Primers for detecting sugarcane rust and detection method thereof
A detection method and technology for rust, which are applied in the fields of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., which can solve the actual needs of difficult to meet the rapid and high-throughput diagnostic detection, time-consuming and labor-intensive, etc. problem, to achieve the effect of rapid detection method and good primer specificity
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Embodiment 1
[0014] Example 1 Design and synthesis of primers
[0015] According to the sugarcane rust genome sequence (sequence accession number JN035303) reported in the GenBank database, the specific primer PM1 / PM2 for amplifying sugarcane rust was designed, and the target fragment size of the amplification was 593bp, and the primer sequence was as follows:
[0016] PM1: 5'-ATCATCTGTGGGTCAAAC-3'
[0017] PM2: 5'-AATAAAGCCAGAGTAAGC-3'
Embodiment 2
[0018] Example 2 Sugarcane leaf tissue total DNA extraction:
[0019] (1) Take by weighing 0.5g leaves with obvious symptoms of sugarcane rust and put them into a mortar, add an appropriate amount of liquid nitrogen to grind to powder and then transfer to a 2ml centrifuge tube;
[0020] (2) Add 600ul of CTAB extract preheated at 65°C to the centrifuge tube containing the grinding material, mix it upside down and put it in a constant temperature bath at 65°C to incubate for 2 hours, and invert the centrifuge tube several times every 20 minutes;
[0021] (3) Take out the centrifuge tube containing the grind and CTAB extract and place it at -20°C for 2 min;
[0022] (4) Add 600ul of chloroform:isoamyl alcohol (24:1), mix well, and centrifuge at 12000rpm / min for 10min;
[0023] (5) Take the supernatant and add 2 / 3 volume (340ul) of isopropanol, mix gently; place overnight at -20°C;
[0024] (6) Centrifuge at 12000rpm / min for 10min at room temperature;
[0025] (7) The supernata...
Embodiment 3
[0029] The establishment of embodiment 3 PCR amplification method:
[0030] Using the total DNA as a template, carry out PCR reaction. 25ul reaction system for PCR reaction, that is, add in a 200ul PCR reaction tube: 2×Taq PCR MasterMix 6.0ul, PM1 (20umol / L) 1.0ul, PM2 (20umol / l) 1.0ul, DNA template 1.0ul, sterilized wxya 2 O 16ul, short-speed centrifugation for 8-10 seconds after adding, put into PCR instrument, pre-denaturation at 94°C for 5min; denaturation at 94°C for 1min, annealing at 53°C for 50s, extension at 72°C for 1min, 34 cycles; extension at 72°C for 5min, 4 cycles Store at ℃.
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