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Anopheles sinensis drug resistance AS-PCR (Polymerase Chain Reaction) detection kit and special primers thereof

A kit and drug resistance technology, which is applied in the field of AS-PCR detection kits and special primers for Anopheles sinensis resistance, can solve the problems of no molecular detection methods and kits for kdr resistance

Active Publication Date: 2012-03-14
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no molecular detection method and kit for KDR resistance against Anopheles sinensis in China

Method used

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  • Anopheles sinensis drug resistance AS-PCR (Polymerase Chain Reaction) detection kit and special primers thereof
  • Anopheles sinensis drug resistance AS-PCR (Polymerase Chain Reaction) detection kit and special primers thereof
  • Anopheles sinensis drug resistance AS-PCR (Polymerase Chain Reaction) detection kit and special primers thereof

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Experimental program
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Embodiment 1

[0069] Embodiment 1, the establishment of the method for detecting the point mutation of the Anopheles sinensis sodium channel gene--AS-PCR method

[0070] 1. AS-PCR detection principle and primer design

[0071] The principle of AS-PCR is: according to the mutation characteristics of the mosquito sodium channel gene, two specific primers (mutant type and non-mutant type) are designed with the base of the mutation site as the 3' end of the primer, respectively matching with the allele of the mutation. The gene DNA sequence and the non-mutated allelic DNA sequence are combined. At the same time, two non-specific outer primers were designed on the upstream and downstream of the mutation site, taking advantage of the lack of 3′→5′ exonuclease activity of Taq enzyme, when using mutant primers to amplify normal DNA templates, the PCR reaction will be caused by phosphate Ester bonds are difficult to form and cannot be extended, and no bands of specific length can be obtained, which...

Embodiment 2

[0097] Embodiment 2, resistance test

[0098] The sensitive strain of Anopheles sinensis laboratory ((susceptible(Ss) mosquito) strain, recorded in Song, F.L., X.M.Cao, T.Y.Zhao, Y.D.Dong and B.L.Lu.2007.Pyrethroid resistance and distribution of kdr allele in Culex pipiens pallens in north China.Int J Pest Manag 53:25-34., available to the public from the Institute of Microbial Epidemiology, Academy of Military Medical Sciences of the Chinese People's Liberation Army): From the insect breeding room of the Institute of Microbial Epidemiology, Academy of Military Medical Sciences, isolated breeding for more than ten years, from Not exposed to any pesticides.

[0099] The natural population of Anopheles sinensis includes: Anopheles sinensis larva resistance group, collected from 5 areas in Jiangsu Province including Xuzhou, Huaiyin, Changshu, Nanjing, and Suzhou; when the number of larvae collected on site is large, the larval bioassay is directly carried out, and the number is n...

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Abstract

The invention discloses an Anopheles sinensis drug resistance AS-PCR (Polymerase Chain Reaction) detection kit and special primers thereof. Primers for detecting kdr gene mutational sites in Anopheles sinensis provided by the invention consist of a primer group A, a primer group B and a primer group C, wherein the primer group A comprises a primer 2 and a primer 3, and the nucleotide sequences ofthe primer 2 and the primer 3 are respectively a sequence 2 and a sequence 3 in a sequence table; the primer group B comprises a primer 2 and a primer 4, and the nucleotide sequences of the primer 2 and the primer 4 are respectively a sequence 2 and a sequence 4 in the sequence table; and the primer group C comprises a primer 2 and a primer 5, and the nucleotide sequences of the primer 2 and the primer 5 are respectively a sequence 2 and a sequence 5 in the sequence table. An experiment of the invention proves that the invention provides a quick, easy and flexible Anopheles sinensis kdr mutational allele detection kit.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an AS-PCR detection kit for drug resistance of Anopheles sinensis and special primers thereof. Background technique [0002] Anopheles sinensis is distributed in all provinces and cities in my country except Qinghai and Xinjiang. It is the dominant mosquito species in plain water networks and rice planting areas in my country. In clumps and bushes, some live in cattle houses and barns. They are partial to animal blood (mainly cattle, horses, pigs and other large livestock), and also suck human blood, overwintering as adult mosquitoes. Anopheles sinensis is an important vector of malaria in my country, especially in areas north of 34° north latitude, where it is the main or only vector. At present, there is no effective vaccine for malaria, and controlling the population of malaria vectors is an important means to prevent and control malaria. Chemical control plays an important role ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 赵彤言谭伟龙李春晓董言德汪中明郭晓霞刘美德张映梅邢丹
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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