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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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to historical reasons, the vast majority of plant quarantine personnel at border ports are majors in pla

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
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Example Embodiment

[0016] Example 1: Specificity test of PCR primers of snails

[0017] 1. Preparation of materials

[0018] The test snails are as follows: scattered snails Helix aspersa Müller, 1774 (Australian specimen); scattered snails Helix aspersa Müller, 1774 (German specimen), small snail Camaenella platyodon ; Wrinkled Snail Camaena cicatricosa ;Samole snail Satsuma stenozona ; Branch snail Bradybaena virgo virgo ; Garden onion snail Cepaea hortensis ; Forest onion snail Cepaea nemoralis ; Tianshui Baby Snail Pupinidius tianshuiensis ; Grey Tip Snail Bradybaena ravida ; Cover the big snail Helix pomatia ; Larvae Plectotropis lithina ; The same type of snail Bradybaena similaris ; Fruit umbilical snail Aegista permellita ; Short spiral snail Bradybaenabrevispira ; Flat pulp Satsuma snail Satsuma uncopila ; Bright big snail Helix lucorum ; Snail Cathaicafasciola fasciola ; Worm Yin's snail Eobania vermiculata ; Pointed-headed spiral snail Cochlicella acuta ; Pointed Bladder...

Example Embodiment

[0031] Example 2: PCR detection sensitivity test of snails

[0032] A 10-fold concentration serial dilution method was used to dilute the snail DNA stock solution (100ng / μL) extracted in Example 1 into 10ng / μL, 1ng / 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 25μL, including 12.5μL of 2×Taq PCR MasterMix mixture, 1μL of upstream and downstream primers, 2μL of DNA template, and sterile ddH for the rest 2 Make up with O, mix well and put it into a PCR amplification instrument for amplification.

[0034] The PCR amplification reaction program is: 94°C pre-denaturation 5min; 94°C 30s, 56°C 30s, 72°C 1min, 30 cycles; 72°C extension 10min, 4°C to end the reaction.

[0035] PCR product detection and identification: Take 5μL of PCR product on a 1.5% agarose gel (containing ethidium bromide) and electrophoresis at 130V for about 30 minutes, and observe with a gel imag...

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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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