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A kind of primer, probe and method for detecting drug resistance gene mutation of Aedes albopictus

An Aedes albopictus, drug-resistant technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. And other issues

Active Publication Date: 2021-08-20
宁波国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional bioassay method is cumbersome, and it is difficult to achieve true standardization from insect source, breeding to measurement, and the repeatability and accuracy of LD50 or LC50 values ​​calculated by LD-p line are low. When the population resistance is low It is difficult to measure accurately when the resistant populations are diverse, so the measured resistance level often has a lag, which is not suitable for early resistance detection and is not conducive to formulating corresponding control strategies

Method used

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  • A kind of primer, probe and method for detecting drug resistance gene mutation of Aedes albopictus
  • A kind of primer, probe and method for detecting drug resistance gene mutation of Aedes albopictus
  • A kind of primer, probe and method for detecting drug resistance gene mutation of Aedes albopictus

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Experimental program
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Effect test

Embodiment 1

[0066] The design of embodiment 1, primer and probe

[0067] 1. Primer and TaqMan-MGB probe design

[0068] For the two point mutations and three alleles of the resistant Aedes albopictus sodium channel kdr, according to the sequence characteristics, three probes were designed using PrimerExpress2.0 software, respectively for the three allele types, in order to improve the annealing Temperature, to increase the specificity of the sequence, increase the annealing temperature of the probe sequence by increasing the MGB group at the 3' end of the probe.

[0069] On the 48-well plate of the quantitative PCR instrument, each sample is designed to be amplified in two parallel tubes. The first tube is added with probes P1, P3 and non-specific primers F and R, and the second tube is added with probes P2, P3 and non-specific primers. Primers F and R, according to the operating procedures of ABI7500fast real-time PCR instrument, the FAM group of the first tube is designed as brown, the...

Embodiment 2

[0083] Example 2. TaqMan-MGB probe detects alleles of three mutation types of Aedes albopictus sodium channel. Genomic DNA of 20 samples of natural populations of Aedes albopictus captured in the field was extracted to obtain sample DNA numbered 1-20 , using the primers and probes designed in Example 1, the reaction system and reaction conditions of Example 1 to carry out double-tube simultaneous TaqMan PCR detection of Group A and Group B using the above-mentioned sample DNA.

[0084] L1014F is the allele containing TTT at the 1014th amino acid of the kdr gene;

[0085] L1014C is the allele containing TGT at the 1014th amino acid of the kdr gene;

[0086] If there is no signal of FAM group and VIC group in the first tube, and there is only signal of FAM group in the second tube, the sample is homozygous for L1014, and the base of the mutation site is TTG;

[0087] If there is a FAM group and no VIC group signal in the first tube, and there is no FAM group and VIC group signa...

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Abstract

The invention provides a primer, a probe and a method for detecting the drug resistance gene mutation of Aedes albopictus. The primers, TaqMan-MGB probe and detection method provided by the present invention can realize fast, simple, sensitive and accurate monitoring of kdr allele type, genotype frequency, etc., and can quickly and accurately grasp Aedes albopictus and pyrethroid Status of KDR alleles associated with ester insecticide resistance to understand the status of resistance genes in the genetic structure of populations. Therefore, the primers, TaqMan-MGB probe, kit and detection method provided by the present invention are a new product and method that can accurately and conveniently predict the occurrence and development of drug resistance in Aedes albopictus.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a primer, a probe and a method for detecting drug resistance gene mutation of Aedes albopictus. Background technique [0002] Aedes albopictus, also known as the Asian tiger mosquito, is a highly aggressive mosquito and an important virus vector that can transmit a variety of pathogens, including dengue fever, Zika and other viruses. At present, dengue fever and Zika have become two important insect-borne infectious diseases worldwide, and their severity is also increasing year by year. Mosquito-borne diseases kill more than 50 million people each year, and there are currently no vaccines or specific treatments to control dengue and Zika. The traditional measure to control arboviruses is to use chemical insecticides to reduce the population of mosquitoes, which are the vectors of transmission, but they do not have a good effect on the control of arboviruses. Che...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6888C12Q2600/124C12Q2600/156C12Q2531/113C12Q2561/101
Inventor 周冬根
Owner 宁波国际旅行卫生保健中心
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