Method for testing and identifying cepaea hortensis through polymerase chain reaction (PCR)

[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]

[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