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Primer, kit and method for identifying walnut bark beetles

A technology of bark beetle and walnut wood, which is applied in the field of identification of bark beetle in walnut wood, can solve the problems of false negative and false positive results, which are sensitive and difficult, and achieve the effect of strong specificity, high accuracy and simple operation

Inactive Publication Date: 2016-02-03
李凯兵
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is difficult to design primers to amplify only one species for closely related species with highly similar sequences; traditional species-specific PCR is sensitive to false negative and false positive results

Method used

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  • Primer, kit and method for identifying walnut bark beetles
  • Primer, kit and method for identifying walnut bark beetles
  • Primer, kit and method for identifying walnut bark beetles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] 1. Take the standard sample of the walnut bark beetle and the sample of its close relative species Pityophthoruslautus Eichhoff for morphological identification. The specific basis for morphological identification is as follows:

[0062] The body is tiny, the total length of the body is 1.5-1.9mm, about 2.8-3.1 times of the body width, the body color of the female is yellowish brown, and the male is nearly black. There is an incarceration between the 1st and 2nd segments of the mallet of antennae. The frontal surface is slightly raised to the edge of the mouth piece, and the side edge protrudes into a semicircle, and the distance from the eye edge is 2-3 times the diameter of the ommatidium; the female insect has a flat or slightly concave frontal surface, smooth surface, fine and well-proportioned markings, and fine hairs Dense, longer hairs near the edge; males have a broad and concave frontal surface, rough inscriptions, short and sparse hairs, and are less conspicuo...

Embodiment 2

[0105] Change some parameters among the embodiment 1, carry out PCR amplification to the standard product Bark beetle, concrete change is as follows:

[0106] 1. The genome concentration of the standard product S. walnut beetle is 50ng / μl;

[0107] 2. The genome concentration of the standard product S. walnut beetle is 200ng / μl;

[0108] 3. The PCR amplification program is: 94°C for 5min; 94°C for 30s, 49°C for 30s, 72°C for 45s, a total of 40 cycles; 72°C for 5min;

[0109] 4. The PCR amplification program is: 95°C for 3min; 95°C for 28s, 51°C for 35s, 72°C for 50s, a total of 38 cycles; 72°C for 8min;

[0110] The above-mentioned four kinds were changed respectively to the standard substance in Example 1, and the results obtained were all consistent with the results of the standard substance in Example 1.

Embodiment 3

[0112] The 10 samples to be tested were detected by the same method as in Example 1, that is, the morphological detection was carried out first, and molecular biological identification was carried out after confirming that it met the morphological characteristics of the walnut bark beetle.

[0113] In the end, 4 of the 10 samples to be detected were S. walnut, that is, the four pairs of PCR primers all amplified the target bands; 6 were non-H. destination strip.

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Abstract

The invention relates to the field of identifying walnut bark beetles, and in particular relates to a primer, a kit and a method for identifying the walnut bark beetles. The primer for identifying the walnut bark beetles consists of a first primer pair, a second primer pair, a third primer pair and a fourth primer pair; and the base sequences of four pairs of primers are shown by SEQ ID No.1-8. The primers are obtained by virtue of multiple times of verification of standard substances according to systematic biology in combination with known sequences of close species of the walnut bark beetles, are strong in specificity, high in sensitivity and good in repeatability, and can be used for identifying the walnut bark beetles very stably and quickly. The kit for identifying the walnut bark beetles, provided by the invention, is convenient and quick, and the method for identifying the walnut bark beetles takes the first primer pair, the second primer pair, the third primer pair and the fourth primer pair as primers to perform PCR (polymerase chain reaction) amplification on a genome extract of a to-be-detected sample, and then detect PCR amplification products, is convenient and quick and is high in reliability.

Description

technical field [0001] The present invention relates to the field of identification of the walnut bark beetle, in particular to a primer, a kit and a method for identifying the walnut bark beetle. Background technique [0002] At present, the identification of the walnut beetle is mainly based on the following morphological characteristics: [0003] (1) The body is tiny, with a total body length of 1.5-1.9mm, about 2.8-3.1 times the body width, the body color of the female is yellowish brown, and the male is nearly black. There is an incarceration between the 1st and 2nd segments of the mallet of antennae. The frontal surface is slightly raised to the edge of the mouth piece, and the side edge protrudes into a semicircle, and the distance from the eye edge is 2-3 times the diameter of the ommatidium; the female insect has a flat or slightly concave frontal surface, smooth surface, fine and well-proportioned markings, and fine hairs Dense, longer hairs near the edge; males ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 李凯兵张永宏吴志毅李海林刘海军谈珺马骏胡学难
Owner 李凯兵
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