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PCR (Polymerase Chain Reaction) primer and identification method of athetis lepigone and sibling species thereof

A technique for the morphological similarity of the two-spotted moth, which is applied in the field of PCR identification primers and its identification, can solve the problems of time-consuming, unfavorable for the rapid identification of the two-spotted spider moth, and high cost of sequencing methods

Inactive Publication Date: 2013-08-28
QINGDAO AGRI UNIV
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  • Description
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AI Technical Summary

Problems solved by technology

[0003] Spodoptera exigua and its morphologically similar species are morphologically similar, and non-professionals cannot distinguish them, and the sequencing method is not only expensive but also time-consuming (Zhu Yanbin, Ma Jifang, Dong Li, etc., 2012. Based on the mitochondrial COI gene sequence of the Chinese Erdian Committee Genetic polymorphism analysis of Spodoptera moth. Acta Entomology, 55(4): 457-465.), which is not conducive to the rapid identification of Spodoptera spp. and its morphologically similar species
However, there is no report on the method of using PCR-RFLP technology to construct a method for distinguishing Spodoptera spp. from its morphologically similar species.

Method used

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  • PCR (Polymerase Chain Reaction) primer and identification method of athetis lepigone and sibling species thereof
  • PCR (Polymerase Chain Reaction) primer and identification method of athetis lepigone and sibling species thereof
  • PCR (Polymerase Chain Reaction) primer and identification method of athetis lepigone and sibling species thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 3

[0035] Spodoptera spp. described in Example 3 and its morphologically similar species were collected in Yantai City, Shandong Province in 2012; Genetic polymorphism analysis. Acta Entomological Sinica, 55 (4): 457-465.) to detect the above-mentioned species of Spodoptera sp. and its morphologically similar species. The samples collected in Weihai City, Shandong Province included Sp. Moths are morphologically similar species.

Embodiment 4

[0036] The two-pointed moth described in Example 4 and its morphologically similar species were collected in many areas of Shandong Province in 2012: A: Liaocheng City, Shandong Province, B: Heze City, Shandong Province, C: Zaozhuang City, Shandong Province, D: Shandong Province Linyi City, Province, E: Zibo City, Shandong Province. According to the method described in (Zhu Yanbin, Ma Jifang, Dong Li, et al., 2012. Analysis of genetic polymorphism of Spodoptera spp. in China based on mitochondrial COI gene sequence. Acta Entomological Sinica, 55(4):457-465.) The samples collected from Weihai City, Shandong Province included Spodoptera bispotentum and its morphologically similar species.

[0037] The RsaI endonuclease described in the examples was purchased from TaKaRa Company, Tris-HCl, ethylenediaminetetraacetic acid, and sodium lauryl sulfate were all purchased from Shanghai Bioengineering Company, and other reagents were all commercially available products.

Embodiment 1

[0039] (1) Extraction of genomic DNA from Spodoptera spp. and its morphologically similar species

[0040] Place the single-headed C. spodoptera and its morphologically similar species in 0.2ml centrifuge tubes containing 60 μl of alkali lysis solution, the alkali lysis solution is: 50mmol L -1 Tris-HCl (pH8.0), 20mmol·L -1 NaCl, 1mmol L -1 EDTA (ethylenediaminetetraacetic acid) and 1% SDS (sodium dodecyl sulfate) are fully ground and homogenized with a sealed gun head, placed in a water bath at 65°C for 15 minutes, and then placed in a water bath at 95°C for 10 minutes to obtain Genomic DNA solution of Spodoptera bispotentum and its morphologically similar species.

[0041] (2) PCR amplification of the COI gene of Spodoptera spp. and its morphologically similar species

[0042] Carrying out PCR amplification with the genomic DNA solution of Spodoptera spp. and its morphologically similar species and the genomic DNA solution of morphologically similar species respectively as ...

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Abstract

The invention relates to a PCR (Polymerase Chain Reaction) primer and an identification method of an athetis lepigone and a sibling species thereof. The identification method comprises the following steps of: (1) extracting the genomic DNA of the athetis lepigone and the sibling species thereof; (2) carrying out PCR amplification on the mitochondrion COI (Cytochrome Oxidase I) gene of the genomic DNA by taking the genomic DNA of the athetis lepigone and the sibling species thereof as a template; (3) carrying out enzyme digestion on a PCR product prepared from the step (2) by using a restriction enzyme RsaI to obtain an enzyme-digested product; and (4) carrying out agarose gel electrophoresis analysis on the enzyme-digested product prepared from the step (3). According to the invention, the restriction enzyme is a common restriction enzyme, a simple, convenient and stable enzyme digestion marker is provided for screening the athetis lepigone and the sibling species thereof, and an identification technology of the athetis lepigone and the sibling species thereof is explored and established, so that the foundation is established for the future identification, population dynamic monitoring and integrated prevention and control of the athetis lepigone and the sibling species thereof.

Description

technical field [0001] The present invention relates to a kind of PCR identification primer and its identification method of Spodoptera spp. and its morphologically similar species, in particular to a PCR-RFLP technology-based primer and identification method for distinguishing Scutellaria spp. and its morphologically similar species, belonging to agriculture field of biotechnology. Background technique [0002] Athetis lepigone belongs to Lepidoptera, Noctuidae, Athetis lepigone. It was first discovered in Hebei Province in 2005 (Jiang Jingyu, Li Xiuqin, Xu Youhui, et al., 2008. Preliminary report on Noctuid moth research of the Second Committee. Plant Protection, 34 (3): 23-26.). In 2011, hazards broke out in 302 districts (counties) of 47 cities in 6 provinces, including Henan and Shandong, covering an area of ​​nearly 2 million hectares, posing a serious threat to the safe production of summer maize (Wang Zhenying, Shi Jie, Dong Jingao, 2012. 2011 Huanghuai Haixia The ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 褚栋李丽莉国栋陶云荔
Owner QINGDAO AGRI UNIV
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