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Primer pair for identifying gall midge melansen and identification method thereof

A Hessian gall mosquito and primer pair technology, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. body damage, etc.

Pending Publication Date: 2022-07-29
NORTHWEST A & F UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The morphological identification method has high requirements on the integrity of the specimen and the professional level of the appraiser, and the main insect states intercepted in the entry transmission are larvae and pupae. The morphological identification method is far from meeting the rapid and accurate requirements of port quarantine. ; In domestic epidemic monitoring, non-target species will be trapped when sexual trapping technology is used, and the captured insects are often damaged, which is not conducive to morphological recognition

Method used

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  • Primer pair for identifying gall midge melansen and identification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] 1. Collection of insect samples

[0018] Collected in Xinjiang wheat fields in 2015, after accurate morphological identification by experts, they were artificially raised in Yining Customs Laboratory.

[0019] 2. DNA extraction

[0020] Extraction using DNA kit

[0021] (1) Collect the sample and place the sample in a sterile 1.5mL centrifuge tube; after freezing in liquid nitrogen, add small steel beads, fully grind with a tissue grinder, and then add 200 μL of GA, and shake until completely suspended;

[0022] (2) Add 20 μL Proteinase K solution, mix well, add it in a water bath at 65°C for 1 hour, invert several times during this period to mix thoroughly;

[0023] (3) Add 200 μL of buffer GB, invert and mix well, place at 70°C for 10 minutes, the solution should become clear, and centrifuge briefly to remove water droplets on the inner wall of the tube lid;

[0024] (4) Add 200 μL of absolute ethanol, fully shake and mix for 15s, and centrifuge briefly;

[0025] ...

Embodiment 2

[0034] The primer pair for identifying Hessian gall mosquito in this example includes an upstream primer HFSP-1 and a downstream primer HFSP-2; the nucleotide sequence of the upstream primer HFSP-1 is shown in SEQ ID NO:1 , the nucleotide sequence of the upstream primer HFSP-2 is shown in SEQ ID NO:2.

[0035] The present embodiment also provides the above-mentioned primer pair identification method for the identification of Hessian gall mosquito, and the method is:

[0036] The DNA of the sample to be tested obtained in Example 1 was amplified by PCR with the upstream primer HFSP-1 and the downstream primer HFSP-2. If a DNA band of 170 bp appeared, the sample to be tested was Hessian gall mosquito;

[0037] The reaction system of the PCR amplification is: 2×PCR Master mix 10 μL, 10 mM upstream primer HFSP-11 μL, 10 mM downstream primer HFSP-21 μL, 1 μL of the DNA of the sample to be tested, ddH 2 0 to make up to 20 μL.

[0038] The PCR amplification reaction procedure was a...

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Abstract

The invention provides a primer pair for identifying gall midge melansen. The primer pair comprises an upstream primer HFSP-1 and a downstream primer HFSP-2, the nucleotide sequence of the upstream primer HFSP-1 is as shown in SEQ ID NO: 1, and the nucleotide sequence of the upstream primer HFSP-2 is as shown in SEQ ID NO: 2. The invention further provides an identification method, the upstream primer HFSP-1 and the downstream primer HFSP-2 are used for carrying out PCR amplification on DNA of a to-be-detected sample, and if a 170bp DNA strip appears, the to-be-detected sample is gall midge melansen. The invention provides a simple and stable molecular marker for identification of gall midge melansen, and solves the technical problem of rapid and accurate identification of gall midge melansen.

Description

technical field [0001] The invention belongs to the technical field of identification of Hessian gall mosquitoes, in particular to a primer pair for identifying Hessian gall mosquitoes and an identification method thereof. Background technique [0002] Mayetiola destructor (Say) belongs to Diptera, Cecidomyiidae, Cecidomyiinae, Mayetiola, and is an important pest of wheat worldwide. my country's entry phytosanitary pests. [0003] Hessian gall mosquitoes can have 1-6 generations a year in various places, and have a facultative diapause habit. The third instar larvae overwintered in the surrounding pupae on the winter wheat plants or the residual wheat stubble. In the spring of the following year, the overwintering larvae develop and pupate. Adults do not feed and live about 3 days. Eggs are mostly produced in the frontal grooves of the host leaves and hatch after 3-10 days. Newly hatched larvae crawl along the vein furrow into the leaf sheath to feed. The feeding of a s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686
Inventor 张皓马琦胡想顺黄卫利陈雨馨
Owner NORTHWEST A & F UNIV
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