A method for detection and identification of Helicodon snails by fluorescent quantitative PCR

A fluorescent quantitative and snail technology, which is applied in the field of molecular biology detection and identification, can solve problems such as fluorescence quantitative PCR of snails that have not yet been seen, and achieve the effect of accurate and convenient result determination and strong specificity

Active Publication Date: 2022-03-22
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
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Problems solved by technology

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

Method used

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  • A method for detection and identification of Helicodon snails by fluorescent quantitative PCR
  • A method for detection and identification of Helicodon snails by fluorescent quantitative PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Primer Specificity Test of Fluorescence Quantitative PCR of Helical Snail

[0019] 1. Preparation of materials

[0020] The snails for testing are as follows: snail with upper spiral teeth, large snail, bright snail, worm Yin's snail, pizza tea snail, Jiangxiba snail, garden onion snail, forest onion snail.

[0021] 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.

[0022] 2. Establishment of fluorescent quantitative PCR method

[0023] 2.1. Design and synthesis of primers: Based on the COI gene sequence of Helicodon snail, the primer design software PrimerExpress3 was used to assist in the design and analysis of primers. After the specificity was tested by ...

Embodiment 2

[0031] Example 2: Detection Sensitivity Test of Fluorescent Quantitative PCR for Helicopterus snail

[0032]The 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, 8 different concentration gradients of 100 fg / μL, 10 fg / μL and 1fg / μL are marked as 1-9 respectively, and curve 10 is the blank control.

[0033] Fluorescence quantitative PCR amplification reaction system: the total volume is 25 μL, including 12.5 μL of 2×Premix Ex TaqTM PCR (ProbeqPCR), 1 μL of upstream and downstream primers and probes, 2 μL of 100 ng / μL DNA template, 0.5 μL of ROXII, and the rest with inert ddH 2 O make up. After mixing, put it into a fluorescent quantitative PCR amplification instrument for amplification.

[0034] The two-step amplification reaction program of fluorescent quantitative PCR is: 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 reaction.

[0035] Judgment of f...

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Abstract

The invention provides a method for detection and identification of snails with fluorescent quantitative PCR, which belongs to the field of detection and identification of molecular biology. The method of the invention comprises the use of specific primers and fluorescent probes to identify the molecular features of the helicoid snail. The method of the invention not only has the advantages of strong specificity, high sensitivity, short time consumption, and accurate and convenient result determination, but also does not require PCR post-processing, can effectively avoid false positives and cross-contamination, and is suitable for rapid detection and identification of upper-spiral snails. The present invention provides a new technical means for the detection and identification of the dangerous pest snails, which have attracted much attention in the international plant quarantine, and is useful for preventing the spread and spread of the snails and protecting human health, agricultural production and ecological environment. important meaning.

Description

technical field [0001] The invention relates to the field of detection and identification of molecular biology, in particular to a method for detection and identification of snails with fluorescent quantitative PCR. Background technique [0002] Spiral snail Helicodonta obvoluta (Müller, 1774) belongs to the order Stylommatopgora, Helicodontidae Kobelt, 1904, the genus Helicodontidae Helicodonta Férussac, 1821, is a dangerous pest of international phytosanitary concern. The snail likes to live under the litter layer and in the crevices of stones in the forest, and feeds on various green plants. It mainly harms the seedlings and tender shoots of crops, leading to lack of seedlings and ridges, reducing yield, seriously endangering vegetables and garden crops, and the silvery white mucus traces left by snails after crawling seriously reduce the commodity value of agricultural products. At the same time, the snail also spreads zoonotic parasitic diseases, threatening the h...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6851
CPCC12Q1/6851C12Q1/6888C12Q2531/113C12Q2563/107
Inventor 王沛胡美玲周卫川
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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