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Kit for amplifying mitochondrial genome of pyralidae insects and application thereof

A technology for amplifying the mitochondrial genome of the moth, which is applied in the biological field, can solve the problems of lack of specificity, high template purity requirements, low amplification efficiency, etc., and achieves the effects of high fidelity, shortened acquisition cycle and strong specificity.

Inactive Publication Date: 2018-11-20
杭州贝英福生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method uses a large number of primers, low amplification efficiency, high requirements for template purity, long time-consuming, and lack of sufficient specificity, which will hinder the rapid accumulation of molecular data of insect mitochondrial genome Phylogenetic study of moths poses difficulties
However, in the prior art, there is no research and report on the general primers for the amplification of long fragments of mitochondrial genomes of insects of Mothidae.

Method used

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  • Kit for amplifying mitochondrial genome of pyralidae insects and application thereof
  • Kit for amplifying mitochondrial genome of pyralidae insects and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: the design of primer

[0017] 1. Based on the mitochondrial genome sequences of different Mothidae insects measured in Genbank, the data comes from the National Center for Biotechnology Information (NCBI, http: / www.ncbi.nlm.gov / );

[0018] 2. Use the ClustalX software to compare the complete mitochondrial genome sequences of different Mothidae insects, and find out the sequence regions that are relatively conservative and have the least base variation; species) as shown in Table 1.

[0019] Table 1 The present invention adopts mitochondrial genome complete sequence data source (part)

[0020] Classification status

[0021] 3. According to the above comparison results, use the primer design software Primer Premier 5 to design 2 pairs of primers in the sequence region with the smallest base variation, F1 (upstream primer): 5'-CGTATAAWGTATGGKTATGCCTGATTTAGCGG-3' (SEQ ID NO: 1 ), R1 (downstream primer): 5'-TGAAYCGTTGCGCTAGCTAGGGTACCTGA-3' (SEQ ID N...

Embodiment 2

[0022] Example 2 Extraction and Detection of Insect Total Genomic DNA

[0023] 1. Material treatment: take the dead adults of brown nest borer, rice stem borer, rice leaf roller, orange black stem borer, and rice stem borer respectively and wash them with distilled water, divide them into 3 parts according to their head, chest, and feet , placed in centrifuge tubes.

[0024] 2. Preparation of lysate: Prepare a sufficient amount of lysate according to 400 μl of each centrifuge tube (add 20 μl of proteinase K).

[0025] 3. Sample grinding: add 200 μl of lysate to the above three centrifuge tubes respectively, grind the sample with scissors and grinding rods until clarified, and then add the remaining lysate into the centrifuge tubes in equal amounts.

[0026] 4. Sample digestion: put the above-mentioned centrifuge tube into a water bath that has been preheated to 55°C, and take a water bath for 3 hours. Shake gently once every 15 minutes.

[0027] 5. Extraction: phenol extrac...

Embodiment 3

[0032] Example 3 Amplification of long fragments of mitochondrial genomes for different Borididae insects

[0033] 1. the genomic DNA of the brown nest borer, rice stem borer, rice leaf roller, orange black stem borer, and rice stem borer extracted in embodiment 2 is carried out with long fragment amplification using the universal primer of the present invention;

[0034] The PCR amplification system is 25 μl, including 5 μl Mg 2+ 10×buffer, 2.5 μl of 2.5mM dNTP, 3 μl of 10 μmol upstream primer, 3 μl of 10 μmol downstream primer, 0.2 μl of 5 U / μl rTaq enzyme, 1 μl of DNA template, and the rest in sterile distilled water ;

[0035] Among them, the amplification program was: 93°C pre-denaturation for 1 min, 92°C denaturation for 8 s, 45°C-55°C annealing for 90 s, 72°C extension for 1 min, a total of 35 cycles, 72°C extension for 6 min, and 4°C storage.

[0036] 2. Use 1% agarose gel electrophoresis to detect the PCR products, observe the results with the UVP ultraviolet gel im...

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Abstract

The invention provides a kit for amplifying mitochondrial genome of pyralidae insects. The kit comprises two pairs of primers, with the sequences being as shown in SEQ ID NO: 1-4, and application of auniversal primer to sequencing of the mitochondrial genome of pyralidae. The universal primer can accurately and rapidly amplify long fragments of mitochondrial genome of various pyralidae insects, and is beneficial to the research on phylogeny and population genetic structures of pyralidae.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for amplifying the mitochondrial genome of the Mothidae insects and an application thereof. Background technique [0002] Boreridae, including Ceratoideae and Boreridae, and Boreridae insects are commonly known as Borer moths. At present, there are at least 15,576 species of borer moths that have been described in the world, and more than 2,000 species have been recorded in my country. Most of the moth superfamily insects are agricultural, forestry and storage pests, especially borer moths, common ones are Chilo suppressalis, Scirpophagaincertulas, and Cnaphalocrocis medinalis , Bean pod borer (Maruca vitrata), etc., which have a wide range of distribution and rich species, and large-scale insect damage has caused serious economic losses to agriculture and forestry. Therefore, the control of borer moth insects has very important economic significance. [0003] In ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2531/113
Inventor 李飞赵蕊
Owner 杭州贝英福生物科技有限公司
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