Establishment of conventional PCR and real-time PCR assays for accurate, rapid and quantitative authentication of four mistletoe species

Highlights

• Four mistletoe species inhabit in Korea.

• Only V. coloratum is permit as an authentic ingredient of food and herbal medicine.

• We developed species-specific primers for mistletoe based on matK sequences.

• Multiplex assay was established for species discrimination in conventional PCR.

• Quantitative real-time PCR assays of high sensitivity were developed.

Abstract

Adulterants in processed food and herbal medicines reduce their safety, quality control, or pharmacological efficacy. Four mistletoe species, including Viscum coloratum, inhabit Korea. Leaves and branches of V. coloratum, defined as edible or medicinal mistletoe species in Korean, are used to prepare Korean herbal medicines as well as leached tea. However, other mistletoe species including Taxillus sutchuenensis var. duclouxii, Korthalsella japonica, and Loranthus tanakae are frequently distributed as authentic V. coloratum in Korean markets because of similarities in the branches morphology and Korean names of these species with V. coloratum. Although herbal medicines and food products prepared from the other mistletoe species are inauthentic, they are sold at high prices because of the rarity of these species. Thus, it is important to distinguish between authentic and inauthentic adulterant mistletoe species. In this study, we developed species-specific primer, based on matK sequences, suitable for both conventional PCR and real time PCR (qPCR) assay. These assays allowed rapid discrimination among all four mistletoe species. Moreover, qPCR assay enabled the detection of trace amounts of adulterant mistletoe species in V. coloratum samples. Furthermore, we used these assays to monitor commercial mistletoe products distributed in Korean markets. Our data suggest that these methods would serve as a reliable genetic tool to prevent adulteration and standardize the quality of commercial V. coloratum products.

Introduction

Processed foods derived from medicinal plants are readily used to prevent disease and maintain optimum health. Viscum coloratum (Kom.) Nakai (a synonym of Viscum album var. coloratum Ohwi, Santalaceae), one of four mistletoe species found in Korea, is a representative medicinal plant used as both a herbal medicine and a processed food. For instance, leached tea prepared from V. coloratum is used to treat hypertension, arteriosclerosis, and arthritis (Shen et al., 2011). The other three mistletoe species are Taxillus sutchuenensis var. duclouxii (Lecomte) H.S.Kiu (Loranthaceae), Korthalsella japonica (Thunb.) Engl. (Santalaceae), and Loranthus tanakae Franch. & Sav (Loranthaceae). Although all four species are referred to as mistletoe in Korea, only V. coloratum is permitted as material for use in food products and herbal medicines as defined in the Korean Food Standards Codex (KFSC) and the Korean Herbal Pharmacopoeia (KHP), respectively. Thus, it is important to verify for the purpose of safety and therapeutic efficacy that authentic species listed in KFSC and KHP are used, because unlisted species do not have the same guarantee of safety and pharmacological efficacy as authentic species. In traditional Korean and Chinese medicine, leaves and branches of V. coloratum are used as Visci Ramulus et Folium (Korea Institute of Oriental Medicine, 2019). In the KFSC of the Ministry of Food and Drug Safety (KFDA), only V. coloratum is defined as an authentic food material, implying that the other three mistletoe species are inauthentic and therefore illegal to sell as commercial products. Nevertheless, adulterants are frequently distributed in markets because of morphological similarities between authentic and inauthentic species, errors in nomenclature, profit, and insufficient authentic resources (Cammà et al., 2012). V. coloratum products are distributed in various processed forms, such as ground powder, dried slices, and leached tea (Carloni et al., 2017; Vassou et al., 2015; Xiong et al., 2018). Because processing using physical, chemical, and/or biochemical treatment alters the appearance of raw materials, visual identification of the species comprising the product is very difficult (Mano et al., 2017).

Molecular approaches, such as DNA markers and PCR amplification, provide a reliable method of species identification using trace amounts of DNA (Kim and Kim, 2018; Moon et al., 2017). Recently, DNA barcoding using short nuclear or organellar DNA sequences, such as internal transcribed spacer (ITS) region, matK and rbcL, has been used for species identification in plants (Chen et al., 2010). Among these sequences, rbcL (1.6 kb) is recommended as a universal DNA barcoding region in the Consortium for the Barcode of Life Plant Working Group (CBOL); however, it provides a low resolution and is relatively large in size (Group, 2009; Kim et al., 2016). By contrast, matK provides high species resolution, although it is also unwieldy in size (1.3 kb). DNA barcoding is based on sequence analysis and involves cumbersome processes, such as PCR amplification, electrophoresis, gel extraction, T-vector cloning, sequencing, and similarity searches using the Basic Local Alignment Search Tool (BLAST) (Moon et al., 2017). Furthermore, DNA barcoding requires a full-length sequence of the target region, which is disadvantageous when highly processed foods need to be authenticated. Compared with DNA barcoding, species-specific primers enable faster discrimination among species and are less time consuming (Kim et al., 2016; Liu et al., 2017). Therefore, DNA barcoding–species specific primers strategy is similar to random amplification of polymorphic DNA (RAPD)–sequence characterized amplified region (SCAR) marker or amplified fragment length polymorphism (AFLP)–sequence-tagged site (STS) marker strategies employed as molecular tools for the identification of species used to prepare processed food and herbal medicine (Kumari et al., 2018; Liu et al., 2017). In conventional PCR, multiplex assay is an advanced technique used to simultaneously discriminate between two or more species, thus serving as an efficient and economical method of species identification (Kim et al., 2016; Moon et al., 2017). Real-time PCR (qPCR), using SYBR Green-based singleplex assay, enables accurate species identification as well as quantitative analysis of trace amounts of adulterants present in authentic samples (Al-Kahtani et al., 2017; Iwobi et al., 2017). To determine the severity of adulteration, qPCR should be preferred over conventional PCR.

In this study, we analyzed matK sequences of all four mistletoe species using universal primers, and designed candidate species-specific primers based on multiple sequence alignment of 17 samples. These species-specific primers were validated using conventional PCR and SYBR Green-based qPCR assays. Additionally, we developed a multiplex PCR assay, which enabled faster and more efficient identification of species than a singleplex PCR assay in conventional PCR. We also demonstrated the utility of conventional PCR and qPCR assays for the identification of adulterant species in commercial products.