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Method for detecting and identifying Helix aspersa MUller through PCR (polymerase chain reaction)

A technology for identifying snails and snails, which is applied in the field of molecular biology detection and identification, can solve the problems of little knowledge about the classification of terrestrial molluscs, and achieve the effects of fast and reliable detection and identification, strong specificity, and guaranteed reliability of results

Active Publication Date: 2014-08-20
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to historical reasons, the vast majority of plant quarantine personnel at border ports are majors in plant protection and know little about the classification of terrestrial molluscs. common technical challenges

Method used

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  • Method for detecting and identifying Helix aspersa MUller through PCR (polymerase chain reaction)
  • Method for detecting and identifying Helix aspersa MUller through PCR (polymerase chain reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: bulk snail PCR primer specificity test

[0017] 1. Preparation of materials

[0018] The snails for testing are as follows: scattered big snails Helix aspersa Müller, 1774 (Australian specimen); loose large snail Helix aspersa Müller, 1774 (German specimen), flat-toothed snail Camaenella playodon ; wrinkled snail Camaena cicatricosa ; Satsuma stenozona ; Zhiba snail Bradybaena virgo virgo ; garden snail Cepaea hortensis ; forest onion snail Cepaea nemoralis ; Tianshui baby snail Pupinidius tianshuiensis ; gray tipped snail Bradybaena Ravida ;Cover large snail Helix pomatia ; Juvenile ring rib snail Plectotropis lithina ;Isomorphic snail Bradybaena similaris ; umbilical snail Aegista permellita ; short-snail Bradybaenabrevispira ; Satsuma uncopila ; bright big snail Helix lucorum ; Tiaohua snail Cathaica fasciola fasciola ; the worm Yin's snail Eobania vermiculata ; pointed spiral snail Cochlicella acuta ; pointed bladd...

Embodiment 2

[0031] Embodiment 2: Sensitivity test of bulk snail PCR detection

[0032] The snail DNA stock solution (100 ng / μL) extracted in Example 1 was diluted to 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 There are 7 different concentration gradients of fg / μL and 10 fg / μL.

[0033] PCR amplification reaction system: the total volume is 25 μL, including 12.5 μL of 2×Taq PCR MasterMix mixture, 1 μL of upstream and downstream primers, 2 μL of DNA template, and the rest with sterilized ddH 2 Make up O, mix well and put it into the PCR amplification instrument for amplification.

[0034] The PCR amplification reaction program was: pre-denaturation at 94°C for 5 min; 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min; extension at 72°C for 10 min, and the reaction was terminated at 4°C.

[0035] Detection and identification of PCR products: 5 μL of PCR products were electrophoresed on 1.5% agarose gel (containing ethidium bromide) at 130 V for about 30 min, and observ...

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Abstract

The invention discloses a method for detecting and identifying Helix aspersa MUller through PCR (polymerase chain reaction). The method comprises the step of identifying the molecular feature of the Helix aspersa MUller by using specific primers, wherein the upstream primer of PCR is HP-(P1): 5'-ACTCTTTGGTATTTGATGCG-3', and the downstream primer is HP-(P2): 5'-ACCTGCTAAAACGGGTAATG-3'. The method can be used for quickly and accurately detecting and identifying the Helix aspersa MUller. The method provides a new technical means for detection and identification of the Helix aspersa MUller in Imported Plant Quarantine Pest Directory of the People's Republic of China, and has significance for preventing the propagation and diffusion of the Helix aspersa MUller.

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 loose snails by PCR. Background technique [0002] loose big snail Helix aspersa Müller, also known as the large scatter snail in 1774, belongs to the mollusk phylum Mollusca in classification, the gastropod class Gastropoda, the handle eye order Stylommatophora, the large snail family Helicidae, and the large snail genus Helix Linne is a quarantine pest that has attracted much attention internationally. The United States, Canada and other countries implement quarantine on it. Scattered snails are nocturnal, often hidden in dark and humid places that are difficult for people to find, and are easy to spread with logs, wooden packaging materials, containers, etc. The characteristic of its transmission is that it does not require a specific host, and any contacted items can be transmitted. In 2007, my ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2531/113
Inventor 王沛周卫川陆军林阳武肖琼崔俊霞
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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