Dendrobium huoshanense specific molecular marker and detection method thereof
A specific technology of Dendrobium Huoshanense, applied in the field of molecular identification of traditional Chinese medicine, can solve the problem of being unable to distinguish Dendrobium Huoshanense
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Embodiment 1
[0045] Embodiment 1, Huoshan Dendrobium specific SNP site
[0046] 1. Sequencing and sequence analysis were performed on 68 species of Dendrobium including all species of the Dendrobium Huoshanense complex, and a SNP molecular marker that only existed in Dendrobium Huoshanense was found on the genome sequence.
[0047] The SNP site and its nearby nucleotides are shown in Sequence 1 of the Sequence Listing, wherein the 190th nucleotide is the SNP site, which is a G / T polymorphism.
[0048] 2. Design primers for the SNP sites in step 1, as shown in Table 1.
[0049] Table 1 Primer Information
[0050]
[0051] 3. Establishment of the method for identifying Dendrobium Huoshanense:
[0052] (1) extract the genomic DNA of the sample to be tested;
[0053] (2) Using the genomic DNA obtained in step (1) as a template, the primer HSSH-1.F and primer HSSH-1.R prepared in step 2 were used for PCR amplification to obtain the amplified product.
[0054] PCR amplification reaction s...
Embodiment 2
[0058] Example 2, Validation of the method for identifying Dendrobium Huoshanense based on specific PCR
[0059] Samples to be tested: 103 Dendrobium materials from known species from different origins shown in Table 2. All the samples in the table are in compliance with the relevant provisions of the "Chinese Pharmacopoeia", "Compendium of Materia Medica" and other medicinal materials. Through the identification, the real medicinal materials of each flavor are consistent with the names, and the quality meets the standards.
[0060] The detection method established in step 3 of Example 1 was used to detect the sample to be tested. The results of electrophoresis are shown in Table 2 and figure 1 . The results of the specific PCR detection by the method of Example 1 are consistent with the actual situation, and the detection accuracy is as high as 100%. figure 1 Electrophoresis results of PCR amplification products of some samples.
[0061] The 213bp target band obtained fr...
Embodiment 3
[0067] Embodiment 3, sensitivity
[0068] The samples to be tested are: randomly select 2 from the 38 samples of Dendrobium huoshanense in Table 2 (numbered 12 and 28 in Table 2), and randomly select 1 from 4 samples of Dendrobium dendrobii in Table 2 (in Table 2 The number is 72), and one is randomly selected from the 2 samples of Dendrobium nobile in Table 2 (the number is 83 in Table 2).
[0069] 1. Extract the genomic DNA of the sample to be tested.
[0070] 2. Use ddH 2 O dilute the DNA solution obtained in step 1 to obtain the respective dilutions.
[0071] 3. Use the dilution obtained in step 2 as a template, and use the primer pair prepared in Example 1 to perform PCR amplification.
[0072] PCR amplification reaction system (25 μL): 10×Taq buffer 2.5 μL, 2.5 mmol / L dNTPs 1 μL, primer HSSH-1.F 0.25 μL, primer HSSH-1.R 0.25 μL, Taq DNA polymerase 1U, DNA Template about 20ng, make up to 25μL with sterilized distilled water. Both primer HSSH-1.F and primer HSSH-1.R w...
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