Authentication of Cordyceps sinensis by DNA Analyses: Comparison of ITS Sequence Analysis and RAPD-Derived Molecular Markers

Abstract

Abstract:Cordyceps sinensis is an endoparasitic fungus widely used as a tonic and medicinal food in the practice of traditional Chinese medicine (TCM). In historical usage, Cordyceps specifically is referring to the species of C. sinensis. However, a number of closely related species are named themselves as Cordyceps, and they are sold commonly as C. sinensis. The substitutes and adulterants of C. sinensis are often introduced either intentionally or accidentally in the herbal market, which seriously affects the therapeutic effects or even leads to life-threatening poisoning. Here, we aim to identify Cordyceps by DNA sequencing technology. Two different DNA-based approaches were compared. The internal transcribed spacer (ITS) sequences and the random amplified polymorphic DNA (RAPD)-sequence characterized amplified region (SCAR) were developed here to authenticate different species of Cordyceps. Both approaches generally enabled discrimination of C. sinensis from others. The application of the two methods, supporting each other, increases the security of identification. For better reproducibility and faster analysis, the SCAR markers derived from the RAPD results provide a new method for quick authentication of Cordyceps.

Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers

“…In our lab, we have successfully characterized a number of plant or fungal species by developing SCAR markers based on RAPD amplicons; namely Dimocarpus longan , Acorus species (Ryuk et al 2014), Lonicera japonica (Yang et al 2014), Trichoderma cf. harzianum (Pérez et al 2014), Cordyceps sinensis (Lam et al 2015), Litchi chinensis , Angelica sinensis , and Ganoderma lucidum (Khan et al 2016).…”

Differentiating Authentic Adenophorae Radix from Its Adulterants in Commercially-Processed Samples Using Multiplexed ITS Sequence-Based SCAR Markers

“…Thus, this method needs only DNA extraction, PCR amplification, gel electrophoresis, and species identification, and can identify the botanical origin of herbal medicines within a few hours. Moreover, the DNA barcoding method involves additional costs for sub-cloning, transformation into the competent cell, and sequencing, which are not necessary in the SCAR method [21]. Judging from our laboratory experience, these additional costs amount to at least $300-$400 USD for the analysis of 10 herbal medicine samples.…”

“…However, the nrDNA-ITS regions have been of limited use because the available primers for these sequences amplify DNA from fungi, other endophytic microorganisms, and multiple copies of the ITS regions. Recently, ITS2 has been proposed as a universal DNA barcode for identifying plant species, herbal medicines, and herbaceous dietary supplements, but it has some disadvantages, such as the complexity of the experiment, the difficulty of interpreting the results, the limitation of sample numbers, and the detection of contaminants [16,20,21]. A sequence-characterized amplified region (SCAR) marker assay can reliably and reproducibly distinguish species based on sequence information obtained from DNA fingerprinting or barcoding in diverse plants and herbal medicines.…”

“…This method amplifies only target-containing samples using specific primers and differentiates positive or negative amplification of target regions, as well as length polymorphisms of target regions by gel electrophoresis of closely related samples [22][23][24]. Therefore, the SCAR method is simple to carry out, its results are simple to interpret, and it produces fewer errors in PCR amplification and sequencing [21]. Furthermore, the efficiency and ability of this assay improved with the combination of individual SCAR markers and multiplex PCR, namely, multiplexed SCAR marker assays [22,25,26].…”

Identification of Ophiocordyceps sinensis and Its Artificially Cultured Ophiocordyceps Mycelia by Ultra-Performance Liquid Chromatography/Orbitrap Fusion Mass Spectrometry and Chemometrics

“…Furthermore, although the polymerase chain reaction (PCR) has been successfully applied in the identification of Ophiocordyceps sinensis ( O.S. ) fruiting bodies [14,15,35], it cannot be used for cultured Ophiocordyceps mycelia, because the integrity of the DNA genome is compromised during the drying process [18,36,37]. …”