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Method to detect dendroctonus valens and detection kit

A detection reagent and target technology, applied in the field of molecular biology, can solve the problems of invasion, misidentification, pathogen infection and pests, etc., and achieve the effect of strong specificity, high sensitivity and high sensitivity

Inactive Publication Date: 2013-06-12
BEIJING FORESTRY UNIVERSITY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] PCR-based technology has been widely used in the identification of pathogenic microorganisms, plant nematodes, and some pests. However, the traditional one-step PCR method may lead to misidentification due to the low quantity of the object to be identified or the low concentration of genomic DNA, which may cause pathogenic infection or pest invasion.

Method used

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  • Method to detect dendroctonus valens and detection kit
  • Method to detect dendroctonus valens and detection kit
  • Method to detect dendroctonus valens and detection kit

Examples

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Embodiment 1

[0039] Example 1 Determination of the DNA sequence of the red beetle COI gene

[0040] The present invention repeatedly compares and screens the COI gene of the red beetle with the nucleotide sequences of other insects published on NCBI, and finds that the homology of the COI gene within the species of the red beetle reaches 99%, while that of other insects is similar to that of other insects. The sequence divergence is large, which can be used to distinguish the red beetle from other species of insects.

Embodiment 2

[0041] The extraction of embodiment 2 insect genome DNA

[0042] The insects involved in this experiment are Scolytus schevyrewiSemenov, Xyleborus artecomans Schedl, Xyleborus interjectus Blandford, Blastophagus minor Hartig , Dendroctonus valens LeConte, provided by Beijing Entry-Exit Inspection and Quarantine Bureau. Insect genomic DNA was extracted using BIOMIGA's EZgene TM Insect gDNA Kit (GD2413-02) kit.

[0043] Specific steps:

[0044] (1) After the insect specimens soaked in pure alcohol were cleaned and dried with an ultrasonic instrument, the heads and elytra of the adults were removed, and the abdominal tissues of the larvae were taken. Tissues <50mg were thoroughly ground in a mortar and placed in a 1.5ml centrifuge tube.

[0045] (2) Add 350ul of Buffer ITL and 25ul of proteinase K (25mg / ml) to the centrifuge tube, mix thoroughly and place in a constant temperature water bath at 60°C for two hours until the tissue is completely digested.

[0046] (3) Add 350...

Embodiment 3

[0055] Embodiment 3 detects the design of the primer of the nest PCR of red fat beetle

[0056] 1. Design of universal primers

[0057] Synthesize a pair of universal primers LCO1490 and primer HCO2198 for amplifying the longhorn COI sequence according to the published insect COI sequence. The primer sequences are as follows:

[0058] LOC1490: 5'-GGTCAACAAATCATAAAGATATTG-3' (SEQ ID NO. 1),

[0059] HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' (SEQ ID NO. 2). The total volume of the PCR amplification reaction is 25 μl, and the amount of each reaction system added is:

[0060]

[0061] After mixing, put it into a PCR machine for amplification. The amplification program is: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, refolding at 53.7°C for 45 seconds, extension at 72°C for 1 minute, 35 cycles, and finally extension at 72°C for 7 minutes. A blank control was made with sterile distilled water for each reaction to eliminate systematic errors. Mi...

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Abstract

The invention provides a method to detect dendroctonus valens and a detection kit. The detection method is a nested polymerase chain reaction (PCR). Nucleotide sequences of a first pair of used primers are shown in SEQ ID NO.1-2, and nucleotide sequences of a second pair of primers are shown in SEQ ID NO.3-4. The invention further provides a kit for detecting the dendroctonus valens. The method to detect the dendroctonus valens and the detection kit have the advantages of being precise in detection, strong in specificity, simple, convenient and quick, can discriminate the dendroctonus valens from four kinds of similar bark beetles including scolytus schevyrewi semenov, round cavity timber beetles, xyleborus interjectus blandfor and adit cutting beetles, and is good in dendroctonus valens detection capacity. The detection sensitivity is 40pg.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a target gene for detecting the red beetle and a primer for detecting the gene, and also relates to a Nest PCR method and a kit for detecting the red beetle by using the primer. Background technique [0002] The red beetle is a borer pest native to coniferous forests in North America. Although it is widely distributed locally, it does not cause serious damage to plants, so it is considered a secondary pest. However, in recent years, it has become a major foreign invasive forestry pest that has invaded our country. After entering my country with the timber trade in the last century, it was first discovered in Shanxi in 1998, and then spread rapidly, and has now expanded to Henan, Hebei, Shaanxi and other provinces. In 2000, the occurrence area in Shanxi Province reached 250,000 hm2, of which 129,000 hm2 caused disasters, involving 54 counties (districts) in 8 prefectures and citi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 石娟陈芳骆有庆李建光赵汗青宗世祥
Owner BEIJING FORESTRY UNIVERSITY
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