A simple and effective method to discern the true commercial Chinese cordyceps from counterfeits

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.

morphological appearance 16 . To ensure the medicinal and edibility safety of Chinese cordyceps products in the marketplace, many detection methods for monitoring and verifying the Chinese cordyceps ingredients have been developed. The traditional methods, including macroscopic and microscopic observation 19,20 , which depend on the tester's subjective judgment and professional experience, could effectively recognise the crude Chinese cordyceps, except as powdered products. 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 PCR 25 , have been developed and used for species identification in recent years. However, none of these methods could clearly differentiate Chinese cordyceps from fermented mycelia in powdered form 16,17,25 . Because Chinese cordyceps is a parasitic complex, both the remains of the host larvae and the fungal sexual stroma are integral parts, as a commodity and raw medicinal material. Neither the fungus O. sinensis nor its host species are equivalent to the TCM Chinese cordyceps. Thus, a scheme that identifies both the O. sinensis fungus and its host species is theoretically reasonable and necessary, and it should more accurately identify Chinese cordyceps.
Duplex PCR technology is a good option for this purpose, because it can not only detect both targets simultaneously but is also simple and reliable. Minor genetic variations in O. sinensis from different geographical regions have been reported 26 , and there are up to 60 host species of the fungus O. sinensis 13,16,27 . Here, degenerate primer pairs were identified and designed, and a duplex PCR technique for Chinese cordyceps detection was developed. Additionally, it was confirmed that this strategy was applicable and could provide a new practical way to identify the TCM Chinese cordyceps.

Results
The development and optimisation of the duplex PCR method. In total, 78 ITS nucleotide sequences of O. sinensis were aligned and showed high identity levels, and they differed from those of the counterfeit species (Fig. 1A). Areas that were conserved and stable among O. sinensis species but differed greatly in the sequences of other species were selected as targets for designing species-specific identification primers. Because a few base variation still existed in the conserved and stable regions within these species, degenerate oligonucleotide primers for the ITS region were designed for good compatibility. The same was true for the host species' mitochondrial cytochrome oxidase c subunit 1 (COI) gene primers (Fig. 1B). Owing to the existence of single-base differences, degenerate primers were designed for the detection of the COI gene. Then, only 1 out of 17 primer pair combinations was selected for the detection of Chinese cordyceps. The CITS primer pair (CITS-F10′: 5′-GTTGCCTCGGCGGGAC-3′/CITS-R10′-2: 5′-CMTTTGCTTGCTTCTTGACTGAG-3′) and the COI primer pair (COI-F: 5′-GGAAATCCHGGATCTTTAATT-3′/COI-R: 5′-GATGCCCCMGARTGTGCAAT-3′), were further tested for their species-specificity and retained for subsequent experiments. The primer pairs, in same PCR reactions, at a 0.3-μM primer concentration and at an annealing temperature of 54 °C always generated clean band of the expected size for O. sinensis (113 bp) and its host Hepialiae (302 bp) in the positive control template. Then, the duplex PCR assay was successfully developed for Chinese cordyceps identification. Fig. 2, the predicted 185-bp amplicon was amplified from all the samples tested, except for the negative control. Thus, DNAs over a wide taxonomic range can be detected, and the validity of the extracted DNAs for PCR can be assessed using the T18S-F/T18S-R primer pair. The amplicon could be used effectively as an analytical control to evaluate the authenticity of the template DNA.

Specificity of the duplex PCR. As shown in
Duplex PCR assays were further performed, and the expected fragments for the ITS (113 bp) and COI gene (302 bp) were successfully amplified from 17 Chinese cordyceps samples using the two primer pairs (CITS-F10′/ CITS-R10′-2 and COI-F/COI-R), and two clear electrophoretic strips displayed the GelRed stain (Fig. 2). A single band of 113 bp appeared in the mycelial samples, and a band of 302 bp appeared in the moth and C. gracilis samples. No PCR products were detected from the other species tested, including the five readily confused varieties [C. hawkesii (C. gunni), C. liangschanensis, C. barnesii and C. militaris], the two counterfeit plants (S. geobombycis and Euphorbia kansui), one sample of a powdered crop mixture and the negative water control. The results showed that when the two target bands appeared from the same reaction, the test must be considered positive, and when none or only one target was detected, the test must be considered negative. In our specificity tests, two target amplicons were detected only in those samples identified preliminarily as Chinese cordyceps by the experts.
Cordyceps gracilis, named "XinJiangChongCao" in Chinese, is shaped like Chinese cordyceps. It is a complex containing the parasitic ergot fungus C. gracilis and its host lepidopteron larvae. The larvae of the moth Hepialus altaicola 28 , which is the host component of "XinJiangChongCao", belongs to a species of Hepialidae family, which is the same as that of the host of Chinese cordyceps. As shown in Fig. 2, only the 302-bp host-specific amplicon appeared in the C. gracilis sample. This result adequately verified and confirmed the assay's specificity.
A sequence analysis showed that the two amplified fragments were more than 99% identical to the corresponding COI gene of Hepialidae species. (GenBank Accession No: KM197522, KF696585, JQ325935, etc.) and the ITS region of O. sinensis (GenBank Accession No: KY321845~321856, MF598754, MF403011, KX082968, etc.), respectively. It was strong evidence for the specificity of the duplex PCR assay. Thus, the duplex PCR method developed in this study specifically identified the Chinese cordyceps.

Sensitivity.
In the sensitivity test, 50.23 ng/μL total genomic DNA was initially extracted from the reference material, powdered Chinese cordyceps, and nine serial DNA dilutions were generated by fivefold serially diluting the initial extract. Then, 2 µL of each DNA dilution was added as the template in a PCR reaction, corresponding to approximately 20,000, 4,000, 800, 160, 32, 6.4, 1.3, 0.25 and 0.05 pg of the specific amplicon of the Chinese cordyceps per PCR reaction. As shown in Fig. 3, both target amplicons were simultaneously detected down to the 32 pg level in all the parallel reactions. The amplicons could not be detected in all the reactions at a lower template amount. Thus, the limit of detection (LOD), with a 95% confidence interval, for the duplex PCR assay was 32 pg in Scientific RepoRtS | (2020) 10:2974 | https://doi.org/10.1038/s41598-020-59900-9 www.nature.com/scientificreports www.nature.com/scientificreports/ accordance with the criteria reported by Žel et al. 29 . Thus, the Chinese cordyceps was detected with an acceptable level of precision and accuracy. These results confirmed that the duplex PCR assay established in this study was a good approach for detecting and authenticating Chinese cordyceps.
Applicability. Some commercial samples were tested. As shown in Table 1, in total nine raw samples (Samples 30-T, 32-T-38-T and 40-T) produced positive amplicons, and the target bands for the ITS from O. sinensis and the COI gene from the host Hepialidae species were synchronously amplified. No bands were detected in sample 31-T and three health-care products (Samples 42-T-44-T). Additionally, a single amplicon of O. sinensis was observed in sample 39-T and two other health-care products (Samples 41-T and 45-T). This confirmed that spurious products exist on the market and that the presence of Chinese cordyceps in the samples could be diagnosed accurately. The duplex PCR method developed in this study could distinguish products made from powdered mycelia of O. sinensis fungi from raw natural Chinese cordyceps. Thus, it is a quick and efficient way to identify and authenticate Chinese cordyceps.

Conclusions and Discussions
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 easily be performed in any molecular biology laboratory. It could specifically detect raw Chinese cordyceps and its primary components in products in which the genomes are undamaged. The results of the specificity analysis were in accordance www.nature.com/scientificreports www.nature.com/scientificreports/ with the preliminary identifications provided by experts. The LOD for Chinese cordyceps DNA could be as low as 32 pg. The accuracy and precision of the established duplex PCR method were confirmed and validated in this study. The applicability test further confirmed that the developed duplex PCR method can reliably identify and authenticate Chinese cordyceps. In addition, the duplex PCR method developed in this study is very simple to operate and cost-effective, which will be beneficial for its popularisation and utilisation. We believe that this method will be useful in the quality control and the identification of Chinese cordyceps and related products. The methodology and developmental strategy could be applied to other TCM materials as well, which will make authenticating TCMs easier. However, the method is not perfect. We were not able to differentiate mixed powders containing O. sinensis fungus mycelia and its insect host caterpillar from powders of the natural Chinese cordyceps using this method, although it was difficult to artificially raise the specific host insects required. In the future, chromatography methods that can detect the specific components of the Chinese cordyceps could be used together with the duplex PCR method. This may increase the reliability and accuracy of the identification of true Chinese cordyceps in any form.  were isolated from stromal tissue of natural Chinese cordyceps specimens (9-XZ-LS) and were cultivated under previously described conditions 30 . The mycelia were then collected and conserved at −80 °C for extraction of genomic DNA. In total, 11 items (30-T~40-T), which were dried raw materials, labelled as Chinese cordyceps, were purchased as test sample from Jingdong Mall (an online shopping platform) (https:// www.jd.com/) and Hehuachi Professional Market for Chinese Herbal Medicine, a local market in Chengdu. Two health-care products (41-T and 42-T) labelled with "DongChongXiaCao" were purchased from local TCM pharmacies in Chengdu, and three healthcare products (43-T-45-T) were purchased from Jingdong Mall. Detailed information regarding all the samples are shown in Table 2.
DNA extraction and purification. All the samples were treated and homogenised in liquid nitrogen.
Genomic DNAs were isolated and purified from all the samples, except for two mycelia specimens, using a Plant Genomic DNA Kit (DP305-02, TIANGEN Biotech Co., Ltd., Beijing, China) in accordance with the manufacturer's instructions. However, before that, the homogenised samples (50-100 mg each) were pre-processed in Proteinase K buffer [20-mM Tris-HCl (pH 8.0), 5-mM ethylenediaminetetraacetic acid (EDTA), 400-mM NaCl, 0.3% sodium dodecyl sulfate and 200 μg/mL Proteinase K (P6556, Sigma-Aldrich, Saint Louis, MO, USA)]. The resultant mixtures were incubated at 55 °C for 30 min, followed by 95 °C for 5 min. An equal volume of phenol/ chloroform (1:1) was added to the suspensions and mixed fully for 5 min, and then, the mixtures were centrifuged at 12,000 × g for 5 min. The supernatants were transferred to fresh tubes and treated once with chloroform.
The total genomic DNAs of the two mycelia samples were prepared using the benzyl chloride method 31

Specificity test.
To determine the specificity of the primer pairs and the obtained method, the duplex PCR was tested three times using both the ITS and COI gene primer mixtures and template DNAs from Chinese cordyceps samples (17), several easily confused varieties (5), counterfeit plants (2), mycelial samples (2), a moth from the family Hepialiae (1) and a powdered mixture of conventional crops seeds (1), as mentioned in Section 2.1. Additionally, DNA from the Chinese cordyceps reference material was used as the positive control, and sterile water was used as the negative control.

Sensitivity analysis.
To further evaluate the sensitivity of the established method, genomic DNA from raw Chinese cordyceps was prepared and fivefold serially diluted using nuclease-free Tris-EDTA buffer to 0.05 pg/ μL, and the gradient dilution technique previously published by Zhang et al. 32 . The duplex PCR assays were performed as described in Section 2.4, and the serial dilutions were used as the templates. All the reactions were repeated three times, with 15 parallel runs for each template DNA, to determine the LOD of the duplex PCR assay based on the positive result criteria 29 .
Applicability test. To test the applicability of the duplex PCR method for analysing practical samples, 16 samples were purchased from a local market or Jingdong Mall, as described in Section 2.1. DNAs were isolated from 11 raw Chinese cordyceps samples and 5 health-care products containing Chinese cordyceps, according to the labels. Then, the duplex PCR assays were performed in triplicate using the obtained DNAs as templates. The genomic DNA isolated from the Chinese cordyceps reference material was used as the positive control. Sterile water was used as the negative control.