Ophiocordyceps sinensis (syn. Cordyceps sinensis) is an extremely rare and precious traditional Chinese medicine (TCM) with multiple medicinal values (Wang and Yao, 2011; Quan et al., 2014). This medicinal material is mainly collected in alpine regions over 4,000 m around mountain snowlines on the Tibetan Plateau, the neighboring provinces of the Tibetan autonomous prefectures and the high Himalayas (Yi et al., 2011). As reported in the New Compilation of Materia Medica, O. sinensis is beneficial to the kidney. Treatment with O. sinensis has also been claimed to have curative effects on several conditions, including erectile dysfunction, bronchial diseases, diabetes, cough and cold, jaundice (Ashok Kumar and Kailash Chandra, 2011; Sirisidthi et al., 2015). The main bioactive components found in O. sinensis are adenosine, cordycepin, cordymin, cordysinin, ergosterol, guanosine, myriocin, melanin, lovastatin, and sitosterol (Hui-Chen Lo et al., 2013). Due to strict environmental requirements, O. sinensis collected in the field is considered much more pharmacologically valuable than which obtained through culture; however, at present, most of the natural materials is collected by local farmers who do not have the expertise to differentiate between O. sinensis and related species. According to one recent study, even some of the O. sinensis materials used for study purposes may contain mycelium from other related species (Dong and Yao, 2010). The increasing rate of adulterated O. sinensis preparations not only harms consumers and the reputation of Traditional Chinese Medicine (Qin et al., 2011) but also hampers scientific research on this product.

The present identification of O. sinensis relies mostly on morphological characteristics, even though this approach has long been controversial. The genus Ophiocordyceps was officially defined by Sung et al. (2007) and Chen et al. (2013) and includes O. sinensis and similar species distributed within the alpine regions such as O. gansuënsis, O. crassispora, O. kangdingensis, O. multiaxialis, O. nepalensis, and others. It is difficult to distinguish these species morphologically (Shrestha et al., 2010) and it is even difficult to differentiate O. sinensis from the closely related adulterants, such as Cordyceps gunnii, Cordyceps cicadae, Cordyceps militaris, Cordyceps liangshanensis and Ophiocordyceps nutans. Chemical methods have also been applied to authenticate O. sinensis (Hu et al., 2015; Zhang et al., 2015); however, these method required relative large amounts of sample material. Genetic methods such as analysis of internal transcribed spacer sequences (ITS) and random amplified polymorphic DNA (RAPD)-derived molecular markers have also been used to identify O. sinensis (Lam et al., 2015). These methods have focused on detection of O. sinensis in untreated fungal material rather than processed materials where DNA degradation or fragmentation can occur (Meissner et al., 2007; Shadi et al., 2011). Therefore, in the present study, a method with specific-species primers was developed in order to increase the probability of detection of O. sinensis and common fungal adulterants even in processed samples.

O. sinensis has a long history of use as a traditional medicine in China. Due to over exploitation in the past decades, O. sinensis has been listed as an endangered species. Naturally produced O. sinensis is worth more than gold and because of this high value, adulterants have emerged frequently in recent years, which leads to market instability and a decline in consumer confidence.

DNA-based identification has become important for the identification of medical plants (Ali et al., 2014) as this technique is convenient, generally accurate and usable by people without taxonomic knowledge. The ITS sequence has been recently selected as the official marker for fugal genetic identification by the Consortium for the Barcode of Life (Das and Deb, 2015). ITS sequences amplification was used to identify fungi from soils or water as an environmental DNA barcode (Bellemain et al., 2010). Dentinger et al. compared the suitability of cytochrome oxidase subunit I (CO1) gene and ITS sequences for mushrooms and fern allies identification and determined that ITS-based identification is superior (Dentinger et al., 2011). Our previous study focused on the identification of raw O. sinensis materials based on ITS sequences (Xiang et al., 2014); however, the suitability for use with processed TCMs was not determined. The current study demonstrated that it is possible to apply PCR-based methodology to determine the presence of O. sinensis DNA in TCMs. Therefore, in another previous study, we proposed a mini-barcoding technique using short barcodes with a relatively high identification specificity for TCM (Liu et al., 2016), demonstrating immediate relevance to both science, industry and consumers. Further studies on mini-barcoding for the identification of TCM are necessary and beneficial. Although ITS sequences are commonly used to identify fungi, the requirement for relatively intact DNA to obtain complete ITS amplicons can make this approach difficult when DNA is extracted from processed samples whose DNA might have been degraded. We hypothesized that amplification of shorter regions of ITS might be possible with DNA from processed samples since Lo et al. were able to amplify a 88-bp fragment from TCM material after it had been boiled for 120 min (Lo et al., 2015). The present study also showed that a 146-bp fragment could be amplified from DNA extracted from processed O. sinensis samples, whereas amplification of the entire ITS region was not possible. For the first time, a specific primer pair is proposed and is proved to be a very efficient tool for the identification of O. sinensis and its adulterants.

The specificity of this primer pair, allows authentication of O. sinensis materials by PCR and amplicon detection along without the need for sequencing. Therefore, analysis times and costs are reduced. The assays can potentially be further simplified and expedited by utilizing isothermal recombinase polymerase amplification and an amplicon detection method that does not involve gel electrophoresis (Del Río et al., 2014). The application of the O. sinensis-specific primer pair along with the five primer pairs targeting DNA from common adulterants should allow determining if a sample said to only contain O. sinensis actually also, or exclusively, contains adulterants added inadvertently or deliberately.

In this study, a species-specific primer pair that amplifies a 146-bp sequence unique to the ITS region of O. sinensis was established. Besides that, five specific primer pairs for common adulterated species were also established. The method developed in this study provides users with an easy authentication method and may make a major contribution to the detection of counterfeit products of O. sinensis in the markets. In conclusion, this method can greatly expand the molecular identification of DNA-degraded materials and result in the rapid authentication of O. sinensis and its common adulterants among all its congeners with high accuracy, specificity and low cost.

JH designed this study. JH and LX provided experimental data. YL analyzed the raw data and drafted the manuscript. All authors helped to finish the manuscript and approved the final manuscript.