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Specific primers and pcr detection method and kit for identifying Heterotrichia spp.

A detection method and kit technology, applied in the field of genetic engineering, can solve problems such as long time, and achieve the effects of strong specificity, good reliability, and protection of agricultural and forestry production safety.

Active Publication Date: 2021-02-09
珠海出入境检验检疫局检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The molecular identification method currently used is mainly by determining the fragment sequence of the first subunit gene (COI) of mitochondrial cytochrome oxidase, and importing the sequencing results into the database for comparison and analysis. However, the time required for this method is relatively long ( 2-3 days)

Method used

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  • Specific primers and pcr detection method and kit for identifying Heterotrichia spp.
  • Specific primers and pcr detection method and kit for identifying Heterotrichia spp.
  • Specific primers and pcr detection method and kit for identifying Heterotrichia spp.

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1, the specificity detection of the apple Heterotrigina

[0024] The experimental specimens are the samples intercepted by the laboratory. Specific detection was carried out on 15 samples (specimen numbers 2-16) of Heterotrichia spp. and its 7 related species. See Table 1 for detailed information on Heterotrichia spp. and other species.

[0025] Table 1 Specimen details

[0026] Chinese name / Latin scientific name Specimen number the host place of origin Thaumatotibia leucotreta 2 Oranges South Africa Thaumatotibia leucotreta 3 fresh grapes South Africa Thaumatotibia leucotreta 4 fresh orange South Africa Thaumatotibia leucotreta 5 fresh orange South Africa Thaumatotibia batrachopa 6 fresh orange South Africa Thaumatotibia sp. 7 Citrus China Epichoristodes acerbella 8 fresh grapes South Africa Apple hazel moth Epiphyas postvittana 9 fresh grapes Australia Codl...

Embodiment 2

[0031] Embodiment 2 specificity test

[0032] Using the established PCR detection system, the detection sensitivity of the PCR reaction was determined by diluting 6 DNA templates with a 10-fold concentration gradient of Specimen No. 3. The result is as image 3As shown, the numbers 1 and 8 in the figure are DL2000marker; the number 2 is 100ng / μl; the number 3 is 10ng / μl; the number 4 is 1ng / μl; the number 5 is 0.1ng / μl; the number 6 is 0.01ng / μl; 7 is 0.001 ng / μl. When the template concentration is 0.1-100ng / μl, a strong detection signal can be amplified; for a DNA template of 0.01ng / μl, the detection amplification product is significantly reduced, and the detection signal is weak; when the DNA template concentration is 0.001ng / μl , it cannot be detected due to weak detection signal. It shows that the detection limit of this method is 0.01ng / μl, and the optimal detection concentration is 0.1-100ng / μl. If the amount of the original DNA extraction solution does not reach the...

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Abstract

The present invention provides a kind of specific primers used for identifying Tortrichia appleus, including such as upstream primer ThL-Its319F and downstream primer ThL-Its732R. Also disclosed are a kit and a detection method for identifying Trichoptera spp., including buffer, Taq DNA polymerase, ddH 2 O and the above-mentioned specific primer pair ThL-Its319F, ThL-Its732R. The routine PCR established by the specific primers and kit has the advantages of rapidity, sensitivity and accuracy, and is suitable for the detection of different insect states of Heterotrichia japonica. Lays the foundation for rapid species identification. The research results can be directly applied to the quarantine and identification of Heterotrichia spp. intercepted in imported fruits, which is of great significance to the protection of agricultural and forestry production safety and the maintenance of ecological balance in our country.

Description

technical field [0001] The invention relates to the technical field of genetic engineering. Background technique [0002] Thaumatotibia leucotreta (Meyrick, 1913) has the same name as Cryptophlebia leucotreta, which belongs to Lepidoptera, Tortricidae, Gapholitini, Thaumatobia leucotreta, and belongs to Lepidoptera. It is an omnivorous pest and its main hosts are avocado Persea americana, citrus Citrus spp., peach Prunus persica, guava Psidium guajava, mango Mangifera sp., corn Zea mays, cotton Gossypium herbaceum, Australia Macadamia spp., Acacia spp., Quercus spp., Eugenia spp., Saccharum officinarum, Litchi chinensis, Solanum melongena, Camelliasinensis, Capsicum spp. and more than 70 kinds of plants in 30 families, are economically important pests. [0003] The worm is similar in shape and host plants to the tortillaria such as Thaumatotibia batrachopa, codling moth Cydia pomonella, pear borer Grapholita molesta, etc. It is often difficult to make rapid and accurate id...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888
Inventor 徐淼锋权永兵黄永辉迟远丽廖力张卫东林伟
Owner 珠海出入境检验检疫局检验检疫技术中心