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Specific gene and molecular identification method of culicolides brevipalpis

A molecular identification and specific gene technology, applied in the field of insect identification, can solve the problems of ITS gene sequence cannot be directly sequenced, ITS mutation speed is fast, and intraspecific gene variation is large, so as to solve the problem of species identification, avoid hybridization or gene penetration, The effect of ensuring accuracy

Pending Publication Date: 2019-05-28
福州国际旅行卫生保健中心
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are reports in the literature that try to use COI and ITS2 genes as molecular markers to identify blood-sucking midges, but the ITS mutation speed is fast, the intraspecific gene variation is large, and the ITS gene sequence cannot be directly sequenced, but can only be obtained by cloning. Combination identification greatly affects the timeliness of identification

Method used

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  • Specific gene and molecular identification method of culicolides brevipalpis
  • Specific gene and molecular identification method of culicolides brevipalpis
  • Specific gene and molecular identification method of culicolides brevipalpis

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1 Obtaining the CO Ⅰ and Cyt b gene sequences of C. breviflora

[0047] In this example, the acquisition of the CO Ⅰ and Cyt b gene sequences of C. brevisiae includes the following steps: extracting genomic DNA from C. breviflora, and design and synthesize primers for the COI and Cyt b genes of C. breviflora. Using the above-mentioned extracted genomic DNA as a template, using the above-mentioned synthetic primers of C. brevisiae CO Ⅰ gene, PCR amplification was carried out to obtain the CO Ⅰ product of C. brevisiae; using the above-mentioned extracted genomic DNA as a template, using the above The synthesized Cyt b gene primer of C. brevifolia was amplified by PCR to obtain the product of C. brevifolia Cyt b. The CO Ⅰ and Cyt b products of C. brevisiae obtained above were detected and sequenced to obtain the CO Ⅰ and Cyt b gene sequences of C. brevisiae.

[0048] specific:

[0049] 1. Preparation of genomic DNA of C. brevifolia

[0050] Take the same batch of other s...

Embodiment 2

[0066] Example 2 Identification of Culicoides to be tested

[0067] The identification of C. midges to be tested includes the following steps:

[0068] S1: Isolate and extract DNA from the Culicoides tissue to be tested;

[0069] (1) The light trap method was used to collect the Culicoides to be tested, and the collected Culicoides to be tested were frozen and sacrificed into specimens and stored directly in 95% alcohol.

[0070] (2) Take out the specimen of C. midges to be tested stored in 95% alcohol, dissected under a dissecting microscope, and make slide specimens for morphological identification except for the chest. After individually numbering the chest of the Culicoides to be tested, make sure to correspond to the slide specimen one by one. If it needs to be stored for a longer period of time, the PCR tube containing the chest tissue part of the Culicoides to be tested should be frozen at -20℃.

[0071] (3) Using a small insect micro-DNA template preparation method, extract th...

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Abstract

The invention relates to the field of insect identification, in particular to a specific gene and a molecular identification method of culicolides brevipalpis. The specific gene is one of a CO I geneand a Cyt b gene, or a combination of the two; the sequence of the CO I gene is SEQ ID NO. 1, the sequence of the Cyt b gene is SEQ ID NO. 2, and the specific gene can be used for accurate identification of the species of the culicolides brevipalpis. The invention provides the molecular identification method of the culicolides brevipalpis, which combines the CO I gene with the Cyt b gene and has high accuracy and short identification time; during homology comparison among DNAs of to-be-detected insects and the CO I and Cyt b genes of the culicolides brevipalpis, the to-be-detected insects arejudged as the culicolides brevipalpis when the homology reaches 98% or above.

Description

Technical field [0001] The invention relates to the field of insect identification, in particular to the specific gene of C. breviflora and its molecular identification method. Background technique [0002] Culicoides belong to the genus Arthropoda, Insecta, Diptera, Ceratopogonidae, and Culicoides in animal classification. It is the largest genus in the midge family and is also in the genus Bloodsucking midges. The genus with the widest distribution, the most species, and the closest relationship with humans and animals, is the vector of many important animal-borne diseases. In recent years, with the global warming, the ability of Culicoides to transmit arboviruses has increased to a certain extent, and the distribution range of Culicoides has expanded to the poles and high altitude areas of the earth. The ability of animals has increased, and some traditional "non-media" species of Culicoides have the ability to spread diseases. Insect-borne diseases such as bluetongue diseas...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/11C12Q1/6869C12Q1/6888
Inventor 郑爱萍杨美琼林妙端陈敏
Owner 福州国际旅行卫生保健中心
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