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Specific primer for amplifying Cytb gene of culicidae insects and method of using specific primer for sequencing

A technology of specificity and primer sequence, which is applied in the field of genetic engineering and molecular biology, can solve the problems of unseen and amplified Cytb gene of mosquitoes, and achieve good amplification performance

Pending Publication Date: 2019-05-10
烟台国际旅行卫生保健中心 +1
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Problems solved by technology

There are general primers for the mitochondrial genome of Diptera insects published, but they cannot specifically amplify the Cytb gene of mosquitoes, and cannot screen whether they are mosquitoes through amplification
Patent search, no patents with "Cytb" and "mosquito" as keywords

Method used

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  • Specific primer for amplifying Cytb gene of culicidae insects and method of using specific primer for sequencing
  • Specific primer for amplifying Cytb gene of culicidae insects and method of using specific primer for sequencing
  • Specific primer for amplifying Cytb gene of culicidae insects and method of using specific primer for sequencing

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Embodiment 1

[0036] 1. Primer design

[0037] First, search for the Cytb gene sequence of Culicidae in the NCBI database, and try to use the NCBI reference sequence. Use the primer premier software to design primers, try to avoid the occurrence of primer dimers, hairpin structures or mismatches, and select the primer with the highest score and the recommended annealing temperature. Enter the primers into primer3plus (http: www.primer3plus.com / ) and NCBI databases, followed by PCR testing. Finally, primers CytbMOSF:TTTCAGCTYGATGAAATTTTGG and CytbMOSR:TATAAAACWGTTAAAAATTTG were selected.

[0038] 2. Extract template DNA from the sample to be tested

[0039] According to the instructions of Qiagen's DNeasy Blood & Tissue kit, the template DNA of the sample to be tested was extracted.

[0040] Table 1 Test sample template DNA concentration and purity

[0041]

[0042] 3. Perform PCR amplification on the extracted template DNA of the sample to be tested

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Abstract

The invention relates to a specific primer for amplifying a Cytb gene of culicidae insects and a method of using the specific primer for sequencing. The sequence of the primer is CytbMOSF: TTTCAGCTYGATGAAATTTTGG and CytbMOSR: TATAAAACWGTTAAAATTTG. The method comprises the steps of 1, extracting template DNAs of a to-be-detected sample; 2, performing PCR amplification on the extracted template DNAsof the to-be-detected sample in step 1; 3, carrying out electrophoresis determination on a PCR amplification product in step 2; 4, carrying out DNA sequencing on the PCR product determined in step 3.The specific primer of the Cytb gene of the culicidae insects is utilized, and a corresponding sequencing method is adopted, so that a gene sequence is quickly and accurately amplified, and a basis is provided for sequent molecular identification and traceability of culicidae insects serving as vector organisms on the Cytb gene.

Description

technical field [0001] The present invention relates to the fields of genetic engineering and molecular biology, especially the molecular identification and traceability of mosquito vectors in the field of border ports or domestic health and safety. Background technique [0002] With the change of natural conditions and the development of international traffic, trade and tourism, biological invasion (including vector biological invasion) has become a serious international ecological and health security problem. According to the report of the Convention on Biological Diversity, biological invasions have caused major economic losses to countries all over the world. Among them, the United States loses US$137 billion a year; India US$117 billion; Brazil US$50 billion; South Africa US$7 billion. [0003] As a unique species of invasive species, vector organisms have attracted international attention because they may carry human susceptible pathogens. More and more vector organi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/12C12Q1/6869
Inventor 王颖段效辉方绍庆钟响李金庆高娟娟
Owner 烟台国际旅行卫生保健中心
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