Method of identifying arabica coffee and robusta coffee
A small seed and coffee technology, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of inaccurate detection results and complicated methods, and achieve the effect of solving difficult identification, accurate identification, and easy operation
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Embodiment 1
[0040] Embodiment 1: method of the present invention
[0041] Extraction of coffee total DNA:
[0042]The total coffee DNA was extracted using a commercially available plant genomic DNA extraction kit, and the operation method is detailed in the kit instructions.
[0043] PCR amplification:
[0044] The PCR amplification system is a 20ul system, including: coffee total DNA 1μL, 10×Buffer 2μL, 2.5mM dNTPs 1.6μL, 25mM MgCl 2 1.2 μL, 0.4 μL of the primer of the sequence shown in SEQ ID NO:1, 0.4 μL of the primer of the sequence shown in SEQ ID NO:2, 0.1 μL of 5U Taq enzyme, ddH 2 O13.3 μL.
[0045] Landing PCR reaction procedure:
[0046] Pre-denaturation at 95°C for 3 minutes;
[0047] Denaturation at 95°C for 30s, renaturation at 68°C for 45s (reduced by 1°C for each cycle thereafter), extension at 72°C for 1min, 15 cycles;
[0048] Denaturation at 95°C for 30s, renaturation at 53°C for 45s, extension at 72°C for 1min, cycle 25 times;
[0049] Extend at 72°C for 5 minute...
Embodiment 2
[0056] Embodiment 2: method of the present invention
[0057] Extraction of coffee total DNA:
[0058] The total coffee DNA was extracted using a commercially available plant genomic DNA extraction kit, and the operation method is detailed in the kit instructions.
[0059] PCR amplification:
[0060] The PCR amplification system is a 20ul system, including: coffee total DNA 1μL, 10×Buffer 2μL, 2.5mM dNTPs 1.6μL, 25mM MgCl 2 1.2 μL, 0.4 μL of the primer of the sequence shown in SEQ ID NO:1, 0.4 μL of the primer of the sequence shown in SEQ ID NO:2, 0.1 μL of 5U Taq enzyme, ddH 2 O13.3 μL.
[0061] General PCR reaction procedure:
[0062] Pre-denaturation at 95°C for 3 minutes;
[0063] Denaturation at 95°C for 30s, renaturation at 57°C for 45s, extension at 72°C for 1min, cycled 35 times;
[0064] Extend at 72°C for 5 minutes and store at 4°C.
[0065] Electrophoresis of PCR amplification products:
[0066] PCR amplification products were detected by electrophoresis usi...
Embodiment 3
[0071] Embodiment 3: Verification of the accuracy of the method of the present invention
[0072] Using the methods of Example 1 and Example 2, 20 samples of medium-grain coffee and small-grain coffee were identified. See Table 1 for details of the medium and small coffee samples used, and see the corresponding electrophoresis test results. figure 1 .
[0073] In Table 1, the test results of the arabica coffee samples
[0074]
[0075]
[0076] It can be seen from the test results that a single-band product of 90bp or 100bp was amplified on the medium-grain coffee sample, and a double-band product of 90bp and 110bp was amplified on the small-grain coffee sample, and the accuracy of the identification result was 100%. %.
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