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Kit for detecting G119S mutation of culex acetylcholinesterase
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A technology of acetylcholinesterase and kits, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve problems such as troubles in daily life and detection troubles in daily life, and achieve the effect of simple operation
Inactive Publication Date: 2012-06-06
INST OF ZOOLOGY CHINESE ACAD OF SCI
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The advantage of this method is that the accurate value of carboxylesterase activity can be obtained, but the disadvantage is that it must be completed with the aid of a microplate reader, which is troublesome in daily life
For target drug resistance (organophosphate), biochemical detection technology (J.Hemingway and C.Smith: Bull.Entomol.Res.76, 559-565(1986)) can be used to detect insensitive acetylcholinesterase, relative to metabolic resistance Microplate detection in sex, which is more accurate and can detect homozygous and heterozygous resistant individual species (Michael COLEMAN and Janet HEMINGWAY: Insecticide resistance monitoring and evaluation in disease transmitting mosquitoes, J.Pestic.Sci. , 32(2), 69-76(2007)) and the use of allele-specific PCR can detect the mutations of the three major targets, but this method is troublesome in daily life detection
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Embodiment 1
[0042] Embodiment 1, the composition of kit
[0043] The kit consists of protein extraction solution, chromogenicreagent, inhibitor I, inhibitor II and substrate tubes.
[0044] Protein extract (1 bottle): composed of 250mM pH7.0PB buffer and Triton X100, the concentration of Triton X100 is 1% (percentage by weight); said 250mM pH7.0PB buffer consists of water, sodium dihydrogen phosphate and hydrogenphosphate disodium composition.
[0046] Inhibitor I (1 bottle): composed of propoxur and ethanol, the concentration of propoxur is 10 -3 M.
[0047] Inhibitor II (1 bottle): composed of propoxur and ethanol, the concentration of propoxur is 10 -1 M.
[0048] Substrate tubes (20 pieces): 1.5ml...
Embodiment 2
[0051] Embodiment 2, the application of kit
[0052] 1. Using the kit prepared in Example 1 to detect the SR strain of Culex mosquito
[0053] 1. Put a single mosquito (Culex spp. SR strain, adult) in a 1.5ml centrifuge tube, add 400ul protein extract, grind the mosquito thoroughly, centrifuge at 10,000rpm for 10min at 4°C, and take the supernatant as the protein to be tested Samples (keep carefully on ice for later use).
[0054] 2. Take a substrate tube, centrifuge a little to make the drug gather at the bottom of the tube, take 1ml of chromogenicreagent and add it to the substrate tube, shake well to get the substrate (put it on ice for later use).
[0055] 3. Prepare three 0.5ml centrifuge tubes (tube No. 1, tube 2 and tube 3), add 100 μl protein sample to be tested in each tube; then add 10 μl absolute ethanol to tube 1, and add 10 μl inhibitor I to tube 2 , Add 10 μl Inhibitor II to No. 3 tube, and place it at room temperature for 15 minutes.
[0056]4. Add 80 μl of ...
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Abstract
The invention discloses a kit for detecting G119S mutation of culexacetylcholinesterase. The kit provided in the invention comprises a protein extracting solution, a developer, an inhibitor I, an inhibitor II and acetylthiocholine iodide. Specifically, the protein extracting solution is composed of a PB buffer solution and Triton X100 with a concentration of 1%; the developer is composed of a PB buffer solution, DTNB (5, 5'-Dithio-bis-2-nitrobenzoic acid) with a concentration of 0.2nM and NaHCO3 with a concentration of 0.35mM; the inhibitor I is composed of propoxur with a concentration of 10<-3>M and ethanol; the inhibitor II is composed of propoxur with a concentration of 10<-1>M and ethanol; a G119S mutantacetylcholinesterase coded gene is as shown in sequence 4 of a sequence table. The kit provided in the invention is simple to operate, and whether a mosquito has acetylcholinesterase G119 S mutation can be known only through visual inspection and color contrast. Being rapid and efficient, the kit of the invention can be used in fields and daily life, and provides guidance for people in terms of pesticide control to mosquitoes.
Description
technical field [0001] The invention relates to a kit for detecting the mutation of Culex acetylcholinesterase G119S. Background technique [0002] Due to its special behavior and physiological characteristics, mosquitoes have become the vectors of various diseases, seriously affecting the tourism and catering industries, restricting the development of sanitary cities, and seriously endangering human health. There are many kinds of mosquitoes, and there are many diseases transmitted by biting. Culex mosquito is the main vector of filariasis, Japanese encephalitis and West Nile virus. Anopheles is the main vector of filariasis and malaria. Aedes mosquito It is the main vector of dengue fever and Japanese encephalitis (Hemingway J and Ranson H. Insecticide resistance in insect vectors of humandisease [J]. Annu. Rev. Entomol. 2000, 45: 371-391.). [0003] At present, physical control, chemical control and biological control are mainly adopted (Xu Chenglong, Jiang Zhikuan. Mosq...
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