Universal primer for ITS2 sequence amplification, method for identifying traditional Chinese medicine materials as well as kit
A technology of sequence amplification and universal primers, which is applied in the fields of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0025] The design of embodiment 1 ITS2 sequence amplification universal primer
[0026] 1. Massive data analysis: Compared with ITS2F / ITS3R published in 2010, the number of ITS sequences in GenBank has increased from about 300,000 to about 800,000. The inventors of the present application downloaded and obtained all about 800,000 sequences from GenBank, sorted and analyzed them, and used them as the basic data for the design of new primers for ITS2, and the data are more representative.
[0027] 2. K-mer modeling analysis: The traditional method of searching for conserved regions through multiple sequence alignment for primer design is not only time-consuming and labor-intensive, but also easily misses important species taxa. The inventor adopts a brand-new primer design idea, first conducts 18-25bp k-mer analysis on the 5.8S rRNA region and 28S rRNA region, and screens candidate k-mer data sets covering all groups through modeling and frequency statistics, and then k-mer -me...
Embodiment 2
[0033] Example 2 ITS2 Sequence Amplification General Primer ITS2F1 / ITS2R1 PCR Amplification of Genomic DNA of Chinese Medicinal Materials
[0034] The PCR amplification of Chinese medicinal material genomic DNA comprises the following steps:
[0035] Step 1, pretreating the Chinese medicinal materials for testing and extracting its genomic DNA;
[0036] For DNA extraction, Tiangen Plant Genomic DNA Extraction Kit (Tiangen Biotech Co., China) was used, and its operation procedure was modified to obtain high-quality DNA. The specific operation steps are as follows:
[0037] a) Weigh about 10 mg of dried leaves, wash them with 70% ethanol, dry them with absorbent paper, put them into EP tubes and grind them on a MM400 ball mill (Retsch, Germany).
[0038] b) Take 700ul 65°C preheated buffer GP1 and quickly add it to the centrifuge tube containing the powder (add mercaptoethanol to the preheated GP1 before the experiment to make the final concentration 0.1%), quickly invert and m...
Embodiment 3
[0055] Example 3 Comparison of PCR amplification efficiency between ITS2 sequence amplification universal primer ITS2F1 / ITS2R1 of the present invention and 2015 "People's Republic of China Pharmacopoeia" recommended primer ITS2F / ITS3R
[0056] In this example, the above two sets of primers ITS2F1 / ITS2R1 and ITS2F / ITS3R were used to perform PCR amplification on the genomic DNA of representative medicinal plants of 67 families respectively, and the success rate of the amplification was compared. The results are shown in the following table :
[0057]
[0058]
[0059]
[0060] From the above results, it can be seen that the PCR amplification success rate of the ITS2 sequence amplification general primer ITS2F1 / ITS2R1 provided by the present invention is as high as 95.52%.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More