Aphid aphid specific ss-coi primer, kit containing the primer and detection method thereof

The invention relates to a technology of aphid and a detection method, which are applied in the field of molecular biology and can solve the problems of inability to accurately assess the pest control effect of natural enemies aphids, and achieve the effects of high sensitivity, high accuracy and simple operation.

Inactive Publication Date: 2015-08-05
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional research methods on the predation of natural enemies cannot accurately evaluate the control effect of natural enemies on each aphid.

Method used

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  • Aphid aphid specific ss-coi primer, kit containing the primer and detection method thereof
  • Aphid aphid specific ss-coi primer, kit containing the primer and detection method thereof
  • Aphid aphid specific ss-coi primer, kit containing the primer and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Amplification Effect of Primers LFSgF / LFSgR on Digemia wheat aphid

[0022] 1) Preparation of the D. wheat aphid genome

[0023] Put the single-headed aphid into a 1.5mL centrifuge tube and grind it, and use the TIANamp Genomic DNA Kit Blood / Cell / Tissue Genomic DNA Extraction Kit (Spin Column Type) (Tiangen Biological Co., Ltd., Beijing) to extract the single-headed aphid genome . The DNA solution was stored at -20°C for later use, and 1 μL of the solution was taken as a DNA template during PCR amplification.

[0024] (2) Synthesis of specific SS-COI primers for the detection of D. wheat aphids

[0025] The specific SS-COI primer sequence is as follows:

[0026] LFSgF: 5'-CATTATCTAATAATATTGCTCATAAC-3' (SEQ ID No. 1)

[0027] LFSgR: 5'-TGATAAAATTAATAAAATAGCTGTG-3' (SEQ ID No. 2)

[0028] 2) PCR amplification

[0029] The reaction system is 20 μL, including: 10×EasyTaq buffer 2.0 μL, dNTP (2.5 mM) 0.4 μL, EasyTaq DNA polymerase (5U / μL) 0.2 μL, forward and ...

Embodiment 2

[0039] Example 2 Determination of the minimum detectable amount of the primer LFSgF / LFSgR on the aphid

[0040] 1) Preparation of the template genome of the two-handed aphid

[0041] 1) Preparation of the template DNA of the two-handed aphid

[0042] According to Example 1, the genomic DNA of the single-headed aphid was extracted, and then the original template solution was diluted with a 10-fold gradient until no bands were detected, and 1 μL was taken as a template for PCR amplification, which was directly added to the PCR reaction system. The system is the same as that described in Example 1.

[0043] Using primers LFSgF / LFSgR to determine the minimum detection amount, PCR amplification was carried out with different dilutions of D. wheat aphid genomic DNA as a template, and the detection limit of DNA concentration was 0.341 ng / μL ( figure 2 ).

[0044] 2) Preparation of Plasmids of D. aphids

[0045] According to the implementation example 1, the genomic DNA of the ap...

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Abstract

The invention relates to a schizaphis graminum specific SS-COI (Species Specific-Cytochrome Oxidase Subunit I) primer and a kit comprising the primer. According to the special mtDNA sequence of the schizaphis graminum, a pair of schizaphis graminum specific SS-COI primers LFSgF and LFSgR (SEQ ID No. 1 and 2) are designed; the pair of primers have specific amplification capability only on the schizaphis graminum; the size of the amplification product is 203 bp; the detection limit of the plasmid concentration is 9.62E+03; the detection limit of the DNA concentration is 0.341 ng / micro L. According to the invention, an SS-COIPCR (Polymerase Chain Reaction) technology is adopted, and consequently, the accuracy of detection is improved and the detection time is saved; the SS-COIPCR technology is suitable for popularization and application in the grassroots level.

Description

technical field [0001] The invention relates to the field of molecular biology, and in particular relates to a two-handed aphid specific SS-COI primer, a kit containing the primer and a detection method thereof. Background technique [0002] The wheat aphid Schizaphis graminum (Rondani) belongs to Hemiptera Aphididae and is distributed all over the country. Its host plants include wheat, barley, oats, sorghum, rice, foxtail, sedge and other gramineous plants. The two-pronged aphid often sucks juice on the front and back sides of wheat leaves or inside and outside the basal leaf sheath, causing wheat seedlings to turn yellow or fall to the ground and fail to joint. In serious cases, wheat plants cannot be eared normally, directly affecting yield, and can also spread wheat yellow dwarf disease. [0003] There are many kinds of predatory natural enemies (such as ladybugs, lacewings, spiders, etc.) in the field. The protection and utilization of natural enemies are important me...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 王倩杨帆陆宴辉杨益众
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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