Deep Sequencing of Plant and Animal DNA Contained within Traditional Chinese Medicines Reveals Legality Issues and Health Safety Concerns Megan L. Coghlan 1 , James Haile 1 , Jayne Houston 1 , Da ´ithı´ C. Murray 1 , Nicole E. White 1 , Paula Moolhuijzen 2 , Matthew I. Bellgard 2 , Michael Bunce 1 * 1 Australian Wildlife Forensic Services and Ancient DNA Laboratory, School of Biological Sciences and Biotechnology, Murdoch University, Murdoch, Australia, 2 Centre for Comparative Genomics, Murdoch University, Murdoch, Australia Abstract Traditional Chinese medicine (TCM) has been practiced for thousands of years, but only within the last few decades has its use become more widespread outside of Asia. Concerns continue to be raised about the efficacy, legality, and safety of many popular complementary alternative medicines, including TCMs. Ingredients of some TCMs are known to include derivatives of endangered, trade-restricted species of plants and animals, and therefore contravene the Convention on International Trade in Endangered Species (CITES) legislation. Chromatographic studies have detected the presence of heavy metals and plant toxins within some TCMs, and there are numerous cases of adverse reactions. It is in the interests of both biodiversity conservation and public safety that techniques are developed to screen medicinals like TCMs. Targeting both the p-loop region of the plastid trnL gene and the mitochondrial 16S ribosomal RNA gene, over 49,000 amplicon sequence reads were generated from 15 TCM samples presented in the form of powders, tablets, capsules, bile flakes, and herbal teas. Here we show that second-generation, high-throughput sequencing (HTS) of DNA represents an effective means to genetically audit organic ingredients within complex TCMs. Comparison of DNA sequence data to reference databases revealed the presence of 68 different plant families and included genera, such as Ephedra and Asarum, that are potentially toxic. Similarly, animal families were identified that include genera that are classified as vulnerable, endangered, or critically endangered, including Asiatic black bear (Ursus thibetanus) and Saiga antelope (Saiga tatarica). Bovidae, Cervidae, and Bufonidae DNA were also detected in many of the TCM samples and were rarely declared on the product packaging. This study demonstrates that deep sequencing via HTS is an efficient and cost-effective way to audit highly processed TCM products and will assist in monitoring their legality and safety especially when plant reference databases become better established. Citation: Coghlan ML, Haile J, Houston J, Murray DC, White NE, et al. (2012) Deep Sequencing of Plant and Animal DNA Contained within Traditional Chinese Medicines Reveals Legality Issues and Health Safety Concerns. PLoS Genet 8(4): e1002657. doi:10.1371/journal.pgen.1002657 Editor: Robert DeSalle, American Museum of Natural History, United States of America Received August 12, 2011; Accepted March 2, 2012; Published April 12, 2012 Copyright: ß 2012 Coghlan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this research was provided by the Australian Research Council (FT0991741) and Murdoch University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: m.bunce@murdoch.edu.au Introduction Traditional Chinese medicines (TCMs) have been an integral part of Chinese culture and the primary medicinal treatment for a large portion of the population for more than 3000 years [1,2]. Outside of Asia there has been, in recent decades, a growing use of TCMs where they are being taken in conjunction with, or as an alternative to, conventional Western medicine [3,4]. The increas- ing popularity of TCM products has seen the monetary value of the industry increase to hundreds of millions of dollars per annum [5], its growth paralleled by the global increase in the use of complementary and alternative medicines. Despite its increased uptake, the therapeutic benefits of only a small number of TCM products have been scientifically validated [6], with their perceived efficacy being based largely on long-standing beliefs [7]. Chinese herbal medicines often contain numerous different plant and animal-derived products that combine to act synergis- tically to affect a desired outcome [8,9]. However, due to the proprietary nature of TCM manufacture, coupled with a lack of industry regulation, the biological origin of contents can be difficult to determine with confidence, leading to questions regarding TCM quality, efficacy and safety [10,11]. Undeclared or misidentified TCM ingredients and adulterants can pose serious health risks to consumers [10,12,13]. These include: allergenic substances [14], plant toxins [7], heavy metals such as mercury, lead, copper and arsenic [15], and pharmaceutically active compounds of undetermined concentration [5]. In the early 1990s the misidentification of the toxic herb Aristolochia fangchi for the anti-inflammatory agent Stephania tetrandra led more than a hundred women to suffer kidney failure, with many later developing cancer of the urinary system [13]. In addition to safety concerns, issues of legality also surround TCMs. These concerns fall into three main categories: matters relating to the trade of endangered species; issues pertaining to honesty of food labelling; and adulteration of samples with drugs. Some TCMs contain plant and animal species [16–18] that fall under the jurisdiction of the Convention on International Trade in Endangered Species (CITES). CITES-listed species (see appendi- PLoS Genetics | www.plosgenetics.org 1 April 2012 | Volume 8 | Issue 4 | e1002657