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Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM)

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      Abstract

      Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.

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      Most cited references 34

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      Basic local alignment search tool.

      A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
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        Search and clustering orders of magnitude faster than BLAST.

         Robert Edgar (2010)
        Biological sequence data is accumulating rapidly, motivating the development of improved high-throughput methods for sequence classification. UBLAST and USEARCH are new algorithms enabling sensitive local and global search of large sequence databases at exceptionally high speeds. They are often orders of magnitude faster than BLAST in practical applications, though sensitivity to distant protein relationships is lower. UCLUST is a new clustering method that exploits USEARCH to assign sequences to clusters. UCLUST offers several advantages over the widely used program CD-HIT, including higher speed, lower memory use, improved sensitivity, clustering at lower identities and classification of much larger datasets. Binaries are available at no charge for non-commercial use at http://www.drive5.com/usearch.
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          UCHIME improves sensitivity and speed of chimera detection

          Motivation: Chimeric DNA sequences often form during polymerase chain reaction amplification, especially when sequencing single regions (e.g. 16S rRNA or fungal Internal Transcribed Spacer) to assess diversity or compare populations. Undetected chimeras may be misinterpreted as novel species, causing inflated estimates of diversity and spurious inferences of differences between populations. Detection and removal of chimeras is therefore of critical importance in such experiments. Results: We describe UCHIME, a new program that detects chimeric sequences with two or more segments. UCHIME either uses a database of chimera-free sequences or detects chimeras de novo by exploiting abundance data. UCHIME has better sensitivity than ChimeraSlayer (previously the most sensitive database method), especially with short, noisy sequences. In testing on artificial bacterial communities with known composition, UCHIME de novo sensitivity is shown to be comparable to Perseus. UCHIME is >100× faster than Perseus and >1000× faster than ChimeraSlayer. Contact: robert@drive5.com Availability: Source, binaries and data: http://drive5.com/uchime. Supplementary information: Supplementary data are available at Bioinformatics online.
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            Author and article information

            Affiliations
            [1 ]Trace and Environmental DNA laboratory, Department of Environment and Agriculture, Curtin University , Kent St, Bentley, WA, 6102, Australia
            [2 ]Separation Science and Metabolomics Laboratory and the Advanced Mass Spectrometry Facility, Murdoch University , South St, Murdoch, WA, 6150, Australia
            [3 ]School of Veterinary and Life Sciences, Murdoch University , South St, Murdoch, WA, 6150, Australia
            [4 ]School of Medical Sciences, The University of Adelaide, Frome Rd , Adelaide, SA, 5005, Australia
            [5 ]Forensic Science SA , Adelaide, SA, 5000, Australia
            [6 ]Centre for Comparative Genomics, Murdoch University , South St, Murdoch, WA, 6150, Australia
            [7 ]LotteryWest State Biomedical Facility Genomics, School of Pathology and Laboratory Medicine, University of Western Australia , 35 Stirling Hwy, Crawley, WA, 6009, Australia
            [8 ]Department of Diagnostic Genomics, Pathwest Laboratory Medicine WA, QEII Medical Centre , Hospital Ave, Nedlands, WA, 6009, Australia
            [9 ]Trace Research Advanced Clean Environment (TRACE) Facility, Department of Physics, Astronomy and Medical Radiation Sciences, Curtin University , Kent St, Bentley, WA, 6102, Australia
            Author notes
            Journal
            Sci Rep
            Sci Rep
            Scientific Reports
            Nature Publishing Group
            2045-2322
            10 December 2015
            2015
            : 5
            26658160
            4675079
            srep17475
            10.1038/srep17475
            Copyright © 2015, Macmillan Publishers Limited

            This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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