppers relatively to the susceptibility of sterilant. Agricultural University Of Nanjing's journal, 2003,26 (2): 29～32.]
At present the monitoring of Provado resistance all is confined to biological assay, and the limitation of biological assay itself makes the detection method of this routine can not detect early stage resistance or low frequency resistance allele about brown paddy plant hopper.Being used to monitor brown paddy plant hopper at present mainly contains topical application, soaks Gen Fa and soaks the stem method the bioassay method of Provado resistance.The ubiquitous shortcoming of biometric techniques has: (1) sensitivity is low: when the very difficult early stage resistance of detection of biological assay or low frequency resistance allele, especially resistance allele are recessive or not exclusively recessive; (2) cycle is long: the inspection of bioassay results generally needs more than the 24h, needs the longer time for this class systemic insecticide of Provado, is generally 48～96h; (3) require very big to examination borer population amount: measuring a typical curve needs 500 examination worms at least, also very high to the requirement that insect is raised.When the Provado use was growing, a kind of quick resistance detection method became very important, and common bioassay method can not satisfy these requirements at all.[Zhuan Yonglin, Shen Jinliang. the detection technique of Nilaparvata lugen (brown planthopper) parathiazine ketone resistance, Agricultural University Of Nanjing's journal, 2000,23 (3): 114～117.]
(3) summary of the invention
Problems such as that technical problem the objective of the invention is is low to the sensitivity of Provado resistance detection method at brown paddy plant hopper in the prior art, cycle length, material requirements height, the molecular detecting method of brown paddy plant hopper to the Provado target resistance is provided, reaches accuracy height, short, highly sensitive purpose of cycle.
The technical scheme brown paddy plant hopper comprises the molecular detecting method of Provado target resistance:
1) Auele Specific Primer: outer primer P:5 '-ACA CGT CCC CAG TGA GCA-3 '
Q：5′-GTC?GGT?GGA?ATG?ATC?TCT?GC-3′
Inner primer A:5 '-ggggggggcc GTT TGG ATC CTG TAC ATC-3 '
B：5′-ggggggggcg?CAT?GAT?TGC?CGT?CGT-3′
2) extraction of single head brown paddy plant hopper genomic dna:
(1) preparation extracting solution A:1% (g/mL) SDS, 50mmol/L TrisHCl, 25mmol/L NaCl, 25mmol/L EDTA, solvent are ultrapure water; Extracting solution B:3mol/L KAC, PH7.2, solvent are ultrapure water.
(2) will try worm and place the 1.0mL centrifuge tube, smash to pieces with the sterilization toothpick the freezing back of liquid nitrogen, adds 60 μ L extracting solution A then, and clean toothpick with other 60 μ LA liquid;
(3) 65 ℃ of water-bath 45min, every the 15min mixing once;
(4) add 120 μ L extracting solution B, ice bath 1～2h behind the mixing;
The centrifugal 15min of (5) 10000 * g transfers to supernatant liquor in another sterilization centrifuge tube;
The centrifugal 10min of (6) 10000 * g adds the dehydrated alcohol of 480 μ L precoolings, and mixing is placed 1～2h for-20 ℃;
The centrifugal 15min of (7) 10000 * g confides all supernatant liquor, and collecting precipitation cleans 1 time with 70% (mL/mL) ethanol;
Repeating step (7), 37 ℃ are dried or the lyophilize precipitation, obtain single head brown paddy plant hopper genomic dna, add 50 μ L distilled water dissolution precipitations, and-20 ℃ of preservations are standby;
3) reaction system 25 μ L:
2.5 μ L10 * Buffer, 1.25U Ex-Taq DNA polymerase, 2.5 μ L single head brown paddy plant hopper genomic dnas, each 0.2mmol/L of dNTPs, MgCl
21mmol/L, each 1 μ mol/L of primer, adding distilled water is 25 μ L to reacting cumulative volume;
4) PCR response procedures:
94 ℃ of 3 min, 20 circulations: 94 ℃ of 30s, 67-58 ℃ of 30s, 1 ℃ of per 2 cycle down, 72 ℃ of 1min; 10 circulations: 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 1min, 72 ℃ of 5min;
5) electrophoresis detection of amplified production:
Get 10 μ L pcr amplification products, on the sepharose of 1% (g/mL), carry out electrophoresis, current stabilization 80mA detects under UV-light behind the 1.5h: if exist length to be about two bands of 900bp (PQ) and 370bp (AQ) size respectively, the individual resistance homozygote that is is described; If exist length to be about two bands of 900bp (PQ) and 540bp (PB) size respectively, the individual responsive homozygote that is be described; If exist length to be about three bands of 900bp (PQ), 540bp (PB) and 370bp (AQ) size respectively simultaneously, the individual heterozygote that is be described.
Beneficial effect
The present invention compares with the prior biological determination techniques, and its advantage and positively effect show:
(1) highly sensitive: the existence of low frequency resistance homozygote or heterozygote and certain screening pressure are the key factors that high-level resistance produces.And the biological assay Monitoring techniques is a kind of extensive detection mode, can not detect early stage resistance or low frequency resistance homozygote or heterozygote, so biometric techniques is difficult to carry out the risk profile that resistance produces.The point mutation that the present invention is relevant according to the anti-Provado strain of brown paddy plant hopper resistance, design is suitable for the Auele Specific Primer of the special allelotrope of two-way pcr amplification (Bi-PASA), through pcr amplification and gel detection, can directly judge individual genotype.By the detection of some amount individuality, can tentatively determine resistance homozygote in certain population, heterozygote and responsive homozygous frequency, for resistance prediction, resistance management and the insect comprehensive regulation provide the most direct evidence.
(2) accuracy height: biometric techniques requires the stdn of examination worm, and the difference between the polypide is very big to result's influence, has caused result's unstable.Operator's operate miss has also strengthened this unstable.And the present invention is based on the detection technique of pcr amplification, because round pcr increasingly mature and promoting and the intelligent development of operation reduced this unstable to a certain extent, make detected result can be used as the ideal material that the brown paddy plant hopper control strategy is formulated.
(3) cycle is short: give birth to the observation time that survey technology needs 24h at least, and also can be longer for the systemic insecticide Provado.And the special allelotrope of two-way pcr amplification (Bi-PASA) technology among the present invention only need just can draw ideal results less than the time of 10h.
(4) material requirements is few: measuring a typical curve in the biometric techniques needs the accurate examination of 500 leaders worm at least, the certain human and material resources of raising need cost of these examination worms.And the present invention only needs examination worm about 100 to the detection of a population.If resistance gene frequency is low especially, as about 1%, only need about 300 examination worm, but that this is a biometric techniques is all non-detectable with how many examination worms.
(4) Figure of description
The special allelotrope result of Fig. 1 individual two-way pcr amplification of different strains
1,2,3-resistance homozygote; 4,5, the responsive homozygote of 6-; 7,8, the 9-heterozygote; The 10-molecule marker
(5) embodiment
The Auele Specific Primer that the special allelotrope of two-way pcr amplification (Bi-PASA) needs has:
Outer primer P:5 '-ACA CGT CCC CAG TGA GCA-3 '
Q：5′-GTC?GGT?GGA?ATG?ATC?TCT?GC-3′
Inner primer A:5 '-ggggggggcc GTT TGG ATC CTG GAC ATC-3 '
B：5′-ggggggggcg?CAT?GAT?TGC?CGT?CGT-3′
The extraction of single head brown paddy plant hopper genomic dna:
(1) get 100 of the firm female adult worm of sprouting wings of single head brown paddy plant hopper, rinsing is 3 times in distilled water, blots with thin water paper;
(2) will try worm and place the 1.0mL centrifuge tube, smash to pieces with the sterilization toothpick the freezing back of liquid nitrogen, adds 60 μ L extracting solution A (1%SDS, 50mmol/L TrisHCl, 25mmol/L NaCl, 25mmol/L EDTA) then, and clean toothpick with other 60 μ LA liquid; (3) 65 ℃ of water-bath 45min, every the 15min mixing once; (4) add 120 μ L extracting solution B (3mol/L KAC, PH7.2), behind the mixing more than the ice bath 1h; The centrifugal 15min of (5) 10000 * g transfers to supernatant liquor in another sterilization centrifuge tube; The centrifugal 10min of (6) 10000 * g adds the dehydrated alcohol of 480 μ L precoolings, and mixing is placed more than the 1h for-20 ℃; The centrifugal 15min of (7) 10000 * g carefully confides all supernatant liquor, and collecting precipitation cleans 1 time with 70% (mL/mL) ethanol; (8) repeating step (7), 37 ℃ are dried or the lyophilize precipitation, add 50 μ L distilled water dissolution precipitations, and-20 ℃ of preservations are standby.
Reaction system (25 μ L):
2.5 μ L 10 * Buffer, 1.25U Ex-Taq DNA polymerase, 2.5 μ L single head brown paddy plant hopper genomic dnas, each 0.2mmol/L of dNTPs, MgCl
21mmol/L, each 1 μ mol/L of primer, adding distilled water is 25 μ L to reacting cumulative volume.
The PCR reaction
94 ℃ of 3min, 20 circulations (94 ℃ of 30s, 67-58 ℃ of 30s (per 2 circulations reduce by 1 ℃), 72 ℃ of 1min), 10 circulations (94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 1min), 72 ℃ of 5min.
The PCR product analysis
Claims (1)
1, brown paddy plant hopper comprises the molecular detecting method of Provado target resistance:
1) Auele Specific Primer: outer primer P:5 '-ACA CGT CCC CAG TGA GCA-3 '
Q：5′-GTC?GGT?GGA?ATG?ATC?TCT?GC-3′
Inner primer A:5 '-ggggggggcc GTT TGG ATC CTG TAC ATC-3 '
B：5′-ggggggggcg?CAT?GAT?TGC?CGT?CGT-3′
2) extraction of single head brown paddy plant hopper genomic dna:
(1) preparation extracting solution A:1% (g/mL) SDS, 50mmol/L Tris HCl, 25mmol/L NaCl, 25mmol/L EDTA, solvent are ultrapure water; Extracting solution B:3mol/L KAC, PH7.2, solvent are ultrapure water;
(2) will try worm and place the 1.0mL centrifuge tube, smash to pieces with the sterilization toothpick the freezing back of liquid nitrogen, adds 60 μ L extracting solution A then, and clean toothpick with other 60 μ L A liquid;
(3) 65 ℃ of water-bath 45min, every the 15min mixing once;
(4) add 120 μ L extracting solution B, ice bath 1～2h behind the mixing;
The centrifugal 15min of (5) 10 000 * g transfers to supernatant liquor in another sterilization centrifuge tube;
The centrifugal 10min of (6) 10 000 * g adds the dehydrated alcohol of 480 μ L precoolings, and mixing is placed 1～2h for-20 ℃;
The centrifugal 15min of (7) 10 000 * g confides all supernatant liquor, and collecting precipitation cleans 1 time with 70% (mL/mL) ethanol;
Repeating step (7), 37 ℃ are dried or the lyophilize precipitation, obtain single head brown paddy plant hopper genomic dna, add 50 μ L distilled water dissolution precipitations, and-20 ℃ of preservations are standby;
3) reaction system 25 μ L:
2.5 μ L 10 * Buffer, 1.25 U Ex-Taq DNA polymerase, 2.5 μ L single head brown paddy plant hopper genomic dnas, each 0.2mmol/L of dNTPs, MgCl
21mmol/L, each 1 μ mol/L of primer, adding distilled water is 25 μ L to reacting cumulative volume;
4) PCR response procedures:
94 ℃ of 3min, 20 circulations: 94 ℃ of 30s, 67-58 ℃ of 30s, 1 ℃ of per 2 cycle down,
72 ℃ of 1min, 10 circulations: 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 1min, 72 ℃ of 5min;
5) electrophoresis detection of amplified production:
Get 10 μ L pcr amplification products, carry out electrophoresis on the sepharose of 1% (g/mL), current stabilization 80mA detects under UV-light behind the 1.5h: if exist length to be about two bands of 900bp and 370bp size respectively, the individual resistance homozygote that is is described; If exist length to be about two bands of 900bp and 540bp size respectively, the individual responsive homozygote that is be described; If exist length to be about three bands of 900bp, 540bp and 370bp size respectively simultaneously, the individual heterozygote that is be described.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154461A (en) * | 2011-01-10 | 2011-08-17 | 南京农业大学 | Molecular detection method for drug resistance of brown paddy plant hopper to phenylpyrazole pesticides |
| CN102382876A (en) * | 2010-08-30 | 2012-03-21 | 高希武 | Method for detecting avermectin pesticide resistance produced by diamondback moth |
| CN103918701A (en) * | 2014-04-25 | 2014-07-16 | 中国计量学院 | Preparation method of Nadiwen-imidacloprid mixed formula preparation |
| CN107058488A (en) * | 2016-12-29 | 2017-08-18 | 南京农业大学 | Brown paddy plant hopper is to Organophosphorus and carbamate pesticides insecticides resistance to the action of a drug molecular detecting method |
| CN107641655A (en) * | 2017-03-21 | 2018-01-30 | 南京农业大学 | A kind of quick detection brown paddy plant hopper is to the drug-fast method of Organophosphorus and carbamate pesticides insecticides |
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2003
- 2003-12-12 CN CN 200310118523 patent/CN1271214C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102382876A (en) * | 2010-08-30 | 2012-03-21 | 高希武 | Method for detecting avermectin pesticide resistance produced by diamondback moth |
| CN102154461A (en) * | 2011-01-10 | 2011-08-17 | 南京农业大学 | Molecular detection method for drug resistance of brown paddy plant hopper to phenylpyrazole pesticides |
| CN102154461B (en) * | 2011-01-10 | 2013-05-01 | 南京农业大学 | Molecular detection method for drug resistance of brown paddy plant hopper to phenylpyrazole pesticides |
| CN103918701A (en) * | 2014-04-25 | 2014-07-16 | 中国计量学院 | Preparation method of Nadiwen-imidacloprid mixed formula preparation |
| CN103918701B (en) * | 2014-04-25 | 2016-03-09 | 中国计量学院 | A kind of taking is opposed surely and the preparation method of Imidacloprid mixing formula preparation |
| CN107058488A (en) * | 2016-12-29 | 2017-08-18 | 南京农业大学 | Brown paddy plant hopper is to Organophosphorus and carbamate pesticides insecticides resistance to the action of a drug molecular detecting method |
| CN107641655A (en) * | 2017-03-21 | 2018-01-30 | 南京农业大学 | A kind of quick detection brown paddy plant hopper is to the drug-fast method of Organophosphorus and carbamate pesticides insecticides |
| CN107641655B (en) * | 2017-03-21 | 2021-06-18 | 南京农业大学 | Method for rapidly detecting drug resistance of brown planthopper to organophosphorus and carbamate insecticides |
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| CN1271214C (en) | 2006-08-23 |
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