Introduction

Improvement in the quality of human life has led to increased use of health-enhancing food products. The Functional Food Center (Dallas, Texas, USA) has defined “functional food” as natural food product that contains known or unknown bioactive compounds that exert health benefits (Martirosyan and Singh 2015). The physiological benefits of functional food may include reduced risks of chronic diseases. The ingredients of functional foods with plant origins have been effective in preventing diseases in humans. For example, the health benefits of cranberries have been demonstrated in the suppression of urinary tract infections (Avorn et al. 1994), and a meta-analysis has indicated that garlic lowers blood pressure (Silagy and Neil 1994). Despite the many advantages of functional foods, the commercial production of these foods has several problems such as food fraud, including the deliberate mislabeling and illegal substitution of food ingredients. The illegal products produced as a result of food fraud might threaten human health. In particular, food ingredients derived from plants have been used in the form of dried powders to produce the commercial foods; it is therefore difficult to discriminate between authentic and spurious raw materials. To protect consumer from food fraud such as mislabeling and intended or unintended mixture, food inspectors need to develop various techniques for detecting illegal food products.

Cynanchum is a genus containing about 300 species, belonging to the family Apocynaceae. Cynanchum wilfordii is listed in the Korea Herbal Pharmacopoeia as the original plant of C. wilfordii Hemsley (KFDA 2011). In addition, C. wilfordii is known to have active components in its root extracts, which are known to be effective for alimentation, improving vigor, and nourishing blood in the form of herbal medicines (Atta-ur-Rahman and Choudhary 1999). Additionally, C. wilfordii has been often used as a health food supplement in Asia, and its importance as a functional food ingredient has increased in Korea. Recently, the root tissues of C. auriculatum Royle ex Wight, which is forbidden to be used as a food ingredient in Korea, has been illegally substituted for those of C. wilfordii to produce food products since it is difficult to distinguish between these two species when used as ingredients in commercial food products. The adulteration of C. wilfordii commercial products has become a particularly serious social problem in Korea since April 2015 (Cheongju, Korea Consumer Agency, http://www.kca.go.kr/). About 60% of C. wilfordii products circulated in the market were detected to contain added C. auriculatum.

Chloroplasts are plant organelles that play an important role in photosynthesis. Most chloroplast genomes have a circular structure and are 120–170 kb in size (Clegg et al. 1994; Shaw et al. 2007). Unlike the diploid nuclear genome, the chloroplast genome is haploid, containing approximately 100 genes (McFadden 2001), and exhibits low levels of substitution. For this reason, it has been used as a useful tool for evolutionary studies in several plant species. For example, the MatureasK (matK) gene, located within the intron region of trnK, is highly conserved in plants and has been used to identify species using methods such as DNA barcoding (Selvaraj et al. 2008). Fuse and Tamura (2000) reported that the variation in the stop codon positions of the matK gene is high among the monocotyledons. In many studies, the matK gene has been used to resolve family-as well as species-level relationships (Steele and Vilgalys 1994; Koch et al. 2001; Tamura et al. 2004).

Polymerase chain reaction (PCR)-based methods are rapid and low cost (Mafra et al. 2008). In particular, quantitative real-time PCR (qRT-PCR) is rapid, accurate, and sensitive, and can be used to detect very low levels of target DNA sequences in foods (Overbergh et al. 2003; Malorny et al. 2008). DNA-based methods are being increasingly used to clarify species-level relationships. In addition, they are used to evaluate food authenticity and safety, owing to their high specificity and sensitivity. For example, many studies have reported successful species identification using these methods in foods such as celery (Pafundo et al. 2009), rice (Hwang et al. 2015), almond (Fuchs et al. 2012), meat (Jonker et al. 2008), and seafood (Herrero et al. 2011). In this study, we developed species-specific molecular markers derived from the matK genes of C. wilfordii and C. auriculatum using single nucleotide polymorphisms (SNPs) and then applied these markers to verify the presence of both species in commercial food products.

Materials and methods

Samples

Standard samples of C. wilfordii and C. auriculatum were obtained from Herbal Medicine Research Division, National Institute of Food and Drug Safety Evaluation (Kim et al. 2015). Nineteen commercially available C. wilfordii products, marketed as health-enhancing food, were purchased from local markets (Table 2).

Genomic DNA extraction

Genomic DNA was extracted from 200 mg each of standard and commercial product sample using the i-genomic Plant DNA Extraction Mini Kit (iNtRON Biotechnology, Seongnam, Korea), according to the manufacturer’s protocol. The quantity of the extracts was measured using a Qubit® 2.0 Fluorometer (Invitrogen, Life Technologies, Grand Island, NY, USA) with a Qubit dsDNA BR Assay Kit (Invitrogen™, Life Technologies), according to the manufacturer’s protocol. Their purity was confirmed using agarose gel electrophoresis with a Molecular Imager® Gel DOC™ XR+ System (Bio-Rad, Hercules, CA, USA), and BioPhotometer Plus UV/Vis Photometer (Eppendorf, NY, USA). DNA concentrations were measured and determined to be in the range of 101–114 ng/µL, and the ratio of the absorbance at 260 and 280 nm (A260/280) of about 1.78 was as expected for pure samples (Glasel et al. 1995).

PCR amplification and DNA sequencing

Chloroplast DNA sequences of matK of C. acutum were downloaded from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) in order to design primer pairs to amplify these regions of both plants. The matK gene in both plants was amplified using appropriate primer pairs (Table 1). The PCR analysis was performed in a final volume of 25 μL using the C1000 Thermal Cycler (Bio-Rad™). The PCR reaction mixture contained 10 ng of DNA, 10× buffer, 25 mM MgCl2, 2.5 mM dNTPs, 10 pmol of each primer, and 0.5 U/μL EX Taq DNA polymerase (Takara Bio Company, Kusatsu, Shiga, Japan). The amplification conditions were as follows: 10 min at 95 °C, followed by 40 cycles of 30 s each at 94 °C, 30 s at 60 °C, and 30 s at 72 °C. The amplifications were confirmed on a 1.5% agarose gel electrophoresis with a 1 kb Plus DNA Ladder (Invitrogen, Life Technologies), and visualized using the Molecular Imager® Gel DOC™ XR+ System (Bio-Rad). The amplicons were cloned using the RBC T&A Cloning Kit (Real Biotech Co., Taipei, Taiwan), according to the manufacturer’s protocol and sequenced by a commercial service (Macrogen, Seoul, Korea).

Table 1 Information of primers used in this study
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Quantitative real-time PCR analysis

The obtained sequences were aligned using ClustalW2 (ftp://ebi.ac.uk/pub/software/clustalw2/) to compare the differences between the sequences of the matK genes of both plants. The primer pairs were designed using Beacon Designer™ (PRIMER Biosoft, Palo Alto, CA, USA) and were synthesized by a commercial service (Macrogen, Seoul, Korea), setting up an amplicon size of 151 bp (Fig. 1B).

Fig. 1
figure 1

Sequence alignment of cloned C. wilfordii and C. auriculatum MatK genes. Species-specific primers were designed on the basis of SNPs of both genes (A). Amplified PCR products were electrophoresed in order to confirm cross-reactivity (B)

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qRT-PCR was performed in a final volume of 20 μL using a CFX Connect™ Real-Time PCR Detection System (BIO-RAD™). The reaction mixture contained 10 ng of DNA, 10 μL of SYBR® Green TOP real qPCR 2xPreMIX (Enzynomics™, Daejeon, Korea), and 10 pmol of each primer. The amplification conditions were as follows: 10 min at 95 °C, followed by 45 cycles of 10 s at 95 °C, annealing time at the appropriate annealing temperature (T m) of each primer pair, and 30 s at 70 °C. PCR products were denatured at 95 °C for 10 s and then annealed at 65 °C for 1 min. This step was followed by a melt curve, ranging from 60 to 95 °C, with temperature increments of 0.5 °C every 30 s (Table S1).

Determination of amplification efficiency, correlation coefficient, and limit of detection (LOD)

To evaluate the correlation between Ct values and DNA concentration, standard curves were obtained using tenfold serially diluted DNA samples of C. wilfordii and C. auriculatum at concentrations of 0.0001, 0.001, 0.01, 0.1, 1, and 10 ng. Correlation coefficient (R 2) was determined using the liner regression method (R 2 ≥ 0.98) (Ramakers and Ruijuter 2003). The slope of the standard curve ranges from −3.6 to −3.1 (Mazzara et al. 2011). Amplification efficiency was calculated based on the standard curve using the equation: E = 10−1/slope and efficiency (%) = (E–1) × 100. The LOD was regarded as the analytical concentration at which the methods detected the presence of a target nucleic acid in at least 95% of true positive biological samples (less than 5% of false negative results) (Ferreira et al. 2016).

Results and discussion

Development of cpDNA markers

We have attempted a qRT-PCR assay to detect C. wilfordii and C. auriculatum cpDNA using species-specific primer pairs. In previous studies, cpDNA markers derived from matK have been used to analyze phylogenetic relationships and to perform plant identifications (Steele and Vilgalys 1994; Tamura et al. 2004). As of September 2015, the complete matK gene sequences of C. wilfordii and C. auriculatum have not been reported, whereas the matK gene sequence of C. acutum has been deposited in the NCBI public sequence database. To identify the matK sequences of C. wilfordii and C. auriculatum, we designed potential genus-consensus primer pairs (Table 1), on the basis of the gene sequences of C. acutum. We successfully amplified 763-bp products in both species (Fig S1). The gene-specific primers were designed on the basis of SNPs of the gene between C. wilfordii and C. auriculatum (Fig. 1A). Primer sets of cpDNA markers for C. wilfordii and C. auriculatum were designed based on SNPs in the fragments. To confirm the allele-specific amplification of each cpDNA marker, we performed PCR on the genomic DNA of the two species with cpDNA markers and visualized the PCR products via gel electrophoresis (Fig. 1B); we determined that each primer set could amplify specific PCR products in one species but not the other. We then sequenced the 151-bp amplicons (Fig. 1B) and confirmed the expected sequences (data not shown). These results suggest that the cpDNA markers could be used for discrimination in both species.

Standard curves and amplification efficiency of SYBR Green PCR systems

To estimate the efficiency and sensitivity of primer sets, we carried out a qRT-PCR assay using tenfold serially diluted total DNA (including chloroplast DNA) and then analyzed the statistical measures of each cpDNA marker using the regression test (Fig. 2). A strong linear correlation (R 2 > 0.99) was obtained between the crossing point values and log DNA concentration for C. wilfordii and C. auriculatum. Linearity was observed (range 10 ng–0.1 pg of total DNA). The slopes of the linear equations were −3.34 for C. wilfordii DNA (Fig. 2A) and −3.54 for C. auriculatum DNA (Fig. 2B), with appropriate amplification efficiencies of 99.35 and 91.52%, respectively. However, the cpDNAs of the counterpart samples were amplified with C t value higher than 30. These results suggest that the developed markers are able to identify C. wilfordii and C. auriculatum samples under appropriate amplification conditions with DNA concentrations of more than 1 pg and C t value of less than 30.

Fig. 2
figure 2

Standard curves obtained by analyzing serially diluted DNAs of C. wilfordii (A) and C. auriculatum (B), respectively. The standard curves obtained are based on efficiency and correlation of coefficient (R 2) of DNA extracted from the plant roots. The x-axis represents log DNA concentration, and the y-axis represents C t value

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Heat treatment is one of the necessary conditions for the manufacturing process. Therefore, the efficiency and sensitivity of primer sets were assessed using qRT-PCR with binary mixtures containing different concentrations of heated and unheated samples (103–10 mg/g) (Fig. 3). The slopes of the linear equations for raw samples were −3.37 for C. wilfordii DNA (Fig. 3A) and −3.26 for C. auriculatum DNA (Fig. 3C), with appropriate amplification efficiencies of 98.03 and 102.69%, respectively. The slopes of the linear equations for heated samples were −3.11 for C. wilfordii DNA (Fig. 3B) and −3.19 for C. auriculatum DNA (Fig. 3D), with appropriate amplification efficiencies of 109.84 and 105.74%, respectively. Ferreira et al. (2016) have reported estimated coffee content in commercial products using SYBR green real-time PCR systems and have suggested that the valid standard curves in a regression equation are essential for accurate quantification and identification of different types of potential adulterants. Therefore, the primer pairs designed in the present study would be useful for accurate quantification and identification for both species in commercial food products.

Fig. 3
figure 3

Standard curves of LOQ, based on triplicate analysis of binary mixtures of flour matrices, spiked with decreasing concentrations of C. wilfordii and C. auriculatum. Non-heated C. wilfordii, heated C. wilfordii, non-heated C. auriculatum, and heat-treated C. auriculatum (130 °C for 15 min), shown in (A–D), respectively. The standard curves obtained are based on efficiency and correlation of coefficient (R 2) of DNAs extracted from both genotypes of roots. The x-axis represents a log DNA concentration, and the y-axis represents the C t value

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Application of cpDNA markers to commercial C. wilfordii products

In order to verify the feasibility of using qRT-PCR systems to detect both species in C. wilfordii commercial products, 19 samples were bought from local markets and were tested using this qRT-PCR system. The procedure is crucial not only because an intended (or unintended) mixture of material from both species could adversely affect health, also because C. wilfordii is a susceptible target for food fraud owing to its higher price (more than twice that of C. auriculatum). Among the samples, 13 were pills, 5 were crude drugs (2 dry matter and 3 powder), and 1 was granular (Table 2). All commercial product samples were amplified using 18S rRNA genes as a positive control for verifying that DNA was extracted from commercial products. The C. wilfordii DNA was detected in 6 pill samples, 2 dry matter samples, and 2 powder sample, but was not detected in 7 pill samples, 1 powder sample, and the 1 granular sample. The C. auriculatum DNA was detected in 11 pill samples, 2 dry matter samples, 2 powder sample, and 1 granula. Both Cynanchum wilfordii DNA and C. auriculatum DNA were both detected in 5 pill samples, and C. auriculatum DNA alone was detected in 3 pill samples. However, DNA from neither species was detected in 1 pill and 1 powder sample.

Table 2 qRT-PCR results on C. wilfordii and/or C. auriculatum with the primer sets in 19 commercial food products containing C. wilfordii
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The DNA-based methods for identification of C. wilfordii and C. auriculatum have been used for species identification in several studies, for example, with sequence-characterized amplified region markers (Moon et al. 2010; Ryuk et al. 2014), single PCR (Kim et al. 2015), multiplex PCR (Moon et al. 2016), and amplification refractory mutation system PCR and high-resolution melt curve analysis (Han et al. 2016). Because sensitivity of the primer sets that were previously developed was not evaluated, it may be difficult to discriminate small amounts of the target species in commercial food products. In addition, few studies showed only application of newly developed primer sets to processed foods. In addition, Li et al. (2013) reported high-performance liquid chromatography (HPLC)-UV analysis for quality assessment and discrimination of the roots of the two plants. Nevertheless, the method requires expensive equipment and experimental skills and entails high operation costs.

Recently, qRT-PCR methods based on SYBR green have proved to be useful tools for species-specific traces and quantification (Pafundo et al. 2009; Sakalar 2013). Many approaches to authenticity verification involve successful qRT-PCR methods with SYBR green in processed foods, such as almond (Pafundo et al. 2009) and coffee (Ferreira et al. 2016). In the present study, we demonstrated sensitivity and accuracy of the primer sets in repeated experiments and in the application to diverse commercial food products containing C. wilfordii (Figs. 2, 3). These results suggest that the method developed in this study may help to verify the authenticity of both Cynanchum in C. wilfordii food products owing to the rapidity and relatively low cost.

In conclusion, we developed primer sets on the basis of SNPs of the matK gene of both C. wilfordii and C. auriculatum for the purpose of authenticity verification of C. wilfordii food products. Specificity and sensitivity of the cpDNA markers were examined using SYBR green-based qRT-PCR with diverse samples. Additionally, the markers were applied successfully to 19 commercial C. wilfordii food products and could prove a useful tool to detect the presence of C. wilfordii and C. auriculatum in commercial products.