System and method for jointly identifying Psilocybe cubensis Sing through multiple DNA sites based on HRM
A site and system technology, applied in recombinant DNA technology, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem of inaccurate hallucinogenic mushrooms, shorten the identification time, improve the identification resolution, specificity, etc. Good results
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Embodiment 1
[0043] In this example, through extensive sampling of wild mushrooms distributed in various parts of China, the species is determined, the species related to the Cuban umbrella is screened, the species-specific verification is carried out, and the identification provided by the present invention is determined by comparing the differences in HRM data between species. Species specificity of methods for the Cuban photic umbrella.
[0044] 1. Screening of multiple DNA sites suitable for the Cuban umbrella;
[0045] According to the data information of NCBI (National Center for Biotechnology Information) Genbank database, the genetic loci related to Cuban umbrella: ITS, ITS1, ITS2, RPB1, PC-PT, PC-13 and tEF1α were screened.
[0046] 2. Design amplification primers or select general amplification primers;
[0047] Use the software Primer Premier 5.0 to design primers for the four DNA sites selected by NCBI. The requirements are as follows: (1) The length of the primer is controlle...
Embodiment 2
[0069] In this example, the multi-site HRM analysis method constructed in Example 1 is used to detect the mixture containing Cuban umbrella. The main purpose is to detect common mixture samples, such as samples mixed with human DNA or industrial hemp DNA sample.
[0070] Mix Cuban canopy and industrial hemp DNA and Cuban canopy and human DNA in equal proportions to make a mixture of 1 ng / μL. The mixture was analyzed by real-time fluorescent PCR under the same HRM analysis conditions. The Cuban umbrellas in the samples were effectively amplified, and the primers in the mixture showed a high affinity for the Cuban umbrellas, which were less interfered by human DNA and industrial hemp DNA, and could accurately identify the Cuban umbrellas in the mixture Umbrella, see the typing map of related HRM figure 2 and image 3 .
[0071] like Figure 2-3 As shown, in the Cuban umbrella sample mixed with industrial hemp DNA, while the Cuban umbrella was identified, the ITS, ITS1 and IT...
Embodiment 3
[0073] In this example, the identification system and method constructed in Example 1 are applied to actual cases, and the standard Cuban umbrella multi-DNA locus HRM combination map is used for comparison with case samples.
[0074] The object of analysis is the 6 suspected Cuban umbrella samples obtained in the case, all of which have been ground into powder, making it difficult to judge the appearance. The above-mentioned detection methods were used for sample identification, and the results were as follows: Figure 4 As shown, all of them can be preliminarily identified as Cuban umbrella samples, and can be further identified as Cuban umbrella samples after gene Sanger sequencing and poison chemical analysis.
[0075] At present, there is a lack of uniformity in the molecular identification standards for the Cuban umbrella at home and abroad, and it mainly relies on the molecular information of the ITS sequence for sequence comparison. The method provided by the present in...
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