Introduction

In 2003, DNA barcoding was proposed1 as a molecular approach for species identification and has become popular since then. DNA barcodes have been successfully used in animal species identification. The region of the mitochondrial cytochrome c oxidase subunit 1 (COI) has been successfully used as the animal barcode, but it is difficult to amplify in fungi and often includes large introns, and it can be insufficiently variable. The internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi2,3. Recently, DNA barcoding approach has been applied to use for detection of adulteration in food and herbal products with success (e.g.4,5,6). However, some limitations of the method mean it is not fully practical in some developing countries like Thailand. The main drawbacks are likely to be that it is time-consuming and its high costs due to outsourced sequencing. To overcome those limitations, DNA barcoding has been applied to be used in combination with High Resolution Melting analysis, so-called Bar-HRM. Bar-HRM is a sequencing free method using fluorescent dye for detection of double-stranded DNA in real-time PCR increasing temperature during the process leads to denaturation of double-stranded DNA into single-stranded DNA and melting temperature (Tm) is measured. The Bar-HRM analysis is not only rapid, cheap (in long term and large scale investigation), and feasible for accurate species discrimination in plants7,8,9,10. It was also was proven to be a good compromise for counterfeiting herbal products and adulteration detection11,12,13,14,15,16,17. As a consequence, the Bar-HRM is one of many promising techniques not only for species identification/discrimination but also for counterfeit/adulterant detection in commercial products sold on the market. Here, the Ophiocordyceps species is the main focus.

A parasitic fungus known as Ophiocordyceps has a long history of use in Asian and is commonly used as a herbal medicine and health supplement. Based on numerous studies, Ophiocordyceps was found to possess anti-cancer, anti-proliferative, and anti-fibrotic18, anti-bacterial19, anti-oxidation20, anti-fatigue, anti-aging and neuroprotective effects21.

In Thailand, two major Ophiocordyceps species are currently popular, Ophiocordyceps sinensis and Ophiocordyceps militaris. Although O. militaris is closely related to O. sinensis and there are some overlap medicinal uses of these two, O. sinensis is rare and expensive. O. sinensis can only grow slowly in high-altitude habitats and cannot be produced by aseptic mycelia cultivation in Thailand. In contrast, O. militaris is now commonly cultivated and thus less expensive than that O. sinensis. In addition, O. militaris has been used to form adulterants which are found in products sold in Asian markets not just Thailand22.

Bar-HRM therefore seems to be a good tool to be used for discrimination between the two closely Ophiocordyceps species. In addition, it is also difficult to identify the species of processed or powdered products such as capsule and tablet. Bar-HRM has been proven to be an efficient and reliable method in doing such difficult task. Here, three cases of species identification and/or discrimination of Ophiocordyceps are presented to show that the Bar-HRM is one promising approach in aiding species identification.

Results and Discussion

Literature Review

Our initial literature search returned 2,181 potentially relevant publications and a refined search allowed us to narrow this to 1,846 relevant publications which focussed on only the two popular species included Ophiocordyceps sinensis and Ophiocordyceps militaris (Fig. 1). Most publications are related to one of three general categories: Pharmacology Pharmacy, Biotechnology Pharmacy or Food Science Technology. Citations indicating ideas across these three general categories were mainly to do with the medicinal properties of the Ophiocordyceps species. We then furthered our search for publications dealing with ‘method or technique’ for identification or authentication. The search returned only 75 publications which a review of abstracts allowed us to narrow to 12 publications reporting the use of DNA. Only 3 out of 12 articles were using DNA barcodes for Ophiocordyceps species identification and/or authentication19,23,24, whilst the rest used DNA in aiding taxonomy or nomenclature. What can be clearly seen in Fig. 1 is the continual growth of studies related to Ophiocordyceps, however only 3 from total of 2,181 publications which accounts for approximately 0.14% of that identification and/or authentication of Ophiocordyceps based on DNA. Few techniques have been used to distinguish between Ophiocordyceps species such as capillary electrophoresis25, high performance liquid chromatography (HPLC)27,27 and microscopic examination28. Although, extensive research has been carried out on methods used for quality control of O. sinensis (see review e.g.22,29,30), no single study exists reporting the use of DNA barcoding coupled with High Resolution Melting analysis (Bar-HRM).

Figure 1
figure 1

Cumulative number of Ophiocordyceps studies over time (1990–2017). Two main Ophiocordyceps species (Ophiocordyceps sinensis and Ophiocordyceps militaris) were focused on. Also the number of publications on a particular research field, method/technique for identification/authentication of Ophiocordyceps were shown.

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Bar-HRM was proved to be a powerful tool for species identification that is capable not only to identify but also to quantitatively detect adulterants. The Bar-HRM analysis is not only rapid, cheap (in long term and large scale investigation), but it is also feasible for accurately species discrimination in various species. Thus, Bar-HRM holds a great potential to be applicable in Ophiocordyceps identification and/or authentication. Research on the Ophiocordyceps identification and/or authentication subject has been mostly restricted to limited comparisons of Pharmacology. Our study could be one to fill in this gap.

Real-time PCR for high resolution melting (HRM) analyses

Three aspects uses of Bar-HRM to identify or authenticate O. sinensis were evaluated here. Firstly, Bar-HRM was used in aiding species discriminating of two related close Ophiocordyceps species. Secondly, Bar-HRM was applied to identify unknown powder. Lastly, we authenticated Ophiocordyceps commercial product sold on the markets by Bar-HRM.

Experiment 1: Differentiation of Ophiocordyceps raw materials

The feasibility of Ophiocordyceps species discrimination in Bar-HRM technique was examined with two Ophiocordyceps species included O. sinensis and O. militaris. Raw materials of the two Ophiocordyceps species were obtained and tested. Genomic DNA was extracted from the samples and taken to be used in HRM analysis with ITS primer pairs. HRM analysis was performed in triplicate on each of the tested species to establish the Tm. The shapes of the melting curves were analysed using Rotor-Gene Q Series Software (v. 2.3.1) to distinguish between the different Ophiocordyceps species. The ITS primer set yielded amplicons of the expected size, approximately 315 base-pairs long. Figure 2 depicts the analysis by means of conventional derivative plots, which show the Tm value for the ITS fragment from each species. The melting temperatures of the O. sinensis were 85.80 ± 0.02 °C, and O. militaris was 87.37 ± 0.05 °O. The two different Ophiocordyceps species could be distinguished by using HRM analysis. A distinct melting curve was generated for each Ophiocordyceps species presenting one inflection point (Fig. 2). It is indicated that the two Ophiocordyceps species could be discriminated with Bar-HRM.

Figure 2
figure 2

The normalised plot shows the differentiation of melting temperature (Tm) of each ITS amplicon from each Ophiocordyceps species, generated by high resolution melting (HRM) analysis.

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Experiment 2: Species identification of unknown powder sample

We received a short notice request from Chiang Mai International Airport’s (Airports of Thailand Public Company Limited: AOT) officers. They wanted us to confirm the species of powder sample carried by a passenger which claimed to be O. militaris. A quick HRM analysis was then performed right after obtaining the sample. Two Ophiocordyceps specimens (O. sinensis and O. militaris) tested in Experment 1 were used as reference species for the analysis. As can be seen in Fig. 3, a melting curve of an unknown sample was similar to the O. militaris’ curve with 96.30% confidence. After about 2.30 hours, we could confirm that the powder sample contained O. militaris as claimed (we also double checked our results with DNA sequencing of the sample, the sequence came two weeks later, sequencing results are shown in Supplementary Data 1). The analysis was carried out in three replicates. Here, it is shown that Bar-HRM is one simple, rapid and efficient method for Ophiocordyceps identification.

Figure 3
figure 3

Melting curve profiles of amplicons obtained from ITS primers from an unknown sample and the two reference Ophiocordyceps (O. sinensis and O. militaris).

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Experiment 3: Authentication of Ophiocordyceps commercial products

The Bar-HRM was performed to authenticate the commercial Ophiocordyceps products sold on markets. It is undeniable that substituting or adulterating of inferior Ophiocordyceps species was reported througout the market in Asia. Here, four Ophiocordyceps products were tested. Two tested products were claimed as O. sinensis (S1 and S2) and other two were claimed as O. militaris (M1 and M2). The examination of the HRM difference curve of both samples revealed that the M1 and M2 produced curves which were the same as O. militaris’s, with a 90% confidence interval, suggesting that the products contain O. militaris (Fig. 4A). Thus, the two O. militaris tested products (M1 and M2) showed no substitution or adulteration. In contrast, results from HRM analysis indicated the substitution/adulteration/contamination of O. militaris in products claimed as O. sinensis (S1 and S2). The melting curve of S1 was distinctly different from the O. sinensis’s and nearly identical to the O. militaris’s (Fig. 4A). Therefore, the species contained in S1 is likely to be O. militaris not the O. sinensis as indicated in the package. In addition, the melting profile of S2 was not similar to any of the two Ophiocordyceps reference curves in which this could be result from mixing of the two species in the product or other species (Fig. 4A). Figures 4B,C represent difference curves of the test products and reference species. O. sinensis was set as a reference species in Fig. 4B and it is clearly shown that none of the tested products (M1-M2 ans S1-S2) produced same melting curves as the O. sinensis. Also, when the O. militaris was set as as a reference species in Fig. 4C, it was found that melting curves of the three tested products (M1-M2 and S1) share similarity with the reference. Both of the O. militaris products tested here were likely to contain the O. militaris as claimed. However, both O. sinensis products were found to be substitued and/or adulteranted with O. militaris. Our results are similar to those reported by Li22. As the results very clearly demonstrate, it is important to have a standard or strict quality control of O. sinensis products.

Figure 4
figure 4

Melting curve profiles of all tested products and the two Ophiocordyceps species which are O. sinensis and O. militaris. (A) Normalised melting curve, (B) Difference melting curves setting O. sinensis as a reference and (C) Difference melting curves setting O. militaris as a reference.

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In Thailand, Ophiocordyceps products are commonly known as ‘Tang Chao’ and are rarely specifically indicated as Ophiocordyceps species in advertisement. As O. sinensis and O. militaris share the same name ‘Tang Chao’, several consumers bought products without realising the species of Ophiocordyceps in the products. Such buyer or consumer behaviour could lead to fraudulent, intentional substitution of inferior Ophiocordyceps species in a product for economic gain.

Conclusion

Most studies in the field of Ophiocordyceps have mainly focused on pharmacology. Few publications about method or technique use in species identification can be found, although this is one of the most important related subjects. Either basic or applied fields such as taxonomy, nomenclature, food science and industry would benefit from having a reliable technique for species identification. Here, the Bar-HRM was proved to be efficient and rapid method for Ophiocordyceps species identification and authentication. In all three aspects of using Bar-HRM to identify or discriminate Ophiocordyceps species in (1) raw materials, (2) unknown sample, and (3) commercial products was successful.

Methods

Literature Review

On 4 April 2018, we conducted a Thomson Reuters Web of Science search using the following search term ‘Ophiocordyceps’. From the search results, we identified publications that focus on ‘sinensis’ or ‘militaris’ species. We then defined the search results into publication year from 1990–2017. Based on articles only, we further refined our search with the term ‘method’ OR ‘technique’ AND ‘identification’ OR ‘authentication’.

Experiment 1: Differentiation of Ophiocordyceps raw materials

DNA was extracted with the Nucleospin Plant II kit (Macherey-Nagel, Germany) following the manufacturer’s instructions. DNA concentrations were adjusted to a final concentration of 20 ng/μL. The DNA was stored at −20 °C for further use. DNA of the two closely related Ophiocordyceps species were then used for Bar-HRM analysis. To determine the characteristic melting temperature (Tm) for each sample that could be used to distinguish the two different species, DNA amplification using real-time PCR was performed using the Rotor-Gene Q 5plex HRM system (Qiagen, Germany). The reaction mixture for the real-time PCR and HRM analysis consisted of a total volume of 10 µl, containing 5 µl of MeltDoctor HRM Master Mix (Applied Biosystems, USA), 0.2 µl of 10 mM forward primer, 0.2 µl of 10 mM reverse primer, 1 µl of 20 ng DNA and 3.6 µl of ddH2O. The nucleotide of forward and reverse primers31 were ITS5 5′-GGAAGTAAAAGTCGTAACAAGG-3′ and ITS2 5′-GCTGCGTTCTTCATCGATGC-3′. Fluorescence dye was used to monitor both the accumulation of the amplified product and the high-resolution melting process in order to derive the Tm value during PCR. The reaction conditions were as follows; an initial denaturing step at 95 °C for 5 min followed by 40 cycles of 95 °C for 30 s, 57 °C for 30 s and 72 °C for 20 s. Melting curves were generated after the last extension step. The temperature for the HRM analysis was increased from 60 to 95 °C at 0.1 °C/s.

Experiment 2: Species identification of unknown powder sample

We received a request from Chiang Mai International Airport (Airports of Thailand Public Company Limited: AOT) to confirm the species contain in powder sample taken from a passenger who carried the sample and claimed that it is the Ophiocordyceps. DNA extraction, DNA amplification using real-time PCR were performed as described in Experiment 1.

Experiment 3: Authentication of Ophiocordyceps commercial products

Two commercial products claimed to be O. sinensis (S1 and S2) and two claimed to be O. militaris (M1 and M2) were purchased and tested. DNA extraction, DNA amplification using real-time PCR were performed as described in Experiment 1.