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Rust-colored palm weevil specific primer and rapid molecular detection method

A specific and palm-based technology, applied in the field of PCR identification, can solve laborious and time-consuming problems, achieve high sensitivity, accurate results, and avoid time-consuming effects

Active Publication Date: 2018-04-13
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to distinguish the rust-colored palm weevil from common weevils in China, a lot of morphological identification work is required, most of which are time-consuming, laborious and require professional knowledge of insect taxonomy
Moreover, weevils in the three states of egg, larva, and pupa are very similar in shape, and can only be carried out when adult samples are obtained. Weevils in the three states of egg, larva, and pupa Insectoids are morphologically very similar, and there are currently no reliable identifying characteristics
There is no rapid molecular identification technology for rusty palm weevil

Method used

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  • Rust-colored palm weevil specific primer and rapid molecular detection method
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  • Rust-colored palm weevil specific primer and rapid molecular detection method

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Experimental program
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Effect test

Embodiment 1

[0033] The design of embodiment 1 primer

[0034] 1. The total DNA of 9 species of weevils was extracted using EZgene from BIOMIGA Company TM Insect gDNA Kit (GD2413-02) kit.

[0035] The 9 species of weevils are: 1-Rusty Palm Weevil Rhynchophorus.ferrugineus, 2-Cryptorrhynchus lapathi, 3-Ditch Orbital Elephant Eucryptorrhynchus.chinensis, 4-Ailanthus Eucryptorrhynchus brandti, 5-Mango Fruit Stone Weevil Odoiporus .longicollis, 6-banana corm weevil Cryptorrhynchus.lapathi, 7-mazon pine horn shin like Shirahoshizo patruelis, 8-corn like Sitophilus zeamais, 9-pine knot like Hyposipalus gigas.

[0036] 2. Amplification of weevil COI gene sequence

[0037] According to literature reports, synthetic weevil insect COI gene sequence amplification universal primers:

[0038] C1-J-2183:5'-CAA CAT TTATTT TGATTT TTT GG-3', (SEQ ID NO.5)

[0039] TL-2-N-3014: 5'-TCC ATT GCA CTA TAC TGC CAT ATT A-3' (SEQ ID NO.6)

[0040] PCR amplification reaction using GreenMaster Mix Kit (Promega...

Embodiment 2

[0056] The establishment of the PCR amplification method of embodiment 2 rapid detection rust color palm weevil

[0057] 1. PCR reaction system

[0058] The total DNA of the rust-colored palm weevil was used as a template for PCR reaction. The 25 μL reaction system is shown in Table 1.

[0059] 2. PCR reaction conditions

[0060] The following conditions were set for the reaction: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 53°C for 45 s, extension at 72°C for 2 min, 30 cycles; final extension at 72°C for 10 min.

[0061] 3. After the reaction, the digested product is judged by agarose gel electrophoresis. When PCR amplification is performed with the primers shown in SEQ ID NO.1-2, if the amplified product is 458bp, the insect sample to be tested is positive. Rusty palm weevil;

[0062] When performing PCR amplification with the primers shown in SEQ ID NO.3-4, if the amplified product is 454bp, the insect sample to be tested is positive, ...

Embodiment 3

[0063] The specificity and the sensitivity of embodiment 3 rust color palm weevil PCR method of the present invention

[0064] With the DNA of 9 kinds of weevil class insects commonly seen in China (see embodiment 1) as template, with the rusty color palm weevil DNA as positive control, the rusty color palm weevil SS-COI primer RFSSF1 / Species specificity of RFSSR1 and RFSSF2 / RFSSR2. Amplify with different concentrations of rust-colored palm weevil DNA standard as a template to test the sensitivity of the primers.

[0065] 1. Preparation of standard products

[0066] After the concentration and purity of the DNA of 9 kinds of weevils were determined, they were prepared into a mother solution with a concentration of 10 ng / μl. And the DNA samples of rust-colored palm weevil were diluted into 10ng / μl, 1ng / μl, 100pg / μl, 10pg / μl, 1pg / μl, 100fg / μl standard solution in sequence according to the ratio (1:10). All standard solutions were stored at 4°C for future use.

[0067] 2. Am...

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Abstract

The invention provides a rust-colored palm weevil specific primer and a rapid molecular detection method. According to the invention, a specific primer pair is designed by analyzing a mitochondrial COI gene of a rust-colored palm weevil, and nucleotide sequences of the specific primer pair are shown in SEQ ID No. 1-2 and SEQ ID No. 3-4 of in a sequence table respectively. The rapid molecular detection method of the rust-colored palm weevil, provided by the invention, takes the total DNA of a sample as a template, performs PCR amplification by using any pair of the two pairs of primers, and judges a result according to agarose electrophoresis. The primer provided by the invention has the advantages of good specificity, and the detection method is quick and simple, high in accuracy and goodin sensitivity, and provides a detection method for the identification of the rust-colored palm weevil.

Description

technical field [0001] The invention relates to biological detection and identification technology, in particular to primers for PCR identification of rust-colored palm weevil and a PCR identification method for rust-colored palm weevil using the primers. Background technique [0002] With the acceleration of global economic integration, the situation of alien species invasion in forestry is becoming increasingly severe. Some major forestry invasive species have caused great losses to my country's forest resources, species diversity and ecological security. [0003] Rusty palm weevil Rhynchophorus ferrugineus (Oliver) belongs to Coleoptera Coleoptera weevil family Curculionidae subfamily Rynchoporinae palm elephant genus Rhynchophorus, also known as red palm weevil, coconut cryptobeak, coconut beetle, Asian palm weevil, India The Rusty Palm Weevil has been included in the List of Imported Plant Quarantine Pests and the List of Forestry Quarantine Pests. The rust-colored pa...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888
Inventor 宗世祥陶静骆有庆任利利阎伟
Owner BEIJING FORESTRY UNIVERSITY
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