Taq mastermix

Two 5 bp tags were designed and added to the 5′ end of the universal ITS2 and psbA-trnH primers (Kress et al., 2005 (link); Chen et al., 2010 (link)) to distinguish the sequences obtained from different regions. Different primers were used to the amplify ITS2 and psbA-trnH regions in the different samples. The designed primers used in the PCR for the detection of the different samples are listed in Supplementary Tables S3, S4. PCR amplifications were performed according to the DNA barcoding protocol recorded in the Chinese Pharmacopoeia and were carried out in an Applied Biosystems Veriti^TM Thermal Cycler (Thermo Fisher Scientific, Inc., United States). For each reaction, 1 μl MgCl₂ (10 mM SBS Genetech Co., Ltd, China) and 2× Taq MasterMix (AidLab Biotechnologies Co., Ltd, China) were added to the PCR master mix. The PCR annealing temperature was increased to 58°C, and 40 cycles were run. A negative control reaction with no DNA template was included in the reactions. The DNA concentrations of the obtained amplicons were assessed via 2% agarose gel electrophoresis, and purification was performed with a QIAquick Gel Extraction kit (Qiagen). The DNA concentrations were measured on an Agilent 2100 bioanalyzer (Agilent Technologies, Inc., United States) with the Qubit platform (Thermo Fisher Scientific, Inc., United States).

Total genomic DNA of CMMs was extracted using a plant genomic DNA extraction kit (Tiangen Biotech Co., Beijing, China) according to the manufacturer’s instructions. Total genomic DNA of TCPMs was extracted in the same way by adding a preprocessing step to rinse with nucleic acid separation solution three times (Xin et al., 2018a (link)). Different pretreatment methods were adopted according to the dosage forms of TCPMs. DNA concentration and purity were determined using a Nanodrop 2000 (Thermo Fisher Scientific Inc., United States).
The amplification of the internal transcribed spacer 2 (ITS2) of nuclear ribosomal genes was performed by the universal primer pair ITS2 2F/3R (2.5 μmol/L) (Table 1). PCRs were performed in 25-µl volumes containing 2 µl of DNA, 12.5 µl of 2x Taq MasterMix (AidLab Biotechnologies Co., Ltd., China), 1 µl of each primer and 8.5 µl of distilled deionized water. The reaction conditions were as follows: 94°C for 5 min; 40 cycles at 94°C for 30 s, 56°C for 30 s, 72°C for 45 s and 72°C for 10 min (Chen et al., 2010 (link)).

Dead larvae or single flies were squashed in 30 uL of squashing buffer containing 10 mM Tris-Hcl (PH 8.0), 1 mM EDTA, 25 mM NaCl, 1 mg/mL proteinase K (Takara, Beijing, China), and incubated at 37 °C for 1 h, followed by heating to 95 °C for 2 min and used as PCR templates. PCR was performed in a 2 x Taq MasterMix (Aidlab Biotech, Beijing, China) under standard PCR procedure, with primers 100–200 bp away from the gRNA targeting site. The PCR products were then digested with T7 Endonuclease I (Viewsolid Biotech, Beijing, China), and T7EI positive F1 PCR products were sequenced for mutants.