接第四篇

4.5. Establishment of a SYBR Green Real-Time PCR Assay

　　Real-time PCR was performed using a Qiagen Rotor-Gene Q thermal cycler (Qiagen, Valencia,CA, USA) with SYBR green, in 20-uL reaction volumes. To generate a standard curve for each species,real-time PCR reactions were performed in 20 uL reaction mixtures containing 1× QuantiNova™SYBR green PCR master mix, templates (one of the following five 10-fold serial dilutions for each species: 15 ng, 1.5 ng, 150 pg, 15 pg, or 1.5 pg), and 0.7 uM of each of the following primer pairs:CM F3/R3 for C. militaris, CP F4/R3 for C. pruinosa, IC F3/R2 for I. cicadae, IT F3/R2 for I. tenuipes,and OS F3/R3 for O. sinensis. gDNAs extracted from pure fungal cultures of each species were used as templates. Negative control reactions were performed in the absence of template DNA. The real-time PCR amplification conditions were as follows: a pre-denaturation step at 95 ℃ for 2 min; followed by 40 cycles of 95 ℃ for 10 s, and 55 ℃ for 20 s. The amplification program finished with a melting curve analysis, i.e., a progressive denaturation of PCR products from 60 ℃ to 99 ℃, at a rate of 1 ℃ every 5 s. The efficiency (E) of the real-time PCR amplification was calculated from the slope of the standard curve using the equation E = (10 - 1/slope - 1) × 100. The threshold cycle (Ct) was calculated from the slope of the standard curve for each species, using Rotor-Gene Q series software(Version 2.1; Qiagen).

4.6. Verification of the SCAR Markers and Real-Time PCR Assay Using Commercial Products

　　To validate the SCAR markers and the real-time PCR assay, 17 commercial Cordyceps and related products were purchased from markets in Korea, Bhutan, and China, and their taxonomic origins were verified. About 15 ng of total gDNA extracted from individual samples was used as a template in 20-uL reaction mixtures. The SCAR PCR amplification was performed under the same conditions as described for developing the SCAR markers, with each of the 0.5 uM primers that are listed in Table 3.As a PCR amplification control, we also amplified nrDNA-ITS regions for all of the 17 commercial products using ITS1 and ITS4 primers. The specificities of the SCAR markers and the taxonomic origins of the 17 commercial samples were confirmed by 1.5% agarose gel electrophoresis, depending on the amplicon size and specificity, and they were reconfirmed by comparing the resulting sequences of the PCR products (SCAR and nrDNA-ITS products) with the control nrDNA-ITS sequences after gel isolation and sub-cloning into the pGEM-T Easy vector (Promega, Madison, WI, USA). BLAST analyses of the SCAR and nrDNA-ITS PCR product sequences were also conducted to confirm the specificities of the PCRs and the identity of the respective species, using BLAST-based comparison and similarity analysis involving the obtained sequences, as well as sequences deposited in the NCBI GenBank and BOLD databases. Real-time amplification was performed as described above, with approximately 15 ng of template DNA, extracted from each of the 17 commercial samples.

4.7. Data Availability

　　The finalized representative nrDNA-ITS sequences obtained from the 18 samples representing the five fungi species (Cordyceps and its related species) were deposited in the NCBI GenBank database under the following accession numbers: O. sinensis, MG833285–MG833287; C. militaris, MG833281–MG833284; C. pruinosa, MG833288–MG833290; I. tenuipes, MG833293–MG833296; I. cicadae,MG833291–MG833292; and B. bassiana, MG833297–MG833298.

5. Conclusions

　　In the current study, we obtained the nrDNA-ITS sequences of five Cordyceps and related medicinal fungal species, O. sinensis, C. militaris, C. pruinosa, I. tenuipes, and I. cicadae. Based on these sequences, we developed two sets of SCAR markers for identifying each species and for detecting the contamination of inauthentic adulterants. Using these species-specific SCAR markers, we also established a real-time PCR assay that is capable of identifying the taxonomic origin, the degree of contamination (purity), and the amount of the adulterants in samples. The SCAR-based conventional and real-time PCR assays were also verified using commercially processed food products and herbal medicines. These assays may be used to identify authentic fungal species and to prevent the agricultural, industrial, and therapeutic applications of inauthentic O. sinenesis. In addition, it may constitute a reliable tool for the quality control and safety assurance of medicinal and food ingredients for Cordyceps and its related materials.

Supplementary Materials: The following are available online. Figure S1, Confirmation of species identification for six fungal species by the construction of a phylogenetic tree based on their nrDNA-ITS sequences; Figure S2, Comparison of nrDNA-ITS sequences and positions of species-specific primers for the development of SCAR markers; Figure S3, Establishment of a real-time PCR assay for distinguishing Cordyceps and its related species, and the results from real-time PCR assays of commercial products; Figure S4, Confirmation of primer specificity by the alignment of species-specific SCAR primer regions used for both conventional and real-time PCR assays.

Author Contributions: B.C.M. designed the experiments, collected and identified the fungal material, analyzed the sequencing data, and wrote the manuscript. W.J.K., I.P., and P.N. performed the experiments, and marker development and validation. G.H.S. collected and identified the fungal material. All authors contributed to the experiments and approved the final manuscript.

Funding: This work was funded by [Ministry of Food and Drug Safety] grant number [16162MFDS060] and [Korea Institute of Oriental Medicine] grant number [K18411]. The APC was funded by [K18411].

Acknowledgments:We thank the Korean Agricultural Culture Collection (KACC) at the National Agrobiodiversity Center, and the Korean Collection for Type Cultures (KTCC) at the Korea Research Institute of Bioscience and Biotechnology, for providing the fungal material. We also thank the Herbarium of Korea Standard Herbal Resources(Index Herbariorum code KIOM) for providing the medicinal material.

Conflicts of Interest: The authors declare no conflicts of interest.