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A method for detecting and identifying forest onion snails by fluorescent quantitative PCR

A fluorescence quantitative and forest technology, applied in the field of molecular biology detection and identification, can solve problems such as fluorescence quantitative PCR and other problems that have not yet been seen, and achieve the effect of accurate and convenient result judgment, high sensitivity and strong specificity.

Inactive Publication Date: 2019-01-11
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
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AI Technical Summary

Problems solved by technology

So far, there is no fluorescent quantitative PCR technology for the detection and identification of forest onion snails

Method used

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  • A method for detecting and identifying forest onion snails by fluorescent quantitative PCR
  • A method for detecting and identifying forest onion snails by fluorescent quantitative PCR

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1: forest onion snail fluorescent quantitative PCR primer specificity test

[0018] 1. Preparation of materials

[0019] The snails for testing are as follows: forest green onion snail, pizza tea snail, bright big snail, worm Yin's snail, cover big snail, Jiangxiba snail, garden green onion snail, scattered big snail.

[0020] The above-mentioned snails were intercepted in the national port quarantine or obtained through domestic investigation and collection. After being confirmed by the National Mollusc Quarantine and Identification Key Laboratory of the former General Administration of Quality Supervision, Inspection and Quarantine, they were stored at -20°C for later use.

[0021] 2. Establishment of fluorescent quantitative PCR method

[0022] 2.1. Design and synthesis of primers: Based on the ITS2 gene sequence of the forest onion snail, the primer design soft Primer Express3 was used to assist in the design and analysis of primers. After the specificit...

Embodiment 2

[0030] Example 2: Sensitivity test of forest onion snail fluorescence quantitative PCR detection

[0031]The forest onion snail DNA stock solution (100 ng / μL) extracted in Example 1 was diluted into 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 There are 8 different concentration gradients of fg / μL, 10 fg / μL and 1fg / μL, marked as 1-9 respectively, and curve 10 is the blank control.

[0032] Fluorescent quantitative PCR amplification reaction system: the total volume is 20 μL, including 10 μL of 2×TaqMan PCR Master Mix, 1 μL of upstream and downstream primers and fluorescent probes, 2 μL of 100ng / μL DNA template, 0.5 μL of ROXII, and the rest with sterilized ddH 2 O make up. After mixing, put it into a fluorescent quantitative PCR amplification instrument for amplification.

[0033] The two-step amplification reaction program of fluorescent quantitative PCR was: pre-denaturation at 95°C for 3 minutes; 40 cycles of 10s at 95°C and 40s at 60°C; and the end of the reactio...

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Abstract

The invention provides a method for detecting and identifying forest onion snails by fluorescent quantitative PCR, belonging to the molecular biological detection and identification field. The methodcomprises the following steps of: applying specific primers and probes to identify molecular characteristics of forest onion snails, wherein the upstream primers of the fluorescent quantitative PCR reaction are CNF: 5 '-CGGCCCACGCATTTGTTAT-3 ', and the downstream prim is CNR: 5'-CCACCTTCCACGAGTCTTCT-3 ', and the fluorescent probe is CNP5'- FAM-CCCACTGCTTGTGGGAGCGCC-BHQ1-3 ', 5'-labeled fluorescence reporter group is FAM, and the 3 '-labeled fluorescence quencher group is BHQ1. The method has the advantages of high detection sensitivity, short detection time, good sensitivity, strong specificity, no post-treatment of PCR, and can effectively avoid false positive and cross-contamination, and is suitable for rapid detection and identification of forest onion snails. The present invention provides a new technical method for detecting and identifying forest onion snails which are dangerous and harmful organisms and are concerned in international plant quarantine, and has important significance for preventing transmission and diffusion of forest onion snails.

Description

technical field [0001] The invention relates to the field of molecular biology detection and identification, in particular to a method for detection and identification of forest onion snails by fluorescent quantitative PCR. Background technique [0002] forest onion snail Cepaea nemoralis (Linnaeu, 1758) belongs to Mollusca Mollusca, Gastropoda, Stylommatopgora, Helicidae, Snail Cepaea , is a dangerous pest that has received much attention in international phytosanitary. The snail is nocturnal and often hides in the dark, and can spread long distances with goods and wood packaging materials in a dormant manner. It not only harms various vegetables, fruits, flowers and other crops, but also is an important harmful organism in agricultural production, and is the intermediate host of many zoonotic diseases, affecting the health of humans and animals. In recent years, ports such as Liaoning, Zhangjiakou, Jiangsu, Shandong, Fujian, Tianjin, Ningbo, and Shanghai have repeatedl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6851
CPCC12Q1/6851C12Q1/6888C12Q2531/113C12Q2563/107
Inventor 胡美玲王沛周卫川林阳武
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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