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PCR-RFLP (Polymerase Chain Reaction-Amplified Fragment Length Polymorphism) assay kit for drug resistance of anopheles sinensis and special primer of same

A kit and reagent technology, applied in the field of Anopheles sinensis drug resistance PCR-RFLP detection kit and its special primers, can solve the problem of no molecular detection method and kit 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
  • Description
  • 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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  • PCR-RFLP (Polymerase Chain Reaction-Amplified Fragment Length Polymorphism) assay kit for drug resistance of anopheles sinensis and special primer of same
  • PCR-RFLP (Polymerase Chain Reaction-Amplified Fragment Length Polymorphism) assay kit for drug resistance of anopheles sinensis and special primer of same
  • PCR-RFLP (Polymerase Chain Reaction-Amplified Fragment Length Polymorphism) assay kit for drug resistance of anopheles sinensis and special primer of same

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Experimental program
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Effect test

Embodiment 1

[0060] Embodiment 1, the establishment of the method for detecting the point mutation of Anopheles sinensis sodium channel gene--PCR-RFLP method

[0061] 1. PCR-RFLP detection scheme and primer design

[0062] According to the Anopheles sinensis genome (kdr sensitive gene cDNA sequence, its partial nucleotide sequence is sequence 5 (the nucleotide sequence of L1014 in this sequence is 1224-1226 TTG), the accession number in GENBANK is JN002364) sequence characteristics, L1014F is a mutation of TTG to TTT, and L1014C is a mutation of TTG to TGT.

[0063] The first base T of the L1014 site has not changed, the second base may be T / G, and the third base may also be T / G, so enzymes are designed for the second and third bases Cutting point, the scheme is as follows:

[0064] 1) CF type scheme design

[0065] The base type of CF type mutation is T(T / G)T, such as figure 1 Design protocol and PCR products for RFLP primers targeting type C / F mutations. A in primer EP1 is replaced ...

Embodiment 2

[0109] Embodiment 2, resistance test

[0110] The susceptible (Ss) mosquito strain of Anopheles sinensis laboratory was 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, never have been exposed to any pesticides.

[0111] 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 not enoug...

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Abstract

The invention discloses a PCR-RFLP (Polymerase Chain Reaction-Amplified Fragment Length Polymorphism) assay kit for drug resistance of anopheles sinensis and a special primer of the same. The invention provides the special primer which is used for detecting a mutational site of a kdr gene in the anopheles sinensis. The special primer consists of a primer set A and a primer set B, wherein the primer set A consists of a primer 1 and a primer 2, nucleotide sequence of the primer 1 is a sequence 1in a sequence table, and that of the primer 2 is a sequence 2 in the sequence table; and the primer set B consists of a primer 3 and a primer 4, the nucleotide sequence of the primer 3 is a sequence 3 in the sequence table, and that of the primer 4 is a sequence 4 in the sequence table. Based on experiments, the invention provides a quick, simple, convenient and flexible assay kit for kdr mutant allelic genes of the anopheles sinensis, which is used for mastering conditions of kdr allelic genes of the anopheles sinensis relative to the resistance of a pyrethroid pesticide.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a PCR-RFLP 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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Patent Type & Authority Applications(China)
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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