Fast and reliable detection of toxic Crotalaria spectabilis Roth. in Thunbergia laurifolia Lindl. herbal products using DNA barcoding coupled with HRM analysis
Data mining and primers used
The amplification of the four selected locus from two ‘Rang Chuet’ medicinal plant
species (T. laurifolia and C. spectabilis) was performed using specific primers corresponding to the matK, rbcL, rpoC and trnL barcode region. All sequences of Thunbergia spp. were extracted from GenBank and the variable characters and average %GC content
was calculated for all samples (Table 3). Data was present for most markers of the target species, except for rpoC. The total number of sequences retrieved for the respective markers were: matK 11 (7 species); rbcL 6 (6 species), trnL 27 (20 species). The absence of rpoC sequences for the target species was resolved by selecting random rpoC sequences from GenBank, which is supported by the high universality of rpoC17], 22]. Two sequences of C. spectabilis were retrieved (matK and trnL).
Table 3. Characteristics of sequences and primers for high resolution melting analysis
For matK 5, rbcL 6, rpoC 5 and trnL 19 sequences were deemed useful for further analysis (Table 4). An alignment of all useful sequences was made, and the primers flanking regions
of each marker ranging from 150 to 170 bp were analyzed (Table 2). Reed and Wittwer 23] found that suitable length for HRM analysis should be 300 bp or less for optimal
results.
Table 4. Sequences of four plastid regions (matK, rbcL, rpoC and trnL) were retrieved from GenBank (NCBI) for each of the species with accession number
Both the sequence length and the nucleotide variation within sequences influence the
dissociation energy of the base pairs and result in different Tm values. The matK amplicon sequences were observed to have higher nucleotide variation than the amplicons
of the other regions, at 30.67%. The relative nucleotide variation within amplicons
was found to be as follows: matK??rpoC??trnL??rbcL (Table 3). The forward and reverse matK primers matched the consensus sequence of the target species at the binding sites
in only 15 out of 26 sites (57.69%) and 19 out 23 of sites (82.61%), respectively
(Table 3). High universality at the initial bases of the primer site is crucial for primer
annealing and subsequent elongation initiation by the DNA polymerase. The matK locus is one of the most variable plastid coding regions and has high interspecific
divergence and good discriminatory power. However, it can be difficult to amplify
with the standard barcoding primers due to high substitution rates at the primer sites
24], 25]. The rbcL, rpoC and trnL primer pairs were expected to be a suitable primer for HRM analysis for discrimination
between the tested plant species. These primers were nearly identical in base similarity
to the mined consensus sequence (Table 3).
The average %GC content of amplicons was calculated in order to predict variation
in melting curves for the different markers. trnL had the lowest average %GC content, with 34.50%, followed by matK, rpoC and rbcL, with 35.20, 41.85, 44.26 and 46.60% respectively (Table 3).
Finding suitable primer pairs for discrimination between T. laurifolia and C. spectabilis
The four primers sets were used for the amplification of DNA-fragments from all seven
samples (two ‘Rang Chuet’ species), and the amplicons were analyzed using HRM to define
Tm. (Table 5). The expected length of amplified products from matK, rbcL, rpoC, and trnL are 160 bp, 150 bp, 170 bp, and 150 bp, respectively. The melting profiles of all
amplicons are illustrated in Fig. 1a-1d. The analysis is presented by means of conventional derivative plots, which show
that the Tm value of each species is represented by a peak. The samples of the two different
species could be easily distinguished using HRM analysis with all four primer pairs.
The melting profiles of seven samples of the two ‘Rang Chuet’ species (T. laurifolia and C. spectabilis) can be divided into two groups. All T. laurifolia samples (T1-T4) are grouped together and the other group contains all C. spectabilis samples (C1-C3). Although the four primer pairs tested could be used to discriminate
T. laurifolia from C. spectabilis, the rpoC region was chosen for further analysis as it would help in demonstrating that Bar-HRM
could work well as a sequencing-free method for plant identification. In addition,
the rpoC region was used as an analytical target in HRM analysis has been shown to be effective
for the detection and quantification of Lens culinaris and Lathyrus clymenum adulterations 18], 22].
Table 5. The values of melting temperature (°C) with standard deviations gaining form high
resolution melting (HRM) analysis using matK, rbcL, rpoC and trnL primers of T. laurifolia and C. spectabilis species
Fig. 1. Melting curve profiles of amplicons obtained from each primer set. The normalized
plot of each primer pair matK (a), rbcL (b), rpoC (c), and trnL (d) shows the differentiation of melting temperature (Tm) of each amplicon from each species, generated by high resolution melting (HRM) analysis
Quantitative detection of T. laurifolia adulterants with Bar-HRM analysis
Detecting limit of adulteration in T. laurifolia products using the developed method with rpoC primers was tested. Figure 2a shows the results of the validation method with T. laurifolia spiked with C. spectabilis in different proportions. These results depict the analysis for one experiment as
all three experiments gave similar results thus showing very good reproducibility.
The process of the T. laurifolia amplicon dissociation reveals the level of contamination resulting from adulteration
as the presence of increasing quantity of C. spectabilis into the T. laurifolia DNA alters the shape and shifts proportionally the melting curve, compared to the
curve of pure T. laurifolia DNA. By applying this approach, we were able to detect adulterations as low as 1
% (Fig. 2a).
Fig. 2. Melting curves obtained by high resolution melting analysis of the two ‘Rang Chuet’species.
a Specific amplicons and applied to reference mixtures containing 50, 25, 12, 6, 3
and 1 % of C. spectabilis in T. laurifolia.b Difference graph of ten commercial herbal products using T. laurifolia as reference species. Data are from a single experiment
Identification of herbal species in commercial products
Constituent species in herbal products bought from markets in Thailand were investigated
to assess the reliability of information regarding their ingredients, as the herbal
products are often sold in processed forms. Ten herbal products labeled as ‘Rang Chuet’
were purchased and examined (Table 1). The HRM analysis using rpoC primers was then performed to identify the species in the products.
The examination of the HRM difference curve of all tested samples using T. laurifolia curve as baseline revealed that seven out of ten samples (COM3-7 and 9–10) produce
curves in which the same as T. laurifolia’s with a 90% confidence interval, suggesting that the products contain T. laurifolia (Fig. 2b). The melting curve of one tested sample (COM8) was found between T. laurifolia and C. spectabilis lines, it could be indicated that the commercial COM8 was probably be admixture of
T. laurifolia and C. spectabilis with around 3% of the toxic C. spectabilis in the product as show in Fig. 2b. However, we cannot rule out the possibility of the COM8 may actually not be contaminated
with the toxic C. spectabilis but other species. In addition, the results of the analysis also reveal that the
two remaining samples (COM1 and COM2) were much likely not contain any of the two
‘Rang Chuet’ species but some other species instead (Fig. 2b). In order to find contaminated or substituted species in COM1 and COM2, DNA barcoding
is one of the best solutions. As can be seen from Newmaster et al26] work, DNA barcoding was performed to detect the adulteration and substitution of
herbal drugs and found that herbal products sold on the markets were contaminated
or substituted with alternative plant species that are not listed on the labels as
they are replaced entirely by powdered rice, wheat and soybean. Thus, DNA sequencing
of rbcL region was carried out to identify species in these two products. The blast result
showed that COM1 and COM2 have a similarity in their sequences to Moringa oleifera and Andrographis paniculata, respectively (Additional file 1: Table S1). The finding provides evidence that substitution in herbal products sold
Thai local market is presented and this substitution could be a serious issue for
consumers. Due to the fact that Bar-HRM has allowed us easily determine herbal species
in processed products sold on the markets within 2 h. Bar-HRM method developed in
this study therefore pose a potential to be a great tool in detection of adulteration
and/or substitution in herbal products especially in processed forms.