Nucleic acid product, kit and method for detecting variety of coriolus versicolor

Abstract

The invention provides a nucleic acid product, a kit and a method for detecting a coriolus versicolor variety. The nucleic acid product is used for detecting an SNP (Single Nucleotide Polymorphism) site of coriolus versicolor, the SNP site is located at the 1835th site in a GenBank sequence with the login number of AY309016.1, and a deoxynucleotide base of the site is T. Through research, it is found that coriolus versicolor and species with confused shapes can be rapidly and efficiently distinguished by detecting the SNP sites. Specifically, when the basic group of the SNP site is T, the sample to be detected can be identified as the coriolus versicolor, and when the SNP site is other basic groups, it can be determined that the sample to be detected does not belong to the coriolus versicolor.

Description

Technical Field

[0001] The present application relates to the technical field of identification of traditional Chinese medicine varieties, and in particular to a nucleic acid product, a kit and a method for detecting versicolor varieties. Background Art

[0002] Trametes versicolor is the dried fruiting body of the Polyporaceae fungus Coriolus versicolor (L. ex Fr.) Quel, also known as Trametes versicolor. Trametes versicolor is a medicinal material recorded in the Chinese Pharmacopoeia. It has the effects of strengthening the spleen and removing dampness, clearing away heat and detoxifying. It can be used to relieve symptoms such as damp-heat jaundice, flank pain, poor appetite, fatigue and weakness.

[0003] The appearance of Yunzhi is usually fan-shaped, semicircular or shell-shaped, and often stacked to form a shingle or rosette shape; the surface is densely covered with gray, brown, blue, purple and black hyphae, forming multi-colored narrow concentric rings with thin edges; the ventral surface is gray-brown, yellow-brown or light yellow; leathery, not easy to break, and the cross-section of the fungus is white; the tube is single-layered, mostly light brown, and the tube mouth is nearly round to polygonal. Other fungi species of the same family and genus as Yunzhi, such as Trametes ochracea and Lenzitesbetulina, are similar in appearance to Yunzhi and are easily confused when collecting.

[0004] Therefore, it is necessary to provide a method for effectively identifying versicolor varieties. Summary of the invention

[0005] Based on this, one or more embodiments of the present application provide a nucleic acid product, kit and method for detecting Versicolor species with strong specificity and high sensitivity.

[0006] According to the first aspect of the embodiments of the present application, a nucleic acid product for detecting the variety of Coriolus versicolor is provided, wherein the nucleic acid product is used to detect the SNP site of Coriolus versicolor, wherein the SNP site is located at position 1835 in the sequence with GenBank accession number AY309011, and the deoxynucleotide base at the site is T.

[0007] In one embodiment, the nucleic acid product includes one or more of the following primer pairs:

[0008] (1) a first primer pair having nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2; and

[0009] (2) A second primer pair having nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 3.

[0010] According to a second aspect of an embodiment of the present application, a kit for detecting Versicolor varieties is provided, the kit comprising the above-mentioned nucleic acid product.

[0011] In one embodiment, the kit further comprises PCR reaction reagents;

[0012] Optionally, the PCR reaction reagents include one or more of PCR buffer, DNA polymerase, magnesium ions and dNTPs.

[0013] In one embodiment, the kit further comprises a DNA extraction reagent.

[0014] According to a third aspect of the embodiment of the present application, a method for detecting versicolor varieties is provided, comprising the following steps:

[0015] The DNA of the sample to be tested is used as a template, and PCR amplification is performed using the above-mentioned nucleic acid product or the above-mentioned kit, and the variety of the sample to be tested is identified based on the obtained amplification result.

[0016] In one embodiment, in the PCR amplification reaction procedure, the annealing temperature is 58°C-69°C.

[0017] In one embodiment, the number of cycles in the PCR amplification reaction procedure is 30 to 40 times.

[0018] In one embodiment, in the PCR amplification reaction system, the concentrations of the forward primer and the reaction primer are independently 0.4 μmol / L to 1.0 μmol / L.

[0019] In one embodiment, the method further comprises the following steps: performing agarose gel electrophoresis on the product amplified by PCR, and identifying the variety of the sample to be tested according to the obtained detection result.

[0020] Compared with the traditional technology, this application has the following beneficial effects:

[0021] The present application has found through research that by detecting the SNP site at position 1835 in the sequence with GenBank accession number AY309016.1, it is possible to quickly and efficiently distinguish between Versicolor and species that are easily confused with each other in appearance. Specifically, when the base of the SNP site is T, the sample to be tested can be identified as Versicolor, and when the SNP site is other bases, it can be determined that the sample to be tested does not belong to Versicolor.

[0022] In addition, the nucleic acid product with a specific nucleotide sequence of the present application has the advantages of high specificity and high sensitivity when used to identify and distinguish Yunzhi and its similar varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 The gel electrophoresis results of amplification using DNA of different species as template by the first primer pair;

[0025] Figure 2 The gel electrophoresis results of the second primer pair amplifying DNA from different species as templates;

[0026] Figure 3 The gel electrophoresis results of amplification by the first primer pair using different concentrations of DNA as template;

[0027] Figure 4 The gel electrophoresis results of the second primer pair amplifying with different concentrations of DNA as template;

[0028] Figure 5 The gel electrophoresis results of the first primer pair amplification at different annealing temperatures;

[0029] Figure 6 The gel electrophoresis results of the second primer pair amplification at different annealing temperatures;

[0030] Figure 7 The gel electrophoresis results of the first primer pair amplification at different annealing temperatures;

[0031] Figure 8 is the gel electrophoresis result of the amplification of the first primer pair at different primer concentrations;

[0032] Fig. 9 The gel electrophoresis results of the second primer pair amplification at different primer concentrations;

[0033] Fig.10 The gel electrophoresis results of the first primer pair amplification under different cycle reaction times;

[0034] Fig.11 The gel electrophoresis results of the second primer pair amplification under different cycle reaction times;

[0035] Fig.12 Shown are the gel electrophoresis results of amplification using different primer pairs. DETAILED DESCRIPTION

[0036] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present application. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present application, etc. can be purchased from the market or can be prepared by existing methods.

[0038] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0039] In the present application, "plurality", "multiple", "multiple times", "multiple", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0040] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0041] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0042] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0043] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0044] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0045] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0046] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1-10, indicating that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0047] In the present application, a "primer pair" refers to a pair of primers that can hybridize with the double strands of a target DNA molecule or can hybridize with the regions of the target DNA molecule located on both sides of the nucleotide sequence to be amplified.

[0048] In a first aspect of the present application, a nucleic acid product for detecting the variety of Coriolus versicolor is provided. The nucleic acid product is used to detect the SNP site of Coriolus versicolor. The SNP site is located at position 1835 in the sequence with GenBank accession number AY309011, and the deoxynucleotide base at the site is T.

[0049] Specifically, when the base of the SNP site is T, the sample to be tested can be identified as Versicolor, and when the SNP site is other bases, it can be determined that the sample to be tested does not belong to Versicolor.

[0050] Based on the analysis of the ITS2 sequence of the medicinal material Coriolus versicolor, the applicant found that by detecting the base type of the above-mentioned SNP site in the Coriolus versicolor genome, Coriolus versicolor can be distinguished from similar varieties quickly and efficiently; using this SNP site as a representative SNP site of the Coriolus versicolor variety has important significance and application value for the variety identification of Coriolus versicolor.

[0051] In some embodiments, the nucleic acid product includes a first primer pair having nucleotide sequences as shown in SEQ ID NO:1 and SEQ ID NO:2.

[0052] In some embodiments, the nucleotide sequences of the second primer pair are shown as SEQ ID NO:1 and SEQ ID NO:3.

[0053] The first primer pair and the second primer pair of the present application both have good specificity and detection sensitivity for the above-mentioned SNP sites of Coriolus versicolor, and can quickly and accurately distinguish and identify Coriolus versicolor and its similar varieties.

[0054] In a second aspect of the present application, a kit for detecting versicolor varieties is provided, the kit comprising the above-mentioned nucleic acid product.

[0055] In some embodiments, the kit further comprises PCR reaction reagents.

[0056] In some specific examples, the PCR reaction reagents include one or more of a PCR buffer, a DNA polymerase, magnesium ions, and dNTPs.

[0057] In some embodiments, the kit further comprises a DNA extraction reagent.

[0058] In some embodiments, the kit further comprises one or more of a positive control, a negative control, a sequencing reagent, and an electrophoresis detection reagent.

[0059] Understandably, the positive control may be a standard sample of Versicolor versicolor; and the negative control may be deionized water or other samples that do not belong to Versicolor versicolor.

[0060] Understandably, in some other specific examples, the above-mentioned kit may not include any of the positive controls, negative controls, PCR reaction reagents, sequencing reagents and electrophoresis detection reagents, and the excluded reagents may be reasonably obtained from the outside.

[0061] In a third aspect of the present application, a method for detecting versicolor varieties is provided, comprising the following steps:

[0062] The DNA of the sample to be tested is used as a template, and PCR amplification is performed using the nucleic acid product of the first aspect or the kit of the second aspect, and the species of the sample to be tested is identified based on the obtained amplification results.

[0063] In some embodiments, a plant DNA extraction kit or CTAB method is used to extract DNA from the sample to be tested.

[0064] In some embodiments, in the PCR amplification reaction procedure, the annealing temperature is 58°C to 69°C.

[0065] As an example, the annealing temperature may be 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C or 69°C; it may also be a value between any two of the above values.

[0066] In some embodiments, in the PCR amplification reaction procedure, the number of cycles is 30 to 40 times.

[0067] As an example, the number of PCR cycles can be 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 times.

[0068] In some embodiments, in the PCR amplification reaction system, the concentrations of the forward primer and the reaction primer are independently 0.4 μmol / L to 1.0 μmol / L.

[0069] As an example, the concentration of the forward primer may be 0.4 μmol / L, 0.5 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 0.8 μmol / L, 0.9 μmol / L or 1.0 μmol / L.

[0070] As an example, the concentration of the reverse primer may be 0.4 μmol / L, 0.5 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 0.8 μmol / L, 0.9 μmol / L or 1.0 μmol / L.

[0071] In a specific example, the reaction procedure of PCR amplification includes: pre-denaturation at 94°C for 90 seconds; denaturation at 94°C for 20 seconds, annealing at 69°C for 20 seconds, extension at 72°C for 20 seconds, and cycle reaction 35 times; and extension at 72°C for 5 minutes.

[0072] In some embodiments, the method for detecting the variety of Coriolus versicolor further comprises the following step: performing agarose gel electrophoresis on the product amplified by PCR, and identifying the variety of the sample to be tested based on the obtained detection result.

[0073] In some embodiments, the step of identifying the species of the sample to be tested according to the obtained test results includes:

[0074] If the amplified product contains a product of 181 bp or 95 bp in size, the sample to be tested is considered to be Versicolor versicolor;

[0075] If the amplified product does not contain a product of 181 bp or 95 bp in size, it is considered that the sample to be tested does not belong to Versicolor versicolor.

[0076] Specifically, the length of the target product obtained by amplification using the first primer pair is 181 bp; the length of the target product obtained by amplification using the second primer pair is 95 bp.

[0077] The above detection method of the present application can be applied to the detection of fresh Coriolus versicolor, Coriolus versicolor spores, Coriolus versicolor medicinal materials, Coriolus versicolor medicinal material powder, and products containing Coriolus versicolor fine powder, etc. The above detection method has the advantages of rapidity, accuracy, high sensitivity and high specificity, and is suitable for promotion and application.

[0078] The present application will be further described below in conjunction with specific examples and comparative examples, but they should not be construed as limiting the scope of protection of the present application. The raw materials involved in the following specific examples, unless otherwise specified, can all be sourced from commercial sources, the instruments used, unless otherwise specified, can all be sourced from commercial sources, and the processes involved, unless otherwise specified, are all conventionally selected by those skilled in the art.

[0079] Example 1: Exploring the specificity of nucleic acid products in detecting versicolor species

[0080] (1) Primer design

[0081] The applicant has found through research that by detecting the SNP site at position 1835 in the sequence with GenBank accession number AY309016.1, it is possible to quickly identify Versicolor and its similar varieties; specifically, when the base of the SNP site is T, the variety to be tested can be identified as Versicolor. Specifically, the nucleotide sequence of the sequence with GenBank accession number AY309016.1 is shown in SEQ ID NO: 4 (the underlined portion is the SNP site).

[0082] SEQ ID NO:4

[0083] t .

[0084] In order to detect the above-mentioned sites, the applicant specifically designed a primer pair with a nucleotide sequence as shown in SEQ ID NO: 1 (forward primer) and SEQ ID NO: 2 (reverse primer); and a primer pair with a nucleotide sequence as shown in SEQ ID NO: 1 (forward primer) and SEQ ID NO: 3 (reverse primer); the primer information is shown in Table 1.

[0085] Table 1

[0086]

[0087] (2) DNA extraction

[0088] The samples of Versicolor, Ganoderma lucidum, Polyporus umbellatus and Poria cocos to be tested were taken and crushed into fine powder. 50 mg was placed in a 1.5 mL centrifuge tube, and 700 μL GP1 buffer was added to the tube and placed in a 56°C water bath overnight. DNA was extracted using a plant genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., model DP-305), diluted to 1 μg / mL, and placed in a 4°C refrigerator for later use. The information of each sample to be tested is shown in Table 2.

[0089] Table 2

[0090]

[0091] (3) PCR amplification

[0092] In a 200 μL centrifuge tube, prepare the reaction system as follows: 10 μL of 2× PCR premix, 0.8 μL each of forward primer (10 μmol / L) and reverse primer (10 μmol / L), 2 μL of DNA template, and 6.4 μL of sterile water; the final concentrations of the forward primer and reverse primer were both 0.4 μmol / L.

[0093] The PCR amplification program was as follows: pre-denaturation at 94°C for 90 seconds; denaturation at 94°C for 20 seconds, annealing at 69°C for 20 seconds, extension at 72°C for 20 seconds, 35 cycles; and extension at 72°C for 5 minutes.

[0094] Take 20 μL of sterile water and perform the same PCR amplification procedure as above as a blank control.

[0095] (4) Electrophoresis detection

[0096] Prepare a 3% agarose gel and add nucleic acid gel stain to the gel; the loading volume of the PCR reaction solution of each sample to be tested is 8 μL, and the loading volume of the DNA Marker is 2 μL (0.5 μg / μL). After the electrophoresis is completed, take the gel slice and take a photo on a gel imager or a UV transilluminator.

[0097] (5) Results analysis

[0098] The electrophoresis diagram of the PCR product obtained by the first primer pair is as follows: Figure 1 As shown, Figure 1 From left to right, the samples corresponding to the channels are: Marker, Yunzhi 01, Yunzhi 02, Yunzhi 03, Ganoderma lucidum, Polyporus umbellatus, Poria cocos and blank control. Figure 1 It can be seen that when the above primers are used for amplification, the Yunzhi samples from different sources can obtain the 181bp target band after amplification, while the Ganoderma lucidum, Polyporus umbellatus and Poria cocos samples did not amplify the above target band; the above results show that the first primer pair has good specificity when used to identify Yunzhi varieties.

[0099] The electrophoresis diagram of the PCR product amplified by the second primer pair is as follows Figure 2 As shown, Figure 2 From left to right, the samples corresponding to the channels are: Marker, Yunzhi 01, Yunzhi 02, Yunzhi 03, Ganoderma lucidum, Polyporus umbellatus, Poria cocos and blank control. Figure 2It can be seen that when the above primers are used for amplification, the Yunzhi samples from different sources can obtain the 95bp target band after amplification, while the Ganoderma lucidum, Polyporus umbellatus and Poria cocos samples did not amplify the above target band; the above results show that the second primer pair also has good specificity when used to identify Yunzhi varieties.

[0100] Example 2: Exploring the sensitivity of nucleic acid products in detecting versicolor varieties

[0101] The DNA samples of Yunzhi 01 with concentrations of 2.2 μg / mL, 1.1 μg / mL, 0.55 μg / mL, 0.275 μg / mL, 0.138 μg / mL, 0.069 μg / mL, 0.034 μg / mL and 0.017 μg / mL were used as amplification templates, and the PCR amplification system, reaction procedure and electrophoresis method were referred to Example 1.

[0102] Result analysis: The electrophoresis diagram of the PCR product obtained by PCR amplification with the first primer pair is as follows Figure 3 As shown, the electrophoresis diagram of the PCR product obtained by PCR amplification with the second primer pair is as shown Figure 4 As shown; Figure 3 and Figure 4 The samples corresponding to the channels from left to right are: Marker, DNA samples of Yunzhi 01 with DNA concentrations of 2.2μg / mL, 1.1μg / mL, 0.55μg / mL, 0.275μg / mL, 0.138μg / mL, 0.069μg / mL, 0.034μg / mL, and 0.017μg / mL. Figure 3~4 It can be seen that when the above primers are used to identify the Versicolor varieties, the minimum detection limit can reach 0.034μg / mL, indicating that the above two pairs of primers have good detection sensitivity.

[0103] Example 3: Exploring the effect of different annealing temperatures on detection

[0104] (1) The method is basically the same as Example 1, except that the DNA sample of Coriolus versicolor 01 is used as a template and the PCR amplification procedure is as follows: pre-denaturation at 94°C for 90 seconds; denaturation at 94°C for 20 seconds, annealing at 67°C, 68°C, 69°C, 70°C, 71°C, and 72°C for 20 seconds, extension at 72°C for 20 seconds, and the reaction cycle is repeated 40 times; and extension at 72°C for 5 minutes.

[0105] Result analysis: The electrophoresis diagram of the PCR product amplified by the first primer pair is as follows Figure 5 As shown, Figure 5 From left to right, the samples corresponding to the channels are: Marker, and the products amplified at annealing temperatures of 67°C, 68°C, 69°C, 70°C, 71°C, and 72°C. Figure 5It can be seen that when the above primers are used for amplification, a clear and bright 181 bp band can be amplified when the annealing temperature is 67°C~69°C, indicating that the annealing temperature of 67°C~69°C can improve the amplification efficiency of the first primer pair.

[0106] The electrophoresis diagram of the PCR product amplified by the second primer pair is as follows Figure 6 As shown, Figure 6 From left to right, the samples corresponding to the channels are: Marker, and the products amplified at annealing temperatures of 67°C, 68°C, 69°C, 70°C, 71°C, and 72°C. Figure 6 It can be seen that when the above primers are used for amplification, a clear and bright 95bp band can be amplified when the annealing temperature is 67°C~70°C, indicating that the annealing temperature of 67°C~70°C can improve the amplification efficiency of the second primer pair.

[0107] (2) Using the DNA sample of Yunzhi 01 as a template, the PCR amplification procedure was as follows: pre-denaturation at 94°C for 90 seconds; denaturation at 94°C for 20 seconds, annealing at 62°C, 64°C, 66°C, 68°C, 70°C, and 72°C for 20 seconds, extension at 72°C for 20 seconds, and a cycle of 40 reactions; and extension at 72°C for 5 minutes.

[0108] Result analysis: The electrophoresis diagram of the PCR product amplified by the first primer pair is as follows Figure 7 As shown, Figure 7 From left to right, the samples corresponding to the channels are: Marker, and the products amplified at annealing temperatures of 62°C, 64°C, 66°C, 68°C, 70°C, and 72°C. Figure 7 It can be seen that when the above primers are used for amplification, a clear and bright 181 bp band can be amplified when the annealing temperature is 62°C to 68°C.

[0109] In summary, an annealing temperature of 62°C~69°C can improve the amplification efficiency of the first primer pair.

[0110] Example 4: Exploring the effects of different primer concentrations on detection

[0111] (1) Basically the same as Example 1, except that: in a 200 μL centrifuge tube, the reaction system was prepared as follows: 10 μL of 2×PCR premix, 2 μL of DNA template of Yunzhi 01, 0.8 μL / 1.2 μL / 1.6 μL / 2 μL of forward primer (10 μmol / L) and reverse primer (10 μmol / L), respectively, and the volume was fixed to 20 μL with sterile water. The final concentrations of the forward primer and reverse primer in the final reaction system were 0.1 μmol / L, 0.2 μmol / L, 0.4 μmol / L, 0.6 μmol / L, 0.8 μmol / L and 1.0 μmol / L.

[0112] The PCR amplification program was as follows: pre-denaturation at 94°C for 90 seconds; denaturation at 94°C for 20 seconds, annealing at 69°C for 20 seconds, extension at 72°C for 20 seconds, 40 cycles; and extension at 72°C for 5 minutes.

[0113] Result analysis: The electrophoresis diagram of the PCR product obtained by the first primer pair is as follows Figure 8 As shown, Figure 8 From left to right, the samples corresponding to the channels are: Marker, and the products amplified when the primer concentrations are 0.1μmol / L, 0.2μmol / L, 0.4μmol / L, 0.6μmol / L, 0.8μmol / L, and 1.0μmol / L. Figure 8 It can be seen that when the above primers are used for amplification, when the primer concentrations are 0.4 μmol / L, 0.6 μmol / L, 0.8 μmol / L and 1.0 μmol / L, the bands in the gel electrophoresis diagram of the amplified product are single, clear and bright.

[0114] The electrophoresis diagram of the PCR product amplified by the second primer pair is as follows Fig. 9 As shown, Fig. 9 From left to right, the samples corresponding to the channels are: Marker, and the products amplified when the primer concentrations are 0.1μmol / L, 0.2μmol / L, 0.4μmol / L, 0.6μmol / L, 0.8μmol / L, and 1.0μmol / L. Fig. 9 It can be seen that when the above primers are used for amplification, when the primer concentrations are 0.4 μmol / L, 0.6 μmol / L, 0.8 μmol / L and 1.0 μmol / L, a single target band of 95 bp in size can be amplified.

[0115] Example 5: Exploring the effects of different cycle reaction times on detection

[0116] (1) The method is basically the same as Example 1, except that: in a 200 μL centrifuge tube, the reaction system is prepared as follows: 10 μL of 2×PCR premix, 2 μL of DNA template of Yunzhi 01, 0.8 μL of each of forward primer (10 μmol / L) and reverse primer (10 μmol / L), and the volume is adjusted to 20 μL with sterile water.

[0117] The PCR amplification program was as follows: pre-denaturation at 94°C for 90 seconds; denaturation at 94°C for 20 seconds, annealing at 69°C for 20 seconds, extension at 72°C for 20 seconds, and the cycle reactions were 20, 25, 30, 35, and 40 times, respectively; and extension at 72°C for 5 minutes.

[0118] Result analysis: The electrophoresis diagram of the PCR product amplified by the first primer pair is as follows Fig.10 As shown, Fig.10From left to right, the samples corresponding to the channels are: Marker, products amplified after 20, 25, 30, 35, and 40 cycles respectively. Fig.10 It can be seen that when the above primers are used for amplification, a single band of 181 bp can be amplified when the number of cycles reaches 30 or more.

[0119] The electrophoresis diagram of the PCR product amplified by the second primer pair is as follows Fig.11 As shown, Fig.11 From left to right, the samples corresponding to the channels are: Marker, products amplified after 20, 25, 30, 35, and 40 cycles respectively. Fig.11 It can be seen that when the above primers are used for amplification, a single band of 95 bp in size can be amplified when the number of cycles reaches 35 or more.

[0120] Example 6: Exploring the effects of different primers on detection

[0121] In order to compare the detection effects of different primer pairs, the primer pairs shown in SEQ ID NOs: 1-2 and the primer pairs shown in SEQ ID NOs: 1 and 3 were used to perform PCR amplification and electrophoresis on the DNA samples of Coriolus versicolor 01, Coriolus versicolor 02 and Coriolus versicolor 03, respectively. The specific steps are as shown in Example 1.

[0122] The electrophoresis results are as follows Fig.12 As shown, Fig.12 The samples corresponding to the channels from left to right are: Marker, the product of Yunzhi 01 amplified by the first primer pair, the product of Yunzhi 01 amplified by the second primer pair, the product of Yunzhi 02 amplified by the first primer pair, the product of Yunzhi 02 amplified by the second primer pair, the product of Yunzhi 03 amplified by the first primer pair, and the product of Yunzhi 03 amplified by the second primer pair.

[0123] Depend on Fig.12 It can be seen that when the first primer pair is used to amplify the DNA sample of Coriolus versicolor, the band is clearer and brighter, indicating that the detection effect of the first primer pair is relatively better.

[0124] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0125] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A nucleic acid product for detecting versicolor species, characterized in that: The nucleic acid product is used to detect the SNP site of Coriolus versicolor, the SNP site is located at the 1835th position in the sequence with GenBank accession number AY309016.1, and the deoxynucleotide base of the site is T.

2. The nucleic acid product for detecting the species of Coriolus versicolor according to claim 1, characterized in that: The nucleic acid product includes one or more of the following primer pairs: (1) a first primer pair having nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2; and (2) A second primer pair having nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO:

3.

3. A kit for detecting varieties of Coriolus versicolor, characterized in that: The kit comprises the nucleic acid product according to any one of claims 1 to 2.

4. The kit for detecting the species of Coriolus versicolor according to claim 3, characterized in that: The kit also includes PCR reaction reagents; Optionally, the PCR reaction reagents include one or more of PCR buffer, DNA polymerase, magnesium ions and dNTPs.

5. The kit for detecting the variety of Coriolus versicolor according to any one of claims 3 to 4, characterized in that: The kit also includes a DNA extraction reagent.

6. A method for detecting the variety of Coriolus versicolor, characterized in that: The steps include: Using the DNA of the sample to be tested as a template, PCR amplification is performed using the nucleic acid product according to any one of claims 1 to 2 or the kit according to any one of claims 3 to 5, and the variety of the sample to be tested is identified based on the obtained amplification results.

7. The method for detecting the variety of Coriolus versicolor according to claim 6, characterized in that: In the PCR amplification reaction procedure, the annealing temperature is 58°C~69°C.

8. The method for detecting the variety of Coriolus versicolor according to claim 6, characterized in that: In the PCR amplification reaction program, the number of cycles is 30 to 40 times.

9. The method for detecting the variety of Coriolus versicolor according to claim 6, characterized in that: In the reaction system of PCR amplification, the concentrations of the forward primer and the reaction primer are independently 0.4 μmol / L to 1.0 μmol / L.

10. The method for detecting the variety of Coriolus versicolor according to any one of claims 6 to 9, characterized in that: The method further comprises the following steps: performing agarose gel electrophoresis detection on the product amplified by PCR, and identifying the variety of the sample to be tested according to the obtained detection result.

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