2.2. DNA extraction and PCR amplification
The DNA of herbal materials was
extracted with a plant genomic DNA extraction kit (Tiangen Biochemical
Technology (Beijing) Co., Ltd, China), and partially optimized regarding
the DNA barcoding principles for traditional Chinese herbal medicine
(Chen et al., 2014a) and previous research (Liu et al., 2017).
Then
four regions, ITS2, psbA-trnH , matK, and rbcL , were
selected for DNA barcodes. Their primers and amplification conditions
were set in accordance with the
previous study (Chen et al., 2010). PCR products were confirmed by gel
electrophoresis on 1.0% agarose gels after staining with GelRed nucleic
acid gel stain (Biotium, USA).
The sample pretreatment and meta-genomic DNA extraction of the mock and
pharmaceutical samples were performed following the previously published
related research protocols (Liu et al., 2021a).100 mg of each sample was
collected for sample pretreatment. DNA concentration was evaluated using
spectrophotometric measurement of absorbance at 260 nm wavelength
(NanoDrop one, Thermo Fisher Scientific
Inc., USA).