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Molecular reaction system for revealing genetic diversity of tephritidae populations

A technology of genetic diversity and molecular response, applied in the field of molecular response system for revealing the genetic diversity of fruit fly populations, can solve the problems of long quarantine period, unfavorable detection and control of quarantine pests, etc., and achieves a promising application prospect. Effect

Inactive Publication Date: 2013-03-06
INST OF PLANT PROTECTION FAAS
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Problems solved by technology

At present, in the port quarantine and identification of fruit flies, the external morphological characteristics of the adults are mainly used as the basis for classification. If the intercepted larvae or eggs, they need to be reared indoors, and then identified after the adults are obtained, resulting in quarantine. The cycle is long, which is not conducive to the discovery and control of quarantine pests

Method used

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  • Molecular reaction system for revealing genetic diversity of tephritidae populations
  • Molecular reaction system for revealing genetic diversity of tephritidae populations
  • Molecular reaction system for revealing genetic diversity of tephritidae populations

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preparation example Construction

[0026] Wherein the preparation of template DNA is as follows:

[0027] Take 5 fruit fly adults from each population and add 800μl extraction buffer (0.1mol / L Tris-HCl, 0.1mol / L NaCl, 0.2mol / L sucrose, 0.05mol / L EDTA, 0.5% SDS), in an ice bath After fully grinding, transfer to 1.5 ml Eppendorf tube, add 0.5mg / ml proteinase K, mix well, put in water bath at 65°C for 1h, add 100μl of 8mol / L KAc, make it fully mix, then ice bath for 1h, 12000r / min at 4°C Centrifuge for 20min, take the supernatant and add 800μl pre-cooled absolute ethanol, mix well, put in ice bath for 2h, centrifuge at 12000r / min for 20min, take the precipitate and wash it once with 70% ethanol, centrifuge at 5000r / min for 5min, and precipitate at 37℃ Dry for 4-6 minutes, finally add 10 μl RNase and 40 μl TE to dissolve, put in water bath at 37°C for 1 hour, and store at 4°C for later use.

Embodiment 1

[0029] The fruit flies used in the experiment were mainly collected from the population trapped in the vegetable base in Zhangzhou, Fujian Province. Reagents Buffer (10×PCR Buffer), dNTP, DNAMarker DL 2000, Taq DNA polymerase, etc. required for the experiment were purchased from Shanghai Bioengineering Co., Ltd.; random primers were synthesized by Shanghai Bioengineering Technology Service Co., Ltd.; Tris, HCl, EDTA, NaCl, Kac, ethanol, etc. are domestic analytical reagents. The specific content is as follows:

[0030] 1. Template DNA preparation and validity verification

[0031] The template DNA is extracted by using the improved DNA extraction method of the present invention, and then the concentration and purity of the fruit fly DNA are measured by an ultraviolet spectrophotometer and gel electrophoresis to verify the validity of the template DNA.

[0032] (1) Ultraviolet spectrophotometer detection

[0033] Use WFZ800-D3B UV-Vis spectrophotometer to detect the concentr...

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Abstract

The invention provides a molecular reaction system for revealing genetic diversity of tephritidae populations. The molecular reaction system comprises 50ng of template DNA, 200mumol / L of dNTP, 2.5mul of 10*PCR Buffer, 0.25mumol / L of primer and 0.5U of Taq enzyme, and finally, ddH2O is added till the total volume is 25mul. An inter-simple sequence repeat (ISSR) amplification reaction program comprises the following steps of: pre-denaturing for 5 minutes at the temperature of 94 DEG C; denaturing for 30 seconds at the temperature of 94 DEG C; annealing for 45 seconds at the temperature of 52 DEG C, and stretching for 90 seconds at the temperature of 72 DEG C; performing 36 cycles; and finally, stretching for 10 minutes at the temperature of 72 DEG C. The constructed molecular reaction system for revealing the genetic diversity of the tephritidae populations can reveal the genetic diversity of the populations, discriminate the affiliation of the interregional tephritidae populations, quickly, accurately and sensitively detect the category of the tephritidae, and overcome the defects of the traditional method.

Description

Background technique [0001] Tephritidae (Tephritidae) is the largest group in Diptera (Diptera). More than 4,500 species of fruit flies have been described in the world, and more than 70 species are considered to be important agricultural pests. threaten. At present, in the port quarantine and identification of fruit flies, the external morphological characteristics of the adults are mainly used as the basis for classification. If the intercepted larvae or eggs, they need to be reared indoors, and then identified after the adults are obtained, resulting in quarantine. The cycle is long, which is not conducive to the discovery and control of quarantine pests. Contents of the invention [0002] The purpose of the present invention is to provide a molecular response system for revealing the genetic diversity of fruit fly populations. [0003] In order to achieve the above object, the technical scheme that the present invention takes is as follows: [0004] The molecular reac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 余德亿姚锦爱胡建峰陈峰
Owner INST OF PLANT PROTECTION FAAS
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