Introduction

Medical herbs have been used in traditional medicine practices for a long time. According to the World Health Organization (WHO) reports, about 80% of the population in developing countries relies on medical herbs for primary health care (World Health Organization (WHO, 2007). In addition, 19% of the population in the United States of America uses medicinal herbs for medical treatment (Gardiner et al., 2007). The American botanical council reported that the sales of traditional herbal medicines in the US reached a total of $7.45 billion in 2016 (Smith et al., 2017). The market value of herbal medicine is expected to grow and reach a global value of $129.68 billion by 2023 (Market Research Future, 2018).

A Chinese herbal medicine, Atractylodes lancea (Tunb), belongs to the Compositae family and has long been used as an important herb in East Asia. In general, A. lancea has been used to reduce acute digestive disorders, rheumatic disease, night blindness, and influenza (Qian et al., 2006). The substances extracted from A. lancea are effective in exerting anticancer, anti-digestive disorders, anti-obesity, and anti-inflammatory properties (Koonrungsesomboon et al., 2014). Among these properties, the anticancer activities of A. lancea, particularly against cholangiocarcinoma cells, have been demonstrated in previous studies, both in vitro and in vivo. The crude ethanolic extract of A. lancea rhizome was shown to reduce potently and selectively the viability of the cholangiocarcinoma cell line (CL-6), with an IC50 (concentration that controls cell growth by 50%) of 24.09 g/mL and a selectivity index of 8.6 (Mahavorasirikul et al., 2010). In addition, A. lancea extract has been shown to delay gastric emptying and stimulate small intestinal motility. The anti-digestive disorder mechanisms of A. lancea extract are related to the inhibition of dopamine D2 and 5-HT3 receptors, the activation of vagal tone, and the inhibition of corticotropin-releasing factor (Zhang et al., 2008).

According to the Ministry of Food and Drug Safety reports, Korea, in 2017, the adulteration of A. japonica, which belongs to the same genus as A. lancea, with Scopolia japonica was reported, as well as adulteration addictive symptoms that were caused by S. japonica. S. japonica belongs to the Solanaceae family and is a highly toxic plant (Okamura et al., 1992). In the Solanaceae family, the two most typical tropane alkaloids, hyoscyamine and scopolamine, have been found (Hashimoto and Yamada, 1987). Hyoscyamine functions as an anticholinergic and antispasmodic agent, while scopolamine (hydrobromide) is an authentic hallucinogen (Heinrich and Gibbons, 2001). The main addictive symptom, hallucination, presents visual, auditory, and tactile symptoms (Magos et al., 1987). However, the morphological similarity between A. lancea, A. japonica, and S. japonica rhizomes can result in adulteration, intentionally or unintentionally. Therefore, the development of a methodology to detect adulteration of S. japonica in medicinal herbs, from the genus Atractylodes, is required for consumer safety.

Various technologies, such as sensory, physicochemical, chromatographic, spectroscopic, and DNA-based assays, have been used to detect the fraudulent ingredients in complicated mixed foods (Hong et al., 2017). Among them, DNA-based methods, such as quantitative real-time PCR (qPCR), multiplex PCR, and PCR–RFLP have been successfully applied to detect food fraud and adulteration due to their economic and time-saving advantages over other approaches (Kane and Hellberg, 2016; An et al., 2018). In particular, DNA from two or more target species in the same reaction can be reliably detected using multiplex PCR. Multiplex PCR has been successfully used to detect species in complex foods, such as meat (Matsunaga et al., 1999; Rodríguez-Plaza et al., 2006; López-Andreo et al., 2006), fish, and seafood (Trotta et al., 2005) products. Furthermore, multiplex PCR has been used as a rapid and convenient detection assay for GM crops, such as maize, soybean, and canola (James et al., 2003; Germini et al., 2004; Forte et al., 2005). Although it has many advantages, multiplex PCR also has many challenges, such as cross-reactivity among the primer combinations, low sensitivity, and lack of efficiency in various food products for broad applications (Xu et al., 2012).

Another DNA-based assay, quantitative real-time PCR assay (qPCR), can be detect complex foods with high specificity and sensitivity. qPCR methods are divided into probe-based real-time PCR (TaqMan assay) and dye-based real-time PCR (SYBR Green I assay) (Arya et al., 2005). In the probe-based real-time PCR assay, the target sequence is specifically amplified with a gene-specific primer set and a fluorescent reporter probe, which are designed to be complementary to a target sequence (López-Calleja et al., 2007). During the extension stage, the probe is broken apart by DNA-polymerase, and then, the fluorescence signals increase in intensity. However, this approach requires many SNPs or indels to design probes, and it is difficult to optimize real-time PCR conditions (Safdar and Junejo, 2015; Şakalar and Kaynak, 2016). On the other hand, SYBR green-based real-time PCR is a more flexible, convenient, and inexpensive method compared to probe-based real-time PCR methods, because the SYBR green dye interacts with double-stranded DNA in a sequence-independent manner (Safdar and Junejo, 2015). However, the drawback of the SYBR green based real-time PCR is that non-specific PCR products might be amplified as a false-positive signal (David, 2000).

The application of nuclear genes as target sequences for a DNA barcode has some problems due to their low copy numbers and low discriminatory power between species (Chase et al., 2007). Internal transcribed spacer (ITS) regions of ribosomal DNA have been used as molecular markers of species discrimination in eukaryotes (Vijayan and Tsou, 2010). It is believed that the sequences of the ribosomal DNA genes are well-conserved, while their spacer sequences possess many inter-species variations. For this reason, the ITS region has good potential for designing DNA markers for species identification (Hirao et al., 2009; Pereira et al., 2008).

Chloroplasts are organelles in plants, and their function is to conduct photosynthesis. The chloroplasts genome is generally a single circular molecule, and its size varies among species, ranging from 107 to 208 kb (Daniell et al., 2016). The chloroplasts genes are generally present in hundreds of copies per cell, and the characteristic two membrane layers protect DNA from degradation during food processing, such as heating, fermentation, and boiling (Garino et al., 2016). The chloroplast genome generally contains 120–130 genes. Certain chloroplast genes e.g., matK, ndhF, ycf1, and ccsA, exhibit higher frequencies of variations, including single-nucleotide polymorphisms (SNPs) and insertion/deletions (indels), than other chloroplast genes (Daniell et al., 2016). Therefore, these chloroplast genes are usually used for plant species identification in food complexes.

In this study, we developed two PCR-based assays, multiplex PCR and SYBR green-based qPCR, to identify A. lancea and S. japonica. We subsequently examined their sensitivity, specificity, and reliability, and the two PCR assays clearly detected adulteration with S. japonica in the medical herb A. lancea. Subsequently, the two developed PCR-based molecular markers were applied to commercial A. lancea products.

Materials and methods

Sample preparation

The A. lancea dried leaves and rhizomes were kindly provided by the National Institute of Food and Drug Safety Evaluation of the Ministry of Food and Drug Safety (Cheongju, Korea). The S. japonica plants and DNAs were kindly provided by the Medical Plant Taxonomy lab from Kangwon National University (Chuncheon, Korea) and the National Institute of Biological Resources (NIBR) (Incheon, Korea), respectively. Both plants’ genomic DNA were isolated from leaves or rhizomes.

Binary A. lancea and S. japonica mixtures

Binary ground rhizomes mixtures that contained different amounts (0.1, 1, 10, and 100%, total 2 g) of A. lancea were mixed with S. japonica (99.9, 99, 90, and 0%). Additionally, to evaluate the applicability of the designed method for processed rhizome products, the reference binary mixtures were treated with three different processing conditions, as follows: (1) dried at 55 °C for 48 h in a dry oven, (2) boiled at 100 °C for 15 min in a water bath, and (3) autoclaved at 121 °C 150 kPa for 20 min.

Blind samples

Blind mixture samples (n = 20) were provided by the National Institute of Food and Drug Safety Evaluation of the Ministry of Food and Drug Safety (Cheongju, Korea). The blind mixture samples were randomly mixed with different percentages of A. lancea and S. japonica ground rhizomes. The ground S. japonica rhizomes were added to those of A. lancea at concentrations of 0–10% (w/w), to prepare final mixtures of 2 g.

Genomic DNA extraction

DNA was isolated from the dried leaves using the DNeasy Plant Pro Kit (QIAGEN, Hilden, Germany), following the manufacturer’s protocol. To effectively isolate genomic DNA from large amounts of binary mixture rhizome samples, a modified large-scale CTAB-based method was used (Minas et al., 2011). DNA quantity and purity were checked using a SPECTROstar Nano reader (BMG Labtech, Ortenberg, Germany). The DNA purity was in the range of 1.7–2.0. If the DNA purity was less than 1.7, it was cleaned using the Wizard DNA Clean-Up system (Promega, Madison, USA).

Sequence analysis and primer design

Target gene sequences, such as matK, ycf1, and ITS2 of A. lancea (NC_037483.1), were downloaded from the National Center for Biotechnology Information (NCBI) and used to design target-specific primer sets. Information on certain target gene sequences, such as matK, ycf1, and ITS2 in S. japonica, were lacking in the NCBI database. To obtain these sequences, conventional primer sets were designed based on their S. parviflora nucleotide sequences (NC_030282.1), which were closely related to those of S. japonica. After PCR using DNA from S. japonica, which was kindly provided by NIBR, the amplicons were sequenced using a commercial service (Macrogen, Seoul, Korea) and nucleotide sequence alignments of both species were conducted using ClustalW2 (EMBL-EBI, Hinxton, Cambridgeshire, UK) and BioEdit 7.2 (Ibis Biosciences, Carlsbad, CA, USA). Target-specific primer sets were designed based on the variable region between A. lancea and S. japonica using Beacon DesignerTM (PRIMER Biosoft, Palo Alto, CA, USA) and commercially synthesized (Macrogen, Seoul, Korea).

Multiplex PCR analysis

Multiplex PCR analysis was carried out using TaKaRa Ex Taq DNA polymerase (TaKaRa Bio Company, Kusatsu, Shiga, Japan), with mixtures of 10 ng/µl DNA and different concentrations (10–50 pmol) of each multiplex primer combination using a C1000 Thermal Cycler (BIO-RAD, California, USA) (Supplementary Table 1). To examine the efficiency of multiplex PCR primer sets, ten-fold dilutions (0.01–10 ng/µl) of DNAs from both target species (A. lancea and S. japonica, 1:1 ratio v/v) were used as templates for multiplex PCR testing. The multiplex PCR amplification conditions were as follows: pre-denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, primer annealing at 60 °C for 20 s, primer extension at 72 °C for 30 s, and final extension at 72 °C for 5 min. Multiplex PCR products were electrophoresed on a 2% agarose gel and then visualized using a Chemi Doc XRS+ (BIO-RAD, California, USA).

Quantitative real-time PCR analysis (qPCR)

qPCR analysis was performed using the GoTaq® qPCR Master Mix kit (Promega, Madison, Wisconsin, USA). The real-time PCR reaction mixture consisted of 10 µL 2X GoTaq® qPCR Master Mix; 0.5 µl of 10 pmol each primer; 1 µl of 10 ng/µl DNA; 0.2 µl of the CXR reference dye; and free water, up to a final volume of 20 µl. Quant studio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was used to perform real-time PCR amplification as follows: pre-denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 s, primer annealing at 55.5–65.0 °C for 60 s according to each primer annealing temperature, and primer extension at 60 °C for 60 s. All qPCR reactions were performed in triplicate.

Construction of real-time PCR standard curve and data analysis

The efficiency of the designed real-time primer sets was confirmed using target and non-target DNA that was diluted ten-fold into five series (10 ng–1 pg/µl). Isolated DNA from each binary mixture was diluted to a final concentration of 10 ng/µL, and a baseline and threshold were set for further analysis. The cycle numbers at the threshold level of log-based fluorescence were defined as the cycle threshold (Ct) numbers, which represented the observed value in the real-time PCR experiments (Yuan et al., 2006). Correlations between diluted DNAs and cycle threshold (Ct) standard curves were confirmed using a default parameter. According to the ENGL documents, the standard curve was calculated as y =  − ax + b (a indicates the standard curve slope and b indicates the y-intercept) (ENGL, 2015). The efficiency of the reaction (E) was calculated as E = (10−1/a), and the efficiency of the primer sets was evaluated as (E − 1) × 100% (ENGL, 2015; Lo and Shaw, 2018). To evaluate the amplification sensitivity of the designed real-time PCR primer sets, two criteria should be confirmed to define an acceptable real-time PCR assay, based on previous reports (Bustin et al., 2009): linear dynamic range and amplification efficiency. The linear dynamic range should ideally extend over four log10 concentrations, with the coefficient of determination (R2) being greater than 0.98. Additionally, the amplification efficiency should be in the range of 110–90%, which corresponds to a slope between − 3.1 and − 3.6 (ENGL, 2015).

To validate the accuracy and sensitivity of the designed target-specific qPCR primer sets, an interlaboratory experiment was performed in two different laboratories. Validation was performed in two laboratories using the same qPCR conditions, with either an Applied Biosystems 7500 Fast Real-Time PCR Instrument System (Applied Biosystems, Foster City, CA, USA) or a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

Results and discussion

Design of the variable region-based DNA markers

First, the A. lancea and S. japonica specific multiplex PCR primer combinations were designed from chloroplast genes, including matK and ycf1. The variable sequences in the chloroplast gene (matK and ycf1) between two species were detected by aligning the target gene sequences. These genes (matK and ycf1) showed high sequence variability between A. lancea and S. japonica. As shown in Supplementary Fig. 1, the target-specific multiplex PCR primers were designed with one common forward primer shared between the two species, and target-specific reverse primers were designed based on target sequences that had polymorphisms and differences in amplicon sizes between the two species (Supplementary Fig. 1 and Supplementary Table 1). The multiplex PCR amplification product size ranged from 279 to 499 (Supplementary Table 1).

Secondly, the target-specific qPCR primer sets were designed from the nuclear ITS region of ribosomal DNA and chloroplast genes, matK and ycf1. Sequence alignments between A. lancea and S. japonica were performed, and we designed the qPCR primer sets based on variable sequences between both plants (Supplementary Fig. 2 and Supplementary Table 1). Food processing, such as drying, heating, and mixing, is known to be harmful and reduces the quality of DNA (Lo et al., 2018). If the PCR amplicon length is long, the efficiency of qPCR with low quality DNA decreases. For these reasons, based on variable sequences between both species, target-specific qPCR primers were designed to amplify short amplicons ranging from 78 to 180 bp (Supplementary Fig. 2 and Supplementary Table 1).

Evaluation and validation of designed DNA markers

Evaluation of the sensitivity with target specific multiplex PCR primer combinations

The multiplex PCRs were carried out with DNA from each species and combined DNA of both species. For the single-species DNA, we found one clear PCR amplicon with multiplex primer combinations. In the combined DNA, two clear PCR amplicons were found with expected sizes and identical sequences to the target genes (Fig. 1A). To determine the optimum combinations of both primer concentrations, each of the reverse target-specific multiplex PCR primer concentrations were diluted to 50–10 pmol, and then, both specific primers were combined to perform the multiplex PCRs. The optimal reverse primer concentrations are shown in Supplementary Table 1.

Fig. 1
figure 1

A Specificity of the target specific multiplex PCR primer sets (AL_M_matK, SJ_M_matK, AL_M_ycf1 and SJ_M_ycf1) used for identification of A. lancea and S. japonica. The PCR amplicons of 499, 321, 396, and 279 bp correspond to the A. lancea and S. japonica. Line M, l kb DNA ladder marker, Line A, DNA of the A. lancea with multiplex primer sets, Line B, DNA of the S. japonica with multiplex primer sets, Line C, DNA of the both species with multiplex primer sets, Line D, NTC. B Sensitivity of target target-specific multiplex primer sets (AL_multi_matK, SJ_multi_matK, and AL_multi_ycf1 and SJ_multi_ycf1). The multiplex PCRs were performed with ten-fold serially diluted DNAs of both target species (10 -0.1 ng/ul). Line M, l kb DNA ladder marker; Line A, 10 ng; Line B, 1 ng; Line C, 0.1 ng; Line D, 0.01 ng

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In addition, to validate the sensitivity of the multiplex primer combinations, we conducted multiplex PCR with ten-fold diluted (10–0.01 ng/µl) DNA templates of both species. As a result, both multiplex primer combinations (AL_M_matK and SJ_M_matk, and AL_M_ycf1 and AL_M_ycf1) can clearly detect both target species DNA until 0.1 ng/µl of both species; however, the combined DNA of 0.01 ng/µl was not clearly detected (Fig. 1B).

Evaluation of the sensitivity with target specific real-time PCR primer sets

The sensitivity of the six primer sets (AL_R_ITS2, AL_R_matK, AL_R_ycf1, SJ_R_ITS2, SJ_R_matK, and SJ_R_ycf1) was evaluated by analyzing standard curves using the tenfold serially diluted DNAs isolated from rhizomes of target species (10–0.001 ng/µl), and regression analysis was performed (Fig. 2). As a result, the slopes the primer sets ranged from − 3.31 to − 3.55, and the correlation coefficients (R2) were higher than 0.99 (Table 1). In addition, the efficiency values ranged from 91.11–100.32% (Table 1), and all criteria satisfied the ENGL guidelines (ENGL, 2015). All A. lancea specific real-time PCR primer sets (AL_R_ITS2, AL_R_matK, and AL_R_ycf1) showed S. japonica DNA amplicons greater than 33 Cts, while all the S. japonica specific real-time PCR primer sets (SJ_R_ITS2, SJ_R_matK, and SJ_R_ycf1) exhibited A. lancea gDNA amplicons greater than 32 Cts (Fig. 2). In addition, the interlaboratory experiments were conducted to evaluate the adaptability of six real-time PCR primer sets with different real-time PCR machines in two independent laboratories. As shown in Supplementary Table 3, the real-time PCR primer sets were found to meet the ENGL criteria (R2 > 0.98 and efficiency ranges of 90.39 –100.62) (Supplementary Table 3).

Fig. 2
figure 2

Sensitivity of target target-specific real-time PCR primers. Standard curve of cycle threshold (Ct) values was obtained on the basis of efficiency and correlation of coefficient (R2) in serial dilution series DNA (A. lancea and S. japonica) using target-specific primer sets. The DNA concentration (log numbers) was represented in the x-axis and the means of Ct value ± SD were represented in the y-axis. A A. lancea specific primer sets (AL_real_ITS2, AL_real_matK, and AL_real_ycf1). Black dots represent ten-fold dilution series of genomic DNA in A. lancea leaves (10 ng–1 pg) and Gray dots represent genomic DNA of S. japonica (10 ng/ul); B S. japonica specific primer sets (SJ_real_ITS2, SJ_real_matK, and SJ_real_ycf1). Gray dots represent ten-fold dilution series of genomic DNA in S. japonica (10 ng–1 pg) and black dot represents the gDNA of A. lancea (10 ng). All real-time PCRs were carried out in triplicate (n = 3)

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Table 1 Evaluation of Slope, R2, and efficiency obtained by real-time PCR system with different material conditions using the designed primer sets threshold cycle (Ct) values obtain from three conditions (dried, heat and autoclaved) with reference binary mixtures
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Based on the evaluation of amplification sensitivity of the two PCR assays, multiplex and real-time PCR primer sets, the designed primer sets were found to be suitable for amplifying the target species DNA in the combined DNA samples of the target species with low DNA concentrations (0.1 ng for multiplex PCR and 0.01 ng for real-time PCR).

Species specificity test of the designed PCR assays

To confirm the cross-reactivity of the designed DNA markers, we conducted a specificity test using multiplex and real-time PCR markers. A total of 16 species, including A. lancea and S. japonica, were used in the specificity test. 18S plant rRNA primer sets were used as a positive control (Allmann et al., 1993), and the PCR products were amplified for less than 20 cycles in the multiplex PCR and before 20 Cts in the real-time PCR. Multiplex PCRs were performed with target-specific multiplex PCR primer combinations (AL_M_matK, AL_M_ycf1, SJ_M_matK, and SJ_M_ycf1) and various PCR cycles, such as 25, 28, 31, 34, and 40 cycles, respectively (Table 1). As a result, the cut-off cycles (28 cycles for AL_M_matK, AL_M_ycf1, and SJ_M_matK and 31 cycles for SJ_M_ycf1) were identified for multiplex PCR. Before the cut-off cycles, PCR products were amplified in target species, that is, A. lancea and S. japonica, but not in the other 15 species. The AL_M_matK primer sets could amplify PCR products from six species that belonged to the Compositae family after more than the cut-off cycles but less than 40 cycles, while the AL M_ycf1 could amplify products from two species of the family. In contrast, the SJ_M_matK could amplify PCR products from two species (I. batatas and Z. mays) after more than the cut-off cycles but less than 40 cycles. However, SJ_M_ycf1 could amplify products only from C. longa after more than the cut-off cycles (Table 2).

Table 2 The results of the species specificity test using the designed multiplex PCR and real-time PCR primer sets
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Subsequently, real-time PCR was performed with the 16 species using the six designed real-time PCR primer sets (Table 1). Five primer sets, except AL_R_ycf1, were not able to amplify PCR products from the other non-targeted 14 species before 40 cycles; however, amplicons were produced with the AL_R_ycf1 from the three species that belonged to the Compositae family after more than the cut-off cycles and before 40 cycles. Collectively, the specificity of the two types of DNA marker tests suggests that all designed primer sets could be useful for detecting the target species without cross-reactivity with at least 14 species, which could be used as medicinal herbs or addition agents in food complexes.

Application of designed real-time PCR assays in samples treated with dry, heat, and autoclave

Generally, the rhizomes of A. lancea undergo three types of food processing, namely dried, heated, and autoclaved food complexes. The food processing processes could cause severe DNA degradation of medical herbs. For example, heat treatment is known to cause strong DNA fragmentation and thus renders the PCR-based marker assays inefficient and unsuitable in verifying the authenticity of food complexes (Hwang et al., 2015). Therefore, the developed real-time PCR assays would be examined in samples treated with the food processing processes to confirm their applicability in the processed food complexes. We examined the slope, R2, and efficiency of the designed real-time PCR assay using dried, heated (100 °C), and autoclaved rhizome reference binary mixtures, to determine their applicability in food complexes. First, standard curves were constructed using tenfold diluted DNA series of each of A. lancea and S. japonica with six real-time PCR primer sets (Table 2). All slopes that were evaluated ranged from − 3.31 to − 3.57, and the six correlation coefficients (R2) were 1.00 for the designed primer sets. The slope efficiencies were estimated to be 91.11% to 100.32% for each target species. Subsequently, DNAs extracted from dried rhizome binary mixtures (0.1–100% w/w) were diluted to the 10 ng/uL and used for the real-time PCR assay. In addition, DNAs of heated and autoclaved rhizome binary mixtures (0.1–100% w/w) were extracted after treatment, respectively. Ct values were obtained from each of the treated samples of three food processing types (Supplementary Table 2). Dried and heated rhizome binary mixtures showed similar low Ct values; however, higher Ct values were found for autoclaved samples than for the dried and heated samples. These results suggest that more severe DNA degradation is present in the autoclaved rhizomes than in the dried and heated rhizomes, which suggests that the application of different Ct values to discern adulteration depends on food processing conditions. For the dried, heated, and autoclaved rhizome binary mixtures, the designed 18 real-time PCR primer sets showed slopes of − 3.18 to − 3.49, R2 > 0.99, and efficiency values of 92.97–109.05 (Table 2). Generally, adulteration of less than 0.1% would not be intended for illegal economic profit. Therefore, in the application of the real-time PCR assay, Ct values of 0.1% target species in all binary mixtures were determined as cut-off cycles for discerning between genuine products and adulteration.

Application of the designed PCR assays for commercial A. lancea products

To screen the adulteration of S. japonica in commercial A. lancea products, the two designed PCR assays, multiplex PCR and real-time PCR, were performed on three types (dried rhizomes, pill, and dried rhizome powder) of 14 A. lancea commercial products purchased from a local market (Supplementary Table 4). First, the 18 s rRNA primer set was used as a positive control to check the amplification ability of the DNA isolated from the commercial products (Allmann et al., 1993). As shown in Table 3, the 18 s rRNA primer sets were amplified with all commercial products, at low Cts that ranged from 13.97 to 16.30. These findings indicate that the DNA from all the commercial products was sufficiently amplifiable. Second, multiplex PCRs with the target-specific multiplex PCR primer combinations were performed until the cut-off cycles. In all of the 14 A. lancea commercial products, A. lancea specific multiplex PCR primer combinations clearly amplified the PCR products, whereas S. japonica specific multiplex PCR primer combinations did not amplify any products before the cut-off cycles (Supplementary Fig. 3, Table 3). Finally, six specific real-time PCR primer sets were examined for the 14 commercial products. In A. lancea real-time PCR primers, Ct values (from 13.83 to 18.96 cycles) lower than the cut-off cycles of each designed primer were found for the 14 commercial products (Table 3). In contrast, the S. japonica specific real-time PCR primer sets presented higher Ct values (from 27.91 to 33.31 cycles) than the cut-off cycles (Table 3). As a result, the 14 A. lancea commercial products purchased from local markets were not adulterated with S. japonica based on the two tested PCR assays. These results indicate that the designed PCR assays can be applied to detect the target DNA (A. lancea or S. japonica) in commercial complex products.

Table 3 Results of the designed PCR assays using A. lancea commercial products
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Application of the designed PCR assays to blind samples

To estimate the reliability of the two PCR assays, a blind test was conducted. Twenty blind samples were mixed randomly by another independent research group with different concentrations of A. lancea and S. japonica. The 18S rRNA plant primer sets were used as positive amplification controls (Allmann et al., 1993) and amplified PCR products with low Cts (13.27–13.93) (Table 4). Subsequently, multiplex PCR was applied to the 20 blind samples with each primer cut-off cycle (Supplementary Fig. 4, Table 4). A. lancea PCR products were amplified in all blind samples; however, S. japonica amplicons were found in 17 blind samples, but not in the others (#1, 5, and 17). These results supported that 17 blind samples were adulterated with S. japonica (at least more than 0.1%), while three samples were genuine products. Finally, a real-time PCR assay was applied to the 20 blind samples. Three A. lancea real-time PCR primer sets could amplify PCR products in less than 18 cycles (13.67 to 17.24), which suggests that all blind samples had A. lancea rhizomes. When amplified with three S. japonica real-time PCR primers, sets lower than the cut-off cycles were found in 17 blind samples, but higher sets were found in three blind samples (#1, 5, and 17) (Table 4). Collectively, both PCR assays produced identical results in the 20 blind samples and can be used commercially to discern adulteration.

Table 4 Results of the blind test for confirming the reliability of the designed PCR assays
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In conclusion, the two PCR assays, multiplex PCR and real-time PCR, that were developed in this study are highly sensitive, fast, and specific methods for detecting A. lancea and S. japonica target species in processed medical herb complexes. To evaluate the sensitivity of the DNA markers, target-specific multiplex PCR primer combinations were amplified with ten-fold serially diluted (10–0.1 ng) DNA template. Standard curves of real-time PCR primer sets were constructed using ten-fold serially diluted DNAs with three conditions (raw, heated, and autoclaved) of binary DNA mixtures. The specificities of the developed DNA markers were evaluated with 14 other species that can be used as medical herbs or other food additives. The reliability of the developed DNA markers was supported by a blind test and their application to commercial A. lancea products. In the above experiments, the DNA markers developed in this study can detect S. japonica through a method other than morphological classification in A. lancea complex products. Thus, these DNA markers could contribute to human health care to identify adulterated products of A. lancea that are mixed with S. japonica and protect consumers from food fraud.