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
0 Cites 8 Cited by

AI-Extracted 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 b...
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

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.

Application Domain

Technology Topic

Forward primerReverse primer +2

Image

  • 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(2)

Example Embodiment

[0016] Example 1: PCR primer specificity test of forest onion snail
[0017] 1. Preparation of materials
[0018] The test snails are as follows: forest onion snail Cepaea nemoralis; Garden onion snail Cepaea hortensis; Scattered snails Helix aspersa; Cover the big snail Helix pomatia; Svenhow Big Snail Nesiohelix swinhoei; Grey Tip Snail Bradybaena ( Acusta ) ravida ravida; Mediterranean white snail Cernuella virgata.
[0019] The above test snails were intercepted during national port quarantine. After being confirmed by the State Key Laboratory of Mollusk Quarantine and Identification of the General Administration of Quality Supervision, Inspection and Quarantine, they should be stored at -20°C for later use.
[0020] 2. Establishment of PCR method
[0021] 2.1. Design and synthesis of primers: Based on the CO I (mitochondrial cytochrome C oxidase subunit I) gene sequence of the forest onion snail, primer design software Primer 5 and Oligo 6 are used to design and analyze primers. After testing their specificity by NCBIBlast, cross Synthesized by Shanghai Shenggong Biotechnology Service Co., Ltd. The primers are as follows:
[0022] The upstream primer is CN-(P1): 5’- ACCTCCTTCCTTTCTACT -3’,
[0023] The downstream primer is CN-(P2): 5'-GTCAACATCTATCCCAAC -3'.
[0024] The size of the amplified fragment is 580bp.
[0025] 2.2, DNA extraction
[0026] 2.2.1. Cyanoguanidine isosulfate method: Take the prepared positive sample (forest onion snail Cepaea nemoralis ) And a negative control sample (garden onion snail Cepaea hortensis; Scattered snails Helix aspersa; Cover the big snail Helix pomatia; Svenhow Big Snail Nesiohelix swinhoei; Grey Tip Snail Bradybaena ( Acusta ) ravida ravida; Mediterranean white snail Cernuella virgata ), cut into pieces, place them in a mortar, add liquid nitrogen to grind into powder, take 50mg, then add 200μL of TE, and mix; add 400μL of lysate, vortex to mix, let stand for 10min; then add 300μL of Tris-saturated phenol and 300μL chloroform: isoamyl alcohol (24:1), shake vigorously for 15s, centrifuge at 13 000r/min for 10 min; take the supernatant, add an equal volume of chloroform: isoamyl alcohol (24:1), shake vigorously for 15s, 13 000r/min Centrifuge for 10min; take the supernatant, add an equal volume of chloroform, shake vigorously for 15s, 13 000r/min, 10min; take the supernatant, add 0.8 times the volume of isopropanol, 12 000r/min, 10min, discard the supernatant; take the precipitate , Add 1mL of 70% absolute ethanol, centrifuge at 13 000r/min, discard the supernatant, dry in a vacuum dryer for 2 minutes, and then add 100μL of sterilized ddH 2 O dissolves. Determine the concentration of DNA with a nucleic acid protein analyzer, and finally dilute the DNA to 100ng/μL with TE solution and store it at -20°C for later use.
[0027] 2.2.2. Kit extraction method: TIANGEN tissue genomic DNA extraction kit, extract DNA according to the instruction manual. Determine the concentration of DNA with a nucleic acid protein analyzer, and finally dilute the DNA to 100ng/μL with TE solution and store it at -20°C for later use.
[0028] 2.3. PCR amplification reaction system: The total volume is 25μL, including 12.5μL of 2×Taq PCR MasterMix mixture, 0.5μL of upstream and downstream primers, 2μL of DNA template, and sterile ddH for the rest 2 O make up. After mixing, put it into a PCR amplification instrument for amplification.
[0029] 2.4. The PCR amplification reaction program is: 95°C pre-denaturation 5min; 95°C 50s, 52°C 30s, 72°C 50s, 35 cycles; 72°C extension 10min, the reaction is terminated.
[0030] 2.5. Detection and identification of PCR products: Take 10μL of PCR products on a 1.5% agarose gel (containing ethidium bromide) at 98V for 40 minutes and observe with a gel imager. If a band is observed at the position of 580bp, it means that the tested snail sample is the forest onion snail. The test results showed that only the forest onion snail sample had amplified bands at 580bp, and no amplified bands appeared in other samples (see figure 1 ), indicating that this primer has strong specificity.
[0031]

Example Embodiment

[0032] Example 2: PCR detection sensitivity test of forest onion snail
[0033] The forest onion snail DNA stock solution (100ng/μL) extracted in Example 1 was diluted to 10 ng/μL, 1ng/μL, 100 pg/μL, 10 pg/μL, 1 pg/μL, 100 using the 10-fold concentration serial dilution method There are 7 different concentration gradients of fg/μL and 10 fg/μL.
[0034] PCR amplification reaction system: The total volume is 25μL, including 12.5μL of 2×Taq PCR MasterMix mixture, 0.5μL of upstream and downstream primers, 2μL of DNA template, and sterile ddH for the rest 2 O make up. After mixing, put it into a PCR amplification instrument for amplification.
[0035] The PCR amplification reaction program is: 95°C pre-denaturation 5min; 95°C 50s, 52°C 30s, 72°C 50s, 35 cycles; 72°C extension 10min, the reaction is terminated.
[0036] PCR product detection and identification: Take 10μL of PCR product on a 1.5% agarose gel (containing ethidium bromide) at 98V for 40 minutes and observe with a gel imager. If a band is observed at the position of 580bp, it means that the concentration can be detected. result( figure 2 ) Shows that there are still faint bands when the DNA concentration is 10fg/μL, indicating that this method has high sensitivity, which can reach 10fg/μL.
[0037] <110> Inspection and Quarantine Technology Center of Fujian Entry-Exit Inspection and Quarantine Bureau
[0038] <120> A PCR method for detecting and identifying forest onion snails
[0039] <160> 2
[0040] <210> 1
[0041] <211> 18
[0042] <212> DNA
[0043] <213> Artificial sequence
[0044] <400> 1
[0045] acctccttcc tttctact 18
[0046]
[0047] <210> 2
[0048] <211> 18
[0049] <212> DNA
[0050] <213> Artificial sequence
[0051] <400> 2
[0052] gtcaacatct atcccaac 18
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Sensitivity10.0
tensileMPa
Particle sizePa
strength10

Description & Claims & Application Information

We can also present the details of the Description, Claims and Application information to help users get a comprehensive understanding of the technical details of the patent, such as background art, summary of invention, brief description of drawings, description of embodiments, and other original content. On the other hand, users can also determine the specific scope of protection of the technology through the list of claims; as well as understand the changes in the life cycle of the technology with the presentation of the patent timeline. Login to view more.
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Similar technology patents

Guiding wheel structure parameter detecting method

InactiveCN104061857AQuick checkReliable resultsUsing optical meansEngineeringImpressions materials
Owner:JIANGXI SAI WEI LDK SOLAR HI TECH CO LTD

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

ActiveCN103993092AReliable resultsResult Reliability GuaranteeMicrobiological testing/measurementMolecular biologyPlant quarantine
Owner:INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU

Classification and recommendation of technical efficacy words

  • Strong specificity
  • Reliable results

Cas9-foki fusion proteins and uses thereof

ActiveUS20150071899A1Strong specificityReduce the possibilityFusion with DNA-binding domainPeptide/protein ingredientsResearch settingNuclease
Owner:PRESIDENT & FELLOWS OF HARVARD COLLEGE

c-MET MODULATORS AND METHOD OF USE

InactiveUS20070179130A1Strong specificityEliminate side effectsBiocideSenses disorderSignal transductionCellular activity
Owner:EXELIXIS INC

Apparatus and method for detection of syncopes

InactiveUS20100249542A1Reliable resultsRisk minimizationInertial sensorsCatheterVasovagal syncopeTreatment unit
Owner:KONINKLIJKE PHILIPS ELECTRONICS NV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products