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LAMP (loop-mediated isothermal amplification) primer group for identifying bark beetles and application thereof

A technology of C. japonica and a primer set, applied in the biological field, can solve the problems of unsuitable grass-roots departments for popularization and application, expensive instruments and equipment, and high identification cost, and achieves the effects of low cost, high sensitivity and strong specificity

Pending Publication Date: 2022-07-29
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the existing PCR molecular identification technology can achieve fast and accurate identification, it requires expensive instruments and equipment, and the cost of identification is high, so it is not suitable for popularization and application in grassroots departments

Method used

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  • LAMP (loop-mediated isothermal amplification) primer group for identifying bark beetles and application thereof
  • LAMP (loop-mediated isothermal amplification) primer group for identifying bark beetles and application thereof
  • LAMP (loop-mediated isothermal amplification) primer group for identifying bark beetles and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1. Acquisition of primer sets and kits for identification of C. beetle

[0084] 1. Design of primer set for identification of C. zebra

[0085] 1. Taking the full-length sequence of COI gene in the mitochondrial genome of C. gracilis as the target gene, the primer set TG-LAMP for identification of P. gracilis was designed and synthesized. The primer sequences of the primer sets are shown in Table 2.

[0086] Table 2

[0087]

[0088] 2. Validation of primer sets used for identification of C. beetle

[0089] 1. Use a micro-sample genomic DNA extraction kit to extract the genomic DNA of the beetle sample to obtain the genomic DNA of the beetle sample. For the test beetle samples, in Table 1, the numbered 1, the numbered 2, the numbered 3, the numbered 3, the numbered 4, the numbered 10 flower Spotted Beetle, Hooked Beetle No. 12, and Hundred-Skin Beetle No. 13.

[0090] 2. Using the genomic DNA of the beetle sample as a template, using the primer set TG-LA...

Embodiment 2

[0099] Example 2. Application of the kit prepared in Example 1 in identifying whether the beetle to be tested is or is a candidate for the beetle

[0100] 1. Identify whether the beetle to be tested is or is a candidate for the beetle by the results of agarose gel electrophoresis

[0101] 1. Use a micro-sample genomic DNA extraction kit to extract the genomic DNA of the beetle to be tested to obtain the genomic DNA of the beetle to be tested.

[0102] 2. Using the genomic DNA of the beetle to be tested as a template, using the primer set TG-LAMP to perform loop-mediated isothermal amplification to obtain the amplification product.

[0103] The reaction system was 25 μL, consisting of 12.5 μL WarmStart LAMP 2X Master Mix, 1 μL template, primer mix, and sterile ultrapure water. The primer mixture is the mixture composed of each primer in the primer set TG-LAMP. In the reaction system, the final concentration of outer primer F3 and outer primer B3 were both 0.2 μM, and the fina...

Embodiment 3

[0135] Example 3. Specificity experiment

[0136] The beetle 1 to be tested is the beetle numbered 1.

[0137] The beetle 2 to be tested is the beetle numbered 2.

[0138] The beetle 3 to be tested is the beetle numbered 3.

[0139] The beetle 4 to be tested is the beetle number 4.

[0140] The beetle 5 to be tested is the beetle number 5.

[0141] The test beetle 6 is the variegated beetle numbered 6.

[0142] The test beetle 7 is the variegated beetle numbered 7.

[0143] Beetle 8 to be tested is the beetle number 8.

[0144] The test beetle 9 is the variegated beetle numbered 9.

[0145] Beetle 10 to be tested is the beetle number 10.

[0146] Beetle 11 to be tested is the beetle No. 11.

[0147] Beetle 12 to be tested is the beetle number 12.

[0148] Beetle 13 to be tested is a hundred strange beetles numbered 13.

[0149]The test beetle 14 is the pseudo-white-bellied beetle numbered 14.

[0150] Perform the following steps for each beetle to be tested:

[015...

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) primer group for identifying bark beetles and application of the LAMP primer group. The LAMP primer group is composed of a prime-F3, a prime-B3, a primer-FIP and a primer-BIP, and nucleotide sequences of the LAMP primer group are sequentially shown as a sequence 1 to a sequence 4 in a sequence table. The LAMP primer group provided by the invention can be used for identifying whether the to-be-detected bark beetles are or are candidate bark beetles, and can also be used for detecting whether a to-be-detected sample contains or is candidate to contain the bark beetles, and distinguishing the bark beetles from other bark beetles. Experiments prove that when the LAMP primer group is used for identifying the bark beetles, high specificity and high sensitivity are achieved, and simple, convenient, rapid and accurate detection can be achieved. The method has great application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a LAMP primer set used for identifying C. beetle and its application. Background technique [0002] Beetles belong to the order Coleoptera, family Dermestidae, and are widely distributed throughout the world. Among them, there are about 130 species of Trogoderma, among which there are more than 20 species of economic significance. It has a wide range of hosts and can seriously harm a variety of animal and plant products. In addition to harming the appearance and quality of the hosts, it also seriously affects foreign trade and leads to huge economic losses. Beetle (non-Chinese species) is a phytosanitary pest entering my country, and its invasion prevention and control is highly concerned by the quarantine department. [0003] Trogoderma granarium is an entry quarantine pest in my country and one of the most important quarantine pests in the world. It is mainly distribu...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6888C12Q1/6844C12Q2600/166C12Q2531/119C12Q2565/125
Inventor 李志红曾令瑜
Owner CHINA AGRI UNIV
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