Detection of Ophiocordyceps sinensis and Its Common Adulterates Using Species-Specific Primers

Abstract

Ophiocordyceps sinensis is a fungus that infects Hepialidae caterpillars, mummifying the larvae and producing characteristic fruiting bodies (stromata) that are processed into one of the most valued traditional Chinese medicines (TCM). The product commands a very high price due to a high demand but a very limited supply. Adulteration with other fungi is a common problem and there is a need to test preparation for the presence of the correct fungus. In the current study, a PCR-based approach for the identification of O. sinensis based on a segment of the internal transcribed spacer (ITS) region was developed. The segments is 146-bp in size and is likely to be amplified even in materials where processing led to DNA fragmentation. Primer development was based on the alignment of sequence data generated from a total of 89 samples of O. sinensis and potential adulterants as well as sequences date from 41 Ophiocordyceps species and 26 Cordyceps species available in GenBank. Tests with primer pair, DCF4/DCR4, demonstrated generation of an amplicon from DNA extracted from O. sinensis stromata, but not from extracts derived from adulterants. Species-specific primer pairs were also developed and tested for detection of the common adulterants, Cordyceps gunnii, Cordyceps cicadae, Cordyceps militaris, Cordyceps liangshanensis and Ophiocordyceps nutans. The collection of primers developed in the present study will be useful for the authentication of preparation claiming to only contain O. sinensis and for the detection of fungi used as adulterants in these preparations.

Establishment of a PCR Assay for the Detection and Discrimination of Authentic Cordyceps and Adulterant Species in Food and Herbal Medicines

“…The identification of authentic species has become a crucial issue for the quality control of food and medicinal sources, because most of these materials are collected from wild habitats or are cultivated on farms [ 34 ]. Since the accurate identification of fungal species is very difficult, depending on the conventional method used, more reliable and objective methods are required in order to discriminate between strains at the species level [ 3 ]. In this study, we developed a simple PCR assay method, and confirmed that the comparative analysis of nrDNA-ITS sequences is one of the most reliable tools for overcoming these difficulties.…”

“…Gmel.) Fr., are repeatedly used as adulterants of authentic Cordyceps [ 1 , 3 ]. Furthermore, only two species, C. militaris and Isaria tenuipes Peck (syn.…”

“…are used as substitutes for C. militaris and I. tenuipes , respectively, because of their morphological similarities, including the color and shape of their fruiting bodies, and their host insect species. Thus, C. militaris , C. pruinosa , I. tenuipes , and I. cicadae are used to adulterate the herbal medicine Cordyceps ( O. sinensis ); C. pruinosa and I. cicadae are used as substitutes of C. militaris and I. tenuipes , respectively, in food products, and they are also used to adulterate the herbal medicine Cordyceps [ 3 , 6 , 7 , 8 ]. The various counterfeited products derived from these inauthentic species are consistently produced, and they adversely affect the safety, quality, and therapeutic efficacy of herbal medicines and dietary supplements [ 9 ].…”

“…However, these O. sinensis -specific PCR assay methods could not identify the species of adulterants and contaminants. Liu et al reported a set of species-specific primers that can identify six Cordyceps and related species, using O. sinensis , C. militaris , and I. cicadae , C. gunnii , C. liangshanensis , and O. nutants [ 3 ]. Although they developed species-specific markers for identifying the six species, including O. sinensis , C. militaris , and I. cicadae , the range of species that are distributed as inauthentic adulterants in the market are, however, different between Korea and China, and the detection limits of the adulterant species were not clarified [ 3 ].…”

“…Liu et al reported a set of species-specific primers that can identify six Cordyceps and related species, using O. sinensis , C. militaris , and I. cicadae , C. gunnii , C. liangshanensis , and O. nutants [ 3 ]. Although they developed species-specific markers for identifying the six species, including O. sinensis , C. militaris , and I. cicadae , the range of species that are distributed as inauthentic adulterants in the market are, however, different between Korea and China, and the detection limits of the adulterant species were not clarified [ 3 ]. Therefore, an additional marker is needed to discriminate between Cordyceps and its related species, including additional adulterant fungal species, and to improve the currently available genetic tools, in order for the markers to be used as simple and rapid assays of commercially processed products in Korea.…”