Multiuse of Bar-HRM for Ophiocordyceps sinensis identification and authentication

Bar-HRM is a hybrid method which combines DNA barcoding and High Resolution Melting analysis. It has proven to be a fast, cost-effective and reliable molecular approach for species identification and authentication. Here, three aspects of the use of Bar-HRM are focused on. First, Bar-HRM is used to discriminate between closely related Ophiocordyceps species. Second, identification of an unknown powder that is claimed to be Ophiocordyceps species using Bar-HRM. Third, authenticating the O. sinensis products sold on the market by the Bar-HRM. Results from HRM analyses with ITS primers shows that the two Ophiocordyceps species (Ophiocordyceps sinensis and Ophiocordyceps militaris) were easily differentiated. Also, an unknown sample was able to be identified in less time compared with using DNA barcoding alone. In addition, the substitution or adulteration of O. sinensis products sold on market was detected via Bar-HRM. The substitution or adulteration of inferior Ophiocordyceps species, particularly O. militaris in high price O. sinensis products has been a concern throughout Asia. Based on our results, the Bar-HRM was again proved to be a promising tool for species identification and authentication.

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 Thailand 22 .
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 authentication 19,23,24 , 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 electrophoresis 25 , high performance liquid chromatography (HPLC) 27,27 and microscopic examination 28 . 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).
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.  was performed in triplicate on each of the tested species to establish the T m . 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 T m 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. 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. 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 Li 22 . As the results very clearly demonstrate, it is important to have a standard or strict quality control of O. sinensis products.
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 (T m ) 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 ddH 2 O. The nucleotide of forward and reverse primers 31 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 T m 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.