Method for detecting and identifying Cernuella virgata Da Costa by PCR
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A snail and reaction technology, applied in the field of molecular biology detection and identification, can solve problems such as little knowledge of terrestrial mollusk classification, and achieve rapid and reliable detection and identification, high specificity, and strong practicability.
Active Publication Date: 2014-07-30
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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Due to historical reasons, the vast majority of plant quarantine personnel at border ports are majors in pla
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Embodiment 1
[0016] Embodiment 1: Mediterranean white snail PCR primer specificity test
[0017] 1. Preparation of materials
[0018] The snails tested are as follows: Mediterranean white snail Cernuella virgata (Da Costa, 1778) (Australian specimen), Mediterranean white snail Cernuella virgata (Da Costa, 1778) (New Zealand specimen), wrinkled snail Camaena cicatricosa , Boling Samo Satsuma stenozona , Zhiba snail Bradybaena virgo virgo , garden snail Cepaea hortensis Tianshui baby snail Pupinidius tianshuiensis , cover big snail Helix pomatia , young stone ring rib snail Plectotropis lithina , isomorphic snail Bradybaena similaris , umbilical snail Aegista permellita ; short-snail Bradybaenabrevispira , Satsuma snail Satsuma uncopila , bright big snail Helix lucorum , Cystosnail Physa acuta , pizza tea snail Theba pisana .
[0019] The above-mentioned snails were intercepted in the national port quarantine or obtained through domestic investigation and col...
Embodiment 2
[0031] Embodiment 2: PCR detection sensitivity test of Mediterranean white snail
[0032] 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 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, 0.5 μL of upstream and downstream primers, 3 μL of DNA template, and the rest with sterilized ddH 2 O make up. After mixing, put it into a PCR amplification instrument for amplification.
[0034] The PCR amplification reaction program was: 94°C pre-denaturation for 5 minutes; 94°C for 30s, 46°C for 30s, 72°C for 1min, 25 cycles; 72°C for 10min, and the reaction was terminated.
[0035] Detection and identification of PCR products: Take 10 μL of PCR products and electrophoresis on 1.5% agarose gel (containing ethidium bromide) at 130V for about 30...
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Abstract
The invention discloses a method for detecting and identifying Cernuella virgata Da Costa by PCR. The method comprises identifying molecular characteristics of the Cernuella virgata Da Costa by specific primers, wherein the upstream primer of PCR reaction is HP- (P1): 5'-GTTATTGTTACTGCTCACGC -3', the downstream primer is HP- (P2): 5'-CTGCTAAGACAGGGAGAGAT-3'. The total volume of the PCR reaction system is 25 muL, wherein the volume of 2*TaqPCRMasterMix is 12.5 muL, the volume of the upstream and downstream primers are respectively 0.5 muL, the volume of DNA template is 3 muL and the rest volume is supplied with sterile ddH2O; PCR reaction program comprises carrying out a denaturation at 94 DEG C for 5 minutes at 94 DEG C for 30 seconds, at 46 DEG C for 30 seconds, at 72 DEG C for 1 minute, circulating for 25 times, extending at 72 DEG C for 10 minutes and completing the reaction. The Cernuella virgata Da Costa can quickly and accurately detected and identified by the method disclosed by the invention. The method provides a new technical means for the detection and identification of the Cernuella virgata Da Costa in the 'List of Quarantine Pests of the People's Republic of China of the Entry of Plants' and is of great significance for preventing the Cernuella virgata Da Costa from spreading and diffusing.
Description
technical field [0001] The invention relates to the field of molecular biology detection and identification, in particular to a method for PCR detection and identification of Mediterranean white snails. Background technique [0002] mediterranean white snail Cernuella virgata (Da Costa, 1778) belongs to Stylommatopgora and Hygromiidae. It is a quarantine pest that has attracted much attention in the world. The United States, Chile, Japan, New Zealand, Australia and other countries and regions implement quarantine on it. In September 2012, the snail was added as a member of the "List of Imported Plant Quarantine Pests of the People's Republic of China". Theba pisana Müller and others are very similar, often difficult to distinguish, and only senior terrestrial mollusc taxonomy experts can identify, and the identification of eggs and young snails is even more difficult. Due to historical reasons, the vast majority of plant quarantine personnel at border ports are majors i...
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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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