Guiding Principles of DNA Barcode Molecular Identification Method for Chinese Medicinal Materials (2)

3.

ITS2 sequence amplification forward primer ITS2F5'-ATGCGATACTTGGTGTGAAT-3';

Reverse primer ITS3R5'-GACGCTTCTCCAGACTACAAT-3'

psbA-trnH sequence amplification forward primer psbAF5'-GTTATGCATGAACGTAATGCTC-3';

The reverse primer trnHR5'-CGCGCATGGTGGATTCACAATCC-3'

COI sequence amplification forward primer HCO21985'-TAAACTTCAGGGTGACCAAAAAATCA-3'; reverse primer LCO14905'-GGTCAACAAATCATAAAGATATTGG-3'

The PCR reaction system is based on 25ul, including 1×PCR buffer (without MgCl), 2.

ITS2 sequence amplification program 94°C for 5 minutes; 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 45 seconds, 35-40 cycles; 72°C for 10 minutes

4.

5.

6.

(1) Sequence splicing: Use professional software with sequence splicing function for sequence splicing on the peak map of bidirectional sequencing, and remove the primer area

(2) Sequence quality and direction: To ensure the reliability of the DNA barcode sequence, it is necessary to remove the weak signal or overlapping peak regions at both ends of the sequencing result.

7.

3.

It should meet the relevant requirements of the 2015 edition of the Chinese Pharmacopoeia, "Guiding Principles for the Verification of Quality Standard Analysis Methods of Traditional Chinese Medicines" (General Principle 9101)

1.

2.

3.
The comparison verification of primitive species takes the leaves of primitive species confirmed by taxonomists as the object.
This method is used to obtain DNA barcode data and compare it with the DNA barcode data produced by corresponding medicinal materials to avoid contamination by endophytic fungi and ensure the accuracy of the results.
:

Four, matters needing attention

(1) The experimental site should have the basic conditions of a molecular biology laboratory
.

(2) This law is temporarily not applicable to situations where the identification of mixtures and processed products and sulfur fumigation cause inapplicability
.

(3) In order to prevent contamination by foreign microorganisms, the experimental utensils must be autoclaved before the experiment, and the surface of the medicinal materials must be wiped with 75% ethanol
.
Some medicinal materials contain endophytic fungi.
If endophytic fungi exist in the outer tissues of the medicinal materials, the internal tissues are used for experimentation
.
If the fungus is spread over the entire medicinal material, the psbA-trnH barcode must be used for the plant medicinal material (the fungus does not contain the gene fragment), and the ITS2 sequence cannot be used
.
In order to further ensure that the experimental results are not contaminated by fungi, the experimenter can use the BLAST method in the GenBank database to test the obtained ITS2 sequence to ensure accurate sequence identification
.

(4) This method is used to identify the original species of medicinal materials, and the medicinal site cannot be determined
.

(5) When necessary, combine with other identification methods to make comprehensive judgments
.

(6) Determination of threshold within species
.
There is a certain range of variation between different samples of the same species, that is, the intra-species variation threshold
.
Different species and different barcode sequences will affect the range of intra-species variation
.
The range of intraspecies variation (intraspecies genetic distance threshold) of each primitive species should be specified under the category of medicinal materials
.

Related Links: Guidelines for DNA Barcode Molecular Identification of Chinese Medicinal Materials (1)