Cordyceps sinensis discrimination method and system

Abstract

The invention discloses a method for distinguishing cordyceps sinensis, which is used for identifying whether artificial cordyceps sinensis exists in wild cordyceps sinensis or not, and comprises the following steps: S1, preparing a cordyceps sinensis sample and acquiring mitochondrial gene sequence data of the cordyceps sinensis sample; s2, the mitochondrial gene sequence is compared with a mitochondrial reference genome of Hepialus xiaojinensis, and the mitochondrial reference genome of Hepialus xiaojinensis is compared with the mitochondrial reference genome of S3, calculating the coverage rate and the consistency of the gene sequence; and S4, if the coverage rate is greater than or equal to 16.1% and the consistency is greater than or equal to 95%, determining that the cordyceps sinensis is artificial cordyceps sinensis. According to the method, the artificial cordyceps sinensis in the wild cordyceps sinensis in the Qinghai region can be quickly judged.

Description

Technical Field

[0001] The present invention relates to the technical field of quality control and identification of traditional Chinese medicines, and in particular to a method and system for identifying cordyceps sinensis. Background Art

[0002] Cordyceps sinensis (Berk.) Sacc., also known as Cordyceps, is one of the more commonly used precious Chinese medicinal materials. It is a complex composed of Cordyceps sinensis (Berk.) Sacc., a member of the Clavicipitaceae family, parasitizing the larvae of the Hepialidae insect bat moth, which lives in the soil of alpine meadows. After vegetative and reproductive growth, the fruiting body of Cordyceps sinensis and the dead insect sclerotium are formed. Cordyceps sinensis is sweet and mild in nature. It has the effects of replenishing deficiency, benefiting essence and qi, nourishing yin and moistening the lungs, stopping bleeding and resolving phlegm, and replenishing the kidneys and strengthening the body. It is often used to treat various diseases caused by kidney deficiency, lung qi deficiency, and kidney and lung deficiency. It is also used as a health product to improve immunity, fight cancer, protect the liver, and prolong life. There are currently 380 known species of Cordyceps in the world, of which Cordyceps sinensis is the most famous. The real Cordyceps sinensis is wild. Wild Cordyceps sinensis has strict requirements on the living environment, a narrow distribution area, and a low natural parasitic rate, which makes its natural resources very scarce. In addition, Cordyceps sinensis has high medicinal value and good effects. There is a great demand for it at home and abroad, so the price is very expensive. As a result, counterfeit products are also emerging in an endless stream. Some counterfeit products are difficult to distinguish from the real ones based on their appearance, color and taste.

[0003] At present, the existing identification technology for wild and artificially cultivated Cordyceps sinensis has the following shortcomings: traditional morphological and microstructural analysis is easily disturbed by environmental factors, is highly subjective and inaccurate (such as intraspecific variation in characteristics such as insect body ring patterns and stroma ratios); chemical component detection methods (such as markers such as adenosine and cordycepin) have a high degree of overlap with the index intervals of artificially cultivated Cordyceps due to significant regional component differences in wild Cordyceps; although DNA barcoding technology (ITS / COI sequence) can distinguish species, it cannot effectively identify wild and artificial strains; although the combined analysis method of stable isotope ratios (δ13C, δ15N) and trace element spectra has identification potential, the detection cost is high and lacks standardized database support. Existing technologies generally have problems such as reliance on large instruments, complex operations, and long identification cycles, which are difficult to meet the market's demand for rapid identification. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for distinguishing Cordyceps sinensis, which can quickly distinguish artificial Cordyceps sinensis from wild Cordyceps sinensis.

[0005] In a first aspect, the present invention provides a method for distinguishing Cordyceps sinensis, which is used to identify whether there is artificial Cordyceps sinensis from wild Cordyceps sinensis, and the distinguishing method comprises the following contents:

[0006] S1. Preparing Cordyceps sinensis samples and obtaining mitochondrial gene sequence data of Cordyceps sinensis samples;

[0007] S2, comparing the mitochondrial gene sequence with the mitochondrial reference genome of Hepialus xiaojinensis;

[0008] S3, calculating the coverage and consistency of the gene sequence;

[0009] S4. If the coverage rate is ≥16.1% and the consistency is ≥95%, the Cordyceps is determined to be artificial Cordyceps sinensis.

[0010] Furthermore, the wild Cordyceps sinensis originates from Northwest China;

[0011] Furthermore, it is the Qinghai Province region of China.

[0012] Furthermore, the Cordyceps sinensis sample includes a wild Cordyceps sinensis sample and / or an artificial Cordyceps sinensis sample.

[0013] Furthermore, in step S2: before comparing with the mitochondrial reference genome of Hepialus xiaojinensis, bacterial DNA sequences are removed in advance.

[0014] Furthermore, the sequencing data were aligned with the mitochondrial sequence of Ophiocordyceps sinensis (accession number NC_022834.1); sequence reads matching the fungal genome were removed, and only insect-derived mitochondrial DNA was retained for coverage and consistency calculations.

[0015] Furthermore, the accession number of the mitochondrial sequence of Ophiocordyceps sinensis is NC_022834.1.

[0016] Furthermore, the accession number of the Hepialus xiaojinensis mitochondrial reference genome is NC_028348.1.

[0017] Furthermore, the coverage threshold is determined by Youden index optimization, and the specific steps include:

[0018] (1) Test the ability of different coverage thresholds from 1% to 100% to distinguish between cultivated Cordyceps and wild Cordyceps;

[0019] (2) Select a threshold that maximizes the sum of sensitivity and specificity.

[0020] In a second aspect, the present invention provides a method for identifying whether there is artificial Cordyceps sinensis from wild Cordyceps sinensis, the discrimination method comprising:

[0021] S1. Preparing Cordyceps sinensis samples and obtaining mitochondrial gene sequence data of Cordyceps sinensis samples;

[0022] S2, comparing the mitochondrial gene sequence with at least one of the sequences shown in SEQ ID NOs 1 to 42;

[0023] S3, calculating the coverage of the gene sequence;

[0024] S4. If the coverage rate is 100%, it is determined that the Cordyceps is artificial Cordyceps sinensis;

[0025] Furthermore, the wild Cordyceps sinensis originates from Northwest China.

[0026] Furthermore, it is the Qinghai Province region of China.

[0027] Furthermore, the Cordyceps sinensis sample includes a wild Cordyceps sinensis sample and / or an artificial Cordyceps sinensis sample.

[0028] In a third aspect, the present invention provides a Cordyceps sinensis identification system, the system comprising:

[0029] (1) Data input module: receiving mitochondrial sequencing data of Cordyceps sinensis samples;

[0030] (2) Sequence alignment module: aligning the sequencing data with the Hepialus xiaojinensis mitochondrial reference genome (accession number: NC_028348.1);

[0031] (3) Coverage calculation module: counts the base coverage of the reference genome;

[0032] (4) Consistency analysis module: calculate sequence consistency;

[0033] (5) Judgment output module: When the coverage rate is ≥16.1% and the consistency is ≥95%, the “artificial Cordyceps sinensis” judgment result is output.

[0034] In a fourth aspect, the present invention provides a Cordyceps sinensis identification system, characterized in that the system comprises:

[0035] (1) Data input module: receiving mitochondrial sequencing data of Cordyceps sinensis samples;

[0036] (2) a sequence alignment module: aligning the sequencing data with at least one of the sequences shown in SEQ ID NOs 1 to 42;

[0037] (3) Coverage calculation module: counts the base coverage of the reference genome;

[0038] (4) Consistency analysis module: calculate sequence consistency;

[0039] (5) Judgment output module: When the coverage rate is 100%, the “artificial Cordyceps sinensis” judgment result is output.

[0040] The beneficial effects of the present invention are: by comparing with the mitochondrial reference genome of Hepialus xiaojinensis, optimizing the coverage threshold, calculating the coverage and consistency, artificial Cordyceps sinensis in the wild Cordyceps sinensis in Qinghai area can be quickly identified. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the agarose gel image of the quality inspection of the Cordyceps sample DNA;

[0042] Figure 2 The mitochondrial sequencing results of live worms in Yushu area;

[0043] Figure 3 The mitochondrial sequencing results of the dead insect in Yushu area;

[0044] Figure 4 ROC curve of the discriminative performance of the long-fragment coverage method. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Example 1

[0047] 1 Experimental methods

[0048] 1.1 Materials

[0049] For the collection of artificial Cordyceps, the main method is procurement. For the collection of natural Cordyceps samples, the first step is to select the collection site, and 18 representative sites were selected.

[0050] 1.2 Database Construction

[0051] After successful DNA extraction, the DNA is amplified in two separate PCR reactions. The PCR amplicons are fragmented using EBLTS and adapter sequences are added. The adapter-attached amplicons are then cleaned in a purification step to remove excess enzymes and reaction components. After purification, the fragmented and tagged amplicons are amplified using a PCR program that adds pre-paired adapters and sequences required for sequencing. Next, the libraries from each 96-well sample plate are mixed into a 1.7 mL tube and purified using magnetic beads. The purified libraries need to be quantified and the library concentration is measured using the Qubit dsDNA HS Assay Kit and diluted as needed to achieve a normalized concentration of 4 nM. The normalized libraries are then diluted to the final loading concentration as required by the sequencing system. Finally, the sequencing run parameters are set according to the sequencing system used, and a sample sheet is created. The sample sheet needs to include information such as sample ID, Index ID, Index, Index2, Lane, and ensure that there is a no-template control sample and a positive control for each index group / lane combination. After all steps are completed, the library is ready for sequencing.

[0052] 1.3 Sequencing

[0053] When using MiSeq Reagent Micro Kit v2 (300-cycles) for chip-on-machine testing, first remove the kit from a -25℃ to -15℃ freezer and thaw it in a room temperature water tank until it is completely thawed. You can also choose to thaw overnight in a refrigerated environment at 2℃ to 8℃, and the reagents are stable for up to one week at this temperature. After thawing, you need to flip the kit ten times to mix the thawed reagents, check whether all positions have been thawed, ensure that the reagents are fully mixed and there is no precipitation, and gently tap the kit to reduce bubbles. After the kit is thawed and mixed, denature and dilute the library if necessary, and add an optional PhiX control. Next, load the prepared library into the LoadSamples slot of the kit, wipe the foil seal with a low-dust laboratory tissue, then pierce the foil seal with a 1 ml pipette and inject 600 microliters of the prepared library. Use MiSeq Control Software (MCS) to set up the run, including restarting the system software, checking the folder location, etc. Complete the steps to load the flow cell and reagents under the guidance of the MCS interface. At the same time, you need to remove the PR2 bottle from the storage environment at 2℃ to 8℃, mix it and load it into the reagent compartment, and ensure that the waste liquid in the waste bottle is properly disposed of. In the reagent compartment, open the reagent cooler door, hold the reagent cartridge and slide it into the reagent cooler, and then close the door. If the software cannot recognize the RFID of the reagent cartridge, you need to check and resolve the read failure. Before starting the run, the system will perform a pre-run check to ensure that all components, disk space, and network connections are ready. After checking, select Start Run to start the sequencing run. During the run, you can monitor the details of the run through the "Sequencing" screen on the instrument. After the sequencing run is completed, perform instrument cleaning, including preparing the cleaning solution with Tween 20 and laboratory-grade water, and performing cleaning steps to ensure that any residual reagents in the instrument's jet lines and pipettes are rinsed out to prevent salt accumulation and crystallization, and avoid cross contamination. When wash is complete, leave the used flow cell, wash tray, and wash bottle containing remaining wash solution on the instrument to prevent the sippers from drying out and air from entering the system.

[0054] 1.4 DNA extraction and purification

[0055] Place the dried herbs in a mortar, add liquid nitrogen to freeze, and quickly grind into powder. Transfer the ground powder to a pre-cooled centrifuge tube, add 2% CTAB extraction buffer (about 700 μL) preheated to 65°C, and gently stir to ensure that the powder is fully in contact with the buffer. Place the centrifuge tube containing the sample in a 65°C water bath for more than 45 minutes, gently shaking or inverting several times every 15 minutes to promote cell lysis. Remove the sample tube from the water bath, add pre-cooled chloroform:isoamyl alcohol (24:1) mixture, and mix upside down for 5-10 minutes. Centrifuge at 10,000 rpm for 10 minutes, and carefully transfer the upper aqueous phase to a new centrifuge tube. Add pre-cooled isopropanol to the supernatant obtained, the amount added is about 2 / 3 of the supernatant volume, and gently shake the centrifuge tube up and down to precipitate the DNA from the solution. Depending on the DNA content, white suspended DNA particles or flocculent DNA suspended in the solution may be observed. Place the sample in a 4°C refrigerator for 1 hour or overnight, or immediately centrifuge at 8,000 rpm for 5 minutes to promote DNA precipitation. After centrifugation, carefully pour off the supernatant, use paper to remove excess solution from the tube wall, and retain the DNA precipitate at the bottom of the tube. Add 70% ethanol (about 600 μL) to wash the precipitate, shake the precipitate for a few seconds, and then centrifuge at 5,000 rpm to remove the supernatant. Repeat the washing step at least once. Dry the DNA precipitate at room temperature until a colorless gel is seen on the tube wall. Add sterile water or 0.1% DEPC-treated water (about 50-100 μL) to dissolve the DNA precipitate. Incubate at 37°C for a period of time to help dissolve the DNA. If the DNA sample contains impurities such as RNA or protein, further purify it by magnetic beads. Use a UV spectrophotometer to detect the content and purity of the DNA, and use agarose electrophoresis to detect the integrity of the DNA.

[0056] 1.5 Insect-derived mitochondrial sequences and first-generation sequencing

[0057] First, the mitochondrial sequence of the bat moth (NC 060512.1) and the fungal genome (NC022834.1) were obtained from the NCBI database. Subsequently, the two sets of sequences were compared using the MEGA11 software, focusing on the sites with large differences in the range of 300bp to 1600bp, and these sites were selected as the main target sequences for comparison. In the process of primer design, a series of basic principles were followed to ensure the effectiveness and specificity of the designed primers. First, the primer length should be controlled between 18 and 25 nucleotides, which can optimize its binding properties. Secondly, the GC content was set between 40% and 60%, which not only helps to enhance the stability of the primers, but also helps to improve the efficiency of the PCR reaction. In addition, the melting temperature (Tm value) of the primers should be as consistent as possible to ensure the consistency of the annealing temperature during the PCR amplification process, thereby improving the success rate of amplification. To achieve the above goals, the Primer-BLAST tool of NCBI was used, which supports the design of primers that can specifically amplify specific splicing variant genes based on the specified template sequence. After the primers are designed, they are verified by PCR technology to ensure the specificity and amplification efficiency of the primers. The composition of the PCR reaction system includes: 10× amplification buffer, dNTP mixture, specific primers, template DNA, Taq DNA polymerase, Mg2+ and double distilled water. The five elements of the PCR reaction include primers, polymerase, dNTP, template and buffer, among which the concentration of Mg2+ has an important influence on the PCR amplification results. In the experimental verification stage, agarose gel electrophoresis was used to analyze the PCR products to evaluate their specificity and amplification efficiency. After confirming that the amplified products meet the design specifications, the PCR products were further subjected to Sanger sequencing.

[0058] 1.6 Sequence alignment and assembly

[0059] The process of aligning and assembling mitochondrial sequences using NGS sequencing data involves multiple steps, including data quality control, sequence alignment, sequence assembly, and result analysis. First, the raw sequencing data needs to undergo quality control to ensure the accuracy and reliability of the data, which usually includes removing low-quality sequence reads, adapter sequences, and contamination sequences. Next, Bowtie2 is used to align the quality-controlled data with the reference mitochondrial genome sequence to identify mitochondrial DNA sequences and determine their locations in the genome. After alignment, MEGAN is used to assemble the mitochondrial sequences in the alignment results to reconstruct a complete mitochondrial genome. In particular, MEANGS software can extract and assemble mitochondrial genomes from animal genome second-generation sequencing data. It uses conserved mitochondrial protein-encoded information to obtain mitochondrial protein-encoded sequences by predicting and assembling reads, and further assembles a complete mitochondrial genome. After assembly, the results are analyzed, including gene annotation, colinearity analysis, phylogenetic tree analysis, and selection pressure analysis, which help to gain a deeper understanding of the structure, function, and evolutionary significance of the mitochondrial genome.

[0060] 1.7 Phylogenetic tree construction

[0061] Use multiple sequence alignment software such as MUSCLE to align the above NGS sequences to ensure that evolutionarily related sites can be aligned. The aligned sequences are used to construct a distance matrix, which shows the genetic distance between different sequences, usually calculated by nucleotide or amino acid differences. Next, select a suitable tree-building method and software to execute the tree-building algorithm, and verify the constructed phylogenetic tree through resampling techniques such as bootstrapping or cohesion to evaluate the node support of the tree and ensure its reliability. Finally, interpret the constructed phylogenetic tree, compare the differences in mitochondrial sequences of different individuals, analyze the length and shape of evolutionary branches, and mainly count the genetic distance as the main indicator for discrimination.

[0062] 2 Results Analysis

[0063] 2.1 Acquisition and quality analysis of Cordyceps sinensis DNA sequences

[0064] like Figure 1As shown in Table 1, the quality inspection conclusions of all samples were mildly degraded, but they still had the ability to construct a second-generation sequencing library. This shows that the overall quality of the extracted DNA is good, especially when the experimental conditions are appropriate. The concentrations in the samples ranged from 14.9 ng / μl (YSCC11) to 190 ng / μl (RG6), and the concentrations of multiple samples were within an acceptable range. The volume was 30 μl, which is suitable for the construction of sequencing libraries. The concentration of the low-concentration sample YSCC11 is only 14.9 ng / μl. Although the library can be constructed, special attention may need to be paid during the library construction process to ensure that the resulting library has sufficient sequencing depth. The high-concentration sample RG6 has a concentration of 190 ng / μl and can usually be given priority for library construction, which may result in higher sequencing coverage.

[0065] Table 1 DNA quality test conclusion

[0066] Sample name Concentration (ng / μl) Volume (μl) Quality inspection conclusion RG1 140 30 Mild degradation RG2 112 30 Mild degradation RG3 120 30 Mild degradation RG4 138 30 Mild degradation RG5 177 30 Mild degradation RG6 190 30 Mild degradation RG7 154 30 Mild degradation YSCC01 182 30 Mild degradation YSCC02 81.4 30 Mild degradation YSCC03 52 30 Mild degradation YSCC04 140 30 Mild degradation YSCC05 188 30 Mild degradation YSCC06 101 30 Mild degradation YSCC07 153 30 Mild degradation YSCC08 162 30 Mild degradation YSCC09 81 30 Mild degradation YSCC10 150 30 Mild degradation YSCC11 14.9 30 Mild degradation YSCC12 77 30 Mild degradation YSCC13 91.6 30 Mild degradation YSCC14 122 30 Mild degradation YSCC15 104 30 Mild degradation YSCC16 148 30 Mild degradation YSCC17 126 30 Mild degradation YSCC18 125 30 Mild degradation

[0067] 2.2 Comparative analysis of artificial Cordyceps and database mitochondrial sequences

[0068] Using the comparison function of NCBIBLAST, taking the artificial Cordyceps produced in Haixi as an example, the similarities of more than 90% are all bat moth mitochondria, as shown in Table 2 below. The most similar is Hepialus xiaojinensis (small golden bat moth), with a coverage rate of 100% and a similarity of 100%. At the same time, combined with the sequencing results of multiple batches of artificial Cordyceps in 2.4, the small golden bat moth is an important feature of the current artificial Cordyceps. Although wild Cordyceps parasitizing the small golden bat moth is not ruled out, the limited number of wild small golden bat moth Cordyceps can be almost ignored because the number of artificial Cordyceps based on the small golden bat moth is too large. It is determined that Cordyceps sinensis with the small golden bat moth as the insect source can be determined as artificial Cordyceps.

[0069] Table 2 Comparison of mitochondrial sequences from artificial Cordyceps sinensis with those from the NCBI database

[0070] name cover Consistency ratio Sequence length Hepialus xiaojinensis mitochondria, complete genome 100％ 100％ 15397 Ahamus yushuensis mitochondria, complete genome 96％ 99.95％ 15336 Hepialus maqenensis mitochondria, complete genome 96％ 99.95％ 15626 Thitarodes sp. XS20 mitochondria, complete genome 95％ 99.87％ 14553 Ahamus maquensis mitochondria, complete genome 96％ 99.84％ 15517 Hepialus lagii mitochondria, complete genome 96％ 99.16％ 15393 Thitarodes damxungensis mitochondria, complete genome 95％ 96.76％ 15362 Thitarodes sp. isolated SD mitochondria, complete genome 96％ 96.09％ 15389 Ahamus yunnanensis mitochondria, complete genome 96％ 95.83％ 15816 Thitarodes gonggaensis mitochondria, complete genome 95％ 93.88％ 15940 Thitarodes xunhuaensis mitochondria, complete genome 95％ 93.62％ 15712 Thitarodes sp. XK-2016 mitochondria, complete genome 95％ 93.51％ 16280 Thitarodes renzhiensis mitochondria, complete genome 95％ 93.51％ 16173 Thitarodes pui mitochondria, complete genome 95％ 93.49％ 15928 Thitarodes sp. mitochondria, complete genome 95％ 93.49％ 16055 Hepialus sp. HL18 mitochondria, complete genome 95％ 93.41％ 15935 Thitarodes pui mitochondria, complete genome 96％ 93.24％ 15064 Thitarodes shambalaensis mitochondria, complete genome 95％ 93.10％ 15612 Thitarodes sp. mitochondria, complete genome 95％ 93.07％ 15496 Thitarodes sejilaensis mitochondria, complete genome 95％ 92.92％ 15290 Triodia sylvina mitochondria, complete genome 95％ 90.48％ 15040 Napialus hunanensis mitochondria, complete genome 95％ 89.97％ 15301 Endoclita signifer mitochondria, complete genome 95％ 89.70％ 15285 Endoclita minanus mitochondria, complete genome 95％ 89.64％ 15248 Endoclita sinensis mitochondria, partial genome 95％ 89.55％ 15606

[0071] 2.3 Sequence comparison of insect-derived mitochondria and bacterial-derived mitochondria

[0072] As shown in Table 3, insect mitochondria are quite different from fungal mitochondria, with the maximum coverage rate being only 28%. The homology is very low and will not affect the specificity of characteristic sequence amplification.

[0073] Table 3 Comparison of mitochondrial sequences of Cordyceps sinensis and Cordyceps genus

[0074]

[0075]

[0076] 2.4 Sequencing characteristics of Cordyceps DNA from different periods

[0077] Live Cordyceps, rigid Cordyceps, Cordyceps with just germinated fruiting bodies, and Cordyceps with a large number of spores were used as samples, representing the early, early, mature, and late stages of Cordyceps sinensis. The purpose was to evaluate the applicability of various species identification methods by studying the residual insect DNA in Cordyceps sinensis at these different growth stages.

[0078] The whole genome of the living insect was sequenced, and then the mitochondrial genome was separated from the whole genome, and a mitochondrial genome with a completeness of 96% was obtained. When an evolutionary tree was established, it was found that the mitochondrial genome was closest to the genome of the jade tree hook bat moth, and the living insect was considered to be the jade tree hook bat moth.

[0079] By sequencing the whole genome of the dead insect, the mitochondrial genome was accurately separated from the whole genome. In this way, a mitochondrial genome with a completeness of 100% was obtained. When an evolutionary tree was established, it was found that the mitochondrial genome of the dead insect and the genome of the small golden bat moth showed extremely high similarity on the evolutionary tree, and it was believed that the insect-derived organism of the dead insect was most likely the small golden bat moth.

[0080] By sequencing the mitochondria of Cordyceps that has just grown out of the stroma, the completeness of its mitochondrial genome can be obtained to reach 11.9%. However, since the evolutionary tree algorithm cannot handle such a fragmented genome sequence, it is impossible to construct an accurate evolutionary tree to reveal its evolutionary relationship. Nevertheless, by using the BLAST method, the species relationship of Cordyceps can still be identified. As shown in Table 5, this sample is closest to the mitochondrial genome of Thitarodes sp.HN19, Thitarodes xunhuaensis or Thitarodes sp.XK-2016.

[0081] When conducting in-depth mitochondrial DNA sequencing analysis on Cordyceps that had entered the spore production stage, it was found that the genetic material of the host insect seemed to have completely disappeared from these samples.

[0082] Table 4 Sequencing results of the complete set of bat moths in Yushu area

[0083]

[0084] Table 5 Results of mitochondrial sequencing of Cordyceps sinensis that had just grown stroma

[0085]

[0086]

[0087] 2.5 Sample short fragment coverage determination method

[0088] Seven batches of cultivated Cordyceps and wild Cordyceps from 18 different locations were used for insect-derived DNA sequencing. The sequencing results are shown in Tables 6 to 30.

[0089] Table 6 Sequencing results of artificial Cordyceps batch 1

[0090]

[0091] All the identified sequences of artificial Cordyceps batch 1 have a high degree of coverage with the standard mitochondrial sequence of Hepialus xiaojinensis.

[0092] Table 7 Sequencing results of artificial Cordyceps batch 2

[0093]

[0094] All the identified sequences of artificial Cordyceps batch 2 had a high degree of coverage with the standard mitochondrial sequence of Hepialus xiaojinensis.

[0095] Table 8 Sequencing results of artificial Cordyceps batch 3

[0096]

[0097]

[0098] All the identified sequences of artificial Cordyceps batch 3 had a high degree of coverage with the standard mitochondrial sequence of Hepialus xiaojinensis.

[0099] Table 9 Sequencing results of artificial Cordyceps batch 4

[0100]

[0101] All the identified sequences of artificial Cordyceps batch 4 had a high degree of coverage with the standard mitochondrial sequence of Hepialus xiaojinensis.

[0102] Table 10 Sequencing results of artificial Cordyceps batch 5

[0103]

[0104]

[0105] All the identified sequences of artificial Cordyceps batch 5 had a high degree of coverage with the standard mitochondrial sequence of Hepialus xiaojinensis.

[0106] Table 11 Sequencing results of artificial Cordyceps batch 6

[0107]

[0108] All the identified sequences of artificial Cordyceps batch 6 have a high degree of coverage with the standard mitochondrial sequence of Hepialus xiaojinensis.

[0109] Table 12 Sequencing results of artificial Cordyceps batch 7 All the identified sequences of artificial Cordyceps batch 7 have a high degree of coverage with the standard mitochondrial sequence of Hepialus xiaojinensis.

[0110] Table 13 Sequencing results of wild Cordyceps batch 1

[0111]

[0112] All the identified sequences of wild Cordyceps batch 1 had high coverage with the standard mitochondrial sequence of Thitarodes damxungensis.

[0113] Table 14 Sequencing results of wild Cordyceps batch 2

[0114]

[0115]

[0116] All the identified sequences of wild Cordyceps batch 2 had high coverage with the standard mitochondrial sequence of Thitarodes sp.XK-2016.

[0117] Table 15 Sequencing results of wild Cordyceps batch 3

[0118]

[0119] All the identified sequences of wild Cordyceps batch 3 had high coverage with the standard mitochondrial sequence of Thitarodes sp.XK-2016.

[0120] Table 16 Sequencing results of wild Cordyceps batch 4

[0121]

[0122] All the identified sequences of wild Cordyceps batch 4 had high coverage with the standard mitochondrial sequence of Thitarodes damxungensis.

[0123] Table 17 Sequencing results of wild Cordyceps batch 5

[0124]

[0125]

[0126] All the identified sequences of wild Cordyceps batch 5 had high coverage with the standard mitochondrial sequence of Thitarodes sp.XK-2016.

[0127] Table 18 Sequencing results of wild Cordyceps batch 6 All the identified sequences of wild Cordyceps batch 6 had high coverage with the standard mitochondrial sequence of Thitarodes sp.XK-2016.

[0128] Table 19 Sequencing results of wild Cordyceps batch 8

[0129]

[0130] All the identified sequences of wild Cordyceps batch 8 had high coverage with the standard mitochondrial sequence of Ahamus yunnanensis.

[0131] Table 20 Sequencing results of wild Cordyceps batch 9

[0132]

[0133] All the identified sequences of wild Cordyceps batch 9 had high coverage with the standard mitochondrial sequence of Thitarodes damxungensis.

[0134] Table 1 Sequencing results of wild Cordyceps batch 10

[0135]

[0136]

[0137] All the identified sequences of wild Cordyceps batch 10 had high coverage with the standard mitochondrial sequence of Thitarodes sp.XK-2016.

[0138] Table 2 Sequencing results of wild Cordyceps batch 11

[0139]

[0140] All the identified sequences of wild Cordyceps batch 11 had high coverage with the standard mitochondrial sequence of Thitarodes gonggaensis.

[0141] Table 23 Sequencing results of wild Cordyceps batch 12

[0142]

[0143] All the identified sequences of wild Cordyceps batch 12 had high coverage with the standard mitochondrial sequence of Thitarodes sp.XK-2016.

[0144] Table 24 Sequencing results of wild Cordyceps batch 13

[0145]

[0146] All the identified sequences of wild Cordyceps batch 13 were highly overlapped with the standard mitochondrial sequence of Thitarodes sp.XK-2016.

[0147] Table 25 Sequencing results of wild Cordyceps batch 14

[0148]

[0149]

[0150] All the identified sequences of wild Cordyceps batch 14 had high coverage with the standard mitochondrial sequence of Thitarodes sp.XK-2016.

[0151] Table 26 Sequencing results of wild Cordyceps batch 15

[0152]

[0153] All the identified sequences of wild Cordyceps batch 15 had high coverage with the standard mitochondrial sequence of Thitarodes damxungensis.

[0154] Table 27 Sequencing results of wild Cordyceps batch 16

[0155]

[0156]

[0157] All the identified sequences of wild Cordyceps batch 16 had high coverage with the standard mitochondrial sequence of Thitarodes sp.XK-2016.

[0158] Table 28 Sequencing results of wild Cordyceps batch 17

[0159]

[0160] All the identified sequences of wild Cordyceps batch 17 had high coverage with the standard mitochondrial sequence of Thitarodes sp.XK-2016.

[0161] Table 29 Sequencing results of wild Cordyceps batch 18

[0162]

[0163]

[0164] All the identified sequences of wild Cordyceps batch 18 had high coverage with the standard mitochondrial sequence of Thitarodes sp.XK-2016.

[0165] Table 30 Discrimination result information table

[0166]

[0167]

[0168]

[0169] Detailed mitochondrial genome analysis was performed on seven batches of cultivated Cordyceps and 18 batches of wild Cordyceps samples. By comparing the sequences of these samples and comparing the comparison results with the NCBI database, the results showed that all Cordyceps samples purchased from JD.com were identified as a specific species, Hepialus xiaojinensis. In contrast, Cordyceps samples collected from the wild showed a richer species diversity. These wild samples were identified as different species, including Thitarodes sp.XK-2016, Thitarodes damxungensis, Thitarodesgonggaensis, and Ahamus yunnanensis. No samples matching Hepialus xiaojinensis were found in the study. No crossover with Hepialus xiaojinensis was found in the wild Cordyceps samples collected in Qinghai Province.

[0170] Therefore, it can be concluded that if the BLAST result shows 100% coverage of the insect-derived DNA component of Hepialus xiaojinensis, it can be identified as artificial Cordyceps. From the above results, all 7 batches of artificial Cordyceps were identified as artificial Cordyceps, and only the 7th batch of 18 batches of wild Cordyceps could not detect the insect-derived component, resulting in failure. The rest of the wild Cordyceps were all identified as Hepialus xiaojinensis, so the accuracy was 17 / 18 = 94.4%, and the specificity was 100%.

[0171] 2.6 Determination of full-length coverage of the reference genome mitochondrial genome

[0172] Seven batches of artificial Cordyceps and 18 wild Cordyceps from different locations were used for insect-derived mitochondrial DNA sequencing. The sequencing results first removed the bacterial DNA sequences, and then the coverage of all insect-derived mitochondrial fragments in the full length of Hepialusxiaojinensis mitochondria was counted. The results are shown in Tables 31-55.

[0173] Table 3 Similarity search results of artificial Cordyceps batch 1

[0174]

[0175]

[0176] Table 32 Similarity search results of artificial Cordyceps batch 2

[0177] Reference genome Average sequencing depth Coverage Species identification results NC_028348.1 5.644866 85.50272798 Hepialus xiaojinensis NC_026903.1 0.399937 23.19467972 Thitarodes gonggaensis NC_032649.1 0.065598 4.957812807 Thitarodes sejilaensis NC_044770.1 0.046934 3.795325825 Thitarodes damxungensis NC_024424.1 0.004967 0.496732026 Napialus hunanensis

[0178] Table 33 Similarity search results of artificial Cordyceps batch 3

[0179] Reference genome Average sequencing depth Coverage Species identification results NC_028348.1 0.553874 34.96362692 Hepialus xiaojinensis NC_026903.1 0.031807 3.180877094 Thitarodes gonggaensis NC_032649.1 0.003401 0.340113807 Thitarodes sejilaensis

[0180] Table 34 Similarity search results of artificial Cordyceps batch 4

[0181] Reference genome Average sequencing depth Similarity Species identification results NC_028348.1 1.647853 51.25357236 Hepialus xiaojinensis NC_026903.1 0.121832 7.880042663 Thitarodes gonggaensis NC_032649.1 0.016481 1.648243835 Thitarodes sejilaensis

[0182] Table 35 Similarity search results of artificial Cordyceps batch 5

[0183] Reference genome Average sequencing depth Similarity Species identification results NC_028348.1 0.42242 23.40867758 Hepialus xiaojinensis NC_026903.1 0.041343 4.13451283 Thitarodes gonggaensis NC_032649.1 0.009549 0.954934921 Thitarodes sejilaensis

[0184] Table 36 Similarity search results of artificial Cordyceps batch 6

[0185] Reference genome Average sequencing depth Similarity Species identification results NC_028348.1 1.096642 40.4260847 Hepialus xiaojinensis NC_026903.1 0.041593 3.21852061 Thitarodes gonggaensis NC_044770.1 0.003125 0.312479656 Thitarodes sejilaensis

[0186] Table 37 Similarity search results of artificial Cordyceps batch 7

[0187] Reference genome Average sequencing depth Similarity Species identification results NC_028348.1 0.186465 16.18602234 Hepialus xiaojinensis NC_032649.1 0.00981 0.981097521 Thitarodes gonggaensis NC_026903.1 0.009159 0.91599222 Thitarodes sejilaensis

[0188] Table 38 Similarity search results of wild Cordyceps batch 1

[0189]

[0190]

[0191] Table 39 Similarity search results of wild Cordyceps batch 2

[0192] Reference genome Average sequencing depth Similarity Species identification results NC_026903.1 0.136951 11.9204467 Thitarodes gonggaensis NC_029873.1 0.01773 1.773096048 Endoclita signifer NC_028348.1 0.087639 1.19 Hepialus xiaojinensis

[0193] Table 40 Similarity search results of wild Cordyceps batch 3

[0194] Reference genome Average sequencing depth Similarity Species identification results NC_026903.1 0.147365 13.31953071 Thitarodes gonggaensis NC_028348.1 0.054 1.26 Hepialus xiaojinensis

[0195] Table 4 Similarity search results of wild Cordyceps batch 4

[0196] Reference genome Average sequencing depth Similarity Species identification results NC_044770.1 4.572777 62.17694161 Thitarodes damxungensis NC_026903.1 0.133375 8.890143673 Thitarodes gonggaensis NC_028348.1 0.107553 7.735775526 Hepialus xiaojinensis NC_032649.1 0.095487 5.624959121 Thitarodes sejilaensis

[0197] Table 42 Similarity search results of wild Cordyceps batch 5

[0198] Reference genome Average sequencing depth Similarity Species identification results NC_026903.1 9.828984 80.75161553 Thitarodes gonggaensis NC_028348.1 0.233747 12.87996882 Hepialus xiaojinensis NC_044770.1 0.037105 2.753726971 Thitarodes damxungensis NC_032649.1 0.039241 2.093008045 Thitarodes sejilaensis

[0199] Table 5 Similarity search results of wild Cordyceps batch 6

[0200] Reference genome Average sequencing depth Similarity Species identification results NC_026903.1 0.467503 32.10364515 Thitarodes gonggaensis NC_028348.1 0.017861 1.786178228 Hepialus xiaojinensis

[0201] Table 6 Similarity search results of wild Cordyceps batch 7

[0202] Species Reads_number Hirsutella rhossiliensis 1277 Ophiocordyceps sinensis 153 Purpureocillium takamizusanense 28 Purpureocillium lilacinum 25 Variovorax paradoxus 23 Hepialus humuli 15 Drechmeria coniospora 15 Neoitamus cyanurus 12 Neonectria neomacrospora 12 Pseudomonas trivialis 11

[0203] No insect-derived ingredients were detected in batch 7 of wild Cordyceps.

[0204] Table 45 Similarity search results of wild Cordyceps batch 8

[0205] Reference genome Average sequencing depth Similarity Species identification results NC_044770.1 2.253678 59.2734848 Thitarodes damxungensis NC_026903.1 0.158093 10.33941904 Thitarodes gonggaensis NC_028348.1 0.096317 8.294362172 Hepialus xiaojinensis NC_032649.1 0.05847 4.310288443 Thitarodes sejilaensis

[0206] Table 46 Similarity search results of wild Cordyceps batch 9

[0207]

[0208] Table 77 Similarity search results of wild Cordyceps batch 10

[0209] Reference genome Average sequencing depth Similarity Species identification results NC_026903.1 2.356336 60.46803438 Thitarodes gonggaensis NC_028348.1 0.038124 2.99428423 Hepialus xiaojinensis NC_032649.1 0.028712 1.929491791 Thitarodes sejilaensis

[0210] Table 48 Similarity search results of wild Cordyceps batch 11

[0211] Reference genome Average sequencing depth Similarity Species identification results NC_026903.1 10.175471 95.76510446 Thitarodes gonggaensis NC_028348.1 0.35416 15.3221616 Hepialus xiaojinensis NC_044770.1 0.028447 1.751188074 Thitarodes damxungensis NC_032649.1 0.016874 0.987638171 Thitarodes sejilaensis

[0212] Table 49 Similarity search results of wild Cordyceps batch 12

[0213] Reference genome Average sequencing depth Similarity Species identification results NC_026903.1 0.063363 5.383022774 Thitarodes gonggaensis NC_026903.1 0.019159 1.38 Hepialus xiaojinensis

[0214] Table 8 Similarity search results of wild Cordyceps batch 13

[0215] Reference genome Average sequencing depth Similarity Species identification results NC_026903.1 0.232058 15.38365017 Thitarodes gonggaensis NC_028348.1 0.017276 1.727721486 Hepialus xiaojinensis NC_044770.1 0.009374 0.937438969 Thitarodes damxungensis

[0216] Table 9 Similarity search results of wild Cordyceps batch 14

[0217] Reference genome Average sequencing depth Similarity Species identification results NC_026903.1 11.780552 66.31532718 Thitarodes gonggaensis NC_028348.1 12.446191 55.76123668 Hepialus xiaojinensis NC_032649.1 0.565925 10.07914187 Thitarodes sejilaensis NC_044770.1 0.374886 6.425362932 Thitarodes damxungensis

[0218] Table 10 Similarity search results of wild Cordyceps batch 15

[0219] Reference genome Average sequencing depth Similarity Species identification results NC_044770.1 28.551686 82.41650934 Thitarodes damxungensis NC_026903.1 0.523777 20.28358115 Thitarodes gonggaensis NC_028348.1 0.475157 16.00415692 Hepialus xiaojinensis NC_032649.1 0.309156 13.44103604 Thitarodes sejilaensis NC_029873.1 0.019562 1.956294164 Endoclita signifer

[0220] Table 11 Similarity search results of wild Cordyceps batch 16

[0221] Reference genome Average sequencing depth Similarity Species identification results NC_026903.1 0.492409 35.47901374 Thitarodes gonggaensis NC_028348.1 0.009742 0.974279034 Hepialus xiaojinensis

[0222] Table 12 Similarity search results of wild Cordyceps batch 17

[0223] Reference genome Average sequencing depth Similarity Species identification results NC_026903.1 15.494103 76.74885501 Thitarodes gonggaensis NC_028348.1 0.39144 11.38607431 Hepialus xiaojinensis NC_044770.1 0.019464 1.946487859 Thitarodes damxungensis NC_032649.1 0.019621 0.981097521 Thitarodes sejilaensis

[0224] Table 55 Similarity search results of wild Cordyceps batch 18

[0225]

[0226] The above data summarize the coverage of different batches of samples in the full length of Hepialus xiaojinensis mitochondria.

[0227] Table 136 Summary of coverage of the full length of mitochondria of Cordyceps sinensis Hepialus xiaojinensis

[0228]

[0229]

[0230]

[0231] Calculate the Youden Index:

[0232] Table 57 Youden Index Calculation Table

[0233] Sensitivity % 95% CI Specificity % 95% CI <1.080 5.882 0.3017% to 26.98% 100.0 64.57% to 100.0% <1.225 11.76 2.090% to 34.34% 100.0 64.57% to 100.0% <1.320 17.65 6.191% to 41.03% 100.0 64.57% to 100.0% <1.555 23.53 9.555% to 47.26% 100.0 64.57% to 100.0% <1.760 29.41 13.28% to 53.13% 100.0 64.57% to 100.0% <2.390 35.29 17.31% to 58.70% 100.0 64.57% to 100.0% <4.015 41.18 21.61% to 63.99% 100.0 64.57% to 100.0% <6.385 47.06 26.17% to 69.04% 100.0 64.57% to 100.0% <8.010 52.94 30.96% to 73.83% 100.0 64.57% to 100.0% <8.995 58.82 36.01% to 78.39% 100.0 64.57% to 100.0% <10.54 64.71 41.30% to 82.69% 100.0 64.57% to 100.0% <12.13 70.59 46.87% to 86.72% 100.0 64.57% to 100.0% <13.84 76.47 52.74% to 90.44% 100.0 64.57% to 100.0% <15.06 82.35 58.97% to 93.81% 100.0 64.57% to 100.0% <15.66 88.24 65.66% to 97.91% 100.0 64.57% to 100.0% <16.10 94.12 73.02% to 99.70% 100.0 64.57% to 100.0% <19.80 94.12 73.02% to 99.70% 85.71 48.69% to 99.27% <29.19 94.12 73.02% to 99.70% 71.43 35.89% to 94.92% <37.70 94.12 73.02% to 99.70% 57.14 25.05% to 84.18% <45.84 94.12 73.02% to 99.70% 42.86 15.82% to 74.95% <53.51 94.12 73.02% to 99.70% 28.57 5.077% to 64.11% <70.63 100.0 81.57% to 100.0% 28.57 5.077% to 64.11% <86.02 100.0 81.57% to 100.0% 14.29 0.7328% to 51.31%

[0234] It can be seen from the table that when the cutoff value is set to 16.1, the best result can be obtained, that is, 100% specificity and 94.12% sensitivity.

[0235] The AUC value of the ROC curve is 0.9580, which indicates that the model has a high ability to distinguish. Generally speaking, an AUC value above 0.9 indicates that the model has a high diagnostic value. The AUC value is close to 0.96, indicating that the model performs well in distinguishing cultivated Cordyceps from wild Cordyceps. The P value is 0.0005, which is much less than 0.05, indicating that the result is statistically significant.

[0236] Table 58 Discrimination performance parameters of the long segment coverage method

[0237]

[0238]

[0239] The only sample with an error was wild Cordyceps batch 14, which was collected from Langdu Village, Gezan Township, Shangri-La City, Yunnan Province, which is geographically close to the natural habitat of the small golden bat moth, so there may be a certain degree of genetic exchange. In contrast, other samples from Qinghai Province and its surrounding areas have very low genetic overlap with the small golden bat moth.

[0240] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for distinguishing Cordyceps sinensis, for identifying whether there is artificial Cordyceps sinensis from wild Cordyceps sinensis, characterized in that: The discrimination method comprises: S1. Preparing Cordyceps sinensis samples and obtaining mitochondrial gene sequence data of Cordyceps sinensis samples; S2, comparing the mitochondrial gene sequence with the mitochondrial reference genome of Hepialus xiaojinensis; S3, calculating the coverage and consistency of the gene sequence; S4. If the coverage rate is ≥16.1% and the consistency is ≥95%, the Cordyceps is determined to be artificial Cordyceps sinensis. The wild Cordyceps sinensis originates from the northwest region of China; further, from the Qinghai Province of China.

2. A method for distinguishing Cordyceps sinensis, for identifying whether there is artificial Cordyceps sinensis from wild Cordyceps sinensis, characterized in that: The discrimination method comprises: S1. Preparing Cordyceps sinensis samples and obtaining mitochondrial gene sequence data of Cordyceps sinensis samples; S2, comparing the mitochondrial gene sequence with at least one of the sequences shown in SEQ ID NOs 1 to 42; S3, calculating the coverage of the gene sequence; S4. If the coverage rate is 100%, it is determined that the Cordyceps is artificial Cordyceps sinensis; The wild Cordyceps sinensis originates from the northwest region of China; further, from the Qinghai Province of China.

3. The identification method according to claim 1 or 2, characterized in that: The cordyceps sinensis samples include wild cordyceps sinensis samples and / or artificial cordyceps sinensis samples.

4. The identification method according to claim 1, characterized in that: In step S2: before alignment with the mitochondrial reference genome of Hepialusxiaojinensis, bacterial DNA sequences were removed in advance.

5. The identification method according to claim 4, characterized in that: The sequencing data were aligned with the mitochondrial sequence of Ophiocordyceps sinensis; sequence reads matching the fungal genome were removed, and only insect-derived mitochondrial DNA was retained for coverage and consistency calculations.

6. The identification method according to claim 1 is characterized in that: The accession number of the mitochondrial sequence of Ophiocordyceps sinensis is NC_022834.

1.

7. The identification method according to claim 1, characterized in that: The accession number of the Hepialus xiaojinensis mitochondrial reference genome is NC_028348.

1.

8. The method according to claim 1, characterized in that The coverage threshold is determined by Youden index optimization, and the specific steps include: (1) Test the ability of different coverage thresholds from 1% to 100% to distinguish between cultivated Cordyceps and wild Cordyceps; (2) Select a threshold that maximizes the sum of sensitivity and specificity.

9. A Cordyceps sinensis identification system, characterized in that: The system comprises: (1) Data input module: receiving mitochondrial sequencing data of Cordyceps sinensis samples; (2) Sequence alignment module: aligning the sequencing data with the Hepialus xiaojinensis mitochondrial reference genome; (3) Coverage calculation module: counts the base coverage of the reference genome; (4) Consistency analysis module: calculate sequence consistency; (5) Judgment output module: When the coverage rate is ≥16.1% and the consistency is ≥95%, the "artificial Cordyceps sinensis" judgment result is output.

10. A Cordyceps sinensis identification system, characterized in that: The system comprises: (1) Data input module: receiving mitochondrial sequencing data of Cordyceps sinensis samples; (2) a sequence alignment module: aligning the sequencing data with at least one of the sequences shown in SEQ ID NOs 1 to 42; (3) Coverage calculation module: counts the base coverage of the reference genome; (4) Consistency analysis module: calculate sequence consistency; (5) Judgment output module: When the coverage rate is 100%, the "artificial Cordyceps sinensis" judgment result is output.

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