Molecular biology method for quickly identifying Heliothis armigera and Helicoverpa assulta

A molecular biology and cotton bollworm technology, applied in the field of molecular biology, can solve the problems of lack of scientific identification methods, inaccurate morphological identification of cotton bollworm and tobacco caterpillar, and achieve the effect of filling gaps in the industry, reducing costs and saving time.

Active Publication Date: 2011-07-27
HONGYUN HONGHE TOBACCO (GRP) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a scientifically effective, simple, quick and accurate molecular biological identificat

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] ——Use standard phenol-chloroform method or DNA extraction kit to extract the total DNA of cotton bollworm and tobacco budworm.

[0027] In order to avoid contamination by fungi and nematodes, the present invention discards the abdomen, wings and antennae, and only extracts DNA from the head, chest and legs. DNA was extracted by referring to the standard phenol / chloroform protocol (the standard phenol / chloroform protocol) or directly using a DNA extraction kit (Shanghai Huashun Bioengineering Co., Ltd.).

Embodiment 2

[0029] —Using DNA barcode primers for PCR amplification of mitochondrial COI gene fragments and sequence determination, specifically including the following steps:

[0030] ①PCR reaction system is 50μl, including about 25ng of template DNA, 1×PCR buffer, 2.5mM Mgcl 2 , 1mM dNTP, 2 μg / μl BSA, 2 pM each of forward and reverse primers, 1 unit of TaqDNA polymerase. Add deionized water to adjust to a final volume of 50 μl, and cover the system with paraffin oil. Reactions were performed on a RoboCycler Gradient 40 (Stratagene) thermal cycler.

[0031] ②PCR reaction conditions are as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 94°C for 1 minute, annealing at 50°C for 1 minute, extension at 72°C for 1 minute. After 35 cycles, an additional 10 min extension was performed at 72°C. The sequencing reaction was electrophoretically sequenced on an ABI 377 automatic sequencer (Applied Biosystems) using Applied's BigDye Terminator kit (V2.0) according to the condition...

Embodiment 3

[0036] —— Species identification based on sequence characteristic variation sites.

[0037] The electrophoresis pattern analyzed by the sequencer was spliced ​​by Seqman in DNASTAR combined with the forward and reverse strands of human work, sorted by Clustal W, and the sequence variation analysis was carried out by MEGA 2.1.

[0038] Table 2 shows the nucleotide sequence characteristic variation sites of cotton bollworm and tobacco budworm. The sequence number of the site is based on the nucleotide position of the fragment. The relevant degenerate bases codes are R=(A / G), Y=(C / T), K=(G / T).

[0039] Table 2.

[0040]

[0041]

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PUM

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Abstract

The invention discloses a method for quickly identifying Heliothis armigera and Helicoverpa assulta, and belongs to the field of molecular biology. The method comprises the following steps of: extracting total DNA by adopting a standard phenol-chloroform protocol or a DNA extraction kit; amplifying a front sequence of mitochondria CO1 by using a DNA barcode primer, and using the amplified sequence as a mark; and performing diagnostic characters of nucleotides. By the method, different biotypes and larvae or incomplete individuals of the same variety of Heliothis armigera and Helicoverpa assulta can be identified; and the method has the characteristics of quickness and convenience, and is more accurate and reliable than the traditional morphologic identification.

Description

technical field [0001] The invention belongs to the field of molecular biology, more specifically, the invention relates to a molecular biology method capable of rapidly distinguishing cotton bollworm and tobacco budworm. Background technique [0002] Tobacco is one of the important pillars of my country's finances, tobacco budworm ( Helicoverpa assault ) and cotton bollworm ( H. armigera ) are the main pests in tobacco production. The two are closely related species occurring in the same domain, and both belong to the Lepidoptera family Noctuidae. Both are very similar in morphology, behavior and ecology, and their identification has been a difficult technical bottleneck in the research of cotton bollworm and tobacco budworm. Accurate species identification is the premise of species distribution investigation and population dynamics research, and is also the basis for in-depth research on biology and integrated control. [0003] The traditional identification methods o...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 段焰青李青青者为王明锋叶灵王坚杨丽萍王文元
Owner HONGYUN HONGHE TOBACCO (GRP) CO LTD
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