Differentiation of medicinal Cordyceps species by rDNA ITS sequence analysis

Chongcao (the sexual stage of Cordyceps sinensis) is an important traditional Chinese fungal drug commonly used as a tonic and/or drug for hundreds of years. Recently, due to an increased demand for Cordyceps sinensis, the isolation of the fungus from the environment has been insufficient, and so other species of Cordyceps or artificially cultured Cordyceps sinensis mycelium as a conidial form (asexual stage) has been used to substitute for wild Cordyceps sinensis. There are more than ten Cordyceps species, and their related asexual species that also carry the name Chongcao on commercial markets. Since the value of each Chongcao may differ by more than 100-fold, the authentication of Chongcao is essential to secure their efficacy. However, the identification of the origin of a particular species, especially of its bio-products on the market, is difficult. Here, DNA sequence analysis of the rDNA ITS (internal transcribed spacer) was exploited to differentiate the medicinal species Cordyceps sinensis from several related Cordyceps species, such as, e. g., C. militaris, C. gunnii and their asexual stages. The data demonstrate that the rDNA ITS sequences of C. sinensis collected from different geographical regions are almost identical and are significantly different from the substitutes, so that they could be easily distinguished at the DNA level. Furthermore, based on the highly divergent ITS region of rDNA, a pair of species-specific primers were designed and used for the rapid identification of Cordyceps sinensis.