Species identification of white false hellebore (Veratrum album subsp. oxysepalum) using real-time PCR

https://doi.org/10.1016/j.forsciint.2017.02.002Get rights and content

Highlights

  • Veratrum album and V. stamineum often cause food poisoning by the accidental ingestion.

  • A TaqMan real-time PCR method was developed to identify V. album and V. stamineum within 30–60 min.

  • The lower detection limit was less than 10 pg of DNA.

  • Mixed samples, cooked samples, and simulated gastric contents were successfully identified.

  • A multiplex assay of two regions was also possible.

Abstract

Food poisoning is frequently caused by the accidental ingestion of toxic plants that possess strong morphological similarities to edible plants. False helleborine (Veratrum album) is one of the most common plants involved in such accidents. In cases of poisoning by toxic plants, rapid and accurate identification, usually based on the morphological or chemical analysis of plant parts, is required for appropriate medical treatment or forensic investigation. However, morphological examinations require experience in systematic botany because the samples are fragmentary, and chemical analysis of natural compounds can be difficult. In this study, we developed a TaqMan real-time PCR method using trnH-psbA and trnL-trnF that could be carried out in 30–60 min. The lower detection limit was less than 10 pg of DNA and the primer sets were specific to V. album and Veratrum stamineum. Mixed samples, cooked samples, and simulated gastric contents were successfully identified, and a multiplex assay of two regions was also possible. These results indicate that the TaqMan real-time PCR analysis is a very effective method to detect small samples of V. album and V. stamineum accurately and rapidly in poisoning cases.

Introduction

Some toxic plants show strong morphological similarities to edible plants or herbs, and poisoning is frequently caused by accidental ingestion [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]. Collecting and eating edible wild plants, both raw and cooked, is common in some countries, and all parts of the plant body, such as the leaf, sprout, stem, and root, are eaten. However, every year, poisoning is caused by misunderstandings regarding wild edible plants [12]. In the case of poisoning by toxic plants, rapid and accurate identification is required for appropriate medical treatment or forensic investigation. If edible plants are contaminated by toxic plants when they are collected, it is assumed that both plants are mixed during cooking and eaten together. In such cases, it is necessary to confirm that edible plants do not interfere with specific detection. Cooked materials or gastric contents also often must be tested. The cause of plant poisoning is conventionally identified by morphological or instrumental analyses of toxic compounds in plant parts. However, morphological examinations require experience in systematic botany because samples are fragmented. Some tissues occasionally show similar characteristics to those of unrelated toxic plants and thus are unsuitable for rapid identification [13], [14]. For instrumental analyses, the identification of natural complex compounds in a short time is difficult and requires experience. The detection limits of such natural compounds are sometimes too high for successful detection [6]. Furthermore, it is sometimes difficult to prepare samples, especially for mixed with food materials [15], [16].

White false hellebore (Veratrum album L. subsp. oxysepalum (Turcz.) Hultén (Melanthiaceae)) and another species of Veratrum (V. stamineum) are the most common causative plants of poisoning [2], [3], [4], [5], [6], [7], [8], [13], [17]. In Europe, V. album is often confused with Gentiana lutea, which is an ingredient in homemade gentian wine [3]. Another type of poisoning caused by the ingestion of V. album roots as herbal tea has been reported [2], [7]. V. album grows primarily in the understories of cool temperate forests or marshy meadows, and contains alkaloids such as protoveratrine, jervine, cyclopamine, and veratramine [4], [13], [18], [19], [20]. Some V. album alkaloids have antihypertensive effects and were used as antihypertensive medicines in the United States of America in the early 1950s [4]. V. stamineum is morphologically similar to V. album and includes the same alkaloids as V. album [13]. Morphological features of the sprouts of V. album and V. stamineum are similar to those of edible Allium victorialis and Hosta montana. In humans nausea, vomiting, and oral paraesthesia develop shortly after ingestion, in some cases resulting in death [4], [13]. Colchicum autumnale, which is another major causative plant of accidental poisoning, also has strong morphological similarities to A. victorialis and H. montana [1], [10], [12], [21], [22]. C. autumnale is currently a major widespread garden plant. This causes difficulties in determining the cause of poisoning.

For chemical analyses, alkaloids of V. album are commonly determined by liquid chromatography–mass spectrometry (LC–MS) [4], [6], [23]. The detection limits for Veratrum alkaloids, especially when mixed with food materials, are relatively high, and detection failure occasionally occurs [6], [16]. The application of other techniques to these samples, for example those based on DNA, may be useful for medical treatment and forensic investigation. However, DNA-based detection methods for V. album have not been reported.

DNA barcoding, which uses short regions of DNA from botanical samples such as trnH-psbA and trnL-trnF for species identification, is regularly performed in forensic science [24], [25], [26], [27]. More and more sequences of DNA barcoding regions, including those for V. album, are being registered in genetic databases, allowing accurate species identification. However, this method is time-consuming, and unsuitable for mixed samples. TaqMan-based real-time PCR (TaqMan PCR) enables accurate identification utilizing target-specific sequences and requires only a small amount of template DNA. TaqMan PCR is also suitable for the analysis of mixed samples [28], [29]. Therefore, this method has been used to identify animal species, bacterioplankton in the body, cannabis, and toxic plants in forensic science [10], [28], [30], [31], [32], [33], [34], [35].

In this study, we developed a sensitive, rapid, and specific detection method for the identification of V. album and V. stamineum. TaqMan PCR was used with the trnH-psbA and trnL-trnF regions. The lowest detection limit was less than 10 pg of DNA and the two primer sets were specific to V. album and V. stamineum. TaqMan PCR could identify not only specimens obtained from botanical gardens, but also products purchased from flower shops. TaqMan PCR could be performed within 30 min–1 h. Mixed samples, cooked samples, and simulated gastric contents were identified as V. album successfully, and a multiplex assay of two regions could be performed. These results indicated that TaqMan PCR is a very effective method to detect small samples of V. album and V. stamineum accurately and rapidly.

Section snippets

Samples and DNA preparation

Target and allied plants are shown in Table 1. Specimens were provided by the Tokyo Metropolitan Medicinal Plant Garden (Nos. 2, 13, 15, and 16), Meiji Pharmaceutical University (Nos. 9, 14, 17, 21, 22, and 23), Nikko Botanical Garden (Nos. 1, 10, 11, 12, 27, and 29), and Tokyo University of Science (Nos. 20, 25, and 26). Market materials were purchased from flower shops in Japan in Aomori Prefecture (Nos. 3–5: 3 samples, 18, 19), Iwate Prefecture (Nos. 6–8: 3 samples), Kyoto Prefecture (No.

Analytical sensitivity and specificity of the real-time PCR assay for V. album and V. stamineum

To identify V. album, two primer sets targeting the trnH-psbA and trnL-trnF regions were designed. The trnH-psbA amplicon was 96 bp and the trnL-trnF amplicon was 178 bp.

The sensitivity of the PCR assay was evaluated using 10-fold serial dilutions of purified genomic DNA (Fig. 1). The detection limits for trnH-psbA and trnL-trnF using V. album were 1 pg and 10 pg, respectively. The linear calibration curve was obtained in a range from 1 pg to 1 ng (trnH-psbA) or from 10 pg to 1 ng (trnL-trnF). Both

Discussion

In the present study, we developed a TaqMan PCR assay that can be used for the rapid, sensitive, and species-specific detection of V. album and V. stamineum. The application to simulated poisoning cases was also tested in detail.

Standard curves with high confidence utilizing the trnH-psbA and trnL-trnF regions were generated (Fig. 1), demonstrating that the quantification of target DNA is achievable. The detection limit for the trnH-psbA primer set (1 pg) was lower than that of the trnL-trnF

Acknowledgments

The authors are grateful to Mr. Takahiko Sugimoto of the Mitsubishi Tanabe Pharma Corporation for technical advice regarding real-time PCR and Mr. Hiroshi Katsumoto of Life Technologies for help with primer design.

The authors thank Prof. Kiyotaka Koyama (Meiji Pharmaceutical University), Dr. Tatsuhiko Suzuki (Tokyo University of Science), and Nikko Botanical Garden for providing samples.

This work was supported by JSPS KAKENHI Grant Number 20392269.

References (47)

  • S. Kanthaswamy et al.

    Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification

    Forensic Sci. Int. Genet.

    (2012)
  • C. Robino et al.

    Evaluation of DNA typing as a positive identification method for soft and hard tissues immersed in strong acids

    Leg. Med. (Tokyo)

    (2015)
  • H. Kato et al.

    Molecular systematics of the Trilliaceae sensu lato as inferred from rbcL sequence data

    Mol. Phylogenet. Evol.

    (1995)
  • D.M. Schieltz et al.

    Analysis of active ricin and castor bean proteins in a ricin preparation, castor bean extract, and surface swabs from a public health investigation

    Forensic Sci. Int.

    (2011)
  • N. Brncic et al.

    Accidental plant poisoning with Colchicum autumnale: report of two cases

    Croat. Med. J.

    (2001)
  • A. Marinov et al.

    Electrocardiographic studies of patients with acute hellebore (Veratrum album) poisoning

    Vutr. Boles.

    (1987)
  • Y. Gaillard et al.

    LC-EI-MS determination of veratridine and cevadine in two fatal cases of Veratrum album poisoning

    J. Anal. Toxicol.

    (2001)
  • B. Zagler et al.

    Dietary poisoning with Veratrum album — a report of two cases

    Wien. Klin Wochenschr.

    (2005)
  • T. Grobosch et al.

    Accidental intoxication with Veratrum album

    J. Anal. Toxicol.

    (2008)
  • C. Rauber-Luthy et al.

    Low-dose exposure to Veratrum album in children causes mild effects — a case series

    Clin. Toxicol. (Phila.)

    (2010)
  • I. Gilotta et al.

    Accidental poisoning with Veratrum album mistaken for wild garlic (Allium ursinum)

    Clin. Toxicol. (Phila).

    (2010)
  • A. Bouziri et al.

    Datura stramonium L. poisoning in a geophagous child: a case report

    Int. J. Emerg. Med.

    (2011)
  • J. Fuchs et al.

    Acute plant poisoning: analysis of clinical features and circumstances of exposure

    Clin. Toxicol. (Phila)

    (2011)
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