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.
Highlights
A total of 314 identified internal transcribed spacer (ITS) sequences of O. sinensis, including 131 sequences generated in the previous study, were obtained with a length of ∼500 bp after alignment; 112 published ITS sequences of 41 different species in Ophiocordyceps and 250 published ITS sequences of 26 species in Cordyceps were downloaded from the GenBank database
The DNA in decoctions boiled for 60 or 90 min was amplified with the universal primer pair 5F/4R, as shown in Figure 2, it appears that the DNA extracted from the O. sinensis decoctions was possibly too fractured or otherwise degraded by boiling for 60 or 90 min to serve as template for amplification of the ITS sequence with the universal ITS primers
polymerase chain reaction (PCR) with the O. sinensis-specific primer pair yielded DNA that could be visualized after gel electrophoresis
Summary
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. Due to strict environmental requirements, O. sinensis collected in the field is considered
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