Method for separating ceratocystis spp from soil
A beak shell and soil technology, which is applied in the field of microorganisms, can solve the problems of enrichment and separation of the long beak shell fungus, difficult to control the dilution ratio of the soil extract, and many experimental equipment and equipment, etc. The effect of separation and purification efficiency, low cost and wide application range
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Embodiment 1
[0036] Embodiment 1: Separation and purification of the long beaked shell fungus
[0037] (1) Rinse fresh and healthy carrots with tap water, remove the skin and sterilize them in ethanol with a volume concentration of 75% for 1 to 2 minutes, dry the surface with clean napkins, and then cut them into thicknesses of 3 to 5 mm And uniform slices, and then use a 2.5cm diameter puncher to take a disc of equal diameter in the center of the carrot slices, and set aside;
[0038] (2) Place the carrot discs in step (1) in a petri dish with a diameter of 9 cm, place 12 carrot discs in each petri dish, overlap each other, and use an electronic balance to take it from Mengzi, Yunnan soil sample, 1g / part; place the weighed soil sample between two overlapping carrot discs, and place a ball of cotton soaked in sterile water in the center of the Petri dish to keep it moist (such as figure 1 ); placed in a constant temperature incubator at 26°C for 3 to 10 days, and observing the enrichment ...
Embodiment 2
[0041] Example 2: Identification of isolates
[0042] (1) Sequence identification
[0043]After the pure bacterium colony of the target bacterium that step (3) obtains is cultivated on the MYEA medium for 2 weeks, use a scalpel (the scalpel handle model is No. 4, and the blade model is No. 24) to scrape the mycelia and The mixture of spores was extracted with the CTAB method for total DNA, and the extracted total DNA was amplified by PCR using the following primer pairs:
[0044] Upstream primer ITS1F: 5'-CTTGGTCATTTAGAGGAAGTAA-3' and
[0045] Downstream primer ITS4: 5'-TCCTCCGCTTATTGATATGC-3',
[0046] The amplified sequence is shown in SEQ ID No.1. The PCR products were detected by 1.5% agarose gel electrophoresis, and then sent to Biomed for sequencing, and the sequencing results were compared and analyzed in the NCBI database to further identify the isolates.
[0047] PCR reaction system:
[0048]
[0049]
[0050] PCR reaction program: pre-denaturation at 95°C ...
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