Primer pair, kit and method for identifying bistort rhizome

Abstract

The invention relates to a primer pair, a kit and a method for identifying bistort rhizome, and relates to the field of identification of traditional Chinese medicinal materials. The primer pair (TF92 / TR237) provided by the invention can be used for simply, quickly and accurately identifying genuine products and adulterants of bistort rhizome at low cost, can be used for distinguishing the adulterants of bistort rhizome (such as other genera except bistort rhizome in polygonaceae or other species except bistort rhizome species in polygonaceae bistort rhizome), and has unexpected technical effects.

Description

Technical Field

[0001] The present invention relates to the field of identification of traditional Chinese medicines, and in particular to a primer pair, a kit and a method for identifying Polygonum multiflorum. Background Art

[0002] Polygonum bistorta L. is a plant of the Polygonaceae family, native to East China, North China, and parts of Central China. The dried rhizome of Polygonum bistorta is listed in the 2020 edition of the Chinese Pharmacopoeia, with functions and indications of clearing heat and detoxifying, reducing swelling, and stopping bleeding. It is used to treat dysentery, hot diarrhea, lung heat cough, carbuncles, scrofula, oral ulcers, vomiting and epistaxis due to blood heat, hemorrhoidal bleeding, and snake and insect bites. The "Zhongyao Zhi" records that Polygonum bistorta has heat-clearing, detoxifying, and astringent properties. It is used to treat enteritis, dysentery, hepatitis, and other conditions. Polygonum bistorta is one of the most astringent traditional Chinese medicines. Therefore, research on the quality control of Polygonum bistorta helps ensure the healthy development of the entire Chinese medicinal materials industry.

[0003] Since the plants of the Polygonaceae family, especially those of the genus Polygonum, such as Polygonum truncatum, Polygonum gracile, Polygonum villosa, Polygonum thunbergii, Polygonum serrata, and Polygonum winged petiole, are particularly similar in properties, it is difficult to identify them by properties. If they are sliced ​​or crushed into powder, it is even more difficult to identify them by properties. In addition, these plants similar to Polygonum truncatum are often confused and sold with Polygonum truncatum in the market, which is extremely detrimental to the healthy development of the Chinese medicinal materials industry.

[0004] Currently, only a few literature reports on the identification of ginseng and its easily confused products. These identification methods, some requiring extensive experience, others relying on large, expensive instrumentation, and limited to identifying only a few species, are unable to distinguish ginseng from other similar species. Therefore, finding simple, rapid, and accurate methods to distinguish genuine ginseng from its confused products, while ensuring it is not confused with other similar species, has become a major challenge in traditional Chinese medicine testing. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions.

[0006] In the first aspect, the present invention provides a primer pair for distinguishing authentic and adulterated Polygonum multiflorum products.

[0007] A primer pair for distinguishing authentic and adulterated Polygonum multiflorum, comprising TF92 and TR237, the sequences of which are:

[0008] TF92: CACAGCGTCGCCCCCA;

[0009] TR237: AGGCGGCACGAGGCTGCCAC.

[0010] In a second aspect, the present invention provides a kit.

[0011] A kit comprising the primer pair for distinguishing authentic Polygonum multiflorum from adulterated products as described in the first aspect.

[0012] In some embodiments, the kit further comprises a PCR polymerase and / or an agarose gel electrophoresis reagent.

[0013] In some embodiments, the agarose gel electrophoresis reagent comprises agarose gel and a nucleic acid gel stain.

[0014] In some embodiments, the nucleic acid gel stain is GelRed TM Nucleic Acid Gel.

[0015] In some embodiments, the PCR polymerase comprises At least one of Max, Taq PCR MasterMix, and PfuPCR Mix.

[0016] In some embodiments, the Max Max.

[0017] In some embodiments, the Taq PCR MasterMix is ​​2×Taq PCR MasterMix.

[0018] In some embodiments, the Pfu PCR Mix is ​​2×Pfu PCR Mix.

[0019] In a third aspect, the present invention provides an application.

[0020] A use of the primer pair described in the first aspect or the kit described in the second aspect in distinguishing authentic Polygonum multiflorum from adulterated products.

[0021] In a fourth aspect, the present invention provides a method for distinguishing authentic Polygonum multiflorum from its adulterated counterparts.

[0022] A method for identifying authentic and adulterated Polygonum multiflorum products using the primer pair for identifying authentic and adulterated Polygonum multiflorum products of the first aspect or the kit of the second aspect.

[0023] In some embodiments, the adulterant comprises genera other than the genus Bistorta in the Polygonaceae family or species other than the species Bistorta in the genus Bistorta in the Polygonaceae family.

[0024] In some embodiments, the adulterant includes any one of Polygonaceae's Bistorta genus, Polygonaceae's Fagopyrum genus, Polygonaceae's Rheum genus, Polygonum genus, Polygonum cuspidatum genus, Polygonaceae's Polygonum genus, and Polygonum multiflorum genus.

[0025] In some embodiments, the confusing products include any one of Polygonum cyrtonema, Polygonum villosa, Sanguisorba officinalis, Polygonum wingtip, Polygonum claspermi, Fagopyrum scabra, Rhubarb, Polygonum hydropiper, Croton tiglium, Polygonum cuspidatum, Polygonum multiflorum, Polygonum multiflorum, and Polygonum multiflorum.

[0026] Beneficial effects

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The primer pair (TF92 / TR237) of the present invention can be used to identify authentic and adulterated products of Polygonum multiflorum at low cost, simply, quickly and accurately, and can distinguish authentic and adulterated products of Polygonum multiflorum from those of Polygonum multiflorum (such as other genera in the Polygonaceae family except the Polygonum genus or other species in the Polygonaceae family except the Polygonum species, etc.), which has unexpected technical effects.

[0029] Terminology

[0030] In the description of the present invention, “plurality” means two or more, unless otherwise clearly defined.

[0031] The term "room temperature" means ambient temperature, which refers to a temperature between about 10°C and about 30°C, or between about 20°C and 30°C, or about 25°C.

[0032] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0033] In the following description, all numerical values ​​disclosed herein are approximate, regardless of whether the word "about" or "approximately" is used. The numerical value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a number having the value of N is disclosed, any number having the value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% is expressly disclosed, where "+ / -" means plus or minus. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1This is the PCR electrophoresis result of the primer pair TF194 / TR456 in Comparative Example 1.

[0035] Figure 2 This is the PCR electrophoresis result of the primer pair TF199 / TR452 in Comparative Example 1.

[0036] Figure 3 This is the PCR electrophoresis result of the primer pair TF227 / TR423 in Comparative Example 1.

[0037] Figure 4 This is the PCR electrophoresis result of the primer pair TF202 / TR401 in Comparative Example 1.

[0038] Figure 5 This is the PCR electrophoresis result of the primer pair TF22 / TR313 in Comparative Example 1.

[0039] Figure 6 This is a graph showing the PCR electrophoresis results of the PCR reaction program conditions in Example 1.

[0040] Figure 7 This is a diagram of the PCR electrophoresis results of different PCR polymerases investigated in Example 2.

[0041] Figure 8 This is a diagram of the PCR electrophoresis results for the repeatability study in Example 3.

[0042] Figure 9 This is a diagram of the PCR electrophoresis results of the specificity investigation in Example 4.

[0043] Figure 10 This is a diagram of the PCR electrophoresis results for the specificity investigation in Example 5.

[0044] Figure 11 This is a diagram of the PCR electrophoresis results for the specificity investigation in Example 5.

[0045] in, Figures 1-6 and Figure 10 In the figure, the sample in lane 1 is QSD, the sample in lane 2 is S01, the sample in lane 3 is S02, the sample in lane 4 is YSLD, the sample in lane 5 is S13, the sample in lane 6 is S14, the sample in lane 7 is ZYLD, the sample in lane 8 is S15, the sample in lane 9 is S16, the sample in lane 10 is CXJD, the sample in lane 11 is S17, the sample in lane 12 is S18, the sample in lane 13 is S19, the sample in lane 14 is S20, the sample in lane 15 is S21, the sample in lane 16 is S22, and the sample in lane N is the negative control;

[0046] Figure 7In the figure, the sample in lane 1 is S01, the sample in lane 2 is S02, the sample in lane P is QSD, and the sample in lane N is the negative control; "2×Pfu PCR Mix" indicates the electrophoresis result using 2×Pfu PCR Mix as the PCR polymerase, Max" means the use of Max is the electrophoresis result of PCR polymerase, and “2×Taq PCRMasterMix” indicates the electrophoresis result when 2×Taq PCR MasterMix is ​​used as the PCR polymerase;

[0047] Figure 8 In the figure, the sample in lane P is QSD, the samples in lanes 1 to 6 are S01, and the sample in lane N is the negative control;

[0048] Figure 9 In the figure, the sample in lane 1 is QSD, the samples in lanes 2 to 13 correspond to S01 to S12, respectively, and lane N is the negative control;

[0049] Figure 11 In the figure, the sample in lane 1 is QSD, the samples in lanes 2 to 13 correspond to S23 to S34, respectively, and lane N is the negative control;

[0050] Figure 1-Figure 5 In the figure, the sample in lane M is DL 500DNAMarker;

[0051] Figures 6-11 In the figure, the sample in lane M is DL 2000DNA Marker;

[0052] The sample information corresponding to each sample number in the above figures is shown in Table 1. DETAILED DESCRIPTION

[0053] In order to enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0054] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0055] 1. Samples and sample information:

[0056] Table 1: Samples and sample information used in the present invention

[0057]

[0058]

[0059] 2. Experimental instruments and reagents

[0060] Experimental instruments: ProFlex 3*32 well PCR instrument (Applied Biosystems (ABI) of the United States); ESP100 electrophoresis instrument (Shanghai Tianneng Technology Co., Ltd.); OSE-260 ultra-micro-volume UV spectrophotometer (Beijing Tiangen Biochemical Technology Co., Ltd.); Tanon 3500 gel imaging system (Shanghai Tianneng Technology Co., Ltd.); Vortex Genie 3 vortex mixer (IKA, Germany); XS-205DU electronic balance (Mettler Toledo); HWS-12 electric constant temperature water bath (Shanghai Yiheng Scientific Instrument Co., Ltd.); 5425 centrifuge (Eppendorf China Co., Ltd.); D1008E handheld centrifuge (Dalong Xingchuang Experimental Instrument (Beijing) Co., Ltd.); M1-L213B microwave oven (Midea Group).

[0061] Main reagents: Plant Genomic DNA Kit was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; DL 2,000 DNA Marker, DL 1,000 DNA Marker, and DL500 DNA Marker were all purchased from Takara Bio (Beijing) Co., Ltd. Max DNA Polymerase was purchased from Takara Bio (Beijing) Co., Ltd.; 2×Taq PCR MasterMix was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; 2×Pfu PCR Mix was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; GelRed TM Nucleic acid gel was purchased from Stain Biotium; agarose was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; 10×Lording Buffer was purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.; and 50×TAE electrophoresis buffer was purchased from Beijing Tianenze Gene Technology Co., Ltd.

[0062] Comparative Example 1: Primer design and screening

[0063] 1. Preparation of template DNA solution

[0064] Approximately 30 mg of the powder of samples QSD, ZYLD, YSLD, CXJD, and S01-S34 in Table 1 were respectively transferred to 2 mL centrifuge tubes. DNA was extracted using the Plant Genome Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to obtain the template DNA solution of each sample. The concentration of the DNA extract was detected using an ultra-micro UV spectrophotometer, and then the solution was aliquoted and stored at -20°C for future use.

[0065] 2. PCR amplification reaction using ITS universal primers

[0066] The DNA template solution of each sample was taken and PCR amplified using plant ITS universal primers for ITS2F / ITS3R. The PCR products were sequenced by a sequencing company (Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd.).

[0067] Universal primer pair: ITS2F (SEQ ID NO: 5): 5'-ATGCGATACTTGGTGTGAAT-3'

[0068] ITS3R (SEQ ID NO:6): 5'-GACGCTTCTCCAGACTACAAT-3'

[0069] PCR amplification system: Max 12.5 μL, 1 μL each of 10 μmol / L ITS2F primer and 10 μmol / L ITS3R primer, 1 μL of template DNA solution (85 ng / μL), and add sterile double-distilled water to 25 μL. For a negative control, replace the DNA template with an equal volume of sterile double-distilled water.

[0070] PCR reaction parameters: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 45 s, 40 cycles; final extension at 72°C for 10 min.

[0071] Gel electrophoresis analysis: Prepare agarose gel with a concentration of 1.5% and add nucleic acid gel dye GelRed to the gel TM Nucleic Acid Gel. Because Max enzyme does not contain Loading Buffer, so it is necessary to add Loading Buffer to the product. Take 9 μL of the PCR product from the test sample, control herb, and negative control, and mix each with 1 μL of 10× Loading Buffer. Finally, take 5 μL of the mixed product and load it on the sample. The loading volume of the DNA molecular weight marker is 5 μL.

[0072] 3. Design of specific primer pairs

[0073] Conventional empirical method for designing specific primer pairs: After PCR amplification of the sample, the product is sequenced and the sequencing sequence is used to design specific primer pairs.

[0074] All the sample genomic sequences obtained by the above sequencing were double-aligned with the sequences uploaded to the NCBI system GenBank database (DNA sequence database of the National Center for Biotechnology Information of the United States) and GPGenome (Global Pharmacopoeia Genome Database). Gene sequences homologous to the sample ITS site DNA sequence and gene sequences of the ITS sites of other homologous species were downloaded. All the measured gene sequences were compared using DNAMAN software to find fragments with variant sites compared to other similar species of Chinese chestnut. It was found that the gene sequences of the ITS sites of Chinese chestnut and other similar species were relatively similar, contained relatively few variant sites, and the number of bases contained in the variant sites was also relatively small. The fragments with variant sites compared to other samples of Chinese chestnut were imported into the primer design software Primer Premier 5.0, and 5 pairs of Chinese chestnut-specific primer pairs were designed (as shown in Table 2 below).

[0075] Table 2: Five pairs of specific primers designed by conventional methods

[0076]

[0077] 4. PCR amplification reaction of ginseng-specific primers

[0078] 4.1 Preparation of template DNA solution

[0079] Template DNA solutions of samples QSD, ZYLD, YSLD, CXJD, S01-S02, and S13-S22 (preparation method see Comparative Example 1) were used for PCR amplification and primer pair screening.

[0080] 4.2 PCR amplification system

[0081] PCR amplification system: 2×Pfu PCR Mix 12.5μL, specific primer pair (1μL of each primer in the specific primer pair was added, and the concentration of each primer was 10μmol / L, that is, 1.0×10 -5 μmol), 1 μL of template DNA solution (concentration 85 ng / μL), and add sterile double-distilled water to 25 μL. For the negative control, replace the DNA template with an equal volume of sterile double-distilled water.

[0082] 4.2 PCR reaction conditions

[0083] Each primer pair in Table 2 was used to prepare a PCR amplification system according to the method under "4.2 PCR amplification system" in Comparative Example 1, and then a PCR reaction was performed. The PCR amplification program reaction parameters were:

[0084] Pre-denaturation at 95°C for 3 min;

[0085] Denaturation at 95°C for 30 s, annealing at 44°C for 1 min, and extension at 72°C for 30 s, for 30 cycles;

[0086] Final extension was performed at 72°C for 5 min.

[0087] 4.3 Gel electrophoresis analysis

[0088] Prepare agarose gel at a concentration of 2% and add nucleic acid gel dye GelRed to the gel TM Nucleic AcidGel. The loading volume of the test sample, control medicinal material and negative control was 10 μL respectively, and the loading volume of DNA molecular weight marker was 10 μL. The results are shown in Figure 1-Figure 5 .

[0089] 5. Conclusion

[0090] Depend on Figure 1-Figure 5 It can be seen that none of the five primer pairs were specific for Polygonum multiflorum compared to other similar species. This indicates that the conventional empirical method of sequencing the PCR amplification products and designing specific primer pairs based on the sequencing sequence is basically unable to design primer pairs that are specific for Polygonum multiflorum.

[0091] Example 1: Investigation of PCR amplification reaction program conditions

[0092] 1. Template DNA solution

[0093] The template DNA solutions of samples QSD, ZYLD, YSLD, CXJD, S01-S02 and S13-S22 (preparation method see Comparative Example 1) were taken and the PCR reaction program conditions were explored according to the conditions shown in Table 4.

[0094] 2. Primer pair design

[0095] The inventors independently designed two pairs of primers shown in Table 3, performed PCR amplification with the primer pairs shown in Table 3, and performed gel electrophoresis analysis.

[0096] Table 3: Two pairs of primers designed independently

[0097]

[0098] 3. PCR amplification system:

[0099] TF92 / TR237 primer pair PCR amplification system: 2×Pfu PCR Mix 12.5μL, 10μmol / L TF92 primer and 10μmol / L TR237 primer were added 1μL each (i.e., 1.0×10 -51 μL of template DNA solution (85 ng / μL) was added with sterile double-distilled water to a volume of 25 μL. For the negative control, replace the DNA template with an equal volume of sterile double-distilled water.

[0100] PCR amplification system for investigation of the TF111 / TR237 primer pair: Follow the above "TF92 / TR237 primer pair investigation" procedures, replacing "add 1 μL each of 10 μmol / L TF92 primer and 10 μmol / L TR237 primer" with "add 1 μL each of 10 μmol / L TF111 primer and 10 μmol / L TR237 primer". Other procedures are the same as those for the above "TF92 / TR237 primer pair investigation".

[0101] 4. PCR reaction program conditions

[0102] The PCR reaction was performed according to the PCR reaction program conditions described in Table 4 using the "TF92 / TR237 primer pair for investigating the PCR amplification system" or the "TF111 / TR237 primer pair for investigating the PCR amplification system" in Example 2.

[0103] Table 4: PCR reaction program conditions

[0104]

[0105] 5. Gel electrophoresis analysis

[0106] Prepare agarose gel at a concentration of 2% and add nucleic acid gel dye GelRed to the gel TM Nucleic Acid Gel. The loading volume of the test sample, control medicinal material and negative control was 10 μL respectively, and the loading volume of DNA molecular weight marker was 10 μL. Figure 6 .

[0107] 6. Conclusion

[0108] The results are as follows Figure 6 As shown in the figure, at positions around 145bp and 818bp for primer pair TF92 / TR237 (number I), two clear, bright target DNA bands were simultaneously amplified in the control and authentic Polygonum multiflorum samples. However, the negative control and the adulterated Polygonum multiflorum samples did not simultaneously produce two target bands at positions around 145bp and 818bp.

[0109] At the positions around 126bp and 837bp of primer pair TF111 / TR237 No.Ⅱ, the control herb, authentic herb, negative control and herb mixed herb could not simultaneously amplify two clear and bright target DNA bands.

[0110] These results demonstrate that the primer pair TF92 / TR237 designed in the present invention exhibits excellent and significant specificity during amplification. The primer pair (TF92 / TR237) designed in the present invention can be used to discriminate authentic and adulterated Polygonum multiflorum products in a cost-effective, simple, rapid, and accurate manner. It can also distinguish Polygonum multiflorum from adulterated Polygonum products (e.g., other genera within the Polygonaceae family or other species within the Polygonaceae genus), demonstrating unexpected technical effectiveness. Therefore, the primer pair TF92 / TR237 was subsequently investigated.

[0111] Example 2: Investigation of PCR polymerase

[0112] 1. Template DNA solution

[0113] Template DNA solutions of S01, S02 and QSD (positive control medicinal material) (preparation method see Comparative Example 1) were respectively taken for PCR amplification and gel electrophoresis analysis.

[0114] 2. PCR amplification system

[0115] PCR polymerase (investigated separately Max, 2× Taq PCR MasterMix, 2× Pfu PCR Mix) 12.5 μL, 10 μmol / L TF92 primer and 10 μmol / L TR237 primer were added 1.0 μL each (i.e., 1.0×10 -5 μmol), 1 μL of template DNA solution (concentration 85 ng / μL), and add sterile double-distilled water to 25 μL. For the negative control, replace the DNA template with an equal volume of sterile double-distilled water.

[0116] 3. PCR reaction program conditions

[0117] Same as Example 1.

[0118] 4. Gel electrophoresis analysis

[0119] Same as Example 1, the results are shown in Figure 7 .

[0120] 5. Conclusion

[0121] Depend on Figure 7 It can be seen that Max, 2×Taq PCR MasterMix, and 2×Pfu PCR Mix can all be used in the method provided by the present invention to distinguish authentic and adulterated Polygonum multiflorum. The preferred PCR amplification system is 2×Pfu PCR Mix, which is beneficial for making the target band clearer and brighter. Therefore, the preferred PCR amplification system is:

[0122] 2×Pfu PCR Mix 12.5μL, 10μmol / L TF92 primer and 10μmol / L TR237 primer were added 1.0μL each (i.e., 1.0×10 -5 μmol), 1 μL of template DNA solution (concentration 85 ng / μL), and sterile double-distilled water to 25 μL. For the negative control, replace the DNA template with an equal volume of sterile double-distilled water.

[0123] Example 3: Repeatability Study

[0124] 1. Template DNA solution

[0125] Six portions of the template DNA solution of S01 (preparation method see Comparative Example 1) and one portion of the template DNA solution of QSD (positive control medicinal material) (preparation method see Comparative Example 1) were taken for PCR amplification and gel electrophoresis analysis.

[0126] 2. PCR amplification system

[0127] 2×Pfu PCR Mix 12.5μL, 10μmol / L TF92 primer and 10μmol / L TR237 primer were added 1.0μL each (i.e., 1.0×10 -5 μmol), 1 μL of template DNA solution (concentration 85 ng / μL), and add sterile double-distilled water to 25 μL. For the negative control, replace the DNA template with an equal volume of sterile double-distilled water.

[0128] 3. PCR reaction program conditions

[0129] Same as Example 1.

[0130] 4. Gel electrophoresis analysis

[0131] Same as Example 1, the results are shown in Figure 8 .

[0132] 5. Conclusion

[0133] Depend on Figure 8 It can be seen that the primer pairs and identification method provided by the present invention have good repeatability, indicating that the identification method provided by the present invention is stable and reliable.

[0134] Example 4: Specificity

[0135] 1. Template DNA solution

[0136] About 30 mg of the powder of samples QSD and S01-S12 in Table 1 were respectively transferred to a 2 mL centrifuge tube. DNA was extracted using the Plant Genome Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to obtain the template DNA solution of each sample. The concentration of the DNA extract was detected using an ultra-micro UV spectrophotometer, and then the solution was aliquoted and stored at -20°C for future use.

[0137] 2. PCR amplification system

[0138] Same as Example 3.

[0139] 3. PCR reaction program conditions

[0140] Same as Example 1.

[0141] 4. Gel electrophoresis analysis

[0142] Same as Example 1, the results are shown in Figure 9 .

[0143] 5. Conclusion

[0144] Depend on Figure 9 The results show that in the electrophoresis diagrams obtained by using the primer pair and identification method provided by the present invention, the two DNA target bands at positions of approximately 145 bp and 818 bp can be clearly, brightly and without any other bands simultaneously amplified for the control medicinal material and the sample of Polygonum multiflorum, which has excellent specificity.

[0145] Example 5: Specificity

[0146] 1. Template DNA solution

[0147] Approximately 30 mg of the powder of samples QSD, ZYLD, YSLD, CXJD, and S01-S34 in Table 1 were respectively transferred to 2 mL centrifuge tubes. DNA was extracted using the Plant Genome Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to obtain the template DNA solution of each sample. The concentration of the DNA extract was detected using an ultra-micro UV spectrophotometer, and then the solution was aliquoted and stored at -20°C for future use.

[0148] 2. PCR amplification system

[0149] Same as Example 1.

[0150] 3. PCR reaction program conditions

[0151] Same as Example 1.

[0152] 4. Gel electrophoresis analysis

[0153] Same as Example 1, the results are shown in Figure 10-11 .

[0154] 5. Conclusion

[0155] Depend on Figure 10-11 The results show that in the electrophoresis diagrams obtained by using the primer pair and identification method provided by the present invention, the two DNA target bands at the positions of about 145bp and 818bp can be clearly, brightly and without any other bands amplified simultaneously for the control medicinal material and the sample of the Polygonum multiflorum. However, the two DNA target bands at the same positions cannot be amplified simultaneously for the control medicinal material and the mixed product of the Polygonum multiflorum. This indicates that the primer pair and identification method provided by the present invention can specifically identify the control medicinal material and the sample of the Polygonum multiflorum, and have excellent specificity.

[0156] The methods of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art will be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and spirit of the present invention to implement and apply the technology of the present invention. Those skilled in the art may refer to the disclosure herein and appropriately modify the process parameters to achieve the desired effect. It is particularly important to note that all similar substitutions and modifications apparent to those skilled in the art are considered encompassed by the present invention.

Claims

1. A primer pair for distinguishing authentic and adulterated Polygonum multiflorum, characterized in that: The primer pair is TF92 and TR237, and its sequence is: TF92: CACAGCGTCGCCCCCA; TR237: AGGCGGCACGAGGCTGCCAC.

2. A kit, characterized in that The invention comprises the primer pair for distinguishing authentic and adulterated Polygonum multiflorum products as described in claim 1.

3. The test kit according to claim 2, further comprising PCR polymerase and / or agarose gel electrophoresis reagent.

4. The kit according to claim 3, wherein the agarose gel electrophoresis reagent comprises agarose gel and a nucleic acid gel dye; and / or the nucleic acid gel dye is GelRed TM Nucleic Acid Gel.

5. The kit according to claim 3, wherein the PCR polymerase comprises At least one of Max, Taq PCRMasterMix, and Pfu PCR Mix; Optionally, the Max is 2× Max; Optionally, the Taq PCR MasterMix is ​​2×Taq PCR MasterMix; Optionally, the Pfu PCR Mix is ​​2×Pfu PCR Mix.

6. Use of the primer pair according to claim 1 or the kit according to any one of claims 2 to 5 in distinguishing authentic and adulterated Polygonum multiflorum products.

7. A method for distinguishing authentic and adulterated Polygonum multiflorum products using the primer pair for distinguishing authentic and adulterated Polygonum multiflorum products according to claim 1 or the kit according to any one of claims 2 to 5.

8. The method according to claim 7, wherein the adulterant comprises genera other than the genus Bistorta in the Polygonaceae family or species other than the species Bistorta in the genus Bistorta in the Polygonaceae family.

9. The method according to claim 7, wherein the adulterant comprises any one of Polygonaceae Bistorta spp., Polygonaceae Fagopyrum spp., Polygonum spp., Polygonum spp., Polygonum spp., Polygonum spp., and Polygonum multiflorum spp.

10. The method according to claim 7, wherein the mixed product comprises any one of Polygonum cyrtonema, Polygonum villosa, Sanguisorba officinalis, Polygonum wing-petal, Polygonum claspermi, Fagopyrum scabra, Rhubarb, Polygonum hydropiper, Carthamus tinctorius, Polygonum cuspidatum, Polygonum multiflorum, Polygonum multiflorum, and Polygonum multiflorum.

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