Specific primers for amplifying aedes albopictus Cytb gene and method for conducting sequencing by means of same

Aedes albopictus, specific technology, applied in the field of molecular biology, can solve the problem of amplifying the Aedes albopictus Cytb gene

Pending Publication Date: 2019-02-12
烟台国际旅行卫生保健中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are general primers published for the mitochondrial genome of dipteran insects, but they...

Method used

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  • Specific primers for amplifying aedes albopictus Cytb gene and method for conducting sequencing by means of same
  • Specific primers for amplifying aedes albopictus Cytb gene and method for conducting sequencing by means of same
  • Specific primers for amplifying aedes albopictus Cytb gene and method for conducting sequencing by means of same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Obtaining and sequence determination of target fragment

[0035] (1) Primer design

[0036] First, search the Aedes albopictus (Aedes albopictus) Cytb gene sequence in the NCBI database, and select the NCBI reference sequence numbered NC_006817. Use the primer premier software to design primers to avoid the occurrence of primer dimers, hairpin structures or mismatches, and select the primer with the highest score and the recommended annealing temperature. The primers were entered into primer3plus and NCBI databases for evaluation, and then PCR tests were performed. Finally, the primers CytbAEDF: 5'-ATTTACCTTTTATTGTATTA-3', as shown in SEQ.ID.NO.1; CytbAEDR: 5'-CCAATTCATGTTAGAAGA-3', as shown in SEQ.ID.NO.2 were selected.

[0037] (2) Extract template DNA from the sample to be tested

[0038] According to the instructions of Qiagen's DNeasy Blood & Tissue kit, the template DNA of the sample to be tested was extracted.

[0039] Table 1 Test sample template D...

Embodiment 2

[0049] Example 2 Verification of Primer Sensitivity and Specificity

[0050] (1) Sensitivity test

[0051] In the test stage, Aedes albopictus was used for sensitivity and repeatability tests. Aedes albopictus was selected for sensitivity test, and 10 -2 That is, 0.19ng / μl DNA template has a clear band around 500bp, as shown in the attached figure 2 shown.

[0052] (2) Specificity test

[0053] Select Aedes albopictus, Aedes aegypti, Aedes dorsalis, Culex palustris, Culex tritaeniorhynchus, Anopheles sinensis, Chrysocephalus chinensis, Transcendina chinensis, Hrophila oblique, human flea, etc. 2 The specificity test was carried out on 10 species of 4 families, 7 genera, and it was found that the primer can successfully amplify the Cytb gene of Aedes albopictus, with a clear band at about 500 bp, but it cannot amplify the Cytb gene of other test samples, and the specificity is ideal. as attached image 3 shown.

[0054] (3) Stability and repeatability test

[0055] Thr...

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Abstract

The invention relates to specific primers for amplifying an aedes albopictus Cytb gene and a method for conducting sequencing by means of the same. For primer sequences, the sequence of a forward primer CytbAEDF is 5'-ATTTTACCTTTTATTGTATTA-3', the sequence of a backward primer CytbAEDR is 5'-CCAATTCATGTTAGAAGA-3', and the method comprises the steps that 1, to-be-determined sample template DNA is extracted; 2, PCR amplification is conducted on the to-be-determined sample template DNA extracted in the step 1; 3, electrophoresis determination is conducted on a PCR amplification product in the step 2; 4, DNA sequence determination is conducted on the PCR product determined in the step 3. By using the specific primers of the aedes albopictus Cytb gene and adopting the corresponding sequencing method, a target gene sequence is amplified fast and accurately, and a reference is provided for fast detecting and authenticating harmful organisms.

Description

technical field [0001] The invention relates to the molecular identification of the mosquito vector insect Aedes albopictus (Aedes albopictus), belonging to the field of molecular biology. 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 137 billion U.S. dollars per year, India 117 billion U.S. dollars, Brazil 50 billion U.S. dollars, and South Africa 7 billion U.S. dollars. [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 organisms can be inte...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/12C12Q1/6869
CPCC12Q1/6869C07K14/43563C12Q2531/113C12Q2565/125
Inventor 王颖张晓龙李晓玉段效辉李磊李金庆方绍庆
Owner 烟台国际旅行卫生保健中心
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