Molecular marker, primers and probe for identification of tricholoma imbricatum
A technology of molecular marking and capping, which is applied in the field of biological identification, can solve the problems of unreliable identification of bacterial species and difficulties in taxonomy, and achieve the effects of short detection time, accurate identification results, and high accuracy
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Embodiment 1
[0034] Example 1: Extraction of Genomic DNA from Capricatus squamosa.
[0035] The fungus samples of Caplet mushroom were collected from the deciduous broad-leaved forest belt of Guancen Mountain in the Kelan area of Xinzhou, Shanxi Province, and were identified as Trichomonas fungus ( Tricholoma imbricatum ).
[0036] Using the tissue separation method, the whole fruit body was wiped and disinfected with 75% ethanol, and small pieces of tissue were cut from the cap, stipe, gills, and root with a scalpel, and the excised tissue was soaked in 75% ethanol for 30 seconds. Rinse them with sterile water, and inoculate them in PDA medium (200 g of potatoes, 20 g of glucose, 15 g of agar, and 1 L of water) sterilized at 121° C. for 30 min. The inoculated culture medium was placed in a constant temperature incubator at 27°C, and the growth of mycelia was checked every 24 hours.
[0037] After 8 days of culture, the hyphae were collected, and the total DNA of the fungi was extracte...
Embodiment 2
[0038] Example 2: Determination of ITS-specific molecular markers.
[0039] Using the total DNA of Lilac mushroom obtained in Example 1 as a template, the DNA was amplified by PCR using universal primers ITS1 / ITS4, and the PCR product was subjected to capillary sequencing to obtain detailed sequence information.
[0040] The sequencing results were compared and analyzed in the whole nucleic acid database in NCBI, and fragments with high conservation were obtained by screening, and the results of different selected sequences were compared and selected, and finally a characteristic sequence in the sequencing results was determined. The amino acid sequence of the specific gene fragment of Tricholoma tricholoma is shown in SEQ ID NO.1.
[0041] After the homology comparison search, it is determined that the selected target sequence is a DNA sequence with high specificity, which can be used as the target sequence for gene chip detection.
Embodiment 3
[0042] Example 3: Design of primers and nucleic acid probes.
[0043] According to the target sequence determined in Example 2 as a molecular marker, according to the primer and nucleic acid probe design principles, design the special primers shown in SEQ ID NO.2 and SEQ ID NO.3, and the nucleic acid probe shown in SEQ ID NO.4 Needle.
[0044] Primer 1: 5'Hex-TTCTTGAATAAGCTTGGTTG-3'.
[0045] Primer 2: 5'-CCAAGCCTAATCAGCTAGAAG-3'.
[0046] Nucleic acid probe: 5'NH 3 -TTTTTTTTTTTTAGGCGTGCACATACATGCTCCGAAGGAG-3'.
[0047] Wherein, the 5' end of the primer 1 is labeled with a fluorescent reporter group Hex; the 5' end of the nucleic acid probe is connected with an amino group, and the nucleic acid probe is subjected to amination treatment.
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