Method of detecting and identifying cernuella virgata da costa through fluorescent quantitation PCR

A fluorescent quantitative and snail technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems that have not yet been seen in fluorescent quantitative PCR, etc., and achieve accurate and convenient result judgment, strong practicability, good practical effect

Inactive Publication Date: 2017-08-18
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Method of detecting and identifying cernuella virgata da costa through fluorescent quantitation PCR
  • Method of detecting and identifying cernuella virgata da costa through fluorescent quantitation PCR

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Embodiment 1: Mediterranean white snail fluorescent quantitative PCR primer specificity test

[0020] 1. Preparation of materials

[0021] The snails tested are as follows: Mediterranean white snail (Australian specimen), Pizza tea snail; Liangda snail;

[0022] 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 General Administration of Quality Supervision, Inspection and Quarantine, they were stored at -20°C for later use.

[0023] 2. Establishment of fluorescent quantitative PCR method

[0024] 2.1. Design and synthesis of primers: Based on the ITS2 gene sequence of Mediterranean white snail, the primer design software PrimerExpress3 was used to assist in the design and analysis of primers. After the specificity was tested by NCBI Blast, the primers were synthesized by Shanghai Sangong B...

Embodiment 2

[0034] Example 2: Sensitivity test of fluorescent quantitative PCR detection of Mediterranean white snail

[0035]The Mediterranean white 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.

[0036] Fluorescent quantitative PCR amplification reaction system: the total volume is 25 μL, including 2×TaqMan PCR Master Mix 12.5 μL, upstream and downstream primers and probes 1 μL each, 100ng / μL DNA template 2 μL, and the rest with sterilized ddH 2 O make up. After mixing, put it into a fluorescent quantitative PCR amplification instrument for amplification.

[0037] The two-step amplification reaction program of fluorescent quantitative PCR is: 95°C pre-denaturation for 5 minutes; 95°C for 10s, 60°C for 40s, 35 cycles; end the reaction.

[0038] Judgment of fluorescent quantitative PCR results: If a typical amplification curve ...

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Abstract

The invention provides a method of detecting and identifying cernuella virgata da costa through a fluorescent quantitation PCR. Molecular characteristics of cernuella virgata da costa can be identified by using a specific primer and a probe, an upstream primer of the fluorescent quantitation PCR is CVF:5'-CGTTTCGGGTGGGAGGGTTA-3', a downstream primer of the fluorescent quantitation PCR is CVR:5'-AAGTCGTCCGAGCTGTTCCG-3', the fluorescent probe is CVP:5'-FAM-AACCCAGGGCGCCTCCGCGG-BHQ-3', wherein a fluorescent reporting group which is marked by a 5' end is FAM, and a fluorescent quenching reporting group which is marked by a 3' end is BHQ. The method can detect and identify cernuella virgata da costa quickly and precisely, the method has the advantages of high specificity and sensitivity, short consuming time, and precise and convenient result determination, PCR post processing is not needed, and a false positive and cross pollution can be avoided effectively.

Description

technical field [0001] The invention relates to a method for detecting and identifying Mediterranean white snails by fluorescent quantitative PCR, which belongs to the technical field of plant quarantine and is suitable for rapid detection and identification of Mediterranean white snails in entry and exit port quarantine and agricultural production. Background technique [0002] mediterranean white snail Cernuella virgata (Da Costa, 1778) belongs to the order Stylommatopgora, Hygromiidae, white snail Cernuella , is a member of the newly added "List of Imported Plant Quarantine Pests of the People's Republic of my country". The snail mainly harms grain crops such as wheat and barley, grapes and citrus fruits, and can crawl onto crop harvesting machines, seriously affecting operations and damaging agricultural tools. Mediterranean White Snail Shells with Pizza Tea Snails Theba pisana are very similar and are often difficult to distinguish, and the identification of eggs ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2563/107C12Q2545/114
Inventor 王沛周卫川王宇杨姗萍林阳武
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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