CN110031623A - Utilize the method for BA-ELISA detection Bemisia tabaci drug resistance - Google Patents
Utilize the method for BA-ELISA detection Bemisia tabaci drug resistance Download PDFInfo
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Abstract
Using the method for BA-ELISA detection Bemisia tabaci drug resistance, it is related to a kind of detection method of Bemisia tabaci drug resistance.The present invention solves the problems, such as that stability and poor repeatability of the existing technology, at high cost, specific and sensibility are bad, and provides a kind of method using BA-ELISA detection Bemisia tabaci drug resistance.The method of the present invention: one, ELISA Plate coating is added after crude enzyme liquid is diluted with homogenate；Two, antibody is added in each hole in ELISA Plate after being diluted with 1%BSA solution, is added reaction buffer and is incubated for；Three, streptavidin is added to be incubated for；Four, color developing agent is added to develop the color.Detection method stability and repeatability of the invention is relatively good, has excellent specificity and sensibility.
Description
Technical field
The present invention relates to a kind of detection methods of Bemisia tabaci drug resistance.
Background technique
Bemisia tabaci (Bemisia tabaci Gennadius) is extremely wide one of the worldwide pest of harm, by straight
It connects piercing and sucking to cause harm, propagate plant virus etc., causes plant physiology disorder, secretion honeydew to induce fungal disease and caused to crop production
Huge economic loss.Bemisia tabaci is in the harm of China's Important Agricultural economic industry such as cotton, vegetables, industrialized agriculture in recent years
Now continuous ascendant trend causes to seriously threaten to increasing peasant income and agricultural sustainable development.With not conforming to for a large amount of chemical agents
Reason uses, and Bemisia tabaci has generated different degrees of resistance to the multiple types insecticide including anabasine.Imidacloprid, thiophene
Worm piperazine is the common medicament of current prevention and treatment Bemisia tabaci, research shows that Bemisia tabaci is main to the resistance mechanism of these anabasine medicaments
It is related to the overexpression of cytochrome P450 gene.Implement the early stage Resistance detecting to Bemisia tabaci, is conducive to formulate rationally effective
Prevention and control measure reduces disaster threat, ensures agricultural safety, promotes increasing peasant income, has positive promotion to social stability and development
Effect.
In the prior art using Inhibition ELISA and double crush syndrome method detection Bemisia tabaci drug resistance, but both
Method stability and repeatability are poor, and specificity and sensibility are bad.
Summary of the invention
The present invention is in order to solve stability and poor repeatability of the existing technology, specificity and sensibility is bad asks
Topic, and provide a kind of method using BA-ELISA detection Bemisia tabaci drug resistance.
The present invention is followed the steps below using the method for BA-ELISA detection Bemisia tabaci drug resistance:
One, Bemisia tabaci adults are ground into crude enzyme liquid, and ELISA Plate coating is added after being diluted with homogenate, is then washed with eluent
It washs 3~5 times；
Two, the antibody of Bemisia tabaci P450 target proteins is diluted to the dilution that concentration is 0.5 μ g/mL with 1%BSA solution,
It is then added in the hole that ELISA Plate described in step 1 is coated, and reaction buffer is added and is incubated for, use eluent later
Washing 3~5 times；
Three, streptavidin is to be added to enzyme mark described in step 2 after 1:200 is mixed according to volume ratio with 1%BSA solution
Plate carries out secondary incubation in the hole through being incubated for, and is then washed 3~5 times with eluent；
Four, color developing agent is added into hole of the ELISA Plate described in step 3 through secondary incubation to develop the color, that is, completes tobacco powder
The detection of lice drug resistance；
Wherein, the benzyl that homogenate described in step 1 is 100mM by 0.01M, pH PBS solution for being 7.4 and concentration
Sulfuryl fluoride ethanol solution is mixed according to the volume ratio of 100:1；Step 1: eluent described in step 2 and step 3 by
The logical volume ratio according to 1000:1 of the PBS solution and Qula that 0.01M, pH are 7.4 forms；Reaction buffer described in step 2 is
According to parts by weight by 50 parts 0.01M, pH be 7.4 PBS solution, 0.05 part of Qula logical, 0.5 part of bovine serum albumin and
0.5 part of N- dodecyl-β-D-Maltose mixes.
There is the principle of very strong affinity according to Avidin (Avidin) and biotin (Biotin), by biotin-avidin
The liquid phase that the enlarge-effect of system (Biotin-Avidin System, BAS) is combined with enzyme-linked immunoassay method (ELISA) method
Biotin-avidin ELISA detection technique improves specificity, the sensibility of ELISA method detection.This method passes through antigen
The combination that the Streptavidin molecule high specific of compound and biotin and enzyme label is formed with biotin antibody, due to
The chromogenic reaction of the multistage amplification of BAS, the enzyme molecule for carrying each antibody dramatically increases, to greatly improve sensitive
Degree.
The present invention be by Bemisia tabaci Cytochrome P450 target gene antigen with label biotin antibody specificity in conjunction with
Compound is formed, and marks the combination of the Avidin and biotin of horseradish peroxidase (HRP), chromogenic substrate is added,
Reaction generates the product of blue under the catalysis of HRP, and antigenic content is higher, and the blue that reaction product is presented is deeper, to reach
Detect the purpose of Bemisia tabaci drug resistance.N- dodecyl-β-D-Maltose (DDM) and Qula are logical in reaction buffer of the present invention
It (Triton) is surfactant, the two, which combines, more effectively prevents non-specific adsorption.
Detection method of the invention provides a kind of connected applications to existing detection resistance technique, to improve and invent one
Kind of new effective detection technique, the period for making up bioassay and molecular biology Resistance detecting is longer, test worm sample size is larger
Or the shortcomings that trivial operations, method of the invention is efficiently, quick, specificity is high, accurate Bemisia tabaci of grasping is to imidacloprid resistance water
Flat detection technique.
Compared with ELISA method and double crush syndrome method, detection method stability of the invention and repeatability compare
Good, testing cost is low, has excellent specificity and sensibility.
Detailed description of the invention
Fig. 1 is the colour developing result that Xinjiang difference Bemisia tabaci uses the method for the present invention to detect；
Fig. 2 is the relative amount of different Bemisia tabaci P450 gene C YP4G68 albumen；
Fig. 3 is the testing result using three groups of different homogenates to Bemisia tabaci CYP4G68；
Fig. 4 is after horseradish peroxidase-labeled Streptavidin (HRP-SA) is diluted using three kinds of different solution to cigarette
The testing result of aleyrodid CYP4G68.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment
Any combination.
Specific embodiment 1: present embodiment is using the method for BA-ELISA detection Bemisia tabaci drug resistance according to following step
It is rapid to carry out:
One, Bemisia tabaci adults are ground into crude enzyme liquid, and ELISA Plate coating is added after being diluted with homogenate, is then washed with eluent
It washs 3~5 times；
Two, the antibody of Bemisia tabaci P450 target proteins is diluted to the dilution that concentration is 0.5 μ g/mL with 1%BSA solution,
It is then added in the hole that ELISA Plate described in step 1 is coated, and reaction buffer is added and is incubated for, use eluent later
Washing 3~5 times；
Three, streptavidin is to be added to enzyme mark described in step 2 after 1:200 is mixed according to volume ratio with 1%BSA solution
Plate carries out secondary incubation in the hole through being incubated for, and is then washed 3~5 times with eluent；
Four, color developing agent is added into hole of the ELISA Plate described in step 3 through secondary incubation to develop the color, that is, completes tobacco powder
The detection of lice drug resistance；
Wherein, the benzyl that homogenate described in step 1 is 100mM by 0.01M, pH PBS solution for being 7.4 and concentration
Sulfuryl fluoride ethanol solution is mixed according to the volume ratio of 100:1；Step 1: eluent described in step 2 and step 3 by
The logical volume ratio according to 1000:1 of the PBS solution and Qula that 0.01M, pH are 7.4 forms；Reaction buffer described in step 2 is
According to parts by weight by 50 parts 0.01M, pH be 7.4 PBS solution, 0.05 part of Qula logical, 0.5 part of bovine serum albumin and
0.5 part of N- dodecyl-β-D-Maltose mixes.
The preparation method of crude enzyme liquid described in present embodiment step 1 is: Bemisia tabaci adults are added in homogenate, are used
Sample broke instrument is crushed 3~10min under the conditions of 4 DEG C to get crude enzyme liquid has been arrived；The wherein weight of Bemisia tabaci adults and homogenate
Than for 1:250~260；Wherein, the phenylmethylsulfonyl fluoride that homogenate is 100mM by 0.01M, pH PBS solution for being 7.4 and concentration
Ethanol solution is mixed according to the volume ratio of 100:1.
It is 7.4 that 1%BSA solution described in present embodiment step 2 and step 3, which is 0.01mol/L, pH by concentration,
PBS solution and bovine serum albumin are formed according to the weight ratio of 100:1.
The antibody of the Bemisia tabaci P450 target proteins of biotin labeling, Bemisia tabaci P450 are added in present embodiment step 2
The antibody of target proteins is Bemisia tabaci CYP4G68 antibody, from Nanjing Genscript Biotechnology Co., Ltd..
Streptavidin is the streptavidin of horseradish peroxidase-labeled in present embodiment step 3, and principle is
The streptavidin of horseradish peroxidase-labeled is added, is combined with biotin labelled antibodies, streptavidin is purchased from R&D
SYSTEMS。
The chromogenic reaction time is 15~20min in present embodiment step 5.
Bemisia tabaci drug resistance described in present embodiment refers to Xinjiang region Bemisia tabaci to the resistance of imidacloprid.
Present embodiment is the antibody specificity by Bemisia tabaci Cytochrome P450 target gene antigen and label biotin
In conjunction with formation compound, and the combination of the Avidin and biotin of horseradish peroxidase (HRP) is marked, colour developing bottom is added
Object, reaction generates the product of blue under the catalysis of HRP, and antigenic content is higher, and the blue that reaction product is presented is deeper, to reach
The purpose of detection Bemisia tabaci drug resistance is arrived.N- dodecyl-the β of present embodiment-D-Maltose (DDM) and Qula are logical
It (Triton) is surfactant, the two, which combines, can more effectively prevent non-specific adsorption.
Compared with using Inhibition ELISA and double crush syndrome method detection Bemisia tabaci drug resistance, present embodiment
Method stability and repeatability are relatively good, and testing cost is low, there is excellent specificity and sensibility.
Specific embodiment 2: the present embodiment is different from the first embodiment in that: crude enzyme liquid in step 1 with
The volume ratio of homogenate is 1:1~125 times.Other steps and parameter are same as the specific embodiment one.
Specific embodiment 3: the present embodiment is different from the first and the second embodiment in that: item is coated in step 1
Part: 37 DEG C in homogenate, coating 60min.Other steps and parameter are the same as one or two specific embodiments.
Specific embodiment 4: present embodiment is unlike specific embodiment three: dilution and anti-in step 2
The volume ratio for answering buffer is 1:1.Other steps and parameter are the same as the specific implementation mode 3.
Specific embodiment 5: present embodiment is unlike specific embodiment one, two or four: incubating in step 2
The condition of educating is 37 DEG C, incubation time 60min.Other steps and parameter are the same as the specific implementation mode 1,2 or 4.
Specific embodiment 6: present embodiment is unlike specific embodiment five: the secondary incubation in step 3
Temperature is 37 DEG C, secondary incubation time is 30min.Other steps and parameter are identical as specific embodiment five.
Specific embodiment 7: present embodiment is unlike specific embodiment one to six: color developing agent in step 4
It is made of tetramethyl benzidine and hydrogen peroxide according to 1:1 volume ratio.Other steps and parameter and one to six phase of specific embodiment
Together.
Specific embodiment 8: present embodiment is unlike specific embodiment one to seven: after developing the color in step 4
It is terminated and is reacted with sulfuric acid.Other steps and parameter are identical as specific embodiment one to seven.
The concentration of sulfuric acid is 0.25M in present embodiment.
Embodiment 1
It is followed the steps below using the method for BA-ELISA detection Xinjiang region Bemisia tabaci drug resistance:
One, it is added in 100 μ L homogenates with the crude enzyme liquid of 20 Bemisia tabaci adults, it is broken with 4 DEG C of sample broke instrument
5min obtains crude enzyme liquid, and ELISA Plate coating is added after then being diluted with homogenate, is coated under the conditions of 37 DEG C in homogenate
60min, then with 300 μ L elution 3 times；Wherein, tetra- kinds of 20 μ L different dilutions times are separately added into ELISA Plate in each hole
Several crude enzyme liquids, the first crude enzyme liquid not being diluted, the diluted crude enzyme liquid of second of 5 times of homogenates, the third 25 times homogenate
The diluted crude enzyme liquid of liquid, the 4th kind of 125 times of diluted crude enzyme liquids of homogenate；Wherein the preparation method of crude enzyme liquid is: Bemisia tabaci at
Worm is added in homogenate, is crushed 3~10min under the conditions of 4 DEG C with sample broke instrument to get crude enzyme liquid has been arrived.
Two, Bemisia tabaci CYP4G68 antibody is diluted to the dilution that concentration is 0.5 μ g/mL with 1%BSA solution, then by 60
The dilution of μ L is added in the hole that ELISA Plate described in step 1 is coated, and is added 60 μ L reaction buffers and is incubated for, is incubated
The condition of educating is 37 DEG C, is incubated for 60min, then is washed 4 times with 300 μ L eluents；
Three, by volume ratio be 1:200 streptavidin and 1%BSA solution with 60 μ L of mixed liquor and reaction buffer
60 μ L, which are added in step 2 in the hole that ELISA Plate described in step 1 is coated, carries out secondary incubation, and incubation conditions are 37 DEG C, incubate
Educate 30miN,Then it is washed 5 times with 300 μ L eluents；
Four, color developing agent is added in each hole of ELISA Plate into step 3 to develop the color, is then terminated with the sulfuric acid of 0.25M
Reaction, that is, complete the detection of Bemisia tabaci drug resistance；Wherein, homogenate described in step 1 is 7.4PBS solution by 0.01M, pH
It is mixed for the phenylmethylsulfonyl fluoride ethanol solution of 100mM according to the volume ratio of 100:1 with concentration；Step 1: step 2 and
Eluent described in step 3 is formed by 0.01M, pH PBS solution for being 7.4 and Qula are logical according to the volume ratio of 1000:1；Step
It is 7.4 PBS that reaction buffer described in two, which is according to parts by weight by 50 parts of 0.01M pH, 0.05 part of Qula is logical, 0.5 part
Bovine serum albumin and 0.5 part of N- dodecyl-β-D-Maltose mix；Color developing agent in step 4 is joined by tetramethyl
Aniline and hydrogen peroxide are formed according to 1:1 volume ratio.
Streptavidin is the streptavidin of horseradish peroxidase-labeled in present embodiment step 3.
Turpan, Xinjiang Ao Yiman Mai Li village's Bemisia tabaci (TP1), Shache county Bemisia tabaci (SC) and field Bemisia tabaci are taken respectively
(HT), Turfan Lv Zongcun Bemisia tabaci (TP2) and Yining Bemisia tabaci (YN) are used as test sample, carry out to Xinjiang region Bemisia tabaci
Resistance (imidacloprid resistance) monitoring, resistance level, which divides range, is: resistant multiple is low-level resistance between 5~10, resistance
Multiple is medium level resistance between 10~40, and resistant multiple is high-level resistance when being greater than 40, and monitoring result shows TP1's
Resistant multiple is 46.1 times, for high-level resistance；The resistant multiple of TP2, SC 1, HT and YN is between 10~20
Etc. horizontal resistances.
It is detected using drug resistance (imidacloprid resistance) of the method for the present invention to TP1, TP2, SC 1, HT and YN, is separately set
Blank group and sensitive strain group, blank group sample are the homogenate in step 1, i.e. blank group sample is by 0.01M, pH
The phenylmethylsulfonyl fluoride ethanol solution that 7.4PBS solution and concentration are 100mM is mixed according to the volume ratio of 100:1, sensitive product
System's group sample does not contact Bemisia tabaci (NJ-S) adult crude enzyme liquid of medicament raising using interior；With the method pair of embodiment 1
The drug resistance (imidacloprid resistance) of each test sample is detected, and testing result is as shown in Figure 1, each population is from a left side in Fig. 1
It is that the diluted crude enzyme liquid of 125 times of homogenates, the diluted crude enzyme liquid of 25 times of homogenates, 5 times of homogenates are dilute respectively to right test sample
The crude enzyme liquid released and the crude enzyme liquid not being diluted, color show that CYP4G68 protein content is higher more deeply feeling, detection detection of the invention
Process is simple, and cost is relatively low, and detection speed is fast, the test period compared with using Inhibition ELISA and double crush syndrome method
Shorten half the time.
Using microplate reader in wavelength 450nm and 630nm, 30 DEG C of measurement reaction product absorbance values, Xinjiang difference Bemisia tabaci
The relative amount of resistant population CYP4G68 albumen is as shown in Figure 2；It can be seen that detection method of the invention in conjunction with Fig. 1 and Fig. 2
Consistent with the result of resistance monitoring, detection side's accuracy of the invention is high, and detection method stability of the invention and repeatability are equal
It is relatively good, in addition, the crude enzyme liquid of Bemisia tabaci adults remains to develop the color when extension rate is 125 times, it follows that side of the present invention
Method remains to complete detection in the case where diluting low concentration, and the method for the present invention has prominent specificity and sensibility.
Embodiment 2
Using three groups of different homogenates, ELISA detection is carried out to indoor raising strain NJ-S using the method for embodiment 1
(Bemisia tabaci CYP4G68) compares the superiority and inferiority of three；Wherein, indoor raising strain NJ-S is the indoor cigarette for not contacting medicament raising
Aleyrodid (NJ-S) adult crude enzyme liquid.
The distinctive points of three groups of experiments are that first group: the homogenate in step 1 is PBS solution (0.01M, pH7.4)；The
Homogenate in two groups of step 1 is the phenylmethylsulfonyl fluoride ethanol solution that 0.01M, pH are 7.4PBS solution and concentration is 100mM
It is mixed according to the volume ratio of 100:1；Third group: the homogenate in step 1 is by PBS, DDM (N- dodecyl-β-D- wheat
Bud sugar), sucrose according to weight ratio be 50:0.5:3.4 ratio form；Separately set blank group, blank group sample is even with second group
Slurries substitute Bemisia tabaci adults crude enzyme liquid, i.e. blank group sample is the benzene that 0.01M, pH are 7.4PBS solution and concentration is 100mM
Methanesulfonyl fluoride ethanol solution is mixed according to the volume ratio of 100:1.Choose the diluted crude enzyme liquid sample of 125 times of homogenates of each group
The testing result of product and the crude enzyme liquid sample not being diluted is compared, as a result as shown in figure 3, in Fig. 3 A behavior be added color developing agent
Sample colour developing as a result, B behavior be added sulfuric acid terminate reaction after sample develop the color result；From figure 3, it can be seen that either reacting
Colour developing result or terminate reaction solution as a result, the ELISA colour developing of three kinds of homogenates processing is all significantly stronger than blank group, wherein
With in second group i.e. embodiment of the present invention 1 homogenate preparation Bemisia tabaci enzyme solution colour developing result it is most obvious, and first group only
ELISA colour developing with the Bemisia tabaci enzyme solution of PBS homogenate preparation is most weak；The obtained detection effect of detection method of the invention is bright
Aobvious, specificity and sensibility are good.
Embodiment 3: in 1 step 3 of embodiment horseradish peroxidase-labeled Streptavidin (HRP-SA) using three kinds not
Same solution dilution, then to the carry out ELISA detection (Bemisia tabaci CYP4G68) of indoor raising strain (NJ-S), compares three
Superiority and inferiority；Wherein, indoor raising strain NJ-S is indoor Bemisia tabaci (NJ-S) adult crude enzyme liquid for not contacting medicament raising.
The distinctive points of three groups of tests are that first group: horseradish peroxidase-labeled strepto- is affine in 1 step 3 of embodiment
Plain (HRP-SA) leads to mixed liquor with 1%BSA and 0.05% Qula and is diluted；The wherein logical volume of 1%BSA, 0.05% Qula
Than for 2000:1；Second group of horseradish peroxidase-labeled Streptavidin (HRP-SA) is diluted with 1%BSA solution；Third group:
In 1 step 3 of embodiment horseradish peroxidase-labeled Streptavidin (HRP-SA) with PBS solution (0.01M, pH7.4),
BSA, Qula are logical and N- dodecyl-β-D-Maltose mixed liquor is diluted；PBS solution, BSA, Qula is led to and 1%N- ten
Dialkyl group-β-D-Maltose is mixed according to weight ratio 50:0.5:0.05:0.5.Test result is as shown in figure 4, A is in figure
The colour developing of color developing agent is added as a result, B is that sulfuric acid terminates the colour developing result after reaction.Separately set blank group, blank group sample is with second
The homogenate of group substitutes Bemisia tabaci adults crude enzyme liquid, i.e. blank group sample is that 0.01M, pH are 7.4PBS solution and concentration is
The phenylmethylsulfonyl fluoride ethanol solution of 100mM is mixed according to the volume ratio of 100:1.From fig. 4, it can be seen that either reacting
Colour developing result or terminate reaction solution as a result, the ELISA colour developing of three groups of tests is all significantly stronger than blank group, wherein with second
(1%BSA, 0.1% Qula is led to and 1%N- dodecyl-β-D- wheat for group (the 1%BSA dilution in embodiment 1) and third group
Bud sugar mixed liquor) color developing effect be higher than first group (1%BSA and 0.05% Qula lead to mixed liquor), second group adds sample
When the diluted concentration of crude enzyme liquid is 125 times, color developing effect is still it is obvious that the sample of third group is led to by 1%BSA, 0.1% Qula
With 1%N- dodecyl-β-D-Maltose composition, in comparison, the cost of 1%BSA dilution of the invention well below
Third group sample.
It is equal to can be seen that detection method stability and repeatability of the invention from the testing result of embodiment 1 and embodiment 2
Relatively good, testing cost is low, in the lower situation of detectable concentration, still there is good detection effect, method tool of the invention
There are specificity and sensibility outstanding.
Claims (8)
1. utilizing the method for BA-ELISA detection Bemisia tabaci drug resistance, it is characterised in that detect the anti-medicine of Bemisia tabaci using BA-ELISA
The method of property follows the steps below:
One, Bemisia tabaci adults are ground into crude enzyme liquid, and ELISA Plate coating is added after being diluted with homogenate, then washs 3 with eluent
~5 times；
Two, the antibody of Bemisia tabaci P450 target proteins is diluted to the dilution that concentration is 0.5 μ g/mL with 1%BSA solution, then
It is added in the hole that ELISA Plate described in step 1 is coated, and reaction buffer is added and is incubated for, wash 3 with eluent later
~5 times；
Three, streptavidin is that the warp of ELISA Plate described in step 2 is added to after 1:200 is mixed according to volume ratio with 1%BSA solution
Secondary incubation is carried out in the hole of incubation, is then washed 3~5 times with eluent；
Four, color developing agent is added into hole of the ELISA Plate described in step 3 through secondary incubation to develop the color, that is, it is anti-completes Bemisia tabaci
The detection of pharmacological property；
Wherein, the benzyl sulphonyl that homogenate described in step 1 is 100mM by 0.01M, pH PBS solution for being 7.4 and concentration
Fluoroethanol solution is mixed according to the volume ratio of 100:1；Step 1: eluent described in step 2 and step 3 by 0.01M,
The logical volume ratio according to 1000:1 of the PBS solution and Qula that pH is 7.4 forms；Reaction buffer described in step 2 is according to weight
It is 7.4 PBS solution, 0.05 part of Qula logical, 0.5 part of bovine serum albumin and 0.5 part that number, which is measured, by 50 parts 0.01M, pH
N- dodecyl-β-D-Maltose mix.
2. the method according to claim 1 using BA-ELISA detection Bemisia tabaci drug resistance, it is characterised in that step 1
In crude enzyme liquid and homogenate volume ratio be 1:1~125 times.
3. the method according to claim 1 or 2 using BA-ELISA detection Bemisia tabaci drug resistance, it is characterised in that step
Condition is coated in one: 37 DEG C in homogenate, coating 60min.
4. the method according to claim 3 using BA-ELISA detection Bemisia tabaci drug resistance, it is characterised in that step 2
The volume ratio of middle dilution and reaction buffer is 1:1.
5. the method according to claim 1,2 or 3 using BA-ELISA detection Bemisia tabaci drug resistance, it is characterised in that step
Incubation conditions in rapid two are 37 DEG C, incubation time 60min.
6. the method according to claim 5 using BA-ELISA detection Bemisia tabaci drug resistance, it is characterised in that step 3
In secondary incubation temperature be 37 DEG C, secondary incubation time is 30min.
7. according to claim 1, utilizing the method for BA-ELISA detection Bemisia tabaci drug resistance described in 2,4 or 6, it is characterised in that
Color developing agent is made of tetramethyl benzidine and hydrogen peroxide according to 1:1 volume ratio in step 4.
8. the method according to claim 7 using BA-ELISA detection Bemisia tabaci drug resistance, it is characterised in that step 4
It is terminated and is reacted with sulfuric acid after middle colour developing.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010068797A (en) * | 2008-08-18 | 2010-04-02 | Sumitomo Chemical Co Ltd | Method for distinguishing biotype in bemisia tabaci |
| AU2008361735B2 (en) * | 2008-11-25 | 2011-08-04 | Jiangsheng Fang | Hydrocarbylidene nitrohydrozinecarboximidamides and a method for making the same, as well as their uses as an insecticide |
| CN102212522A (en) * | 2011-04-29 | 2011-10-12 | 山东省农业科学院高新技术研究中心 | Method for preventing and treating bemisa babaci by utilizing RNA (Ribose Nucleic Acid) interference technology |
| CN102937652A (en) * | 2012-10-30 | 2013-02-20 | 浙江大学 | Dot enzyme-linked immunosorbent assay (ELISA) method for detecting bemisia tabaci carrying tomato yellow leaf curl virus and application of method |
| WO2013182613A1 (en) * | 2012-06-08 | 2013-12-12 | Bayer Cropscience Ag | Detection system for the identification of insecticide resistance |
| CN104031991A (en) * | 2014-05-21 | 2014-09-10 | 中国农业科学院蔬菜花卉研究所 | Fluorescent quantitative PCR detection method of resistance of bemisia tabaci on thiamethoxam and kit thereof |
| CN105123734A (en) * | 2015-08-01 | 2015-12-09 | 华中农业大学 | Preparation method of mixed insecticide composed of avermectin, nitenpyram, and indoxacarb |
| WO2017042187A1 (en) * | 2015-09-09 | 2017-03-16 | Endura S.P.A. | Substituted dihydrobenzofuran compounds and their use as synergists |
| CN107043753A (en) * | 2017-02-27 | 2017-08-15 | 中国农业科学院蔬菜花卉研究所 | Bemisia tabaci Diacloden resistant gene CYP6DZ7 and its promoter |
| CN107873054A (en) * | 2014-09-09 | 2018-04-03 | 博德研究所 | The method and apparatus based on droplet for compound single-cell nucleic acid analysis |
| CN107894510A (en) * | 2017-12-13 | 2018-04-10 | 中国农业科学院生物技术研究所 | Quantitatively detect the enzyme linked immunological kit and its detection method of insect resistance protein Vip3Aa20 in transgenic corns |
-
2019
- 2019-05-10 CN CN201910386833.9A patent/CN110031623B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010068797A (en) * | 2008-08-18 | 2010-04-02 | Sumitomo Chemical Co Ltd | Method for distinguishing biotype in bemisia tabaci |
| AU2008361735B2 (en) * | 2008-11-25 | 2011-08-04 | Jiangsheng Fang | Hydrocarbylidene nitrohydrozinecarboximidamides and a method for making the same, as well as their uses as an insecticide |
| CN102212522A (en) * | 2011-04-29 | 2011-10-12 | 山东省农业科学院高新技术研究中心 | Method for preventing and treating bemisa babaci by utilizing RNA (Ribose Nucleic Acid) interference technology |
| WO2013182613A1 (en) * | 2012-06-08 | 2013-12-12 | Bayer Cropscience Ag | Detection system for the identification of insecticide resistance |
| CN102937652A (en) * | 2012-10-30 | 2013-02-20 | 浙江大学 | Dot enzyme-linked immunosorbent assay (ELISA) method for detecting bemisia tabaci carrying tomato yellow leaf curl virus and application of method |
| CN104031991A (en) * | 2014-05-21 | 2014-09-10 | 中国农业科学院蔬菜花卉研究所 | Fluorescent quantitative PCR detection method of resistance of bemisia tabaci on thiamethoxam and kit thereof |
| CN107873054A (en) * | 2014-09-09 | 2018-04-03 | 博德研究所 | The method and apparatus based on droplet for compound single-cell nucleic acid analysis |
| CN105123734A (en) * | 2015-08-01 | 2015-12-09 | 华中农业大学 | Preparation method of mixed insecticide composed of avermectin, nitenpyram, and indoxacarb |
| WO2017042187A1 (en) * | 2015-09-09 | 2017-03-16 | Endura S.P.A. | Substituted dihydrobenzofuran compounds and their use as synergists |
| EP3346837B1 (en) * | 2015-09-09 | 2019-01-09 | Endura S.p.a. | Substituted dihydrobenzofuran compounds and their use as synergists |
| CN107043753A (en) * | 2017-02-27 | 2017-08-15 | 中国农业科学院蔬菜花卉研究所 | Bemisia tabaci Diacloden resistant gene CYP6DZ7 and its promoter |
| CN107894510A (en) * | 2017-12-13 | 2018-04-10 | 中国农业科学院生物技术研究所 | Quantitatively detect the enzyme linked immunological kit and its detection method of insect resistance protein Vip3Aa20 in transgenic corns |
Non-Patent Citations (7)
| Title |
|---|
| L GUO ETAL: ""Detoxification enzymes of Bemisia tabaci B and Q: biochemical characteristics and gene expression profiles"", 《PEST MANAGEMENT SCIENCE》 * |
| NINA YANG ETAL: ""Transcriptomic and Proteomic Responses of Sweetpotato Whitefly,Bemisia tabaci,to Thiamethoxam"", 《PLOS ONE》 * |
| 吕世静 等: "《临床免疫学检验考试指南》", 31 October 2017, 中国医药科技出版社 * |
| 寿佩勤: "《免疫检验技术》", 31 March 2012, 人民军医出版社 * |
| 张灿 等: ""烟粉虱热点问题研究进展"", 《应用昆虫学报》 * |
| 王震宇: ""烟粉虱吡虫啉抗性的监测与机理"", 《中国优秀博硕士学位论文全文数据库(博士)农业科技辑 2012年 第06期》 * |
| 贾尊尊 等: ""新疆主要农区烟粉虱生物型鉴定"", 《新疆农业科学》 * |
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