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Molecular biological method for identifying diptera insects

A molecular biology and dipteran technology, applied in the field of molecular biology, to achieve the effect of low equipment requirements, cost reduction, and filling gaps in the industry

Inactive Publication Date: 2014-10-29
SICHUAN AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the use of DNA barcodes as molecular markers to identify B. dorsalis, Drosophila and blowfly at home and abroad.

Method used

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  • Molecular biological method for identifying diptera insects
  • Molecular biological method for identifying diptera insects
  • Molecular biological method for identifying diptera insects

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Embodiment 1

[0026] 1. The DNA extraction kit (Shanghai Sangon Bioengineering Co., Ltd.) was used to extract the total DNA of the Diptera insects Bactrocera dorsalis, Drosophila and Calliphora. For the identification of larvae, DNA extractions were all assayed from individual larvae of several Diptera.

[0027] 2. Using DNA barcode universal primers to PCR amplify the front sequence of the mitochondrial COI gene and determine the sequence, specifically including the following steps:

[0028] ①The primers used for PCR amplification and sequencing are:

[0029]

[0030] ②The PCR reaction system is 50ul, including about 25ng template DNA, 25ul2×Taq PCRMasterMix, 2pM forward and reverse primers, and deionized water (ddH 2 O) adjusted to a final volume of 50ul; the PCR reaction was completed on a RoboCycler Gradient40 (Stratagene) thermal cycler.

[0031] ③PCR reaction conditions are as follows: pre-denaturation at 95°C for 2 minutes, denaturation at 94°C for 45 seconds, renaturation at 50...

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Abstract

The invention belongs to the field of molecular biology and particularly discloses a molecular biological method for identifying diptera insects. The method comprises the steps: extracting total DNAs (Deoxyribonucleic Acids); amplifying forepart sequences of mitochondrion COI (Cytochrome C oxidase I) genes of the diptera insects by using DNA barcode universal primers, and taking the amplified forepart sequences as marks; carrying out sequence characteristic comparison for species identification, thereby carrying out species identification. The method disclosed by the invention can be used for identifying different biotypes and larvae or incomplete individuals of three kinds of pests, namely bactrocera dorsalis hendel, fruit fly and calliphoridae, and has the characteristics of accuracy, quickness, convenience and low cost; compared with the traditional morphological characterization methods, the time is saved and the cost is reduced; the method disclosed by the invention has lower requirements for instrument equipment used, identification can be carried out in general molecular biology laboratories and is more accurate and reliable than the traditional morphological identification, the current difficult problem that accurate identification is relatively difficult to carry out during the quarantine of bactrocera dorsalis hendel larvae, fruit fly larvae and calliphoridae larvae is fundamentally solved, and the gap in the industry is filled up, so that the method has important significance.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a molecular biology method for identifying dipteran insects. Background technique [0002] Bactrocera dorsalis Hendel is a world-threatening quarantine pest. It has a wide range of hosts and can eat more than 250 kinds of fruits in 46 families such as bananas, citrus, carambola, guava, mango, eggplant, and pepper. and vegetables. This species is mainly harmed by larvae. Adults lay eggs under the fruit skin. After hatching, the larvae develop by feeding on the pulp. Lost food value. Because the insect has a wide range of hosts, high fecundity, strong adaptability, and great harm, it has caused serious economic losses to the fruit and vegetable industry and the flower industry. Many countries have listed it as a key quarantine object. Flies are classified as Class II quarantine pests. [0003] The English common name of fruit fly is fruit fly or vinegar fly, which widely exists...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q2600/156
Inventor 李地艳杨明耀吴楠徐怀亮胡耀东姚永芳
Owner SICHUAN AGRI UNIV
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