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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Problems solved by technology

However, there is no report on the use of DNA barcodes as molecular mar

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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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[0025] Example 1

[0026] 1. Use DNA extraction kit (Shanghai Shenggong Biological Engineering Co., Ltd.) to extract the total DNA of Diptera insects Bactrocera dorsalis, Drosophila and blowfly. In order to identify the larvae, all DNA extractions were performed on individual larvae of several Diptera insects.

[0027] 2. PCR amplification of mitochondrial COI gene pre-sequence and sequence determination using DNA barcode universal primers include the following steps:

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

[0029]

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

[0031] ③The PCR reaction conditions were 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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