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
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  • 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 by experienced terrestrial shellfish taxonomists, and the identification of eggs and juvenile snails is even more difficult
Due to historical reasons, at present, most of the plant quarantine officers at border ports are majors in plant protection, and they know little about the classification of terrestrial molluscs. technical problems

Method used

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  • 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

Embodiment 1

[0016] Example 1: Forest onion snail PCR primer specific test

[0017] 1. Preparation of materials

[0018] Test snails are as follows: Forest onion snails Cepaea Nemoralis Garden onion snail Cepaea hortensis ; Sanda snail Helix aspersa ; Cover the big snail Helix pomatia ; Sven Hao's big snail NESIOHELIX SWINHOEI ; Bradybaena (( Acusta Cure ravida ravida ; Mediterranean white snail Cernuella Virgata Essence

[0019] The above-mentioned trial snails are intercepted in the nationwide port quarantine. After confirming the key laboratory of the State Inspection and Quarantine of the State Inspection and Quarantine of the Quality Inspection and Quarantine, it is stored at -20 ° C for later use.

[0020] 2. The establishment of the PCR method

[0021] 2.1, the design synthesis of primers: based on forest -based onion snail CO i (mitochondrial cytochromes C oxidase sub -meal I) gene sequence, design and analyze primers with primers of PRIMER 5 and Oligo 6, and submit it after testing it...

Embodiment 2

[0032] Example 2: Forest onion snail PCR test sensitivity test

[0033] Use the 10x concentration series to dilute method to dilute the forest snail DNA raw liquid (100NG / μL) extracted from the Example 1 to 10ng / μL, 1ng / μL, 100 PG / μL, 10 PG / μL, 1 PG / μL, 100FG / μL and 10 FG / μL have 7 different concentration gradients.

[0034] PCR amplification reaction system: The overall accumulation is 25 μL, of which 2 × TAQ PCR MasterMIX mixed solution is 12.5 μl, the upstream and downstream primers are 0.5 μl, the DNA template is 2 μL, and the remaining sterilizer DDH 2O complement.Put it in the PCR amplifier for amplification after mixing.

[0035] The PCR amplification reaction program is: 95 ° C premium 5min; 95 ° C 50s, 52 ° C 30s, 72 ° C 50s, 35 cycles; 72 ° C extension for 10 minutes, ending the reaction.

[0036] PCR product testing and identification: Take PCR products 10 μL at 1.5%agarose gel (containing bromide ingot) with 98V electrophoresis 40min, and observe with gel imaging instr...

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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

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Application Information

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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
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