All experiments were performed in accordance with the guidelines for the use and care of animals of the Center for Laboratory Animal Care, China Academy of Chinese Medical Sciences. All experimental protocols were approved by the Research Ethics Committee of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China. A total of 269 samples, including 47 original animals (dry samples from local stores), 19 specimens (dry and dissected samples from Kunming Institute of Zoology, Chinese Academy of Sciences), and 203 medicinal materials belonging to 16 species of G. gecko and its adulterants were collected from localities, such as Anguo herb market and Bozhou herb market (see Fig. 1 and Supplementary Table S1). A total of 274 COI sequences were analyzed, including 269 sequences obtained from this study and five sequences downloaded from GenBank. All the samples of this study were deposited as Voucher at the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, and the obtained 269 COI sequences have been submitted to GenBank.
In addition, 15 batches of five kinds of related products were purchased from online stores and drugstores.
DNA extraction, amplification, and sequencing
In 75% ethanol 10-30 mg of underlying muscles of specimens were rinsed then pulverized using a DNA Extraction Grinder (Sceintz Biotech Co., China). Genomic DNA was extracted using an Animal Genomic DNA Kit (Tiangen Biotech Co., Ltd, Beijing, China). COI sequences were amplified with universal primers LCO1490/HCO2198 according to Standard DNA Barcodes of Chinese Materia Medica in Chinese Pharmacopoeia [11, 28, 29]. The PCR products were examined using 1.0% agarose gel electrophoresis. The purified PCR products were bi-directionally sequenced using an ABI 3730 XL sequencer (Applied Biosystems Inc.).
Sequence analysis and primers design
The attained trace files were assembled and trimmed using CodonCode Aligner V 3.0 (CondonCode Co., USA). Then the 269 COI sequences were verified in the NCBI database and DNA Barcoding System for Identifying Herbal Medicine (http://www.tcmbarcode.cn/en/). A total of 274 COI sequences were analyzed using MEGA 5.0 software, including calculating intra- and inter-specific distances and constructing a neighbor-joining (NJ) tree based on the Kimura 2-Parameter model. Sequentially, two pairs of specific primers of G. gecko were designed based on COI regions of G. gecko (658 bp) using Primer 5.0 software: COISF2: 5ꞌ-GCCTCCGCGAGTGTG-3ꞌ, COISR2: 5ꞌ-CGTGTATTGGGTTATGCTTGG-3ꞌ; and COISF3: 5ꞌ-AAAATGAAAACCCCAAG-3ꞌ, COISR3: 5ꞌ-TACGAACGGTCAACAA-3ꞌ. A reaction mixture comprising of 12.5 μL 2 × Taq PCR Master Mix (Aidlab Biotechnologies Co., China), 1 μL of each primer (2.5 μM), 1 μL genomic DNA, and 9.5 μL ddH2O was prepared. The PCR amplification conditions of COISF2/COISR2 were initially set up as 94 ℃ for 5 min, followed by 40 cycles of 94 ℃ for 30 s, 61 ℃ for 30 s, and 72 ℃ for 30 s, and a final extension at 72 ℃ for 10 min. The amplification of COISF3/COISR3 started with 94 ℃ for 5 min, followed by 40 cycles of 94 ℃ for 30 s, 50 ℃ for 30 s, and 72 ℃ for 30 s, and a final extension temperature at 72 ℃ for 5 min.
The genomic DNA extracted from G. gecko was diluted to a series of concentrations (100 ng/μL, 10 ng/μL, 1 ng/μL, 100 pg/μL, 10 pg/μL, and 1 pg/μL) and then amplified with the specific primers to determine the minimum amplifiable concentration.
Distinguishing G. gecko from related products
Fifteen batches of functional foods and Chinese patent medicines, including three batches of functional foods (Renshen Gejie Powder), twelve batches of Chinese patent medicines (three batches of Gejie Dingchuan Pill, Gejie Dingchuan Capsule, Shenge Pingchuan Capsule, and Gejie Dangshen Syrup each), allegedly containing G. gecko were collected from online stores and drugstores. DNA was extracted from all samples in triplicates. Using nuclear separation liquid (100 mM Tris-HCl (pH 8.0), 20 mM EDTA (pH 8.0), 700 mM NaCl, 2% PVP-40, and 0.4% β-mercaptoethanol), 0.5-1.0 g of powdered samples, 5-6 g of pills, and 10 g of syrup were rinsed 1-5 times until the supernatant liquid became lightly colored or colorless . Genomic DNA of these functional foods and Chinese patent medicines was extracted using an Animal Genomic DNA Kit (Tiangen Biotech Co., Ltd, Beijing, China) and amplified with universal primers of COI region and specific primers to examine the existence of G. gecko.