Method for identifying Frankliniella occidentalis species by utilizing PCR (Polymerase Chain Reaction)-RFLP (Restriction Fragment Length Polymorphism) technology
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A technology of PCR-RFLP and western flower thrips, applied in the field of agricultural biology
Inactive Publication Date: 2012-03-28
INST OF PLANT PROTECTION SHANDONG ACAD OF AGRI SCI
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But utilize PCR-RFLP technology to construct the method for distinguishing two strains of Western flower thrips as yet not yet reported
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Embodiment 1
[0033] Analysis of the sample material of Thrips occidentalis
[0034] (1) Extraction of Western Flower Thrips Genomic DNA
[0035] Place a single thrips individual in a 0.2ml centrifuge tube containing 60 μl of alkaline lysate: 50 mmol L -1 Tris-HCl (pH8.0), 20mmol·L -1 NaCl, 1mmol L -1 EDTA and 1% SDS were thoroughly ground and homogenized with a sealed pipette tip, placed in a water bath at 65°C for 15 minutes, and then at 95°C for 10 minutes to prepare a genomic DNA solution of Thrips occidentalis.
[0036] (2) PCR amplification of the COI gene of Thrips occidentalis
[0037] The COI gene was amplified by PCR using the genomic DNA of Thrips occidentalis as a template to obtain the PCR product;
[0040] 10mM dNTP: 1 μl; ddH 2 O supplemented to 50 μl;
[0041] Amplification primer sequences are as follows:
[0042]Sense primer: 5'GGAT...
Embodiment 2
[0048] A kind of method utilizing PCR-RFLP technology to distinguish western flower thrips strain, the steps are as follows:
[0049] (1) Extraction of Western Flower Thrips Genomic DNA
[0050] Place a single thrips individual in a 0.2ml centrifuge tube containing 60 μl of alkaline lysate: 50 mmol L -1 Tris-HCl (pH8.0), 20mmol·L -1 NaCl, 1mmol L -1 EDTA and 1% SDS were thoroughly ground and homogenized with a sealed pipette tip, placed in a water bath at 65°C for 15 minutes, and then at 95°C for 10 minutes to prepare a genomic DNA solution of Thrips occidentalis.
[0051] (2) PCR amplification of the COI gene of Thrips occidentalis
[0052] The COI gene was amplified by PCR using the genomic DNA of Thrips occidentalis as a template to obtain the PCR product;
[0053] The PCR amplification system is:
[0054] Genomic DNA 2μl, 20μM primer 0.4μl, 5U / μl Taq enzyme 0.2μl, 10×Taq Buffer 2μl,
[0055] 0.4 μl of 10 mM dNTPs, ddH 2 O supplemented to 20 μl;
[0056] Amplificati...
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Abstract
The invention relates to a method for identifying Frankliniella occidentalis species by utilizing PCR (Polymerase Chain Reaction)-RFLP (Restriction Fragment Length Polymorphism) technology, comprising the following steps: (1) extracting Frankliniella occidentalis genome DNA; (2) conducting PCR amplification to the mitochondrion COI gene of the Frankliniella occidentalis genome DNA by taking the Frankliniella occidentalis genome DNA as a template; (3) conducting enzyme cutting to the PCR product prepared in step (2) by adopting restriction enzyme EarI to obtain an enzyme cutting product; and (4) conducting agar gel electrophoresis analysis on the enzyme cutting production prepared in step (3). The restriction enzyme is the common restriction enzyme, therefore, the method provides a simple, convenient and stable enzyme cutting marker for screening two types of Frankliniella occidentalis, and simultaneously explores and establishes an identification technology for the two types of Frankliniella occidentalis, and lays a foundation for identification on the population dynamics of the two types of Frankliniella occidentalis and research on biology and invasion mechanisms.
Description
technical field [0001] The invention relates to a method for identifying thrips occidentalis strains by using PCR-RFLP technology, and belongs to the field of agricultural biotechnology. Background technique [0002] Western flower thrips (Frankliniella occidentalis), which belongs to Thysanoptera (Thysanoptera) Thripidae (Thripidae) flower thrips (Frankliniella), originally originated in the western United States, and began to spread rapidly to all continents of the world in the 1980s. It has been reported in 69 countries and regions, and it is one of the most serious worldwide pests to crops (Kirk, 2001; Kirk and Terry, 2003). The pest not only feeds on the juice of the host plant, forms scars on the surface of the fruit, and reduces the quality of the fruit, but also can spread tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV) in a persistent manner (Kritzman A, 2002 ). Moreover, the economic loss caused by the spreading virus is far greater than...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 于毅褚栋段惠生张安盛国栋
Owner INST OF PLANT PROTECTION SHANDONG ACAD OF AGRI SCI
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