Specific primers, kits, methods and applications for identifying Morchella sclerotiorum
A kind of technology of Morchella latifolia and test kit, which is applied in the field of biology and achieves the effects of improving identification efficiency, shortening reaction time and short reaction time
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Embodiment 1
[0069] Example 1: Preparation of primers for Morchella serrata
[0070] 1. Sample source
[0071] Information on the source of morels samples
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[0074] 2. Experimental instruments and reagents
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[0078] 3. Experimental method
[0079] 3.1. DNA extraction
[0080] Take about 30 mg of the fruiting body of the dried Morchella fungus, grind it on an MM400 ball mill, and use the high-efficiency plant genomic DNA kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the total DNA, and the extract is divided and placed at -20 ℃ for later use .
[0081] 3.2. ITS universal primer PCR amplification reaction
[0082] Using the fungal universal ITS sequence as the Morel DNA barcode, the nucleotide sequence is:
[0083] ITS5F: 5'-GGAAGTAAAAGTCGTAACAAGG-3';
[0084] ITS4R: 5'-TCCTCCGCTTATTGATATGC-3'.
[0085] PCR amplification system: 2x PrimeSTAR Max premix 12.5μL, 10μM / L fungal universal primers ITS5F / ITS4R 1μL...
Embodiment 2
[0092] Example 2: PCR amplification conditions and procedure studies
[0093] 1. Study on annealing temperature (60℃ / 62℃ / 64℃ / 66℃ / 68℃)
[0094] Select the nucleotide sequence: MI-4F: 5'-GTTATGATTCTGACGTCGGC-3'; MI-4R: 5'-CACCAGGGCTAGTAGCTTTAC-3', as the ladder-specific primer pair, according to Tm=4℃(G+C)+2 The Tm value of the primer pair was calculated as 57.59°C in °C (A+T). Taking the Tm value of the primer as a reference, the annealing temperature gradient test was set, and the temperature was 60°C / 62°C / 64°C / 66°C / 68°C.
[0095] PCR amplification system: 2x PrimeSTAR Max premix 12.5μL, 10μM / L MI-4F / MI-4R primers 1μL each, DNA template 1μL (concentration 50ng / μL), ddH 2 O make up to 25 μL.
[0096] The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 45sec, annealing at 60°C / 62°C / 64°C / 66°C / 68°C for 45sec, extension at 72°C for 1min, 35 cycles; extension at 72°C for 10min.
[0097] The PCR products were analyzed by agaro...
Embodiment 3
[0115] Example 3: Durability Study
[0116] 1. Different template amounts (5ng / μL / 1ng / μL / 0.5ng / μL / 0.1ng / μL / 0.05ng / μL)
[0117] PCR amplification system: 12.5 μL of 2x PrimeSTAR Max premix, 1 μL of 10 μM / L MI-4F / MI-4R primers, 1 μL of DNA template diluted 10 times / 50 times / 100 times / 500 times / 1000 times at a concentration of 50ng / μL , ddH 2 O make up to 25 μL.
[0118] The PCR amplification program was as follows: pre-denaturation at 98 °C for 1 min; denaturation at 98 °C for 15 sec, annealing at 68 °C for 15 sec, extension at 72 °C for 10 sec, 35 cycles; extension at 72 °C for 5 min.
[0119] Agarose gel electrophoresis: PCR product 6μL+1μL 10x Loading Buffer, voltage 110V, time 35min, a unique band can be obtained between 100-250bp.
[0120] The initial concentration range of the DNA template amount of the morel-specific primers was 50ng / μL, and the DNA template amount of the morel-specific primers was diluted 10 times / 50 times / 100 times / 500 times / 1000 times. The result i...
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