A method for identifying authenticity of cordyceps sinensis and its products based on fluorescent duplex PCR
By designing specific primer combinations on the SYBR Green fluorescent PCR platform, the amplification compatibility bottleneck caused by the difference in thermodynamic characteristics between the ITS sequence of Cordyceps sinensis and the COI sequence of Hepialus larvae was solved, enabling efficient and accurate identification of Cordyceps sinensis and its products, which is applicable to the identification of raw Cordyceps and its deep-processed products.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JING BRAND
- Filing Date
- 2026-05-13
- Publication Date
- 2026-07-10
AI Technical Summary
In existing technologies, the thermodynamic differences between the high GC content of the Cordyceps sinensis ITS sequence and the low GC content of the Hepialus larvae COI sequence in the dual PCR system prevent the achievement of balanced and specific dual amplification on the SYBR Green fluorescent PCR platform, resulting in problems such as amplification inhibition, non-specific interference, and primer dimer interference.
A set of specific primer combinations was designed. The primer pairs obtained through systematic screening achieved balanced and specific synergistic amplification of the Cordyceps sinensis ITS sequence and the Hepialus larvae COI gene sequence in the same SYBR Green reaction system at a universal annealing temperature of 60℃. The results were determined by the SYBR Green dye method, avoiding the need for electrophoresis and the use of fluorescent probes.
It enables efficient and accurate identification of Cordyceps sinensis and its products, avoids cross-contamination of amplification products, reduces costs, and improves detection efficiency and accuracy. It is applicable to the identification of raw Cordyceps sinensis and its deep-processed products.
Smart Images
Figure CN122357771A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular identification technology of traditional Chinese medicine materials, specifically to a method for identifying the authenticity of Cordyceps sinensis and its products based on fluorescence doublet PCR. Background Technology
[0002] Cordyceps sinensis is a fungus belonging to the Clavicipitaceae family, specifically the Cordyceps militaris fungus (Cordyceps sinensis). Ophiocordyceps sinensis Cordyceps sinensis, a parasitic fungus complex formed within the larvae of insects in the Hepialidae family, is mainly produced in the high-altitude regions of the Qinghai-Tibet Plateau. It is a traditional and precious tonic in Chinese medicine, possessing various pharmacological activities such as kidney and lung tonification, immune regulation, and anti-fatigue effects. In recent years, with the continuous growth of market demand, Cordyceps sinensis resources have become increasingly scarce. Some unscrupulous merchants, seeking exorbitant profits, use closely related species (such as Cordyceps militaris and Cordyceps liangshanense) or other imitations (such as Cordyceps militaris and Cordyceps militaris) to impersonate Cordyceps sinensis, even deceiving consumers with artificially pressed "fake Cordyceps" or deep-processed products containing no Cordyceps sinensis components. This seriously infringes upon consumers' legitimate rights and interests and endangers public health. Therefore, establishing a detection method that can accurately and efficiently identify the authenticity of Cordyceps sinensis and its products has significant market supervision and industrial application value.
[0003] Traditional morphological identification methods rely on the physical characteristics of intact Cordyceps sinensis individuals, which are completely ineffective for highly processed products such as powders and capsules that have lost their morphological features. Physicochemical analysis methods such as near-infrared spectroscopy and high-performance liquid chromatography can detect specific chemical components, but due to significant variations in component content between different origins and batches of Cordyceps sinensis, and the possibility of counterfeit products having target components artificially added, they are difficult to use as definitive criteria for determining authenticity. Molecular biological methods, based on the stability and specificity of genetic material itself, have become the most authoritative means of species identification.
[0004] Among the currently disclosed molecular identification methods for Cordyceps sinensis, CN103233062A discloses a single-tube dual PCR combined with agarose gel electrophoresis detection method, which determines the authenticity of the product by simultaneously amplifying the characteristic housekeeping genes of the Cordyceps sinensis fungus and its host moth, the black-hearted ghost moth. CN110894537A discloses a method for designing specific primers in the ITS region and identifying the product through PCR amplification and agarose gel electrophoresis. Both of these methods rely on agarose gel electrophoresis to detect the amplified products. However, electrophoresis is difficult to accurately distinguish between non-specific amplified fragments and primer dimers of similar length, posing a risk of misjudgment. Furthermore, these methods involve cumbersome operations such as manual gel preparation, sample loading, electrophoresis, staining, and imaging. Experimental repeatability is affected by multiple factors such as gel concentration, buffer solution, and electrophoresis conditions. The steps are time-consuming and have low throughput, making them unsuitable for rapid screening of large batches of samples. Moreover, the tube-opening operation can easily lead to cross-contamination of the amplified products. CN102086471A discloses a detection scheme based on the fluorescent probe method (TaqMan method). Although this method does not require an electrophoresis step, it requires the design and synthesis of fluorescently labeled probes for each target. The probe design involves the optimization of complex parameters such as annealing temperature, and the cost of probe synthesis is much higher than that of ordinary primers, making it difficult to promote and apply in grassroots laboratories and large-scale rapid screening scenarios.
[0005] At a deeper level, the applicant's extensive experimental research revealed that simply migrating the conventional dual-phase PCR system from the gel electrophoresis platform to the SYBR Green fluorescent PCR platform is not a simple replacement of the detection method. The SYBR Green dye method demands extremely high specificity from PCR amplification. Any non-specific amplification products or primer dimers will bind to the dye, generating fluorescence signals and exhibiting melting curve peaks, severely interfering with result interpretation. The GC content of the ITS sequence region of *Cordyceps sinensis* is over 50%, resulting in a high Tm value for its specific primers, making amplification efficiency easily low or even unsuccessful under universal annealing conditions. Conversely, the GC content of the COI gene sequence region of *Hepialus hepiali* larvae is only about 30%, resulting in a low Tm value for its specific primers, making non-specific amplification highly likely at lower annealing temperatures. When these two types of primers with significantly different thermodynamic properties coexist in the same reaction tube and need to achieve specific and balanced amplification simultaneously under a universal annealing temperature, problems such as primer dimer interference, amplification imbalance, and complete inhibition of amplification by one primer become unique technical obstacles hindering the successful implementation of SYBR Green dual-phase fluorescent PCR. Existing technical literature does not suggest how to solve the above-mentioned specific technical problems, nor does it disclose primer combinations and supporting methods that can successfully achieve balanced and specific dual amplification of the Cordyceps sinensis ITS sequence and the host COI gene sequence in the SYBR Green fluorescent PCR system.
[0006] Therefore, there is an urgent need in this field to develop a fluorescent double PCR method that can overcome the above-mentioned technical obstacles, has the advantages of simple operation, high accuracy, and controllable cost, and is applicable to the authenticity identification of Cordyceps sinensis raw grass and deep-processed products. Summary of the Invention
[0007] In view of this, the present invention proposes a method for identifying the authenticity of Cordyceps sinensis and its products based on fluorescent duplex PCR, aiming to solve the technical problem in the prior art where the thermodynamic characteristics of the dual PCR system conflict between the high GC content of fungal ITS sequences and the low GC content of insect COI sequences, resulting in the inability to achieve balanced and specific dual amplification in the SYBR Green fluorescent PCR platform.
[0008] The technical solution of this invention is implemented as follows: This invention provides a method for identifying the authenticity of Cordyceps sinensis and its products based on fluorescence duplex PCR, comprising the following steps: (1) Extract total nucleic acid from the sample to be tested; (2) Using the total nucleic acid extracted in step (1) as a template, fungal-specific primer pairs and insect-specific primer pairs are used in the same PCR reaction tube to perform double PCR amplification by SYBR Green dye method and collect fluorescence signals; the fungal-specific primer pairs target the ITS sequence of Cordyceps sinensis, and the insect-specific primer pairs target the COI gene sequence of the host moth larvae. (3) Determine the results based on the amplification curve and melting curve: If both the fungal-specific primer pair and the insect-specific primer pair show specific amplification curves, and the melting curve shows two independent specific product peaks, then the sample to be tested is determined to be genuine Cordyceps sinensis or contains its components.
[0009] The core concept of this invention lies not in simply combining dual PCR technology with a fluorescence detection platform. The applicant discovered in its research that the extreme differences in base composition between the ITS region (GC content >50%) of Cordyceps sinensis and the COI region (GC content <35%) of Hepialus larvae make the primer design space extremely narrow, simultaneously meeting the three requirements of "high specificity amplification," "Tm values of both primer pairs compatible with the same annealing temperature," and "sufficient separation of Tm values of the amplification products of the two targets" within this specific sequence region. Conventional primer design software and principles are ineffective in this scenario, and multiple primer combinations designed according to conventional approaches failed in the dual SYBR Green system. Through a systematic primer screening strategy, the applicant successfully obtained a primer combination from multiple candidate primers that can achieve balanced and specific synergistic amplification of the ITS and COI targets at a universal annealing temperature of 60℃ in the same SYBR Green reaction system, thus overcoming the amplification compatibility bottleneck caused by the significant differences in the base composition of the two targets. The primer combination was not obtained through simple deduction from conventional primer design rules, but rather through a non-obvious creative screening result specifically for this high / low GC pooled detection system. This primer combination makes it possible to simultaneously and accurately identify the dual genetic markers of the Cordyceps sinensis "insect-fungus complex" in a low-cost, electrophoresis-free, and probe-free manner.
[0010] In some embodiments, the fungal-specific primer pairs and the insect-specific primer pairs can achieve balanced amplification of the ITS sequence and the COI gene sequence at an annealing temperature of 60°C, and the melting curve peaks of the two target amplification products are independent of each other, without primer dimer interference peaks. This clear definition distinguishes the method of this invention from existing methods that fail due to simple substitutions: simultaneously satisfying the three conditions of "general annealing temperature of 60°C," "balanced amplification," and "independent and interference-free melting curve peaks" constitutes the necessary technical guarantee for the successful implementation of this method on the SYBR Green platform. "Balanced amplification" means that the amplification efficiency of the two primer pairs on their respective templates matches in the same reaction system, avoiding a situation where one target amplifies strongly while the amplification of the other target is inhibited; "independent melting curve peaks" means that the two amplification products have different Tm values, which can be accurately distinguished in melting curve analysis, thus providing dual confirmation for result determination.
[0011] In some embodiments, the fungal-specific primer pair is: Upstream primer F1: 5′-GGTGGTGAGTGAAGAAGGC-3′ Downstream primer R1: 5′-CCCGCTGACCTCATCCTT-3′; The insect-specific primer pair is: Upstream primer F2: 5′-TATGTTGAGCAGTAGCAGGA-3′, Downstream primer R2: 5′-GCTGGTGGTGAAGTTGTAG-3′.
[0012] This specific primer combination is the preferred implementation obtained by the applicant through extensive experimental screening and verification. During the screening process, the applicant performed dual SYBR Green qPCR tests on multiple sets of fungal candidate primers and multiple sets of insect candidate primers in pairs. It was found that most combinations produced problems such as amplification inhibition, disappearance of fluorescence signal, or abnormal peaks in melting curves due to the mismatch of thermodynamic parameters between primers. The above-mentioned preferred primer combination is the primer pair that was finally determined from the 10 primer combinations tested. It can achieve balanced and specific amplification of ITS sequence and COI gene sequence at a universal annealing temperature of 60℃. The Tm values of the amplification products of the two targets can form clear and distinguishable independent melting curve peaks, providing a reliable primer tool basis for electrophoresis-free and probe-free dual identification based on SYBR Green dye method.
[0013] In some embodiments, the reaction system for the dual PCR amplification in step (2) is as follows: 25 ng DNA template, 10 μL SYBR Green qPCR Master Mix, 1 μL each of the upstream and downstream primers of the fungal-specific primer pair, 1 μL each of the upstream and downstream primers of the insect-specific primer pair, and 20 μL made up with sterile double-distilled water. The proportions of each component in this reaction system are optimized to provide a relatively balanced amplification environment for the two primer pairs while ensuring amplification sensitivity, avoiding non-specific amplification due to excessive primers or decreased amplification efficiency due to insufficient primers.
[0014] In some embodiments, the duplex PCR amplification procedure in step (2) is as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 5 s; 60℃ annealing and extension for 30 s, for a total of 40 cycles. The above amplification procedure adopts a two-step PCR strategy, combining annealing and extension into a single 60℃ step, simplifying the thermal cycling process and shortening the overall detection time. The 60℃ annealing and extension temperature is one of the key parameters of the method of this invention: this temperature can ensure the effective binding and extension of fungal ITS primers with high GC content to the template, and can also avoid non-specific binding of insect COI primers with low GC content at this temperature, thereby achieving optimal primer compatibility.
[0015] In some implementations, step (2) further includes a melting curve analysis procedure: 95℃ for 15s, 60℃ for 60s, and 95℃ for 15s. The melting curve analysis procedure is executed after the amplification cycle is completed. By programmatically heating the PCR products and collecting fluorescence signals in real time, the characteristic Tm values of the amplified products are obtained, thereby confirming the specificity of the amplified products. The melting curve analysis of the SYBR Green dye method provides product typing capability for dual PCR—fungal ITS products and insect COI products have different Tm values due to their differences in base composition and length, which appear as two independent fluorescence decline peaks in the melting curve. Compared with the detection method that can only reflect fragment size by gel electrophoresis, it can more accurately distinguish between specific products and non-specific products and primer dimers.
[0016] In some embodiments, in step (3), the Tm value of the amplification product of the fungal-specific primer pair is 87.5℃±1.0℃, and the Tm value of the amplification product of the insect-specific primer pair is 77.5℃±1.0℃. This explicit definition of the Tm value provides an objective and quantitative criterion for result determination, avoiding subjective errors that exist when manually interpreting gel images. The approximately 10℃ difference between the Tm values of the two target points ensures sufficient separation of the two specific product peaks in the melting curve, making result interpretation more accurate and reliable.
[0017] In some embodiments, the sample to be tested is raw Cordyceps sinensis, Cordyceps sinensis powder, Cordyceps sinensis capsules, Cordyceps sinensis tablets, Cordyceps sinensis oral liquid, or a compound preparation containing Cordyceps sinensis components. The method of this invention can be widely applied to Cordyceps sinensis products of different forms and processing degrees. For deeply processed products such as powders and capsules, the DNA in the sample has often undergone varying degrees of degradation, and the proportion of target DNA in the total nucleic acid may be low, requiring the method to have high sensitivity and strong anti-interference ability. The high amplification efficiency of the specific primer combination and the high sensitivity of the SYBR Green dye method used in this invention enable the effective detection of trace amounts and partially degraded DNA in deeply processed products, thereby greatly expanding the applicability of molecular identification methods in the market supervision and product quality control of Cordyceps sinensis.
[0018] In some implementations, the method is performed entirely in closed tubes, eliminating the need for agarose gel electrophoresis and fluorescently labeled probes. This closed-tube operation means that no further processing is required after PCR amplification; the amplification signal and melting curve are directly acquired and analyzed on a quantitative PCR instrument, fundamentally eliminating the risk of cross-contamination in the laboratory caused by the diffusion of amplified products upon opening the tube. Furthermore, compared to the TaqMan probe method, this method only requires the design and synthesis of ordinary PCR primers, eliminating the need for probe synthesis and condition optimization, significantly reducing reagent costs and making large-scale identification of numerous samples more economically feasible.
[0019] The present invention has the following advantages over the prior art: This invention provides a method for identifying the authenticity of Cordyceps sinensis and its products based on SYBR Green fluorescent duplex PCR technology. Its core contribution lies in providing a set of specific primers obtained through systematic screening. This primer set overcomes the thermodynamic conflict caused by the high GC content of the Cordyceps sinensis ITS sequence and the low GC content of the Hepialus larvae COI sequence. For the first time, it achieves balanced, specific, and simultaneous amplification of the dual genetic markers of the "Cordyceps sinensis-Fungus complex" in the same SYBR Green reaction tube at a universal annealing temperature of 60℃. This solves the technical problems commonly encountered when directly transferring existing duplex PCR systems to the SYBR Green fluorescent platform, such as amplification inhibition, non-specific interference, and primer dimer interference. Compared with conventional duplex PCR methods relying on agarose gel electrophoresis, this invention's method involves a completely closed-tube operation, eliminating cumbersome steps such as gel preparation, sample loading, electrophoresis, staining, and imaging, avoiding the risk of contamination caused by opening the tube, significantly improving detection efficiency. The result determination is based on the objective quantitative index of Tm value, eliminating subjective errors from manual interpretation, and significantly improving accuracy and repeatability. Compared to TaqMan probe-based fluorescent PCR methods, the method of this invention eliminates the need for designing and synthesizing expensive fluorescent probes, significantly reducing costs and making system optimization more convenient. This makes it more suitable for widespread application in grassroots laboratories and large-scale rapid screening scenarios. Furthermore, thanks to the high-efficiency amplification performance of the specific primer combination and the high sensitivity of the SYBR Green dye method, this invention can be successfully applied to the authenticity identification of Cordyceps sinensis raw materials and various deep-processed products such as powders and capsules. It overcomes the limitations of traditional morphological methods and conventional PCR electrophoresis in identifying deep-processed products, providing a novel technical means that combines accuracy, convenience, and economy for market supervision, enterprise quality control, and third-party testing of Cordyceps sinensis. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0021] Figure 1 The image shows the fluorescence amplification curve of dual SYBR Green qPCR using a combination of fungal primer 1 and insect primer 3.
[0022] Figure 2 The melting curve of a double SYBR Green qPCR using a combination of fungal primer 2 and insect primer 4 is shown.
[0023] Figure 3 This is a double SYBR Green qPCR fluorescence amplification curve of the preferred primer combination of the present invention (fungal primer 7 + insect primer 7).
[0024] Figure 4 This is a fluorescence amplification curve of a Cordyceps sinensis sample detected by the method of this invention.
[0025] Figure 5 This is a melting curve of the amplification products of fungal-specific primer pairs for Cordyceps sinensis samples.
[0026] Figure 6 This is a melting curve of the amplification products of the insect-specific primer pair for Cordyceps sinensis samples.
[0027] Figure 7 The amplification curves of Cordyceps militaris, Cordyceps liangshanensis, and Cordyceps militaris samples were obtained by the method of this invention.
[0028] Figure 8 The amplification curves of commercially available Cordyceps sinensis products (capsules) with different sample sizes were detected by the method of this invention. Detailed Implementation
[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0031] Example 1: Design and Screening of Specific Primers The key to this invention lies in obtaining a primer combination capable of achieving balanced and specific synchronous amplification of the Cordyceps sinensis ITS sequence and the Hepialus larvae COI gene sequence in a SYBR Green fluorescent duplex PCR system. Because the GC content of the fungal ITS region is as high as 50% or more, its primer Tm value is relatively high, leading to low amplification efficiency or even failure at the universal annealing temperature. Conversely, the GC content of the insect COI region is only about 30%, resulting in a lower primer Tm value, which easily leads to non-specific amplification at lower annealing temperatures. When these two types of primers with significantly different thermodynamic properties coexist in the same reaction tube and are subjected to duplex PCR at the universal annealing temperature, problems such as primer dimer interference, amplification imbalance, or even complete inhibition of amplification of one primer type commonly occur. Therefore, not any combination of ITS and COI primers is suitable for the SYBR Green fluorescent duplex PCR system.
[0032] To address the aforementioned technical issues, this embodiment first performs bioinformatics analysis on the conserved regions of the ITS sequence of Cordyceps sinensis and the COI gene of Hepialus larvae. The analysis shows that the usable primer design windows within the ITS conserved region generally have high GC content (>50%), while the usable windows within the COI conserved region have low GC content (<35%). Directly designing primers according to conventional primer design rules (such as Tm value matching and avoiding 3' end complementarity) cannot solve the compatibility problem between the two targets at the same annealing temperature due to the significant difference in GC content, requiring extensive trial-and-error screening within a limited sequence space. Therefore, this application designs multiple sets of candidate primers and uses a single-factor method to combine fungus-specific primers and insect-specific primers in pairs. Dual SYBR Green qPCR tests are performed on each combination under a uniform annealing temperature of 60℃ to screen primer combinations that can overcome the aforementioned thermodynamic conflicts. The candidate primer sequences and test results are shown in Table 1.
[0033] Table 1. Primer combination screening test results
[0034] As shown in Table 1, among the 10 primer combinations tested, the amplification was severely inhibited and no fluorescence signal was observed when fungal primer 1 was combined with insect primer 3 (see Appendix). Figure 1 This indicates a serious compatibility problem between the two primer pairs, possibly due to primer dimerization competing for reaction system resources, or the Tm values of the two primer pairs being insufficient to simultaneously satisfy the effective binding of their respective templates at the common annealing temperature of 60℃. When fungal primer 2 is combined with insect primer 4, the melting curve shows a significant abnormal peak in the low-temperature region of approximately 65-70℃ (see appendix). Figure 2The peak was identified as a primer dimer melting peak, and such non-specific signals directly interfere with accurate interpretation of results in the SYBR Green dye method. Most other combinations failed to meet the primer combination requirements of SYBR Green fluorescent duplex PCR due to factors such as large differences in Tm values, uneven amplification efficiency, overlapping melting peaks at the two target sites, or the presence of non-specific shoulder peaks.
[0035] After multiple rounds of screening, among the 10 primer combinations tested, the 7th primer combination—the combination of fungal-specific primer pair F7 / R7 and insect-specific primer pair F7 / R7—was finally determined. This combination achieved balanced amplification of both targets in the universal system, and the melting curve showed two independent, sharp peaks of specific products, with no primer dimer interference peaks (see appendix). Figure 3 The sequence is shown in Table 2.
[0036] Table 2 Preferred specific primer pairs of the present invention
[0037] The primer combination was experimentally verified to have well-matched Tm values. At a universal annealing temperature of 60℃, both primers exhibited high amplification efficiency and specificity for their respective targets. The Tm values of the amplified products from the two targets differed by approximately 10℃, and the melting curve peaks were completely separated. This provides a reliable primer basis for electrophoresis-free and probe-free dual identification based on the SYBR Green dye method. This optimized primer combination was used in the dual PCR experiments in subsequent examples.
[0038] Example 2: Identification of the authenticity of Cordyceps sinensis This embodiment uses Cordyceps sinensis and closely related species / common counterfeits as test objects to verify the specificity of the method of the present invention.
[0039] 2.1 Total Nucleic Acid Extraction from Samples Approximately 50 mg of each of the following samples were weighed: *Cordyceps sinensis* (collected from Yushu, Qinghai), *Cordyceps militaris* (collected from Guangxi), *Cordyceps liangshanensis* (collected from Liangshan, Sichuan), and *Cordyceps militaris* (commercially available artificially cultured product). All samples underwent morphological identification and molecular confirmation using DNA barcoding technology. The ITS sequence of the *Cordyceps sinensis* sample showed 100% similarity to the reference sequence of *Ophiocordyceps sinensis* in GenBank (accession number: JX968028), and its COI sequence was identified as belonging to the Hepialidae family. The species of *Cordyceps militaris*, *Cordyceps liangshanensis*, and *Cordyceps militaris* samples were also confirmed by corresponding molecular identification. Total nucleic acids were extracted using a modified CTAB method. The main steps are as follows: After grinding each sample into powder with liquid nitrogen, transfer it to a centrifuge tube containing preheated CTAB extraction buffer and incubate at 65°C; add an equal volume of chloroform / isoamyl alcohol for extraction, centrifuge and collect the supernatant; precipitate with isopropanol, wash with ethanol, dissolve the obtained nucleic acid precipitate in an appropriate amount of TE buffer, and store at -20°C for later use.
[0040] 2.2 Dual SYBR Green fluorescent PCR amplification Using the total nucleic acid extracted from each sample as a template, double PCR amplification was performed in the same PCR tube using the fungal-specific primer pairs and insect-specific primer pairs shown in Table 2.
[0041] The PCR reaction system consisted of: 25 ng DNA template, 10 μL ChamQ Universal SYBR qPCR Master Mix, 1 μL each of fungal-specific upstream and downstream primers, 1 μL each of insect-specific upstream and downstream primers, and sterile double-distilled water to a final volume of 20 μL.
[0042] The amplification program was as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 5 s; 60℃ annealing and extension for 30 s (fluorescence signals were collected during the annealing and extension stages), for 40 cycles. Subsequently, the melting curve program was run: 95℃ for 15 s, 60℃ for 60 s, 95℃ for 15 s, with continuous fluorescence signal acquisition at a heating rate of 0.05℃ / s.
[0043] 2.3 Result Determination The amplification results of Cordyceps sinensis samples are attached. Figure 4 Both fungal-specific primer pairs and insect-specific primer pairs exhibited typical S-type fluorescence amplification curves. (See attached image.) Figure 5 and attached Figure 6As shown, melting curve analysis revealed two independent, sharp, specific product peaks: the fungal ITS product had a Tm value of 87.5℃±1.0℃, and the insect COI product had a Tm value of 77.5℃±1.0℃. No primer dimer interference peak was observed between the two peaks. This result indicates that the sample simultaneously contains DNA from both Cordyceps sinensis and the ghost moth larvae, consistent with the biological characteristics of an "insect-fungus complex."
[0044] The amplification results of Cordyceps militaris, Cordyceps liangshanense, and Cordyceps militaris samples are attached. Figure 7 None of the counterfeit samples showed simultaneous positive results for both primer pairs; some samples showed weak amplification signals for a single target, but their Ct values were significantly higher and the melting curve peaks were abnormal, failing to meet the criteria for simultaneous positive results for two targets and independent peaks in the melting curve.
[0045] The above results demonstrate that the method of this invention exhibits good specificity against closely related species and common counterfeits, avoiding false positives. Under the preferred criteria, genuine Cordyceps sinensis can only be identified when both the fungal and insect primer pairs exhibit typical amplification curves, and the melting curves show independent specific peaks corresponding to the two products respectively. Compared to single-target identification methods, this method provides a more rigorous basis for authenticity determination from the dual genetic dimension of the "fungus-insect complex." Compared to dual PCR methods relying on agarose gel electrophoresis, this method further verifies the specificity of amplified products through melting curve Tm value genotyping, accurately distinguishing specific products from non-specific products or primer dimers of similar length, significantly improving identification accuracy.
[0046] Example 3: Identification of Deep-Processed Cordyceps Sinensis Products This embodiment uses commercially available compound capsule preparations as the test object to verify the applicability of the method of the present invention to deep-processed Cordyceps sinensis products.
[0047] 3.1 Total Nucleic Acid Extraction from Samples Weigh out 10mg, 15mg and 25mg of the contents of commercially available Cordyceps sinensis, ginseng, deer antler, Dendrobium officinale and Ligustrum lucidum capsules respectively, and extract total nucleic acid according to the modified CTAB method described in Example 2.
[0048] 3.2 Double SYBR Green fluorescent PCR amplification Using the total nucleic acid extracted from each sample as a template, double PCR amplification was performed using the same preferred primer combination, reaction system, and amplification procedure as in Example 2.
[0049] 3.3 Result Judgment As attached Figure 8As shown, the capsule contents samples with different sample amounts of 10mg, 15mg and 25mg all showed typical fluorescence amplification curves for both fungal-specific primer pairs and insect-specific primer pairs. This indicates that the method of the present invention can effectively extract and detect trace amounts and partially degraded DNA of Cordyceps sinensis and Hepialus larvae in complex compound deep-processed products, and reliable identification results can be obtained with a minimum sample amount of only 10mg.
[0050] Compared to the limitations of existing morphological methods and conventional PCR electrophoresis in identifying deep-processed products, the method of this invention benefits from the highly specific primer combination and the detection sensitivity of the SYBR Green dye method. It can overcome the amplification challenges caused by complex matrix interference and DNA degradation, and successfully expand the application scope of molecular identification from raw Cordyceps sinensis to powders, capsules and other deep-processed products. It has important practical value for market supervision, enterprise quality control and third-party testing scenarios.
[0051] In summary, this invention, through a systematic primer screening strategy, successfully obtained a primer combination capable of achieving balanced and specific synergistic amplification of the Cordyceps sinensis ITS sequence and the Hepialus larvae COI gene sequence in the same SYBR Green reaction system at a universal annealing temperature of 60℃. This overcomes the amplification compatibility bottleneck caused by the significant differences in the base composition of the two targets. The method of this invention is performed entirely in closed tubes, requiring neither gel electrophoresis nor fluorescently labeled probes. Results are determined based on objective quantitative criteria of Tm values, offering a comprehensive advantage of high accuracy, ease of operation, and low cost. It is suitable for the authenticity identification of raw Cordyceps sinensis and its processed products.
[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for identifying the authenticity of Cordyceps sinensis and its products, characterized in that, Includes the following steps: (1) Extract total nucleic acid from the sample to be tested; (2) Using the total nucleic acid extracted in step (1) as a template, fungal-specific primer pairs and insect-specific primer pairs are used in the same PCR reaction tube to perform double PCR amplification by SYBR Green dye method and collect fluorescence signals; the fungal-specific primer pairs target the ITS sequence of Cordyceps sinensis, and the insect-specific primer pairs target the COI gene sequence of the host moth larvae. (3) Determine the results based on the amplification curve and melting curve: If both the fungal-specific primer pair and the insect-specific primer pair show specific amplification curves, and the melting curve shows two independent specific product peaks, then the sample to be tested is determined to be genuine Cordyceps sinensis or contains its components.
2. The method according to claim 1, characterized in that, The fungal-specific primer pair and the insect-specific primer pair can achieve balanced amplification of the ITS sequence and the COI gene sequence at an annealing temperature of 60°C, and the melting curve peaks of the amplification products of the two targets are independent of each other and there is no primer dimer interference peak.
3. The method according to claim 2, characterized in that, The fungal-specific primer pair is: Upstream primer F1: 5′-GGTGGTGAGTGAAGAAGGC-3′ Downstream primer R1: 5′-CCCGCTGACCTCATCCTT-3′; The insect-specific primer pair is: Upstream primer F2: 5′-TATGTTGAGCAGTAGCAGGA-3′, Downstream primer R2: 5′-GCTGGTGGTGAAGTTGTAG-3′.
4. The method according to claim 3, characterized in that, The reaction system for the double PCR amplification in step (2) is as follows: 25 ng DNA template, 10 μL SYBR Green qPCR Master Mix, 1 μL each of the upstream and downstream primers in the fungal-specific primer pair, 1 μL each of the upstream and downstream primers in the insect-specific primer pair, and 20 μL made up with sterile double-distilled water.
5. The method according to claim 4, characterized in that, The procedure for duplex PCR amplification in step (2) is as follows: pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 5 s; annealing and extension at 60℃ for 30 s; for a total of 40 cycles.
6. The method according to claim 3, characterized in that, In step (3), the Tm value of the amplification product of the fungal-specific primer pair is 87.5℃±1.0℃, and the Tm value of the amplification product of the insect-specific primer pair is 77.5℃±1.0℃.
7. The method according to any one of claims 1-6, characterized in that, The samples to be tested are raw Cordyceps sinensis, Cordyceps sinensis powder, Cordyceps sinensis capsules, Cordyceps sinensis tablets, Cordyceps sinensis oral liquid, or compound preparations containing Cordyceps sinensis.
8. The method according to any one of claims 1-6, characterized in that, The method is a closed-tube operation throughout, requiring no agarose gel electrophoresis step and no fluorescently labeled probes.
9. A specific primer combination for identifying the authenticity of Cordyceps sinensis and its products, characterized in that, include: Fungal-specific primer pair: upstream primer F1: 5′-GGTGGTGAGTGAAGAAGGC-3′, downstream primer R1: 5′-CCCGCTGACCTCATCCTT-3′; The insect-specific primer pair is as follows: upstream primer F2: 5′-TATGTTGAGCAGTAGCAGGA-3′, downstream primer R2: 5′-GCTGGTGGTGAAGTTGTAG-3′.
10. A reagent kit for identifying the authenticity of Cordyceps sinensis and its products, characterized in that, It includes the specific primer combination as described in claim 9.