A specific primer pair, method and application for detecting Trametes versicolor.

Abstract

This invention discloses a specific primer pair, method, and application for detecting *Trametes versicolor*. The specific primer pair is used to amplify gene fragments of *Trametes versicolor*, including primer pair 1 and / or primer pair 2, with the following nucleotide sequences: Primer pair 1: SEQ ID NO:1 and SEQ ID NO:2; Primer pair 2: SEQ ID NO:3 and SEQ ID NO:4. This invention employs polymerase chain reaction (PCR) to amplify specific gene fragments of *Trametes versicolor* by designing specific primers and amplification conditions, followed by detection by gel electrophoresis. This method can accurately identify samples containing *Trametes versicolor*, including medicinal materials, processed slices, and cultures. The identification results are accurate, and the method has high specificity, which is of great significance for ensuring the quality of *Trametes versicolor* pharmaceuticals and the safety of clinical applications.

Description

Technical Field

[0001] The present invention relates to the technical field of Coriolus versicolor detection, and more particularly, to a specific primer pair, method and application for detecting Coriolus versicolor. Background Art

[0002] Coriolus versicolor is a large medicinal fungus, also known as Variegated Coriolus and Versicolor Fomes in biological taxonomy. The species origin of Coriolus versicolor included in the "List of Chinese Biological Species 2023" is Trametes versicolor of the genus Fomes in the family Polyporaceae, also known as Coloured Velvet Bracket Coriolus versicolor (L.ex Fr.) Quel. Due to different strains and growth environments, different traits are presented on the caps of Coriolus versicolor, including white-edge type, blue-gray type, blue-gray fine-line type, blue-gray annulus type, gray-yellow edge type, gray-yellow annulus type, purple-copper annulus type, brown annulus type, gray-white edge type, thick hair bundle type, blue-black type, blue-black fine-line type, black annulus type, multi-color type, etc.

[0003] Coriolus versicolor is also a traditional Chinese medicine, with the effects of strengthening the spleen and promoting diuresis, clearing heat and detoxifying. It is clinically used in traditional Chinese medicine for damp-heat jaundice, hypochondriac pain, anorexia, fatigue and other symptoms, with remarkable effects. The "Chinese Pharmacopoeia" 2025 edition includes Coriolus versicolor medicinal materials, which are derived from the dried fruiting bodies of the fungus Coloured Velvet Bracket Coriolus versicolor ) (L.ex Fr.) Quel. The "Sichuan Provincial Chinese Medicine Pieces Processing Specification" 2015 edition includes Coriolus versicolor pieces, and the processing method is to remove impurities, wash, dry and cut into pieces.

[0004] Artificial fermentation can obtain Coriolus versicolor cultures. Coriolus versicolor capsules prepared from Coriolus versicolor cultures are a kind of Chinese patent medicine, with the content being brown powder, having the effect of adjusting immune function, and mainly used for the treatment of chronic viral hepatitis and early liver cirrhosis. Clinical studies have shown that when Coriolus versicolor capsules are combined with adriamycin to treat patients with advanced primary liver cancer, the decline amplitude of alpha-fetoprotein and the improvement rate of Karnofsky score in the treatment group are significantly better than those in the control group using only adriamycin. This medicine can enhance the curative effect of chemotherapy and reduce adverse reactions such as bone marrow suppression.

[0005] At present, for the identification of Coriolus versicolor medicinal materials, Coriolus versicolor pieces and Coriolus versicolor cultures used as medicines, microscopic identification and physicochemical identification methods are adopted to identify the authenticity, quality and superiority of drugs. Microscopic identification is carried out by crushing the drug into fine powder and then observing its cell characteristics under a microscope. Physicochemical identification is to carry out a precipitation reaction between the aqueous extract of the drug and alkaline tartaric acid copper. The principle is that the reducing polysaccharide contained in Coriolus versicolor reduces Cu 2+The reduction to orange-red cuprous oxide produces a precipitate and a color reaction. The specificity of microscopic and physicochemical identification methods for *Trametes versicolor* is poor, making it difficult to distinguish medicinal *Trametes versicolor* from other fungi in the same family and genus. A more specific method for identifying *Trametes versicolor* is needed.

[0006] In view of the above, this application is hereby submitted. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a specific primer pair, method, and application for detecting *Trametes versicolor*. Employing polymerase chain reaction (PCR), specific gene fragments of *Trametes versicolor* are amplified by designing specific primers and amplification conditions, followed by detection using gel electrophoresis. This method can accurately identify samples containing *Trametes versicolor*, including medicinal materials, processed slices, and cultures. The identification results are accurate, and the method has high specificity, which is of great significance for ensuring the quality of *Trametes versicolor* pharmaceuticals and the safety of their clinical applications.

[0008] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a specific primer pair for detecting *Trametes versicolor*, said specific primer pair being used to amplify a gene fragment of *Trametes versicolor*, the nucleotide sequence of which is as follows: Primer pair 1: SEQ ID NO:1 and SEQ ID NO:2.

[0009] This invention is achieved through the following technical solution: Secondly, the present invention provides a specific primer pair for detecting *Trametes versicolor*, wherein the specific primer pair is used to amplify a gene fragment of *Trametes versicolor*, and its nucleotide sequence is as follows: Primer pair 2: SEQ ID NO:3 and SEQ ID NO:4.

[0010] Thirdly, the present invention provides a specific primer pair for detecting *Trametes versicolor*, said specific primer pair being used to amplify gene fragments of *Trametes versicolor*, comprising primer pair 1 and primer pair 2, the nucleotide sequences of which are as follows: Primer pair 1: SEQ ID NO:1 and SEQ ID NO:2; Primer pair 2: SEQ ID NO:3 and SEQ ID NO:4.

[0011] Molecular biological identification is a method that determines the origin of a species or an individual organism by analyzing its molecular structure or gene sequence. It has the advantage of precise identification. Polymerase chain reaction (PCR) is a commonly used molecular biological identification method. PCR uses specifically designed primers and DNA polymerase to amplify specific gene fragments to a certain concentration in vitro, thereby enabling rapid detection and identification of target species.

[0012] By employing polymerase chain reaction (PCR) and designing specific primers and amplification conditions, specific gene fragments of *Trametes versicolor* can be amplified, followed by detection using gel electrophoresis. This method can accurately identify samples containing *Trametes versicolor*, including medicinal materials, processed slices, and cultures. The identification results are accurate, and the method has high specificity, which is of great significance for ensuring the quality of *Trametes versicolor* medicines and the safety of their clinical applications.

[0013] Fourthly, the present invention provides a method for detecting *Trametes versicolor* using polymerase chain reaction (PCR), wherein a sample containing *Trametes versicolor* is added to a polymerase reaction system for polymerase chain reaction, and the polymerase reaction system contains the specific primer pair for amplifying *Trametes versicolor* gene fragments as described in any one of claims 1 to 3.

[0014] In one specific embodiment, the polymerase reaction system includes: 25ul of 1×Easy Taq PCR premix, 1ul of Yunzhi genomic nucleic acid solution, 1ul of upstream primer solution, 1ul of downstream primer solution, and 22ul of Nuclease-free Water.

[0015] In one specific embodiment, the reaction conditions of the polymerase reaction system are as follows: Initial denaturation: 94℃, 5 min; Cyclic amplification: denaturation, 94℃, 30s; annealing, 52-68℃, 30s; extension, 72℃, 1min; 34 cycles; 3) Final extension: 72℃, 10min.

[0016] In a specific embodiment, the method for detecting Trametes versicolor using polymerase chain reaction (PCR) includes the following steps: (1) Genomic nucleic acid extraction was performed on the samples containing Trametes versicolor using a genomic extraction kit; (2) Prepare the PCR reaction system; (3) Mix the extracted genomic nucleic acid with PCR reagents and perform PCR reaction; (4) After the reaction is complete, electrophoresis is performed, and gel imaging analysis is used to determine whether there is Ganoderma lucidum in the sample.

[0017] Fifthly, the present invention provides the application of the specific primer pair and the method for detecting Ganoderma lucidum using polymerase chain reaction in the identification of samples containing Ganoderma lucidum.

[0018] In one specific embodiment, the sample containing Ganoderma lucidum includes Ganoderma lucidum medicinal material, Ganoderma lucidum processed slices, Ganoderma lucidum mycelium powder, and Ganoderma lucidum fermentation culture.

[0019] In a sixth aspect, the present invention provides a reagent for detecting Trametes versicolor, comprising the specific primer pair 1 and / or primer pair 2.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention provides a specific primer pair, method, and application for detecting Coriolus versicolor. The method uses polymerase chain reaction (PCR) to amplify specific gene fragments of Coriolus versicolor by designing specific primers and amplification conditions, and then detects them by gel electrophoresis. This method can accurately identify samples containing Coriolus versicolor, such as Coriolus versicolor medicinal materials, Coriolus versicolor processed slices, and Coriolus versicolor cultures. The identification results are accurate and the method has high specificity, which is of great significance for ensuring the quality of Coriolus versicolor medicines and the safety of clinical applications. 2. The present invention provides a specific primer pair, method and application for detecting Coriolus versicolor. By comparing and analyzing the Icc1 and RPB2 gene sequences of Coriolus versicolor with those of related species, a specific region of the Icc1 gene of Coriolus versicolor was discovered. A corresponding primer pair for specific amplification of Coriolus versicolor was designed for this specific region, which can specifically amplify the nucleic acid of Coriolus versicolor, while showing obvious amplification differences for related species, thus realizing the distinction between Coriolus versicolor and closely related species. 3. The specific primer pair, method and application for detecting Coriolus versicolor provided in the embodiments of the present invention also have a certain ability to distinguish different Coriolus versicolor subtypes. When primer pair 1 is used, the amplification bands of different Coriolus versicolor subtypes are slightly different between 1500bp and 2000bp. When primer pair 2 is used, the amplification bands of different Coriolus versicolor subtypes are slightly different between 350bp and 500bp. By comparing these subtle differences, the nucleic acid amplification method using two primer pairs has a certain ability to distinguish different Coriolus versicolor subtypes. 4. The present invention provides a specific primer pair, method and application for detecting Trametes versicolor. Based on the amplification band position of the two primers, it can be used as one of the bases for determining whether the strain typing of Trametes versicolor powder is consistent with strain CGMCC No.42378. 5. The present invention provides a specific primer pair, method and application for detecting Ganoderma lucidum, which can easily and quickly detect and identify samples containing Ganoderma lucidum, such as Ganoderma lucidum medicinal materials, Ganoderma lucidum decoction pieces, Ganoderma lucidum mycelium powder, and Ganoderma lucidum fermentation culture. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is an electrophoretic gel imaging analysis diagram of PCR products obtained using different primer pairs, provided in Example 1 of the present invention. Figure 2 This is an electrophoretic gel imaging analysis diagram of PCR products obtained by detecting different raw materials using the primer pairs of the present invention, provided in Example 2 of the present invention; Figure 3 The different origins of Ganoderma lucidum provided in Embodiment 3 of the present invention; Figure 4 This is an electrophoretic gel imaging analysis diagram of PCR products obtained by using the primer pairs of the present invention to detect Ganoderma lucidum medicinal materials and Ganoderma lucidum mycelium powder from different origins in Example 3 of the present invention. Figure 5 The sequencing results (partial) of the nucleic acid amplification fragments from the primer pair 1 provided in Example 3 of the present invention (for the primer pair 1 provided in Example 3 of the present invention). Figure 6 The sequencing results (partial) of the nucleic acid amplification fragments from the primer pair 2 provided in Example 3 of this invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are only used to explain this invention and are not intended to limit this invention.

[0024] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that these specific details are not necessary to practice the invention. In other embodiments, well-known materials or methods have not been specifically described in order to avoid obscuring the invention.

[0025] Throughout this specification, references to “an embodiment,” “an example,” or “an example” mean that a particular feature, structure, or characteristic described in connection with that embodiment or example is included in at least one embodiment of the invention. Therefore, the phrases “an embodiment,” “an example,” “an example,” or “an example” appearing in various places throughout the specification do not necessarily refer to the same embodiment or example. Furthermore, specific features, structures, or characteristics can be combined in one or more embodiments or examples in any suitable combination and / or sub-combination. The term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0026] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60–120 and 80–110 are listed for a specific parameter, it is understood that ranges of 60–110 and 80–120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0027] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the method may also include step (c), indicating that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0028] Example 1 This invention provides specific primer pairs for detecting Trametes versicolor and a method for designing those primer pairs, as detailed below: 1. Primer pair design The Icc1 and RPB2 gene sequences of *Trametes versicolor* were searched in the GenBank database and combined with the *Trametes versicolor* sequencing results. These sequences were then compared with those of other *Trametes versicolor* species to identify specific regions of the Icc1 and RPB2 genes. Primers were designed for these specific regions, including two primer pairs for Icc1 (primer pair 1 and primer pair 2) and two primer pairs for RPB2. Primer information is shown in Table 1. After Primer-Blast comparison, the designed primers and probes matched only the relevant *Trametes versicolor* sequences.

[0029] Table 1 Primers for PCR detection of Trametes versicolor .

[0030] 2. Genomic nucleic acid extraction from *Trametes versicolor* mycelium powder Animal and microbial genome extraction kits (containing RNase A) were used from Beijing TransGen Biotech Co., Ltd. EasyPure ® Genomic DNA Kit (containing RNase A) Cat. NO. EE101-01; Lyticase: Nanjing Dulai Biotechnology, Lyticase, biotechnology grade; for genomic nucleic acid extraction from *Trametes versicolor* powder. The extraction method used was the same as described in the instructions for treating Yeast Cells, extracting genomic nucleic acid from approximately 40 mg of *Trametes versicolor* powder to obtain a *Trametes versicolor* genomic nucleic acid solution.

[0031] Among them, the Yunzhi mycelium powder sample came from Gansu Guangyao Baiyunshan Traditional Chinese Medicine Technology Co., Ltd., and the strain was CGMCC No. 42378.

[0032] 3. PCR reaction PCR reactions were performed using the four primer pairs from step 1 and the extracted genomic nucleic acid from the *Trametes versicolor* powder from step 2.

[0033] PCR reagent: 2×EasyTaq PCR premix (2× EasyTaq ® PCR SuperMix (+dye)), Beijing TransGen Biotech Co., Ltd., Cat. NO. AS111-12; PCR reaction volume was 50 μL. This included 25 μL of 1×EasyTaqPCR premix, 1 μL of *Trametes versicolor* genomic nucleic acid solution, 1 μL (0.2 μm) of upstream primer solution, 1 μL (0.2 μm) of downstream primer solution, and 22 μL of Nuclease-free Water.

[0034] Gradient PCR was used for nucleic acid fragment amplification under the following conditions: Initial denaturation: 94℃, 5 min; Cyclic amplification (1) Denaturation, 94℃, 30S; (2) Annealing: 52-68℃ (gradient setting, 12 wells, from 52 degrees to 68 degrees, 4 primer pairs in 4 PCR tubes, 4 PCR tubes placed in wells 2, 5, 8 and 11 respectively, for a total of 16), 30s; (3) Extension: 72℃, 1min; (4) Repeat 34 times.

[0035] 3) Final extension: 72℃, 10 min; Take 5 μL of PCR product, add 1 μL of 6×Landing Buffer for spotting, and add DNA Marker (Trans2K® DNA Marker, Beijing TransGen Biotech Co., Ltd., Cat. NO. BM101-01) for spotting to label the fragment size of the PCR product. Electrophoresis results were recorded using a gel analysis imaging system, as shown below. Figure 1 As shown.

[0036] Figure 1 In the image, from left to right, lane 1 is the DNA marker; lanes 2-5 contain PCR products using the TV-F1 and TV-R1 primer pairs (primer pair 1); lanes 6-9 contain PCR products using the TV-F2 and TV-R3 primer pairs (primer pair 2); lanes 10-13 contain PCR products using the TVRPB2-F1 and TVRPB2-R1 primer pairs (primer pair 3), with no visible bands; lanes 14-17 contain PCR products using the TVRPB2-F1 and TVRPB2-R2 primer pairs (primer pair 2), with no visible bands on electrophoresis. Figure 1 As can be seen, both primer pairs 1 and 2 of this invention can produce electrophoretic bands, indicating that primer pairs 1 and 2 successfully amplified the nucleic acid of *Trametes versicolor*. However, primer pairs 3 and 4 cannot produce electrophoretic bands, indicating that the nucleic acid amplification was not achieved.

[0037] The primer pairs 1 and 2 for the Icc1 gene designed and screened in this embodiment of the invention have good specificity and can specifically amplify the nucleic acid of Ganoderma lucidum, thereby accurately identifying samples such as Ganoderma lucidum medicinal materials, Ganoderma lucidum decoction pieces, and Ganoderma lucidum fermentation cultures.

[0038] Example 2 This invention provides a method for PCR detection of *Trametes versicolor* powder, *Trametes versicolor* slices, and related species such as *Trametes versicolor*, *Trametes orientalis*, *Cryptocoryne*, *Trametes versicolor ... and *Trametes versicolor* using primer pairs 1 and 2 from Example 1. The specific method is as follows: 1. Primer pair selection Primer pair 1 (TV-F1, TV-R1) and primer pair 2 (TV-F2, TV-R3) targeting the Icc1 gene of *Trametes versicolor* were used.

[0039] 2. Genomic nucleic acid extraction Using the Animal and Microbial Genome Extraction Kit (containing RNase A) from Beijing TransGen Biotechnology Co., Ltd., and following the extraction method for Yeast Cells as described in the instructions, genomic nucleic acid was extracted from eight samples: Yunzhi mycelium powder, Yunzhi medicinal slices, Baisuojun, Dongfangsuojun, Cryptoporum, Maofengjun, Antrodia camphorata, and Bainangpaozhijun. Genomic nucleic acid solutions were obtained for each sample.

[0040] Among them, the Yunzhi mycelium powder sample came from Gansu Guangyao Baiyunshan Traditional Chinese Medicine Technology Co., Ltd., and the strain was CGMCC No. 42378. The Yunzhi processed slices, Baishuanjun, Dongfangshuanjun, Yinkongjun, Maofengjun, Niuzhangzhi, and Bainangpaozhijun came from Sichuan Gulin Hongan Pharmaceutical Co., Ltd.

[0041] 3. PCR reaction PCR reactions were performed using the two primer pairs from step 1 and the genomic nucleic acids extracted from each sample in step 2.

[0042] PCR reagents: 2×EasyTaq PCR premix (Beijing TransGen Biotech Co., Ltd., Cat. NO. AS111-12) was used, with a PCR reaction volume of 50 μL. This included 25 μL of 1×EasyTaq PCR premix, 1 μL of *Trametes versicolor* genomic nucleic acid solution, 1 μL (0.2 μm) of upstream primer solution, 1 μL (0.2 μm) of downstream primer solution, and 22 μL of Nuclease-free Water.

[0043] Gradient PCR was used for nucleic acid fragment amplification under the following conditions: 1) Initial denaturation: 94℃, 5 min; 2) Cyclic amplification (1) Denaturation, 94℃, 30S; (2) Annealing: 50-60℃ (gradient setting, 12 wells, from 50 degrees to 60 degrees, the PCR corresponding to primer pair 1 is placed in the 4th column well at 52.5 degrees, the PCR corresponding to primer pair 2 is placed in the 9th column well at 58 degrees), 30s; (3) Extension: 72℃, 1 min; (4) Repeat 35 times.

[0044] 3) Final extension: 72℃, 10 min; Take 5 μL of PCR product, add 1 μL of 6×Landing Buffer for spotting, and add DNA Marker (Trans2K® DNA Marker, Beijing TransGen Biotech Co., Ltd., Cat. NO. BM101-01) for spotting to label the fragment size of the PCR product. Electrophoresis results were recorded using a gel analysis imaging system, as shown below. Figure 2 As shown.

[0045] Figure 2 In the middle, from left to right, lanes 1 and 18 are DNA markers; lanes 2 to 9 are PCR products of primer pairs TV-F1 and TV-R1 (primer pair 1), namely *Bacillus subtilis*, *Bacillus orientalis*, *Cryptococcus*, *Cryptococcus fuciformis*, *Antrodia camphorata*, *Bacillus thunbergii*, *Trametes versicolor* medicinal material, and *Trametes versicolor* powder; lanes 10 to 17 are PCR products of primer pairs TV-F2 and TV-R3 (primer pair 2), namely *Bacillus subtilis*, *Bacillus orientalis*, *Cryptococcus*, *Cryptococcus fuciformis*, *Antrodia camphorata*, *Bacillus thunbergii*, *Trametes versicolor* medicinal material, and *Trametes versicolor* powder.

[0046] As can be seen from the figure, *Bacillus thunbergii* has no bands, while *Bacillus thunbergii* has a relatively dark band around 750 bp; *Bacillus orientalis* has multiple relatively dark bands ranging from over 100 bp to over 1000 bp; *Cryptocoryne*, *Hymenopterus xanthipes*, and *Antrodia camphorata* have no obvious bands; *Trametes versicolor* medicinal material has a very dark band close to 500 bp, while *Trametes versicolor* powder has obvious bands around 500 bp.

[0047] The electrophoresis results showed that primer pair 1 and primer pair 2, which amplify the specific nucleic acid of *Trametes versicolor*, can specifically amplify the nucleic acid of *Trametes versicolor*. The amplification of *Trametes versicolor* species in the same genus is very different from that of *Trametes versicolor*. Therefore, it is possible to accurately distinguish *Trametes versicolor* from closely related species in the same genus, thereby enabling rapid and accurate detection and identification of samples such as *Trametes versicolor* medicinal materials, *Trametes versicolor* processed slices, and *Trametes versicolor* mycelium powder.

[0048] Example 3 This invention provides a method for PCR detection of *Trametes versicolor* mycelium powder and *Trametes versicolor* medicinal materials from different origins using primer pair 1 and primer pair 2 from Example 1. The specific method is as follows: 1. Materials and Reagents The sample of *Trametes versicolor* mycelium powder was obtained from Gansu Guangyao Baiyunshan Traditional Chinese Medicine Technology Co., Ltd., and the strain was CGMCC No. 42378. *Trametes versicolor* medicinal materials from different origins (see...) Figure 3 1-Produced in Leshan, Sichuan; 2-Produced in Luzhou, Sichuan; 3-Produced in Linzhi, Tibet (A); 4-Produced in Zhaotong, Yunnan (A); 5-Produced in Gulin, Luzhou, Sichuan; 6-Produced in Linzhi, Tibet (B); 7-Produced in Zhaotong, Yunnan (B)) originates from Gulin Hongan Pharmaceutical Co., Ltd., Sichuan.

[0049] Plant genomic DNA extraction kit: Beijing TransGen Biotech Co., Ltd.; Animal and microbial genomic DNA extraction kit (containing RNase A) EasyPure ® Genomic DNA Kit (containing RNase A) Cat. NO. EE101-01; Lyticase: Nanjing Dulai Biotechnology, Lyticase, biotechnology grade; PCR reagent: 2×EasyTaq PCR premix (2× EasyTaq ® PCR SuperMix (+dye)) Cat.NO.AS111-12; 2. Primers Primer pair 1 (TV-F1, TV-R1) and primer pair 2 (TV-F2, TV-R3) targeting the Icc1 gene of Ganoderma lucidum.

[0050] 1.3 Genomic nucleic acid extraction Genomic nucleic acid extraction was performed on eight samples, including Yunzhi mycelium powder and Yunzhi medicinal materials from different origins, using the animal and microbial genome extraction kit (containing RNase A) from Beijing TransGen Biotechnology Co., Ltd. The extraction method followed the Yeast Cells treatment method described in the instructions.

[0051] 1.4 PCR reaction The PCR reaction volume was 50 μL, using 2×EasyTaq PCR premix (Beijing TransGen Biotech Co., Ltd., Cat. NO. AS111-12). This included 25 μL of 1×EasyTaq PCR premix, 1 μL of *Trametes versicolor* genomic nucleic acid solution, 1 μL (0.2 μm) of upstream primer solution, 1 μL (0.2 μm) of downstream primer solution, and 22 μL of Nuclease-free Water.

[0052] Gradient PCR was used for nucleic acid fragment amplification under the following conditions: Initial denaturation: 94℃, 5 min; Cyclic amplification (1) Denaturation, 94℃, 30S; (2) Annealing: 50-60℃ (gradient setting, 12 wells, from 50 degrees to 60 degrees, the PCR corresponding to primer pair 1 is placed in the 4th column well at 52.5 degrees, the PCR corresponding to primer pair 2 is placed in the 9th column well at 58 degrees), 30s; (3) Extension: 72℃, 1 min; (4) Repeat 35 times.

[0053] 3) Final extension: 72℃, 10 min; Take 5 μL of PCR product, add 1 μL of 6×Landing Buffer for spotting, and add DNA Marker (Trans2K® DNA Marker, Beijing TransGen Biotech Co., Ltd., Cat. NO. BM101-01) for spotting to label the fragment size of the PCR product. Electrophoresis results were recorded using a gel analysis imaging system, as shown below. Figure 4 As shown. Sequencing results of the amplified nucleic acid fragments from *Trametes versicolor* powder using primer pair 1 and primer pair 2 are as follows. Figure 5 and Figure 6 As shown.

[0054] Figure 4 In the image, the first lane on the left and the first lane on the right are DNA markers, with 100bp, 250bp, 500bp, 750bp, 1000bp, and 2000bp PCR products from bottom to top. The left side shows the PCR product of primer pair 1, and the right side shows the PCR product of primer pair 2. Left and right sides (from left to right): the second to eighth bands correspond to *Trametes versicolor*, and... Figure 3 The order is consistent. The ninth band corresponds to the mycelium powder of *Trametes versicolor*, and the tenth band is the blank control.

[0055] As shown in the figure, the PCR product of primer pair 1 is located between 1500bp and 2000bp, while the PCR product of primer pair 2 is located between 350bp and 500bp. In some strains, the PCR product is not obvious at one corresponding position of primer pair 1 or primer pair 2, indicating low amplification efficiency; however, at least one primer pair shows good amplification results. By comparing these subtle differences, the nucleic acid amplification method using two primer pairs demonstrates a certain ability to distinguish between different *Trametes versicolor* strains.

[0056] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A specific primer pair for detecting Trametes versicolor, characterized in that, The specific primer pair used to amplify the gene fragment of *Trametes versicolor* has the following nucleotide sequence: Primer pair 1: SEQ ID NO:1 and SEQ ID NO:

2.

2. A specific primer pair for detecting Trametes versicolor, characterized in that, The specific primer pair used to amplify the gene fragment of *Trametes versicolor* has the following nucleotide sequence: Primer pair 2: SEQ ID NO:3 and SEQ ID NO:

4.

3. A specific primer pair for detecting Trametes versicolor, characterized in that, The specific primer pairs used to amplify the gene fragment of *Trametes versicolor* include primer pair 1 and primer pair 2, whose nucleotide sequences are as follows: Primer pair 1: SEQ ID NO:1 and SEQ ID NO:2; Primer pair 2: SEQ ID NO:3 and SEQ ID NO:

4.

4. A method for detecting *Trametes versicolor* using polymerase chain reaction (PCR), characterized in that, The sample to be tested containing *Trametes versicolor* is added to a polymerase reaction system for polymerase chain reaction, wherein the polymerase reaction system contains the specific primer pair for amplifying the *Trametes versicolor* gene fragment as described in any one of claims 1 to 3.

5. The method for detecting *Trametes versicolor* using polymerase chain reaction (PCR) according to claim 4, characterized in that, The polymerase reaction system includes: 25ul of 1×Easy Taq PCR premix, 1ul of Yunzhi genomic nucleic acid solution, 1ul of upstream primer solution, 1ul of downstream primer solution, and 22ul of Nuclease-free Water.

6. The method for detecting *Trametes versicolor* using polymerase chain reaction (PCR) according to claim 4, characterized in that, The reaction conditions for the polymerase reaction system are: Initial denaturation: 94℃, 5 min; Cyclic amplification: denaturation, 94℃, 30s; annealing, 52-68℃, 30s; extension, 72℃, 1min; 34 cycles; 3) Final extension: 72℃, 10min.

7. The method for detecting *Trametes versicolor* using polymerase chain reaction (PCR) according to claim 4, characterized in that, Specifically, the steps include the following: (1) Genomic nucleic acid extraction was performed on the samples containing Trametes versicolor using a genomic extraction kit; (2) Prepare the PCR reaction system; (3) Mix the extracted genomic nucleic acid with PCR reagents and perform PCR reaction; (4) After the reaction is complete, electrophoresis is performed, and gel imaging analysis is used to determine whether there is Ganoderma lucidum in the sample.

8. The application of the specific primer pair described in any one of claims 1 to 3 or the method for detecting Ganoderma lucidum using polymerase chain reaction as described in any one of claims 4 to 7 in the identification of samples containing Ganoderma lucidum.

9. The application according to claim 8, characterized in that, The samples containing Ganoderma lucidum include Ganoderma lucidum medicinal materials, Ganoderma lucidum processed slices, Ganoderma lucidum mycelium powder, and Ganoderma lucidum fermentation culture.

10. A reagent for detecting Trametes versicolor, characterized in that, Includes the specific primer pair described in any one of claims 1 to 3.

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