Method for amplifying ITS break sequence in Bactrocera dorsalis rDNA by nesting PCR

A kind of orange fruit fly, nested technology, applied in the field of biological DNA

Inactive Publication Date: 2010-09-08
SHANGHAI ACAD OF AGRI SCI
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Problems solved by technology

There are few research reports on species identification and population differentiation of Bactrocera dorsalis using molecular biology techniques in my country

Method used

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  • Method for amplifying ITS break sequence in Bactrocera dorsalis rDNA by nesting PCR
  • Method for amplifying ITS break sequence in Bactrocera dorsalis rDNA by nesting PCR
  • Method for amplifying ITS break sequence in Bactrocera dorsalis rDNA by nesting PCR

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Experimental program
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Embodiment Construction

[0067] Production of a set of nested PCR amplified ITS interrupted sequences in Bacteralis dorsalis rDNA:

[0068] 1. Test materials and methods:

[0069] 1.1 Test samples and source of insects

[0070] Cut off the outer skin of citrus with a diameter of 3-4 cm and a thickness of 2 mm, put them in an incubator, take one female and one male Bactrocera dorsalis bred to adults in the laboratory, and put them in the incubator until the females lay eggs. Extract total genomic DNA from citrus.

[0071] 1.2 Genomic DNA extraction (CTAB method)

[0072] The peeled part of the citrus was cut into squares with a length and width of 0.5 cm and a thickness of 0.2 cm for genomic DNA extraction. DNA extraction and quality detection methods refer to the molecular cloning manual. DNA samples were stored in a -20°C refrigerator. The details are as follows: freeze the plant material with liquid nitrogen (about 0.05g per eppendorf tube), grind it with a grinding stick (quickly), then add 50...

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Abstract

The invention discloses the amplification of ITS break sequence in Bactrocera dorsalis rDNA by nesting PCR, comprising the following steps: amplifying the specific ITS fragment of the Bactrocera dorsalis rDNA by designing a nesting specific primer; using the specific ITS fragment of the amplified Bactrocera dorsalis rDNA to accurately and quickly identify actrocera dorsalis flyblow in fruit, and determining the difference of the amplification Bactrocera dorsalis flyblow and relative species by sequences. The method has reliable result, can quickly detect the existence of Bactrocera dorsalis flyblow in fruit and determines the species evolutionary relationship through sequence analysis.

Description

technical field [0001] The invention belongs to biological DNA technology, in particular to a method for nested PCR amplification of ITS interrupted sequence in Bactrocera dorsalis rDNA. Background technique [0002] Bactrocera dorsalis (Hendel) is a quarantine pest that directly endangers the production of fruits and vegetables and has an important impact on export trade. Dangerous fruit and vegetable pests are one of the main targets of plant quarantine in my country. At present, the port quarantine and identification of Bactrocera dorsalis is mainly based on the external morphological characteristics of the adults, and the larvae or eggs are often intercepted at the port. The usual method is to rear them indoors to obtain adults before identification. [0003] With the rapid development of molecular biology, polymerase chain reaction (PCR) technology, nucleic acid sequence analysis technology and other molecular technologies have been widely used in taxonomy, systematics,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 孔秋莲戚文元叶军岳玲陈志军
Owner SHANGHAI ACAD OF AGRI SCI
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