Method for identifying helix pomatia by PCR (polymerase chain reaction) detection
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A snail and cover technology, 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 results of guaranteed reliability, high sensitivity and reliable results
Active Publication Date: 2014-04-16
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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[0002]The large cover snail Helix pomatia (Linnaeus,1758) is the "People's Republic of China Entry Plant Quarantine Pests" promulgated in 2007 The members of the list, whose shells are very similar to those of the same genus Helix aspersa (Müller), Helix lucorum (Linnaeus, 1758), etc., are often difficult to identify. Differentiation, and only senior terrestrial mollusc taxonomy experts can identify, the identification of eggs and young snails is more difficult
Due to historical reasons, the vast majority of plant quarantine officers at border ports are majors in plant protection and know little about the classification of terrestrial molluscs. common technical challenges
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Embodiment 1
[0016] Embodiment 1: the PCR primer specificity test of cover cover big snail
[0017] 1. Preparation of materials
[0018] The snails for testing are as follows: cover big snail Helix pomatia (Dutch specimen), the large cap snail Helix pomatia (German specimen), garden onion snail Cepaea hortensis , forest onion snail Cepaea nemoralis , loose big snail Helix aspersa Tianshui baby snail Pupinidius tianshuiensis , Ciba snail Bradybaena sequiniana , Mediterranean white snail Cernuella virgata , gray pointed snail Brady Baena ( Acusta ) ravida ravida , bright big snail Helix lucorum Tiaohua snail Cathaica ( Cathaica ) fasciola fasciola , Yongan ring rib snail Plectotropis yonganensis , Sven Miller's snail Nesiohelix swinhoei , flat snail Brady Baena ( Brady Baena ) uncopila , Satsuma snail Satsuma stenozona .
[0019] The above-mentioned snails were intercepted in the national port quarantine or obtained through domestic investigation ...
Embodiment 2
[0031] Embodiment 2: PCR detection sensitivity test of the large snail with cover
[0032] The 10-fold concentration serial dilution method was used to dilute the DNA stock solution (100 ng / μL) of the snail snail extracted in Example 1 to 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, There are 7 different concentration gradients of 100 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 (10 μM), 2 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: pre-denaturation at 95°C for 5 minutes; 30 cycles at 95°C for 30 s, 45°C for 30 s, and 72°C for 1 min; extension at 72°C for 10 min, and the reaction was terminated.
[0035] Detection and identification of PCR products: Take 10 μL of PCR products and elec...
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Abstract
The invention discloses a method for identifying helix pomatia by PCR (polymerase chain reaction) detection, which uses specific primers to identify molecular characteristics of helix pomatia. The forward primer for the PCR reaction is HP-(P1): 5'-TTCGTTGAGAGTTAGG-3', and the reverse primer is HP-(P2): 5'-ATAGTAATAGCACCAGC-3'. The total volume of the PCR reaction system is 25 mu L, wherein the 2*TaqPCRMasterMix is 12.5 mu L, the forward and reverse primers are respectively 0.5 mu L, the DNA (deoxyribonucleic acid) template is 2 mu L, and the rest is sterilized ddH2O. The PCR reaction process comprises the following steps: pre-denaturating at 5 DEG C for 5 minutes; treating at 95 DEG C for 30 seconds, 45 DEG C for 30 seconds and 72 DEG C for 1 minute, and repeating 30 times; and extending at 72 DEG C for 10 minutes, and finishing the reaction. The method disclosed by the invention can quickly and accurately detect and identify helix pomatia. The invention provides a new technical means for detection and identification of helix pomatia in the Catalogue of Quarantine Pest for Import Plants to the People's Republic of China, and has important meanings for preventing helix pomatia from spreading and diffusion.
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 large snails. Background technique [0002] cover big snail Helix pomatia (Linnaeus, 1758) is a member of the "List of Imported Plant Quarantine Pests of the People's Republic of China" issued by my country in 2007. Helix aspersa (Müller), bright big snail Helix lucorum (Linnaeus, 1758), etc. are very similar, often difficult to distinguish, and only senior terrestrial mollusc taxonomy experts can identify, and the identification of eggs and juvenile snails is even more difficult. Due to historical reasons, the vast majority of plant quarantine officers at border ports are majors in plant protection and know little about the classification of terrestrial molluscs. common technical challenges. In the past two decades, molecular systematics has developed rapidly, that is, the comparison of gene seq...
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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 周卫川王沛林阳武肖琼
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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