Discriminatory Power Evaluation of Nuclear Ribosomal RNA Barcoding Sequences Through Ophiocordyceps sinensis Related Samples.

Ping Zhang,Bin Liu,Feng Wei,Shenghui Cui,Xiu Ren,Shuangcheng Ma,Shuai Kang

doi:10.3389/fmicb.2018.02498

Abstract

Since the cost of Ophiocordyceps sinensis has increased dramatically and the counterfeits may have adverse effect to health, a rapid and precise species-level DNA barcoding identification system could be a potent approach and significantly enhance the regulatory capacity. The discrimination power of three subunits sequences from nuclear ribosomal RNA gene cluster were determined by Simpson’s index of discrimination using 43 wild O. sinensis fruiting bodies, pure cultures, commercial mycelium fermented powder and counterfeits. The internal transcribed spacer (ITS) sequences showed the highest variance and discrimination power among 43 samples, as determined by Simpson’s index of discrimination (D = 0.972), followed by large subunit (LSU; D = 0.963) and small subunit (SSU; D = 0.921). ITS-2 sequences showed the highest discrimination power for 43 samples among ITS-1, ITS-2, and 5.8S region of ITS sequences. All O. sinensis samples were grouped into a unique ITS sequence cluster under 95% similarity and two O. sinensis samples and six non-O. sinensis samples showed false claims. Our data showed that the ITS region could provide accurate species identification for O. sinensis samples, especially when macroscopic and microscopic method could not be applied in the highly processed commercial products. Since the authentication of O. sinensis related products is essential to ensure its safety and efficacy, identification of O. sinensis through ITS sequence comparison or unique PCR amplification of the species specific target, such as the ITS region, should be considered in the next revision of Chinese pharmacopeia.

Highlights

Ophiocordyceps sinensis, as a well-known Chinese caterpillar fungus, has been used in tonic and healthy food among Asia countries from the 15th century since it contains multiple valuable medicinal components determined by modern pharmacological science (Sun et al, 2017; Tsuk et al, 2017; Wang et al, 2017)
Three subunits from nuclear ribosomal RNA gene cluster, including nuclear ribosomal internal transcribed spacer (ITS), large subunit (LSU), and small subunit (SSU) regions in O. sinensis, have been widely used in fungi identification (Xu, 2016) and the ITS region was formally recommended by the International Fungal Barcoding Consortium as the primary fungi barcode (Schoch et al, 2012)
An Ophiocordyceps reference material was obtained from the National Institutes of Food and Drug Control (NIFDC), and two reference strains were obtained from the China General Microbiological Culture Collection Center (CGMCC) (Table 1)

Summary

Introduction

Ophiocordyceps sinensis, as a well-known Chinese caterpillar fungus, has been used in tonic and healthy food among Asia countries from the 15th century since it contains multiple valuable medicinal components determined by modern pharmacological science (Sun et al, 2017; Tsuk et al, 2017; Wang et al, 2017). Discriminatory Power Evaluation for O. sinensis dramatically because of the contradiction between limited natural resource and increasing demand. Other Ophiocordyceps related fungi and the conidial form of the artificially cultured O. sinensis fermentation mycelia have been used as substitutes in Chinese medicine and healthy food (Zhou et al, 2014; Cao et al, 2015). Identification of O. sinensis through DNA barcoding has been reported (Xiang et al, 2013), but limited data were available to characterize the discrimination power of different nuclear ribosomal DNA barcoding regions for O. sinensis

Methods

Results

Discussion

Conclusion