Method for testing and identifying cepaea hortensis through polymerase chain reaction (PCR)

A snail and forest technology, applied in the field of molecular biology detection and identification, can solve problems such as difficult to distinguish, difficult to identify eggs and young snails, little knowledge about the classification of terrestrial molluscs, etc., to achieve good practicability, rapid and reliable detection and Strong identification and practical effect

Active Publication Date: 2013-03-06
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
View PDF0 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002]The forest onion snail Cepaea nemoralis (Linnaeus) is a dangerous pest of international phytosanitary concern, and its shells are similar to garden species of the same genus. The onion snail Cepaea hortensis Müller and other related species are so similar that they are often difficult to distinguish and can only be identified b

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for testing and identifying cepaea hortensis through polymerase chain reaction (PCR)
  • Method for testing and identifying cepaea hortensis through polymerase chain reaction (PCR)

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0016] Example 1: PCR primer specificity test of forest onion snail

[0017] 1. Preparation of materials

[0018] The test snails are as follows: forest onion snail Cepaea nemoralis ; Garden onion snail Cepaea hortensis ; Scattered snails Helix aspersa ; Cover the big snail Helix pomatia ; Svenhow Big Snail Nesiohelix swinhoei ; Grey Tip Snail Bradybaena ( Acusta ) ravida ravida ; Mediterranean white snail Cernuella virgata .

[0019] The above test snails were intercepted during national port quarantine. After being confirmed by the State Key Laboratory of Mollusk Quarantine and Identification of the General Administration of Quality Supervision, Inspection and Quarantine, they should be stored at -20°C for later use.

[0020] 2. Establishment of PCR method

[0021] 2.1. Design and synthesis of primers: Based on the CO I (mitochondrial cytochrome C oxidase subunit I) gene sequence of the forest onion snail, primer design software Primer 5 and Oligo 6 are used to design and an...

Example Embodiment

[0032] Example 2: PCR detection sensitivity test of forest onion snail

[0033] The forest onion snail DNA stock solution (100ng / μL) extracted in Example 1 was diluted to 10 ng / μL, 1ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 using the 10-fold concentration serial dilution method There are 7 different concentration gradients of fg / μL and 10 fg / μL.

[0034] PCR amplification reaction system: The total volume is 25μL, including 12.5μL of 2×Taq PCR MasterMix mixture, 0.5μL of upstream and downstream primers, 2μL of DNA template, and sterile ddH for the rest 2 O make up. After mixing, put it into a PCR amplification instrument for amplification.

[0035] The PCR amplification reaction program is: 95°C pre-denaturation 5min; 95°C 50s, 52°C 30s, 72°C 50s, 35 cycles; 72°C extension 10min, the reaction is terminated.

[0036] PCR product detection and identification: Take 10μL of PCR product on a 1.5% agarose gel (containing ethidium bromide) at 98V for 40 minutes and observe with a gel image...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Sensitivityaaaaaaaaaa
Login to view more

Abstract

The invention discloses a method for testing and identifying cepaea hortensis through polymerase chain reaction (PCR), and the method comprises the following steps that a specific primer is applied to identify the molecular features of cepaea hortensis; a forward primer of the PCR is CN-(P1):5'-ACCTCCTTCCTTTCTACT-3', and a reverse primer is CN-(P2):5'-GTCAACATCTATCCCAAC-3'. The total volume of a PCR system is 25mu.L, including 12.5mu.L of 2*TaqPCRMasterMix, respectively 0.5mu.L of the forward and reverse primers, 2mu.L of a deoxyribonucleic acid (DNA) template, and the rest is sterilizing ddH2O; and PCR procedures are as follows: predegeneration is carried out for 5 minutes at 5DEG C, 50 seconds at 95DEG C, 30 seconds at 52DEG C and 50 seconds at 72DEG C, and the process is repeated for 35 times; and extension is carried out for 10 minutes at 72DEG C, and the reaction is over. According to the method for testing and identifying cepaea hortensis through PCR, the cepaea hortensis can be quickly and accurately tested and identified, a novel technical means is provided for the testing and the identification of the dangerous and harmful creature cepaea hortensis, and important significance in preventing the cepaea hortensis from being spread and diffused is realized.

Description

Technical field [0001] The present invention involves the field of molecular biology testing and appraisal, and specifically involves a method of PCR testing and identification of forest onion snails. Background technique [0002] Forest onion snail Cepaea Nemoralis (Linnaeus) is a dangerous and harmful creature that has attracted much attention in international plant quarantine. Cepaea hortensis Müller and other similar similarity are extremely similar, often difficult to distinguish, and only senior land -based shellfish classification experts can identify, and the appraisal of eggs and snails is even more difficult.Due to historical reasons, most of the plant quarantiners in the border ports are mostly plant protection majors, and they know very little about the classification of terrestrial software animals. This situation has caused passiveness to the actual quarantine work of our country.Technical problems.In the past two decades, the rapid development of the molecular syst...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/68
Inventor 周卫川肖琼邵碧英林阳武
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap