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Abstract
The Chinese cordyceps, a complex of the fungus Ophiocordyceps sinensis and its species-specific host insects, is also called “DongChongXiaCao” in Chinese. Habitat degradation in recent decades and excessive harvesting by humans has intensified its scarcity and increased the prices of natural populations. Some counterfeits are traded as natural Chinese cordyceps for profit, causing confusion in the marketplace. To promote the safe use of Chinese cordyceps and related products, a duplex PCR method for specifically identifying raw Chinese cordyceps and its primary products was successfully established. Chinese cordyceps could be precisely identified by detecting an internal transcribed spacer amplicon from O. sinensis and a cytochrome oxidase c subunit 1 amplicon from the host species, at a limit of detection as low as 32 pg. Eleven commercial samples were purchased and successfully tested to further verify that the developed duplex PCR method could be reliably used to identify Chinese cordyceps. It provides a new simple way to discern true commercial Chinese cordyceps from counterfeits in the marketplace. This is an important step toward achieving an authentication method for this Chinese medicine. The methodology and the developmental strategy can be used to authenticate other traditional Chinese medicinal materials.
Highlights
The ascomycetes fungus Ophiocordyceps sinensis[1], which is synonymous with Cordyceps sinensis in the historical classification system[2], colonises and mummifies the larvae of moth species in the family Hepialidae in the winter[1,2]
Molecular biology techniques based on the internal transcribed spacer (ITS) sequence of Chinese cordyceps, such as DNA barcoding[17,21,22], DNA barcoding coupled with high resolution melting[23], loop-mediated isothermal amplification[24] and nested PCR25, have been developed and used for species identification in recent years
Degenerate primer pairs targeting the ITS region of O. sinensis and the COI gene of its species-specific host were designed, and a duplex PCR assay for Chinese cordyceps was successfully established that can be performed in any molecular biology laboratory
Summary
The ascomycetes fungus Ophiocordyceps sinensis (anamorph: Hirsutella sinensis)[1], which is synonymous with Cordyceps sinensis in the historical classification system[2], colonises and mummifies the larvae of moth species in the family Hepialidae in the winter[1,2]. Molecular biology techniques based on the internal transcribed spacer (ITS) sequence of Chinese cordyceps, such as DNA barcoding[17,21,22], DNA barcoding coupled with high resolution melting[23], loop-mediated isothermal amplification[24] and nested PCR25, have been developed and used for species identification in recent years. None of these methods could clearly differentiate Chinese cordyceps from fermented mycelia in powdered form[16,17,25]. It was confirmed that this strategy was applicable and could provide a new practical way to identify the TCM Chinese cordyceps
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