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A set of primers used to identify stored grain pests of pseudocereals and their applications

A technology of pseudo-grass and red pseudo-grass, which is applied to primers for identifying grain-storing insects of pseudograss and their applications, primers and their application fields, and achieves the effects of short time consumption, high sensitivity and intuitive results.

Active Publication Date: 2017-01-04
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Up to now, there is no application of specific primer PCR technology for the 6 species of common Tribeia cerevisiae in the world, namely Tribeipes red, Tribeips variegata, Tribeipes brown, Tribeipes black, Tribeipes freundii and Tricornis brevicornus. Molecular identification of stored grain pests

Method used

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  • A set of primers used to identify stored grain pests of pseudocereals and their applications
  • A set of primers used to identify stored grain pests of pseudocereals and their applications
  • A set of primers used to identify stored grain pests of pseudocereals and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1, design and synthesis of specific primers for 6 kinds of pseudo-grain beetles stored grain pests

[0061] 1. Extract the thorax (including the leg segments) or individual thoraxes (including leg segments) of each single-headed T. spp. from the red T. spp., the mixed T. ts. Genomic DNA from the entire body of a non-adult worm.

[0062] 2. Use each genomic DNA obtained in step 1 as a template, and use the universal primer pair LCO1490 and HCO2198 as primers to perform PCR amplification to obtain the mtDNA COI gene sequence of each formosanus, and the length of the different amplification products is about 700bp.

[0063] PCR amplification system (total volume is 25 μL): 2×Taq PCR MasterMix (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.) 12.5L, ddH 2 O10.5 μL, 1 μL of template with a concentration of 50 ng / μL, and 0.5 μL each of LCO1490 and HCO2198 with a concentration of 10 μM.

[0064] PCR amplification program: pre-denaturation at 94°C for 3 m...

Embodiment 2

[0071] Example 2. Specific detection of primers specific to the mtDNA COI gene sequence of 6 kinds of pseudo-grain beetles stored grain pests

[0072] 1. Specific detection of Tca33F26 and Tca346R24

[0073] (1) Extracting Erythrina chinensis (involving the following geographical populations: Prague (Czech Republic) population, Rakovník (Czech Republic) population, Osijek (Croatia) population, Bordeaux (France) population, Kansas (United States) population, Guangdong Guangzhou (China) population , Henan Xuchang (China) populations, Guangxi Beihai (China) populations, Xinjiang Aksu (China) populations) total DNA from the thorax of a single forebear adult or the whole body of a single non-adult state, and the hybrids were extracted in the same way Trichobeetias (involving the following geographic populations: Prague (Czech Republic) population, Herink (Czech Republic) population, Kyjov (Czech Republic) population, Bordeaux (France) population, Kansas (United States) population),...

Embodiment 3

[0101] Example 3. Sensitivity detection of specific primers for the mtDNA COI gene sequence of 6 kinds of pseudo-grain beetles stored grain pests

[0102] 1. Sensitivity detection of Tca33F26 and Tca346R24

[0103] (1) Genomic DNA was extracted from the thorax of a single adult or single non-adult state of the thorax of the red Tribetium (Guangzhou (China) population of Guangdong), and the genomic DNA was diluted to 100 ng / μL, 10 ng / μL, 1ng / μL, 0.1ng / μL, 0.01ng / μL and 0.001ng / μL.

[0104] (2) Use the genomic DNA of each dilution obtained in step (1) as a template, and use Tca33F26 and Tca346R24 as primers to perform PCR amplification to obtain each PCR amplification product, and simultaneously use ddH 2 O replaced the template as a negative control.

[0105] The PCR amplification system is as in Example 2.

[0106] The PCR amplification procedure was as in Example 2.

[0107] (3) Each PCR amplification product is carried out to agarose gel electrophoresis, the result is as...

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Abstract

The invention discloses a group of primers for identifying the forebear stored grain pests and the application thereof. The invention discloses a set of primers, which are at least one of the following primer pairs: (1) a primer pair composed of DNA molecules shown in SEQ ID No.3 and SEQ ID No.4; (2) SEQ ID No. .5 and a pair of primers consisting of DNA molecules shown in SEQ ID No.6; (3) a pair of primers consisting of DNA molecules shown in SEQ ID No.7 and SEQ ID No.8. The primers of the present invention are used to detect the method of 6 kinds of common grain beetle class stored-grain pests. Compared with the traditional morphological identification method, the method does not need the basis of morphological taxonomy and has realized the identification of the non-adult state, while compared with other molecular Compared with biological identification methods, this method simplifies the molecular detection steps for the identification of forebears stored grain pests, and has the characteristics of simple operation, short time consumption, high sensitivity and stability, and intuitive results. It laid the foundation for the study of closely related species.

Description

technical field [0001] The invention relates to a set of primers and the application thereof; in particular to a set of primers for identifying the forebear stored grain pests and the application thereof, belonging to the field of biotechnology. Background technique [0002] Tribolium belongs to Coleoptera, Tenebrionidae, and Tribolium. There are more than 30 species of this genus recorded at home and abroad, of which about 10 species are harmful to stored grain. , the six species of T. chinensis, T. spp., T. spp. brown, T. spp. black, T. freundii and S. shorthorns are widely distributed in the world. [0003] The genus Tribolium Macleay1825 is a group of Coleoptera stored-grain pests that are widely distributed, more harmful, and of great economic significance. About 10 of the known species of the forebear are stored-grain pests, which can damage flour, Cereals, corn, beans, oil crops, etc. Except for the 3 species that have been reported in our country (Trypsis spp., P. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 李志红王一椒
Owner CHINA AGRI UNIV
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