PCR detection method, kit and use thereof

Abstract

The application provides a PCR detection method, a kit and a use, relates to the technical field of nucleic acid molecule detection, and comprises the following steps: step one, a connecting probe combination and a primer combination are designed for a target sequence or a complementary sequence of a known mutation site; the known mutation site is set as at least one SNP site; step two, nucleic acids in a sample to be detected are extracted, and the connecting probe combination, the primer combination, a DNA polymerase and a DNA ligase are used for reaction to obtain a PCR product; and step three, the PCR product is detected. The application establishes a molecular detection method which can be used for highly degraded short fragment nucleic acids (including DNA and RNA) and can be used for quality control of products related to a traditional Chinese medicine whole industry chain.

Description

Technical Field

[0001] This invention relates to the technical field of nucleic acid molecular detection, and to a PCR detection method, kit, and application, particularly to a PCR detection method, kit, and application for products across the entire traditional Chinese medicine industry chain. Background Technology

[0002] The quality of traditional Chinese medicine (TCM) is a crucial prerequisite for the development of the TCM industry. Traditional identification methods (morphological identification, microscopic identification, physicochemical identification, etc.) all have certain limitations and often fail to accurately identify the authenticity of medicinal materials. Molecular identification of TCM is one of the most promising technologies for accurately identifying the origin of medicinal materials. Molecular identification involves detecting genetic material DNA, which can accurately identify the origin of medicinal materials. With the development of molecular biology technology, many molecular detection techniques are used in the identification and research of TCM materials. For example, DNA barcoding technology has been included in the Chinese Pharmacopoeia and has become a legally recognized method for the identification of TCM materials. The polymerase chain reaction-restriction endonuclease length polymorphism (PCR-RFLP) identification method for medicinal materials such as Fritillaria cirrhosa, Zaocys dhumnades, Dendrobium nobile (Dendrobium huoshanense), Bungarus fasciatus, and Agkistrodon halys has also been included in the Chinese Pharmacopoeia. In addition, multiplexed probe amplification-melting curve method (MLPA-HRM), random amplified peptide DNA labeling (RAPD), simple sequence repeat (ISSR) DNA sequence, high-resolution melting curve (HRM), loop-mediated isothermal amplification (LAMP) site-specific PCR, and other methods have been applied to the identification of medicinal materials. However, they are currently difficult to apply to the quality control of traditional Chinese medicine preparations. Most traditional Chinese medicine preparations undergo long-term processing such as decoction and drying, resulting in severe DNA degradation, making it difficult to apply existing molecular identification methods. Summary of the Invention

[0003] This invention is a molecular detection method for highly degraded nucleic acids developed to solve the quality control problems of traditional Chinese medicine. It can be used for quality control and detection of traditional Chinese medicine and related products containing bio-based ingredients.

[0004] This invention provides a PCR detection method, comprising the following steps:

[0005] Step 1: Design ligation probe and primer combinations targeting the known mutation site or its complementary sequence;

[0006] The known mutation site is set to at least one SNP site;

[0007] When there is only one SNP site, the ligation probe combination consists of a left probe LLP and a right probe RLP, and the primer combination consists of a forward primer FP and a reverse primer RP.

[0008] When there are two or more SNP sites, the ligation probe combination consists of a left probe LLP, a middle probe MLP and a right probe RLP, and the primer combination consists of a forward primer FP and two or more reverse primers RP.

[0009] The left probe LLP has two parts, the left extension sequence X and the hybridization sequence M1, from the 5' end to the 3' end. The hybridization sequence M1 of the left probe is the same as the upstream sequence adjacent to the SNP1 site.

[0010] The 5' to 3' ends of the intermediate probe MLP1 are the hybridization sequence N1, the linking sequence Z, and the hybridization sequence M2, respectively. The hybridization sequence N1 is the same as the downstream sequence adjacent to the SNP1 site, and the hybridization sequence M2 is the same as the upstream sequence adjacent to the SNP2 site. The SNP1 site is located at the 3' end of the left probe hybridization sequence M1 or the 5' end of the intermediate probe hybridization sequence N1. The design of other intermediate probes follows the same pattern. The hybridization sequence N is the same as the downstream sequence adjacent to the previous SNP site, and the hybridization sequence M is the same as the upstream sequence adjacent to the next SNP site.

[0011] The right probe RLP has two parts, the hybridization sequence Nn and the right extension sequence Y, from the 5' end to the 3' end. The hybridization sequence Nn of the right probe is the same as the downstream sequence adjacent to the SNPn site. The SNPn site is located at the 3' end of the hybridization sequence Mn of the intermediate probe or the 5' end of the hybridization sequence Nn of the right probe.

[0012] All hybridization sequences are 5-18 nucleotides in length;

[0013] The 5' ends of all intermediate probes MLP and right probe RLP were phosphorylated.

[0014] The forward primer FP has two parts, the extended sequence E and the sequence XM, from its 5' end to its 3' end, or it may only have the sequence XM; the sequence XM is partially or completely identical to the left probe LLP.

[0015] The reverse primer RPn has two parts, the extension sequence Fc and the complementary sequence YcNc, from the 5' end to the 3' end, or only the complementary sequence YcNc. The complementary sequence YcNc is complementary to part or all of the sequence of the right probe RLP.

[0016] Simultaneously, for each intermediate probe, a reverse primer is synthesized, namely RP1, RP2 to RPn-1, whose sequence is complementary to part or all of the sequence of the intermediate probe;

[0017] Step 2: Extract nucleic acid from the sample to be tested, and react it with the ligation probe combination, primer combination, DNA polymerase, and DNA ligase to obtain PCR products;

[0018] Step 3: Detect the PCR products.

[0019] Furthermore, the specific process for obtaining the PCR product in step two is as follows:

[0020] S2.11 Extract nucleic acid from the sample to be tested, add ligation probe combination and DNA ligase, and prepare ligation reaction system;

[0021] S2.12. Heating at 90-98℃ for at least 5 seconds to denature, followed by a linkage reaction for several minutes; repeating the heating denaturation and linkage reaction one to several times, or not repeating, to obtain the linkage product;

[0022] S2.13. Take the ligation product, add the primer combination and DNA polymerase, prepare the reaction system, perform PCR reaction, and obtain the PCR product.

[0023] Furthermore, the specific process for obtaining the PCR product in step two can also be as follows:

[0024] S2.21 Extract nucleic acid from the sample to be tested, add the ligation probe assembly, heat at 90-98℃ for no less than 5 seconds, and then cool.

[0025] S2.22. Add primer combination, DNA ligase, and DNA polymerase to prepare the reaction system. After the ligation reaction takes several minutes, heat at no less than 90°C for at least 5 seconds, and then continue the PCR reaction to obtain the PCR product.

[0026] Furthermore, for SNPs with two or more mutation sites, the parameters for step two during the PCR reaction are set as follows:

[0027] S2.13. Take the ligation product, add the forward primer, reverse primer RP1 and DNA polymerase to prepare PCR reaction system 1; take the ligation product, add the intermediate probe MLP1, reverse primer RP2 and DNA polymerase to prepare PCR reaction system 2; and so on up to the intermediate probe MLPn-1 and reverse primer RPn and DNA polymerase to prepare PCR reaction system n; after each reaction system is PCR reacted separately, the PCR reaction solutions are combined and the PCR reaction is continued to obtain the PCR product.

[0028] Furthermore, the melting temperature T of the left probe LLP and the right probe RLP mentioned in step one... m Not lower than 65℃;

[0029] The melting temperature T of the portion of sequence XM in the forward primer FP that is identical to the portion of sequence LLP in the left probe. m Not lower than 65℃;

[0030] The melting temperature T of the complementary sequence YcNc in the reverse primer RP and the complementary portion of the right probe RLP sequence.m Not lower than 65℃;

[0031] Furthermore,

[0032] In step two, when performing the PCR reaction, the reaction is first cycled at a lower annealing temperature at least once, and then cycled at an annealing temperature at a higher temperature of 3°C or more at least once.

[0033] Furthermore, the mutation site is two or more SNP sites; in step one, for each SNP site, a corresponding linker template LMB is designed and synthesized, the sequence of which is complementary to the target sequence containing the SNP site.

[0034] Step two involves adding 1 to n-1 linker templates (LMBs) to the linker reaction system.

[0035] Furthermore, in step three, the PCR products are detected by electrophoresis, which is set to any one of agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis.

[0036] Furthermore, in step two, a fluorescent dye is added to the PCR reaction system, wherein the fluorescent dye is any one of SYBR Green I, Eva Green, LC Green or Solis Green; in step three, the PCR product is detected using the fluorescence signal as an indicator.

[0037] Furthermore, in step one, either the forward primer FP or the reverse primer RP, a fluorescent group and a quenching group are also connected during the design and synthesis. In step three, the PCR product is detected using the fluorescence signal as an indicator.

[0038] Furthermore, the forward primer FP described in step one is designed and synthesized with a fluorescent group attached to its 5' end; at the same time, a quenching primer QP is synthesized, with a quenching group attached to its 3' end, and its sequence is complementary to the 5' end of the forward primer FP. In step two, the quenching primer QP is added to the PCR reaction system. In step three, the PCR product is detected using fluorescence signal as an indicator.

[0039] Furthermore, step one also includes designing and synthesizing a TaqMan fluorescent probe, the sequence of which is partially identical to the sequence of the left probe LLP and is located downstream of the forward primer. The TaqMan fluorescent probe is connected to a fluorescent group and a quenching group. Step two involves adding the TaqMan fluorescent probe to the PCR reaction system. Step three involves detecting the PCR product using the fluorescence signal as an indicator.

[0040] Furthermore, the primer combination described in step one also includes the tag primer TagEXc and the detection template MB;

[0041] The 5' to 3' ends of the tag primer TagEXc are the tag sequence Tag and the sequence EXc, respectively. The sequence EXc is partially or completely complementary to the extended sequence EX of the forward primer FP.

[0042] The 5' to 3' ends of the detection template MB are respectively the probe sequence T and the extension sequence G. The probe sequence T is connected to a fluorescent group and a quenching group, and the extension sequence G contains a sequence complementary to the tag sequence Tag.

[0043] Step two involves adding the tag primer TagEXc and the detection template MB to the PCR reaction system. Step three involves detecting the PCR products using fluorescence signals as an indicator.

[0044] The present invention also provides a kit comprising the ligation probe combination and primer combination used in the PCR detection method described above.

[0045] The present invention also provides a PCR detection method as described above for the purpose of detecting mutation sites.

[0046] Furthermore, it includes the following steps:

[0047] Step 1: Select specific SNP sites and perform fluorescence detection using the PCR detection method described in any one of claims 9-13 to obtain the copy number A;

[0048] Step 2: Select a conserved sequence as an internal reference sequence, take any base site within the internal reference sequence as a SNP, and perform fluorescence detection using the PCR detection method described in any one of claims 9-13 to obtain the copy number B of the internal reference sequence;

[0049] Step 3: Calculate the ratio R of A / B.

[0050] Furthermore, the specific SNP site includes any one of the following:

[0051] (1) The SNP site is the 15th base C of the sequence 5'-ACCCATCATGTGTACGCTCCCCATAATATG-3', which is used for the detection of products containing Astragalus membranaceus and / or Astragalus membranaceus DNA.

[0052] (2) The SNP site is the 15th base G in the sequence 5'-GAACCCTCAACTCCGTCCGCCTTTGTTGGG-3', which is used for the detection of products containing Poria cocos DNA.

[0053] (3) The SNP site is one of the 16th base G and the 20th base C in the sequence 5'-TAACAATACCGGGCTGATTCAGTCTGGTAATTGG-3', which is used for the detection of products containing ginseng DNA;

[0054] (4) The SNP site is the 16th base T in the sequence 5'-CCAACCCATCACTCCTTTGCGGGAGTCGAG-3', which is used for the detection of products containing American ginseng DNA;

[0055] (5) The SNP site is the 15th base A in the sequence 5'-ACGACCCGCGAACAAGTTACAATACCGGGT-3', which is used for the detection of products containing Panax notoginseng DNA.

[0056] (6) The SNP site is the 16th base C of the sequence 5'-TTCAGTGCGCCCCCGCCGGCCCGGAGACGG-3', which is used for the detection of products containing licorice DNA.

[0057] (7) The SNP site is one of the 13th base C, 14th base A, or 15th base A in the sequence 5'-ACCGTTGCCCGACAACAATTGCCTCGCGAT-3', and is used for the detection of DNA products containing Glycyrrhiza glabra and / or Glycyrrhiza inflata.

[0058] (8) The SNP site is the 15th base A in the sequence 5'-GAACATGTTTAACCACATCGGGACCGTGGT-3', which is used for the detection of products containing Rehmannia glutinosa DNA;

[0059] (9) The SNP site is the 15th base G in the sequence 5'-TTGAATTGTACGTCGGCATCCCGTTAGCGG-3', which is used for the detection of products containing Angelica sinensis DNA;

[0060] (10) The SNP site is the 15th base T in the sequence 5'-CGGGCATCGGCGTCTTTCCAAAATACAACG-3', which is used for the detection of products containing Angelica dahurica DNA;

[0061] (11) The SNP site is one of the 16th base C and the 17th base A in the sequence 5'-CTGTCGTGCGTGCCGCAGTCGCCAGCGCGA-3', which is used for the detection of products containing Cornus officinalis DNA;

[0062] (12) The SNP site is one of the 16th base C and the 17th base C of the sequence 5'-GTGACCACCTGTGGGCCTTCCCTTTGGCAT-3', which is used for the detection of products containing Codonopsis pilosula DNA;

[0063] (13) The SNP site is the 16th base T in the sequence 5'-TAATGGCCCTCGTATTGAGCCGTGCGTCGC-3', which is used for the detection of products containing Atractylodes macrocephala DNA.

[0064] (14) The SNP site is one of the 16th base C and the 17th base A in the sequence 5'-GACTCCCGACGATCGCACCGTGGGGCGGCG-3', which is used for the detection of products containing Pinellia ternata DNA;

[0065] (15) The SNP site is one of the 16th base G and the 19th base A in the sequence 5'-GGTGTGTGTCTGGGGGTTAAGGCGTGTCTTGA-3', which is used for the detection of products containing Forsythia DNA;

[0066] (16) The SNP site is the 15th base A in the sequence 5'-CCTCGTGCGGGCCCATCCTGGCCCATTAAC-3', which is used for the detection of products containing Sophora flavescens DNA.

[0067] (17) The SNP site is one of the 15th T and 16th T bases in the sequence 5'-CGAGGCCTTTTAGGTTGAGGGCACATCTGCT-3', which is used for the detection of products containing Houttuynia cordata DNA;

[0068] (18) The SNP site is one of the 15th base G or the 16th base A in the sequence 5'-CCGAAAACAGAGACGACGGCAACGGACGTC-3', which is used for the detection of products containing Eucommia ulmoides DNA;

[0069] (19) The SNP site is the 16th base T in the sequence 5'-GAAAACCAAAAGAGATCGTCCCCCCTCCGT-3', which is used for the detection of products containing Scutellaria baicalensis DNA;

[0070] (20) The SNP site is one of the 15th base A and the 16th base G in the sequence 5'-GCCCCCGTTGCCCCAGCTTTGGGATGCGCG-3', which is used for the detection of products containing peony DNA;

[0071] (21) The SNP site is the 15th base C of the sequence 5'-CGTGAGCCTCTCCTCCATCCCATGTCCGGT-3', which is used to detect products containing Paeonia lactiflora DNA.

[0072] (22) The SNP site is the 15th base G in the sequence 5'-AATGCTCGGGCTGAGGGAAGGCGTGAGCCT-3', which is used for the detection of products containing peony DNA.

[0073] (23) The SNP site is the 15th base T in the sequence 5'-AGTATAAACCCAATTCACTGGATCCTTTGC-3', which is used for the detection of products containing Alisma plantago-aquatica DNA;

[0074] (24) The SNP site is the 15th base G in the sequence 5'-CTCGCGTGCTGTCGGGCGCCAAGGCGTCGT-3', which is used for the detection of products containing Scutellaria barbata DNA.

[0075] (25) The SNP site is the 15th base G in the sequence 5'-TTATCGTGAATTCTGCGATCATTACATATA-3', which is used for the detection of products containing yam DNA;

[0076] (26) The SNP site is one of the 13th base T and the 16th base C in the sequence 5'-CTCACTAAACACTCCCGAATTTCCAATATC-3', which is used for the detection of products containing donkey DNA;

[0077] (27) The SNP site is the 15th base G in the sequence 5'-CCGACTCACTATCAGCACCCCTACTAGTTC-3', which is used for the detection of products containing pig DNA.

[0078] (28) The SNP site is the 15th base T in the sequence 5'-GGAATCCCATCCGATATGGACAAAATCCCA-3', which is used for the detection of products containing horse DNA;

[0079] (29) The SNP site is the 15th base T in the sequence 5'-TTAGAAAGCCAAATTTCAGGATACTGTTCT-3', which is used to detect products containing cattle or zebu DNA.

[0080] (30) The SNP site is the 15th base A of the sequence 5'-TTATTCTACCCACCATTATAGCAATCACAG-3', which is used for the detection of products containing sika deer DNA.

[0081] (31) The SNP site is the 15th base G in the sequence 5'-TGATGATATGGACGGACAGACGCCAACACAGCAG-3', which is used for the detection of products containing sheep DNA.

[0082] (32) The SNP site is the 15th base C of the sequence 5'-TATCCGCTCTCCTACTAACATCCGGCCTCA-3', which is used for the detection of products containing goat DNA;

[0083] (33) The SNP site is the 15th base C of the sequence 5'-TACTATCTGTCCGACAAAGCGGTCTCAACTC-3', which is used to detect products containing Lonicera grahami DNA.

[0084] (34) The SNP site is the 15th base T in the sequence 5'-AAATGAGGATGCAGTGTCGGGAATGGTCGG-3', which is used for the detection of products containing Lonicera japonica DNA from South China.

[0085] (35) The SNP site is the 16th base T in the sequence 5'-CGTGTACAAATGAACTTCTTTGAGTAAGGAATC-3', which is used to detect products containing Lonicera japonica DNA.

[0086] (36) The SNP site is the 15th base A in the sequence 5'-CGGGTCCTGATTGTATTTTGGGCTTTCATTT-3', which is used for the detection of products containing honeysuckle DNA.

[0087] (37) The SNP site is the 15th base A in the sequence 5'-GCGCACGTCCTGCGATGGGTTGGCTCTCGTG-3', which is used for the detection of products containing Codonopsis pilosula or Codonopsis pilosula DNA.

[0088] (38) The SNP site is one of the 15th base C and the 16th base A in the sequence 5'-CCTGAGCGATGGTGCAGGCTCTCGTGACCCTG-3', which is used for the detection of products containing Platycodon grandiflorus DNA;

[0089] (39) The SNP site is one of the 16th base T, 17th base T, 18th base C, or 19th base T in the sequence 5'-GGGATTACCTACTCTTTCTTAACGGTCAAAGCGA-3', and is used for the detection of products containing Bupleurum chinense DNA.

[0090] (40) The SNP site is the 16th base T in the sequence 5'-TACTCTGTGAGCAACTGCGACCCTTTGGCGC-3', which is used for the detection of products containing Bupleurum chinense DNA.

[0091] (41) The SNP site is one of the 16th base T and the 17th base A in the sequence 5'-GGTGGAAGGCACTACTAACCTCTTGCCATCTTG-3', which is used to detect products containing Bupleurum chinense DNA;

[0092] (42) The SNP site is the 16th base C of the sequence 5'-CCGGCTCAAGTAGGTCCACCAACTAAATAAAG-3', which is used for the detection of products containing Danshen DNA;

[0093] (43) The SNP site is one of the 16th base C and the 17th base A in the sequence 5'-GGGGATGCGTTCCATCAGGGGCGGAGACTGGTC-3', which is used for the detection of products containing Costus Root DNA;

[0094] (44) The SNP site is the 16th base A in the sequence 5'-ACTTTAAGACGGCCTAGTGTCATGTTGCCCC-3', which is used for the detection of products containing *Inula japonica* DNA.

[0095] (45) The SNP site is one of the 15th base T and the 16th base G in the sequence 5'-TGGTCTCCCGTGCCTGCGGTGTGGTTGGCC-3', which is used for the detection of products containing Sichuan costus DNA;

[0096] (46) The SNP site is the 15th base A in sequence 5'-TCGGGTGCCCATCGACCGACGAAACCAACC-3', which is used for the detection of products containing Notopterygium incisum DNA;

[0097] (47) The SNP site is the 15th base T in the sequence 5'-GTGCCTTGCGGCGCTGCTGGCCCAAAAGCG-3', which is used for the detection of products containing Notopterygium incisum DNA.

[0098] (48) The SNP site is the 15th base T of the sequence 5'-CTACTTGGCCCCATTTTGGTGGCCGACTGA-3', which is used for the detection of products containing Amomum villosum DNA.

[0099] (49) The SNP site is the 16th base C of the sequence 5'-TTTATTTCCAATTCACTTTCAATCTAAAATAGA-3', which is used for the detection of products containing safflower DNA.

[0100] (50) The SNP site is one of the 16th base C and the 19th base G in the sequence 5'-CGCCAAGGAACACTTCTTGGAAACGCCGTCGCGGCC-3', which is used for the detection of products containing saffron DNA;

[0101] (51) The SNP site is the 15th base G in the sequence 5'-GCCGCGCGGTGCAAGCCCGTGGGACCATAC-3', which is used for the detection of products containing cinnamon DNA.

[0102] (52) The SNP site is one of the 16th T and 17th T bases in the sequence 5'-CCCCCAGGGCTGTCCTTGGACGGCGCCCAC-3', which is used for the detection of products containing Corydalis DNA;

[0103] (53) The SNP site is one of the 15th base G or the 18th base C in the sequence 5'-GATAGTACCCATTTGGGCAACGTCCAGTGCCAATG-3', which is used for the detection of products containing Aristolochia DNA;

[0104] (54) The SNP site is the 18th base G in the sequence 5'-CAAGGATTTGGAATTACGTGTATGCCATTCTCATC-3', which is used for the detection of products containing Asarum DNA;

[0105] (55) The SNP site is one of the 19th base T and the 20th base G in the sequence 5'-CGCCAGTCTTAGTGTATATGAGTAGGAGTTATTGAAAAAT-3', which is used for the detection of products containing Gelsemium DNA;

[0106] (56) The SNP site is the 16th base T in the sequence 5'-TAAGACCCCGGTCTTTTTGTATAGGAGTTATTGAAA-3', which is used for the detection of products containing Strychnos nucifera DNA.

[0107] (57) The SNP site is one of the 15th base T and the 16th base C in the sequence 5'-ATCCTCCAAAGACATCAAGACGCGTCGTCCTC-3', which is used for the detection of products containing Aconitum DNA;

[0108] (58) The SNP site is the 16th base C of the sequence 5'-CCATGGATGGGAACTCATGATTGGCTCTGTCT-3', which is used for the detection of products containing Corydalis DNA;

[0109] (59) The SNP site is the 15th base T in the sequence 5'-TGTGGATCGGCCTTTGGTGTGATAATTGTCTA-3', which is used for the detection of products containing Poria cocos DNA.

[0110] (60) The SNP site is the 16th base C of the sequence 5'-GAAAAGACTAGTTGACAAGGCTTCTATGTTC-3', which is used for the detection of products containing shiitake mushroom DNA;

[0111] (61) The SNP site is the 15th base T in the sequence 5'-AGGCATGTGCACGCTCTGCTCATCCACTCT-3', which is used for the detection of products containing Yunzhi DNA.

[0112] (62) The SNP site is one of the 14th base T and the 15th base G in the sequence 5'-TTCTAACGGTCTCTGTATGGAGACAAAGCT-3', which is used for the detection of products containing Ganoderma lucidum DNA;

[0113] (63) The SNP site is the 16th base T in the sequence 5'-CAAGAACACTAATGATCAATACCCGAAAAACC-3', which is used for the detection of products containing red deer DNA.

[0114] (64) The SNP site is the 16th base G in the sequence 5'-ACAAGGCATCCCCCTGTCATCAGGCCTAATCC-3', which is used for the detection of products containing deer DNA.

[0115] Compared with the prior art, the present invention has the following beneficial effects:

[0116] (1) This invention establishes a molecular detection method for highly degraded short fragment nucleic acids (including DNA and RNA), which can be used for quality control of products related to the entire Chinese medicine industry chain, including Chinese medicinal materials, Chinese medicinal decoction pieces, Chinese medicine formula granules, Chinese medicine preparations, and extracts derived from biological materials. It can also be used for products containing biological elements such as food, health food, beverages, cosmetics, and agricultural, forestry, animal husbandry and fishery products. It can also be used for nucleic acid detection of clinical samples such as pathogens and disease DNA markers, as well as various biological samples, and can detect copy number variations (deletion or duplication) and methylation status of genes.

[0117] (2) Suitable for the detection of highly degraded DNA samples, requiring short target DNA fragments. The left and right probe hybridization sequences designed in this invention are 5-18 bp, and the target DNA length is 10-36 bp, which is suitable for the detection of highly degraded DNA samples, especially traditional Chinese medicine compound preparations, food and other deep-processed products that have undergone long-term high-temperature extraction and drying.

[0118] (3) High specificity, accurately detecting SNP sites. DNA ligase can only catalyze the ligation of left and right probes when they hybridize with the same target DNA in the sample, are completely paired and adjacent. If there are gaps or incorrect bases, they will not be ligated. At the same time, with a high PCR annealing temperature (above 65℃), the hybridized sequence is less likely to bind to the wrong template, which can effectively avoid the occurrence of non-specific amplification.

[0119] (4) High sensitivity: The use of heat-resistant DNA ligase allows the DNA ligase to continue ligating the left and right probes during PCR amplification, further improving the sensitivity. It can even detect as low as 1 copy.

[0120] (5) Multiple detection can be performed. When using the fluorescent probe method, the PCR product can be designed to have different Tm values ​​for different SNP sites. After PCR, melting curve analysis is performed to measure Tm. Different Tm values ​​represent different SNP sites, thus enabling one fluorescent channel to detect multiple SNP sites. Combined with the design of probes with different fluorescent groups, up to 30 SNP sites can be detected simultaneously.

[0121] (6) The analysis cost is low. When using a multi-SNP design, template sequences can be flexibly added for bridging, and a single reagent can be used to detect multiple different SNP sites.

[0122] (7) A unique PCR detection method for traditional Chinese medicine preparations can be established. A traditional Chinese medicine compound is composed of multiple Chinese herbs. Specific SNP sites of each Chinese herb are selected. For these multiple SNP sites, a ligation reaction is carried out in combination with overlap extension PCR technology to fuse the products containing multiple SNP sites into one. This full-length PCR product, including all SNP sites, represents this traditional Chinese medicine compound preparation. Multiple herbs, or even all herbs, in this traditional Chinese medicine compound preparation can be detected by a single PCR test.

[0123] (8) Flexible detection methods. For multiple SNP sites, probes and primers can be designed for each SNP and LPCR detection can be performed simultaneously. As long as one SNP is present, it can be amplified and detected. Alternatively, overlapping extension PCR technology can be combined, so that the full-length PCR product contains multiple SNP sites.

[0124] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the figures. Attached Figure Description

[0125] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0126] Figure 1 This is an overall schematic diagram of a PCR detection method in an embodiment of the present invention;

[0127] Figure 2(a) is a schematic diagram of the design and detection principle of the first fluorescent probe in an embodiment of the present invention;

[0128] Figure 2(b) is a schematic diagram of the design and detection principle of the second fluorescent probe in an embodiment of the present invention;

[0129] Figure 2(c) is a schematic diagram of the design and detection principle of the third fluorescent probe in an embodiment of the present invention;

[0130] Figure 2(d) is a schematic diagram of the design and detection principle of the fourth fluorescent probe in an embodiment of the present invention;

[0131] Figure 3 This is a schematic diagram of the PCR detection amplification curve of Astragalus membranaceus in an embodiment of the present invention (wherein, 1 represents the schematic diagram of the PCR detection amplification curve of Astragalus membranaceus slices, 2 represents the schematic diagram of the PCR detection amplification curve of Zhenqi Fuzheng Granules, and 3 represents the schematic diagram of the PCR detection amplification curve of Guhan Yangsheng Essence).

[0132] Figure 4 This is a schematic diagram of the melting peak curve of Astragalus PCR detection in an embodiment of the present invention (wherein, 4 represents the schematic diagram of the melting peak curve of Astragalus slices PCR detection, 5 represents the schematic diagram of the melting peak curve of Zhenqi Fuzheng Granules PCR detection, 6 represents the schematic diagram of the melting peak curve of Guhan Yangsheng Essence PCR detection, and 7 represents the schematic diagram of the melting peak curve of blank and each negative sample PCR detection).

[0133] Figure 5 These are gel electrophoresis images of Astragalus PCR detection in embodiments of the present invention (wherein, 8 represents the gel electrophoresis image of Astragalus slices, 9 represents the gel electrophoresis image of Guhan Yangshengjing, 10 represents the gel electrophoresis image of Zhenqi Fuzheng Granules, 11 represents the gel electrophoresis image of Astragalus slices, 12 represents the gel electrophoresis image of Zhenqi Fuzheng Granules negative sample lacking Astragalus, 13 represents the gel electrophoresis image of Guhan Yangshengjing negative sample lacking Astragalus, 14 represents the gel electrophoresis image of negative control template, and 15 represents the gel electrophoresis image of blank water). Detailed Implementation

[0134] To make the above-mentioned objectives, features, and advantages of the present invention clearer and easier to understand, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that the accompanying drawings of the present invention are all in a simplified form and use non-precise proportions, and are only used to facilitate and clearly assist in illustrating the implementation of the present invention; the "several" mentioned in the present invention are not limited to the specific number shown in the examples in the accompanying drawings; the orientations or positional relationships indicated by terms such as "front," "middle," "rear," "left," "right," "up," "down," "top," "bottom," and "center" mentioned in the present invention are all based on the orientations or positional relationships shown in the accompanying drawings of the present invention, and do not indicate or imply that the device or component referred to must have a specific orientation, nor should they be construed as limitations on the present invention.

[0135] The PCR detection method provided by this invention includes the following steps:

[0136] Step 1: Design ligation probe and primer combinations targeting the known mutation site or its complementary sequence;

[0137] The known mutation site is set to at least one SNP site;

[0138] When there is only one SNP site, the ligation probe combination consists of a left probe LLP and a right probe RLP, and the primer combination consists of a forward primer FP and a reverse primer RP.

[0139] When there are two or more SNP sites, the ligation probe combination consists of a left probe LLP, a middle probe MLP and a right probe RLP, and the primer combination consists of a forward primer FP and two or more reverse primers RP.

[0140] The left probe LLP has two parts, the left extension sequence X and the hybridization sequence M1, from the 5' end to the 3' end. The hybridization sequence M1 of the left probe is the same as the upstream sequence adjacent to the SNP1 site.

[0141] The 5' to 3' ends of the intermediate probe MLP1 are the hybridization sequence N1, the linking sequence Z, and the hybridization sequence M2, respectively. The hybridization sequence N1 is the same as the downstream sequence adjacent to the SNP1 site, and the hybridization sequence M2 is the same as the upstream sequence adjacent to the SNP2 site. The SNP1 site is located at the 3' end of the left probe hybridization sequence M1 or the 5' end of the intermediate probe hybridization sequence N1. The design of other intermediate probes follows the same pattern. The hybridization sequence N is the same as the downstream sequence adjacent to the previous SNP site, and the hybridization sequence M is the same as the upstream sequence adjacent to the next SNP site.

[0142] The right probe RLP has two parts, the hybridization sequence Nn and the right extension sequence Y, from the 5' end to the 3' end. The hybridization sequence Nn of the right probe is the same as the downstream sequence adjacent to the SNPn site. The SNPn site is located at the 3' end of the hybridization sequence Mn of the intermediate probe or the 5' end of the hybridization sequence Nn of the right probe.

[0143] All hybridization sequences are 5-18 nucleotides in length;

[0144] The 5' ends of all intermediate probes MLP and right probe RLP were phosphorylated.

[0145] The forward primer FP has two parts, the extended sequence E and the sequence XM, from its 5' end to its 3' end, or it may only have the sequence XM; the sequence XM is partially or completely identical to the left probe LLP.

[0146] The reverse primer RPn has two parts, the extension sequence Fc and the complementary sequence YcNc, from the 5' end to the 3' end, or only the complementary sequence YcNc. The complementary sequence YcNc is complementary to part or all of the sequence of the right probe RLP.

[0147] Simultaneously, for each intermediate probe, a reverse primer is synthesized, namely RP1, RP2 to RPn-1, whose sequence is complementary to part or all of the sequence of the intermediate probe;

[0148] Step 2: Extract nucleic acid from the sample to be tested, and react it with the ligation probe combination, primer combination, DNA polymerase, and DNA ligase to obtain PCR products;

[0149] Step 3: Detect the PCR products.

[0150] Its detection principle is as follows:

[0151] Artificially designed and synthesized left and right probes hybridize with the same complementary target DNA in the sample. Using DNA ligase, the 5' phosphate ends and 3' hydroxyl ends of adjacent left and right probes are linked by phosphodiester bonds. Then, PCR technology is used to amplify and detect the ligated left and right probes. (See [link to relevant documentation]). Figure 1 As shown.

[0152] This invention utilizes a shorter hybridization sequence of 5-18 nucleotides, whose T... m The temperature is typically below 45℃, and combined with a higher PCR annealing temperature (above 60℃), the hybridization sequence is less likely to bind to the incorrect template, effectively avoiding non-specific amplification. Furthermore, because the hybridization sequence is short, the target sequence length required for detection is only 10-36 nucleotides, making it suitable for detecting highly degraded short nucleic acid fragments.

[0153] One of the differences between this invention and MLPA (Multiple Linked Probe Amplification) is that MLPA probes have longer hybridization sequences, requiring no less than 21 nucleotides, and recognize target sequences of 50 to 100 nucleotides in length, making them unsuitable for detecting short target sequences.

[0154] The second difference lies in the fact that MLPA uses universal primers with fluorescent groups but no quenching groups, and directly detects the length of single-stranded and double-stranded DNA products using capillary electrophoresis. In contrast, the fluorescence detection method in this invention uses primers or probes with both fluorescent and quenching groups. It exhibits no fluorescence during non-amplification but generates fluorescence during PCR amplification due to the formation of double-stranded DNA, and uses a quantitative real-time PCR instrument for product detection.

[0155] The third difference is that MLPA uses universal primer sequences, while this invention can use universal primers or specific primers, including hybridization sequences. Using specific primers can further improve the specificity of detection.

[0156] The fourth difference is that in MLPA, when using universal primers for PCR amplification, the remaining left probe in the system will affect the amplification. Therefore, the left probe cannot have a fluorescent group, and a small amount of ligation product needs to be taken out for a separate PCR reaction. However, the PCR detection method provided in this application allows the forward primer and the left probe to be the same, and the forward primer can be used to replace the left probe. The ligation reaction and PCR amplification reaction can be carried out continuously in the same tube.

[0157] Example 1:

[0158] The above-mentioned objective of this invention is achieved through the following technical solution:

[0159] In a first aspect, the present invention proposes a PCR detection method for detecting specific nucleic acid fragments contained in a sample, comprising the following steps:

[0160] Step 1: Select a specific SNP site in the nucleic acid. Based on the sequence containing this SNP site or its complementary sequence, design and synthesize a left probe (LLP) and a right probe (RLP). The left probe consists of two parts from its 5' end to its 3' end: a left extension sequence (X) and a hybridization sequence (M). The hybridization sequence M of the left probe is identical to the upstream sequence adjacent to the SNP site. The right probe consists of two parts from its 5' end to its 3' end: a hybridization sequence (N) and a right extension sequence (Y). The hybridization sequence N of the right probe is identical to the downstream sequence adjacent to the SNP site. The SNP site is located at the 3' end of the hybridization sequence M of the left probe or the 5' end of the hybridization sequence N of the right probe. The 5' end of the right probe is phosphorylated. The length of both hybridization sequences M and N is 5-18 nucleotides.

[0161] Step 2: Design and synthesize forward primer FP and reverse primer RP. Forward primer FP consists of two parts from its 5' end to its 3' end: the extension sequence E and sequence XM, or only sequence XM. Sequence XM is partially or completely identical to the left probe LLP from Step 1. Reverse primer RP consists of two parts from its 5' end to its 3' end: the extension sequence Fc and the complementary sequence YcNc, or only the complementary sequence YcNc. The complementary sequence YcNc is partially or completely complementary to the right probe RLP from Step 1.

[0162] Step 3: Extract nucleic acid from the sample to be tested, add the left probe LLP, right probe RLP, and DNA ligase designed and synthesized in Step 1, prepare the ligation reaction system, heat at 90-98℃ for at least 5 seconds to denature, and perform the ligation reaction for several minutes. Repeat the above denaturation and ligation steps 1 to several times, or not repeatedly, to obtain the ligation product. Take this ligation product, add the forward and reverse primers designed and synthesized in Step 2, and DNA polymerase to prepare the PCR reaction system, and perform the PCR reaction.

[0163] Step 4: Detect the PCR products from Step 3.

[0164] Furthermore, the specific process of step 3 is as follows:

[0165] Nucleic acid is extracted from the sample to be tested. The left probe LLP and right probe RLP designed and synthesized in step 1 are added. The mixture is heated at 90-98℃ for more than 5 seconds, cooled, and then the forward and reverse primers, DNA ligase, and hot-start DNA polymerase designed and synthesized in step 2 are added to prepare the reaction system. After the ligation reaction has been going on for several minutes, the mixture is heated at 90℃ for more than 30 seconds to continue the PCR reaction.

[0166] The invention is characterized by the fact that it does not require a second opening of the lid to add reagents, and the ligation reaction and PCR reaction can be carried out continuously.

[0167] Furthermore, the melting temperatures T of the left probe LLP and the right probe RLP are... m Not lower than 65℃. The melting temperature T of the portion of the forward primer FP where the sequence XM is identical to that of the left probe LLP. m The melting temperature T of the complementary sequence YcNc in the reverse primer RP and the complementary portion of the right probe RLP sequence is not lower than 65℃. m The PCR reaction should be carried out at an annealing temperature of not less than 65°C in step 3.

[0168] As a further embodiment, the PCR product detection method in step 4 includes the following three methods:

[0169] 1) PCR products are detected using electrophoresis to determine their length. Preferred electrophoresis methods include agarose gel electrophoresis, polyacrylamide gel electrophoresis, and capillary electrophoresis.

[0170] 2) A fluorescent dye is used. A fluorescent dye is also added to the PCR reaction system in step 3 to detect the fluorescence of the PCR products. The fluorescent dye is preferably one of SYBR Green I, Eva Green, LC Green, or Solis Green.

[0171] 3) Use probes or primers modified with fluorescent groups and quenching groups to perform fluorescence detection on PCR products.

[0172] The following four methods can be used for probe or primer design and detection:

[0173] ① One of the forward primers FP or reverse primers RP in step 2 is designed and synthesized to connect a fluorescent group and a quencher group. Before amplification, it is in a randomly coiled state, with the fluorescent group R and the quencher group Q relatively close together, resulting in a weak fluorescence signal. A further optimized design is a scorpion-shaped probe containing a reverse complementary stem-loop structure of 8-12 consecutive bases. With the fluorescent group R and the quencher group Q located at the base of the stem-loop structure, the fluorescence signal is even weaker, reducing the influence of the fluorescence background. After PCR amplification, when double-strand extension is initiated, it forms an extended state, causing the fluorescent group R and the quencher group Q to move further apart, generating a fluorescence signal. See Figure 2(a).

[0174] ② The 5' end of the forward primer is connected to a fluorescent group R, and the 3' end of the complementary quenching primer QP is connected to a quenching group Q. Before amplification, the fluorescent group R and the quenching group Q are relatively close, resulting in a weak fluorescence signal. After PCR amplification, when double strand synthesis is initiated, the quenching primer is hydrolyzed, and the fluorescent group R and the quenching group Q are thus separated, generating a fluorescence signal. See Figure 2(b).

[0175] ③ The detection template MB is linked to a fluorescent group R and a quencher group Q. Before amplification, it is in a randomly coiled state, with the fluorescent group R and the quencher group Q relatively close together, resulting in a weak fluorescence signal. During PCR amplification, the Taq enzyme cleaves and releases the tag sequence Tag. The released tag sequence Tag hybridizes with the detection template MB and initiates double-strand extension, forming an extended state. As a result, the fluorescent group R and the quencher group Q are separated, generating a fluorescence signal. See Figure 2(c).

[0176] ④ The TaqMan fluorescent probe method is used. The TaqMan fluorescent probe is connected to a fluorophore and a quencher group. As the forward primer extends, the TaqMan fluorescent probe is hydrolyzed, releasing the fluorophore and generating a fluorescent signal. See Figure 2(d).

[0177] The indicators used for fluorescence detection are Ct value and / or melting temperature T. m .

[0178] The fluorescent group is preferably one of FAM, TET, HEX, VIC, ROX, CY3, CY5, CY5.5, CY7, JOE, TexasRed, Quasar 570, Quasar 670, TAMRA, or AlexaFluor 633; ​​the quenching group is preferably one of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL, MGB, or Eclipse.

[0179] Example 2:

[0180] Because traditional Chinese medicine (TCM) compound preparations contain multiple herbs, each with specific SNP sites, testing of these preparations typically targets at least two SNP sites. Detection of multiple SNP sites can be achieved using overlap extension PCR technology, which fuses multiple fragments after the ligation reaction into a single long DNA chain. This long DNA chain allows for the simultaneous detection of multiple SNP sites. The specific method is as follows:

[0181] Step (I): Select two or more specific SNP sites in the nucleic acid, namely SNP1, SNP2 to SNPn. Design and synthesize a DNA left probe LLP, a middle probe MLP and a right probe RLP based on the sequence containing the SNP site or the complementary sequence. The left probe has two parts from the 5' end to the 3' end, namely the left extension sequence X and the hybridization sequence M1. The hybridization sequence M1 of the left probe is the same as the upstream sequence adjacent to the SNP1 site.

[0182] The intermediate probe MLP1 consists of hybridization sequence N1, linking sequence Z, and hybridization sequence M2 from its 5'-end to its 3'-end. Hybridization sequence N1 is identical to the downstream sequence adjacent to SNP1, and hybridization sequence M2 is identical to the upstream sequence adjacent to SNP2. SNP1 is located at the 3' end of the left probe hybridization sequence M1 or the 5'-end of the intermediate probe hybridization sequence N1. Other intermediate probes are designed in the same way. Hybridization sequence N is identical to the downstream sequence adjacent to the previous SNP, and hybridization sequence M is identical to the upstream sequence adjacent to the next SNP. There are a total of n-1 intermediate probes.

[0183] The right probe RLP consists of two parts from the 5' end to the 3' end: the hybridization sequence Nn and the right extension sequence Y. The hybridization sequence Nn of the right probe is the same as the downstream sequence adjacent to the SNPn site. The SNPn site is located at the 3' end of the hybridization sequence Mn of the intermediate probe or the 5' end of the hybridization sequence Nn of the right probe.

[0184] All intermediate and right probes were phosphorylated at their 5' ends. All hybridization sequences were 5–18 nucleotides in length.

[0185] Step (II): Design and synthesize forward primer FP and reverse primer RP. Forward primer FP is the same as in step two of Example 1. For each intermediate probe and right probe in step (I), a reverse primer is synthesized, namely RP1, RP2 to RPn, whose sequence is complementary to the intermediate probe or right probe.

[0186] Step (III): Extract nucleic acid from the sample to be tested, add the left probe, right probe, all intermediate probes and DNA ligase designed and synthesized in step (I), prepare the ligation reaction system, heat at 90-98℃ for at least 5 seconds to denature, and perform the ligation reaction for several minutes. Repeat the above denaturation and ligation steps 1 to several times, or not repeatedly, to obtain the ligation product. Take this ligation product, add the forward primer and all reverse primers designed and synthesized in step (II), and DNA polymerase to prepare the PCR reaction system, and perform the PCR reaction.

[0187] Alternatively, take the ligation product, add the forward and reverse primers RP1 and DNA polymerase designed and synthesized in step (II) to prepare PCR reaction system 1, intermediate probe MLP1 and reverse primer RP2 and DNA polymerase to prepare PCR reaction system 2, and so on up to intermediate probe MLPn-1 and reverse primer RPn and DNA polymerase to prepare PCR reaction system n. Each reaction system is PCR reacted separately, and then the systems are combined for PCR reaction.

[0188] Step (IV): Detect the PCR products from step (III).

[0189] Furthermore, in step (I), for each SNP site, a corresponding DNA template MB can be designed and synthesized, namely MB1, MB2 to MBn, whose sequence is complementary to the sequence containing the SNP site. In step (III), 1 to n-1 of the DNA templates MB designed and synthesized in step (I) are added to the ligation reaction system according to detection needs. Using this invention, a single reagent set can be used to detect different SNPs.

[0190] Furthermore, in step (II), when designing complementary reverse primers for each intermediate probe, the Tm of the complementary reverse primer is 3°C lower than that of the complementary reverse primer RPn designed for the right probe. In step (III), PCR can be performed at a higher temperature. First, the reaction is cycled at a lower annealing temperature for 1 to several times, and then cycled at an annealing temperature 3°C or higher for 1 to several times to improve the efficiency of obtaining full-length DNA products.

[0191] Example 3:

[0192] This invention also provides a kit comprising a combination of ligation probes and primers used in the PCR detection method described above. Specifically, the kit includes DNA ligase, DNA polymerase, primers, probes, and / or templates.

[0193] Furthermore, the present invention also provides the use of the PCR detection method as described above for detecting mutation sites.

[0194] The steps for quantitative analysis of SNP sites using the PCR detection method described above are as follows:

[0195] Step (i) Select a specific SNP site and perform fluorescence detection on this SNP site using the method described above to obtain the copy number A.

[0196] Step (ii): Select a conserved sequence as an internal reference sequence, take any base site within the internal reference sequence, and perform fluorescence detection using the same method as described above to obtain the copy number B of the internal reference sequence.

[0197] Step (iii) Calculate the ratio R of A / B as an indicator for quality control.

[0198] In all the methods described above, the DNA ligase is preferably a heat-resistant DNA ligase, more preferably a 9°N DNA ligase and / or a Taq DNA ligase, and the ligation temperature is preferably not lower than 45°C.

[0199] Furthermore, the technology of the present invention can also detect RNA in samples, such as microRNA, and the DNA ligase is preferably an RNA template-mediated DNA ligase, such as PBCV DNA ligase (Splint R ligase).

[0200] Experimental Example 1:

[0201] The specific process for detecting Astragalus membranaceus using the PCR detection method described above is as follows:

[0202] (a) Synthesizing primer combinations and ligation probe combinations;

[0203] Targeting the 15th base C of the SNP site 5'-ACCCATCATGTGTACGCTCCCCATAATATG-3', we designed and synthesized the ligation probe combination, primer combination, and control template shown in Table 1, which were used to detect Astragalus membranaceus slices, Zhenqi Fuzheng Granules, Guhan Yangsheng Essence, and other products containing Astragalus membranaceus.

[0204] Table 1: Ligation probe combinations, primer combinations, and control template sequences for the 15th base C of the SNP site at sequence 5'-ACCCATCATGTGTACGCTCCCCATAATATG-3'

[0205]

[0206]

[0207] (II) DNA extraction;

[0208] Take the samples to be tested: Astragalus membranaceus slices, Astragalus rubra slices, Zhenqi Fuzheng granules, and Zhenqi Fuzheng granules (negative samples lacking Astragalus membranaceus), grind them into fine powder, take 50mg and place them in a 1.5mL centrifuge tube, add 700μL GP1 buffer and incubate overnight at 56℃. Then extract DNA using a plant genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd., model DP-305), dilute to 1μg / ml, and use as the DNA detection template solution.

[0209] Take 2 ml each of Gu Han Yang Sheng Jing and Gu Han Yang Sheng Jing (negative sample lacking Astragalus membranaceus), and extract DNA using a plant genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd., model DP-305) to serve as the DNA detection template solution.

[0210] (III) Probe ligation and PCR amplification;

[0211] Take the DNA template solutions extracted in step (II) and the negative control template solutions, and prepare the ligation reaction solution according to the following system: 0.2 μL template DNA, 8.1 μL ddH2O, 0.5 μL 9N DNALigase (40 U / μL) (Harbin Xinhai Gene Testing Co., Ltd., model D3003), 1 μL 10×9N DNALigase Buffer, and 0.1 μL each of the left probe LLP (10 μM) and right probe RLP (10 μM) from step (I) above.

[0212] The ligation reaction procedure is as follows: pre-denaturation at 95°C for 60 seconds; denaturation at 95°C for 10 seconds; ligation reaction at 60°C for 80 seconds; repeat the above two steps of denaturation and ligation reaction 20 times in total to obtain the ligation product for later use.

[0213] Prepare the PCR reaction solution according to the following system: Take 2.5 μL of each ligation product, 0.8 μL of ddH2O, 5 μL of 2×SYBR GreenqPCRMix (Biosharp, model BL697A), 0.1 μL of 50 mM MgCl2, and 0.8 μL each of the forward primer FP (10 μM) and the reverse primer RP (10 μM) from step (I) above.

[0214] Perform PCR amplification according to the following procedure: pre-denaturation at 95℃ for 5 minutes; denaturation at 95℃ for 10 seconds; annealing and extension at 72℃ for 30 seconds; repeat the above two-step reaction of denaturation and annealing for a total of 40 times.

[0215] Use sterile water instead of the ligation product to prepare the PCR reaction solution, and perform PCR amplification simultaneously as a blank control.

[0216] (iv) Test results;

[0217] The results were obtained using a quantitative real-time PCR instrument (Xi'an Tianlong Technology Co., Ltd., model Gentier 96E). The Ct values ​​were: Astragalus membranaceus slices 21.293, Zhenqi Fuzheng granules 23.262, and Guhan Yangshengjing 27.551, indicating that Astragalus membranaceus DNA was detectable in all three samples. Amplification curves are shown below. Figure 3 As shown.

[0218] Melting curve analysis was performed according to the procedure. The Tm value of Astragalus membranaceus slices was 80.66℃, the Tm value of Zhenqi Fuzheng granules was 81.15℃, and the Tm value of Guhan Yangshengjing was 80.87℃, which are basically close, indicating that Astragalus membranaceus DNA can be detected in all three samples. See [link to relevant documentation]. Figure 4 As shown.

[0219] The following samples—Hongqi decoction pieces, Zhenqi Fuzheng granules (a negative sample lacking Astragalus), Guhan Yangshengjing (a negative sample lacking Astragalus), negative control template, and blank water—did not show amplification curves, Ct values, or Tm values, indicating that Astragalus DNA was not detected in any of these samples.

[0220] Take each PCR product and perform electrophoresis as follows: Prepare a 3% agarose gel and add nucleic acid gel staining agent to the gel; load 8 μL of PCR reaction solution and 2 μL of DNA Marker (0.5 μg / μL) for each sample. After electrophoresis, take a picture of the gel on a gel imaging system. Astragalus membranaceus slices, Zhenqi Fuzheng granules, and Guhan Yangshengjing all showed bands at the same position (approximately 84 bp), while Astragalus membranaceus slices, Zhenqi Fuzheng granules (a negative sample lacking Astragalus membranaceus), Guhan Yangshengjing (a negative sample lacking Astragalus membranaceus), negative control template, and blank water all showed no band at 84 bp. See [link to relevant documentation]. Figure 5 As shown.

[0221] Experimental Example 2:

[0222] The specific process for detecting Poria cocos using the PCR detection method described above is as follows:

[0223] (A) Synthesizing primer combinations and ligation probe combinations;

[0224] Primers and probes were designed and synthesized targeting the 15th base G at the SNP site 5'-GAACCCTCAACTCCGTCCGCCTTTGTTGGG-3' to detect Poria cocos slices and products containing Poria cocos such as Liuwei Dihuang Pills and Huoxiang Zhengqi Water.

[0225] Table 2: Linkage probe combinations, primer combinations, and control template sequences for the 15th base G at the SNP site of sequence 5'-GAACCCTCAACTCCGTCCGCCTTTGTTGGG-3'.

[0226]

[0227]

[0228] (B) DNA extraction;

[0229] Take the samples to be tested: Poria cocos slices, Liuwei Dihuang pills, and Liuwei Dihuang pills (negative samples lacking Poria cocos), grind them into fine powder, take 50mg and put them into a 1.5mL centrifuge tube, add 700μL GP1 buffer and incubate overnight at 56℃, then extract DNA using a plant genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd., model DP-305) as the DNA detection template.

[0230] 2 ml each of Huoxiang Zhengqi Water and Huoxiang Zhengqi Water (negative sample lacking Poria cocos) were used to extract DNA using a plant genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd., model DP-305) as a template for DNA detection.

[0231] (C) Probe ligation and PCR amplification;

[0232] Take the DNA template solutions extracted in step (B) and the negative control template solutions, and prepare the ligation reaction solution according to the following system: 8.3 μL template DNA, 0.5 μL 9.N DNALigase (40 U / μL) (Harbin Xinhai Gene Testing Co., Ltd., model D3003), 1 μL 10×9N DNALigase Buffer, and 0.1 μL each of the left probe LLP (10 μM) and right probe RLP (10 μM) from step (A) above.

[0233] The ligation reaction procedure is as follows: pre-denaturation at 95°C for 60 seconds; denaturation at 95°C for 10 seconds; reaction at 45°C for 350 seconds; repeat the above two steps of denaturation and ligation reaction a total of 5 times to obtain the ligation product for later use.

[0234] Prepare the PCR reaction solution according to the following system: Take 3.9 μL of each ligation product, 5 μL of 2×SYBR Green qPCRMix (Biosharp, model BL697A), 0.1 μL of 50 mM MgCl2, and 0.5 μL each of the forward primer FP (10 μM) and the reverse primer RP (10 μM) from step (A) above.

[0235] Perform PCR amplification according to the following procedure: pre-denaturation at 95℃ for 5 minutes; denaturation at 95℃ for 10 seconds; annealing and extension at 70℃ for 30 seconds; repeat the above two-step reaction of denaturation and annealing for a total of 40 times.

[0236] Use sterile water instead of the ligation product to prepare the PCR reaction solution, and perform PCR amplification simultaneously as a blank control.

[0237] (D) Test results;

[0238] The results were obtained by using a real-time PCR instrument (Xi'an Tianlong Technology Co., Ltd., model Gentier 96E). The Ct values ​​were as follows: Poria cocos slices: 25.160; Liuwei Dihuang pills: 28.309; and Huoxiang Zhengqi water: 26.402. This indicates that Poria cocos DNA can be detected in all three samples.

[0239] Melting curve analysis was performed according to the procedure. (Poria cocos slices T) m The value was 82.50℃, and the temperature of Liuwei Dihuang Pills (T) was... m The Tm value was 82.26℃, and the Tm value of Huoxiang Zhengqi Water was 82.53℃. These values ​​are essentially the same, indicating that Poria cocos DNA could be detected in all three samples.

[0240] Liuwei Dihuang Pills (negative sample lacking Poria cocos), Huoxiang Zhengqi Water (negative sample lacking Poria cocos), negative control template, and blank water all showed no amplification curves, Ct values, or T values. m The value indicates that no Poria cocos DNA was detected in these samples.

[0241] Take each PCR product and perform electrophoresis detection as follows: Prepare a 3% agarose gel and add nucleic acid gel staining agent to the gel; the loading volume of PCR reaction solution for each sample is 8 μL, and the loading volume of DNA Marker (0.5 μg / μL) is 2 μL. After electrophoresis, take a picture of the gel on a gel imaging system and record it. Poria cocos slices, Liuwei Dihuang pills, and Huoxiang Zhengqi water all have bands at the same position (about 99 bp), while Astragalus membranaceus slices, Liuwei Dihuang pills (negative sample without Poria cocos), Huoxiang Zhengqi water (negative sample without Poria cocos), negative control template, and blank water all have no band at 99 bp.

[0242] Experimental Example 3:

[0243] The specific process for detecting Poria cocos using the PCR detection method described above is as follows:

[0244] (a) Synthesizing primer assemblies and ligation probe assemblies;

[0245] Based on the SNP site at the 15th base G of the sequence 5'-GAACCCTCAACTCCGTCCGCCTTTGTTGGG-3', the following primers and probes were designed and synthesized for the detection of Poria cocos and products containing Poria cocos.

[0246] Table 3: Linkage probe and primer sequences for the SNP site at the 15th base G of sequence 5'-GAACCCTCAACTCCGTCCGCCTTTGTTGGG-3'

[0247]

[0248] (b) Other steps were the same as in Experiment 2. Detection was performed using a real-time PCR instrument. The results were as follows: the Ct value of Poria cocos slices was 27.154, the Ct value of Liuwei Dihuang pills was 29.354, and the Ct value of Huoxiang Zhengqi water was 28.314, indicating that Poria cocos DNA could be detected in all three samples.

[0249] Experiment Example 4:

[0250] The specific process for detecting Poria cocos using the PCR detection method described above is as follows:

[0251] The primer and probe combinations and DNA extraction were designed using the same methods as steps (A) and (B) in Experiment Example 2;

[0252] Probe ligation and PCR amplification;

[0253] Take the DNA sample template solution extracted in step (B), add 3.2 μL of template DNA, 0.1 μL each of the left probe LLP (10 μM) and right probe RLP (10 μM) from step (A), mix well, pre-denature at 95℃ for 60 seconds, cool, and add 0.5 μL of 9.N DNALigase (40 U / μL) (Harbin Xinhai Gene Testing Co., Ltd., model D3003), 5 μL of 2×SYBR Green qPCR Mix (Biosharp, model BL697A), 0.1 μL of 50 mM MgCl2, and 0.5 μL each of the forward primer FP (10 μM) shown in SEQ ID NO.11 and the reverse primer RP (10 μM) shown in SEQ ID NO.12.

[0254] After reacting at 45℃ for 30 minutes, PCR amplification was performed according to the following procedure: pre-denaturation at 95℃ for 5 minutes; denaturation at 95℃ for 10 seconds; annealing and extension at 68℃ for 30 seconds. The above two-step denaturation and annealing and extension reactions were repeated a total of 40 times.

[0255] Test results;

[0256] The results of the quantitative real-time PCR test were as follows: the Ct value of Poria cocos slices was 29.544, the Ct value of Liuwei Dihuang pills was 29.874, and the Ct value of Huoxiang Zhengqi water was 29.785, indicating that Poria cocos DNA could be detected in all three samples.

[0257] Experimental Example 5:

[0258] The specific process for detecting Poria cocos using the PCR detection method described above is as follows:

[0259] Synthesize primer assemblies and ligation probe assemblies;

[0260] Table 4: Forward primer and left probe sequences for the 15th base G of the SNP site in sequence 5'-GAACCCTCAACTCCGTCCGCCTTTGTTGGG-3'

[0261]

[0262] The other primers and probes are the same as in Experiment 2.

[0263] DNA extraction was performed using the DNA extraction steps described in Experimental Example 2.

[0264] Probe ligation and PCR amplification;

[0265] Except for preparing the PCR reaction solution according to the following system, all other steps are the same as in Experiment Example 2 (C).

[0266] PCR reaction system: Take 5 μL of each ligation product, 0.1 μL of Accurate Taq HSDNA polymerase (5 U / μL) (Hunan Aike Rui Biotechnology Co., Ltd., model AG11206), 2 μL of 10×Taq PCR Buffer, 1 μL of 50mM MgCl2, 0.4 μL of 10mM dNTP Mix, 9.9 μL of ddH2O, and 0.8 μL each of the forward primer FP (10 μM) shown in SEQ ID NO.20 and the reverse primer RP (10 μM) shown in SEQ ID NO.12.

[0267] Test results;

[0268] The results of the quantitative real-time PCR test were as follows: the Ct value of Poria cocos slices was 25.124, the Ct value of Liuwei Dihuang pills was 27.247, and the Ct value of Huoxiang Zhengqi water was 27.744, indicating that Poria cocos DNA could be detected in all three samples.

[0269] Experimental Example 6:

[0270] The specific process for detecting Poria cocos using the PCR detection method described above is as follows:

[0271] Synthesize primer assemblies and ligation probe assemblies;

[0272] Table 5: Forward primer and left probe sequences for the 15th base G of the SNP site 5'-GAACCCTCAACTCCGTCCGCCTTTGTTGGG-3'

[0273]

[0274] All other conditions are the same as in Experiment 5.

[0275] The results of the quantitative real-time PCR test were as follows: the Ct value of Poria cocos slices was 24.245, the Ct value of Liuwei Dihuang pills was 25.454, and the Ct value of Huoxiang Zhengqi water was 25.672, indicating that Poria cocos DNA could be detected in all three samples.

[0276] Experiment Example 7:

[0277] The specific process for detecting Poria cocos using the PCR detection method described above is as follows:

[0278] Synthesize primer assemblies and ligation probe assemblies;

[0279] Table 6: Left probe and quenching primer sequences for the SNP site at the 15th base G of sequence 5'-GAACCCTCAACTCCGTCCGCCTTTGTTGGG-3'

[0280]

[0281] The other primers and probes are the same as in Experiment 2.

[0282] DNA extraction was performed using the DNA extraction steps described in Experimental Example 2.

[0283] Probe ligation and PCR amplification;

[0284] Except for the PCR reaction solution being prepared according to the following system, all other conditions were the same as in Experiment 2.

[0285] PCR reaction system: Take 5 μL of each ligation product, 0.1 μL of Accurate Taq HS DNA polymerase (5 U / μL) (Hunan Aike Rui Biotechnology Co., Ltd., model AG11206), 2 μL of 10×Taq PCR Buffer, 1 μL of 50 mM MgCl2, 0.4 μL of 10 mM dNTP, 8.9 μL of ddH2O, 0.8 μL each of the forward primer FP (10 μM) shown in SEQ ID NO.11 and the reverse primer RP (10 μM) shown in SEQ ID NO.12, and 1.0 μL of the quenching primer QP (10 μM) shown in SEQ ID NO.23.

[0286] Test results;

[0287] The results of the quantitative real-time PCR test were as follows: the Ct value of Poria cocos slices was 23.457, the Ct value of Liuwei Dihuang pills was 24.487, and the Ct value of Huoxiang Zhengqi water was 24.356, indicating that Poria cocos DNA could be detected in all three samples.

[0288] Experiment Example 8:

[0289] The specific process for detecting Poria cocos using the PCR detection method described above is as follows:

[0290] Synthesize primer assemblies and ligation probe assemblies;

[0291] Table 7: Tag primer TagEXc and detection template MB sequence for the SNP site at the 15th base G of sequence 5'-GAACCCTCAACTCCGTCCGCCTTTGTTGGG-3'

[0292]

[0293]

[0294] The other primers and probes are the same as in Experiment 2.

[0295] DNA extraction was performed using the DNA extraction steps described in Experimental Example 2.

[0296] Probe ligation and PCR amplification;

[0297] Except for the PCR reaction solution being prepared according to the following system, all other conditions were the same as in Experiment 2.

[0298] PCR reaction system: Take 5 μL of each ligation product, 0.1 μL of Accurate Taq HSDNA polymerase (5 U / μL) (Hunan Aike Rui Biotechnology Co., Ltd., model AG11206), 2 μL of 10×Taq PCR Buffer, 1 μL of 50 mM MgCl2, 0.4 μL of 10 mM dNTP, 8.9 μL of ddH2O, 0.8 μL each of the forward primer FP (10 μM) shown in SEQ ID NO.11 and the reverse primer RP (10 μM) shown in SEQ ID NO.12, 1.0 μL of TagEXc (10 μM) shown in SEQ ID NO.24, and 1.0 μL of the detection template MB (10 μM) shown in SEQ ID NO.25.

[0299] Test results;

[0300] The results of the quantitative real-time PCR test were as follows: the Ct value of Poria cocos slices was 22.647, the Ct value of Liuwei Dihuang pills was 23.654, and the Ct value of Huoxiang Zhengqi water was 23.681, indicating that Poria cocos DNA could be detected in all three samples.

[0301] Experiment Example 9:

[0302] The specific process for detecting Poria cocos and Rehmannia glutinosa using the PCR detection method described above is as follows:

[0303] Synthesize primer assemblies and ligation probe assemblies;

[0304] Based on the SNP sites at the 15th base G of sequence 5'-GAACCCTCAACTCCGTCCGCCTTTGTTGGG-3' and the 15th base A of sequence 5'-GAACATGTTTAACCACATCGGGACCGTGGT-3', the following primers and probes were designed and synthesized for the detection of products containing both Poria cocos and Rehmannia glutinosa.

[0305] Table 8: Sequences of probe and primer combinations for connecting SNP sites at the 15th base G of sequence 5'-GAACCCTCAACTCCGTCCGCCTTTGTTGGG-3' and SNP sites at the 15th base A of sequence 5'-GAACATGTTTAACCACATCGGGACCGTGGT-3'.

[0306]

[0307]

[0308] DNA extraction;

[0309] Samples to be tested: Liuwei Dihuang Pills, Liuwei Dihuang Pills (negative sample lacking Poria cocos), and Liuwei Dihuang Pills (negative sample lacking Rehmannia glutinosa) were extracted according to step (B) in Experimental Example 2.

[0310] Probe ligation and PCR amplification;

[0311] Take the DNA sample template solutions extracted in step (B) and prepare the ligation reaction solution according to the following system: 8.2 μL template DNA, 0.5 μL 9.N DNALigase (40 U / μL) (Harbin Xinhai Gene Testing Co., Ltd., model D3003), 1 μL 10×9N DNALigase Buffer, and 0.1 μL each of the left probe LLP (10 μM) shown in SEQ ID NO.28, the middle probe MLP (10 μM) shown in SEQ ID NO.30, and the right probe RLP (10 μM) shown in SEQ ID NO.29.

[0312] The ligation reaction procedure is as follows: pre-denaturation at 95°C for 60 seconds; denaturation at 95°C for 10 seconds; reaction at 45°C for 350 seconds; repeat the above two steps of denaturation and ligation reaction a total of 5 times to obtain the ligation product for later use.

[0313] Prepare the PCR reaction solution according to the following system: Take 3.5 μL of each ligation product, 5 μL of 2×ApexHF FS PCR MasterMix (Hunan Aike Rui Biotechnology Co., Ltd., model AG12202), and 0.5 μL each of the forward primer FP (10 μM) shown in SEQ ID NO.31, the reverse primer RP1 (10 μM) shown in SEQ ID NO.32, and the reverse primer RP2 (10 μM) shown in SEQ ID NO.33.

[0314] Perform PCR amplification according to the following procedure: pre-denaturation at 95℃ for 1 minute; denaturation at 95℃ for 10 seconds; annealing and extension at 65℃ for 30 seconds.

[0315] The two-step reaction of denaturation and annealing extension is repeated 10 times, followed by a two-step reaction of denaturation at 95°C for 10 seconds and annealing extension at 72°C for 30 seconds, for a total of 30 times.

[0316] Use sterile water instead of the ligation product to prepare the PCR reaction solution, and perform PCR amplification simultaneously as a blank control.

[0317] Test results;

[0318] Take each PCR product and perform electrophoresis detection as follows: Prepare a 3% agarose gel and add nucleic acid gel staining agent to the gel; the loading volume of PCR reaction solution for each sample is 8 μL, and the loading volume of DNA Marker (0.5 μg / μL) is 2 μL. After electrophoresis, take a picture of the gel on a gel imaging system. Liuwei Dihuang Pills showed a band at approximately 117 bp, while Liuwei Dihuang Pills (negative sample lacking Poria cocos), Liuwei Dihuang Pills (negative sample lacking Rehmannia glutinosa), and blank water showed no band at 117 bp, indicating that the Liuwei Dihuang Pills sample contains both Poria cocos and Rehmannia glutinosa.

[0319] Experimental Example 10:

[0320] The specific process for detecting Poria cocos and Rehmannia glutinosa using the PCR detection method described above is as follows:

[0321] The same primer and ligation probe combinations and DNA extraction steps as in Experiment Example 9 were used for primer and ligation probe design and DNA extraction.

[0322] The probe ligation system and procedure were performed in the same manner as in Experiment 9, and PCR reaction solutions 1 and 2 were prepared according to the following systems.

[0323] Preparation of PCR reaction solution 1: Take 4.0 μL of each ligation product, 5 μL of 2×ApexHF FS PCRMaster Mix (Hunan Aikerui Biotechnology Co., Ltd., model AG12202), and 0.5 μL each of the forward primer FP (10 μM) shown in SEQ ID NO.31 and the reverse primer RP1 (10 μM) shown in SEQ ID NO.32.

[0324] Preparation of PCR reaction solution 2: Take 4.0 μL of each ligation product, 5 μL of 2×ApexHF FS PCRMaster Mix (Hunan Aike Rui Biotechnology Co., Ltd., model AG12202), 0.5 μL each of the intermediate probe MLP (10 μM) shown in SEQ ID NO.30 and the reverse primer RP2 (10 μM) shown in SEQ ID NO.33.

[0325] PCR reaction solutions 1 and 2 were both subjected to PCR amplification according to the following procedure: 95℃ pre-denaturation for 1 minute; 95℃ denaturation for 10 seconds, 65℃ annealing and extension for 30 seconds, and the above two-step reaction of denaturation and annealing and extension was repeated 10 times. Then PCR reaction solutions 1 and 2 were combined and the two-step reaction of 95℃ denaturation for 10 seconds and 72℃ annealing and extension for 30 seconds was repeated 30 times.

[0326] Test results;

[0327] Take each PCR product and perform electrophoresis detection as follows: Prepare a 3% agarose gel and add nucleic acid gel staining agent to the gel; the loading volume of PCR reaction solution for each sample is 8 μL, and the loading volume of DNA Marker (0.5 μg / μL) is 2 μL. After electrophoresis, take a picture of the gel on a gel imaging system. The Liuwei Dihuang Pill sample has a band at approximately 117 bp, while Liuwei Dihuang Pill (poria-deficient negative sample), Liuwei Dihuang Pill (rehmannia-deficient negative sample), and blank water have no band at 117 bp, indicating that the Liuwei Dihuang Pill sample contains both poria and rehmannia.

[0328] Experimental Example 11:

[0329] The specific process for detecting Rehmannia glutinosa using the PCR detection method described above is as follows:

[0330] Synthesize primer assemblies and ligation probe assemblies;

[0331] Table 9: FLMB sequences for detecting Poria cocos with SNP sites at the 15th base G of sequence 5'-GAACCCTCAACTCCGTCCGCCTTTGTTGGG-3' and SNP sites at the 15th base A of sequence 5'-GAACATGTTTAACCACATCGGGACCGTGGT-3'

[0332]

[0333] The design of the remaining primers and probes is the same as in Experiment Example 9.

[0334] DNA extraction;

[0335] Samples to be tested: Rehmannia glutinosa, Liuwei Dihuang Pills, and Liuwei Dihuang Pills (negative sample lacking Rehmannia glutinosa) were extracted according to step (B) in Experimental Example 2.

[0336] Probe ligation and PCR amplification;

[0337] In addition, 0.1 μL of the Poria cocos detection template FLMB (10 μM) shown in SEQ ID NO.34 was added to the ligation reaction system, and the rest were the same as in Experiment 9.

[0338] Test results;

[0339] The PCR products were taken and electrophoresis was performed using the method in Example 9. The Rehmannia glutinosa and Liuwei Dihuang pill samples showed a band at approximately 117bp, while Liuwei Dihuang pill (a negative sample lacking Rehmannia glutinosa) and blank water showed no band at 117bp, indicating that the Rehmannia glutinosa and Liuwei Dihuang pill samples contained Rehmannia glutinosa DNA.

[0340] Experimental Example 12:

[0341] The specific process for detecting Astragalus membranaceus using the PCR detection method described above is as follows:

[0342] Synthesize primer assemblies and ligation probe assemblies;

[0343] The following probes, primers, and control templates were designed and synthesized to detect Astragalus membranaceus products containing Astragalus membranaceus, targeting the SNP site at position 15 (C) of sequence 5'-ACCCATCATGTGTACGCTCCCCATAATATG-3' and the internal reference SNP site at position 12 (A) of sequence 5'-GGTGTCCTAAGATGAGCTCAACGA-3'.

[0344] Table 10: Sequences of the linker probes, primers, and control templates for the SNP site of Astragalus membranaceus: 5'-ACCCATCATGTGTACGCTCCCCATAATATG-3' (base C at position 15) and the internal reference SNP site (base A at position 12) of the sequence 5'-GGTGTCCTAAGATGAGCTCAACGA-3'.

[0345]

[0346] The DNA extraction from Astragalus membranaceus was performed using the same steps as in Experiment 1 (II).

[0347] Probe ligation and PCR amplification;

[0348] Take the DNA sample template solution extracted in step (II) and prepare the ligation reaction solution according to the following system: 0.2 μL template DNA, 7.9 μL ddH2O, 0.5 μL 9N DNALigase (40 U / μL) (Harbin Xinhai Gene Testing Co., Ltd., model D3003), 1 μL 10×9N DNALigase Buffer, and 0.1 μL each of the two left probes LLP (10 μM) shown in SEQ ID NO.37 and SEQ ID NO.38 and the two right probes RLP (10 μM) shown in SEQ ID NO.39 and SEQ ID NO.40.

[0349] The ligation reaction procedure is as follows: pre-denaturation at 95°C for 60 seconds; denaturation at 95°C for 10 seconds; ligation reaction at 60°C for 60 minutes to obtain the ligation product for later use.

[0350] Prepare the PCR reaction solution according to the following system: Take 5 μL of ligation product, 0.1 μL of Accurate Taq HSDNA polymerase (5 U / μL) (Hunan Aike Rui Biotechnology Co., Ltd., model AG11206), 2 μL of 10×Taq PCR Buffer, 1 μL of 50 mM MgCl2, 0.4 μL of 10 mM dNTP Mix, 9.1 μL of ddH2O, and 0.8 μL each of the two forward primers FP (10 μM) shown in SEQ ID NO.41 and SEQ ID NO.42 and the reverse primer RP (10 μM) shown in SEQ ID NO.43.

[0351] Perform PCR amplification according to the following procedure: pre-denaturation at 95℃ for 5 minutes; denaturation at 95℃ for 10 seconds; annealing and extension at 65℃ for 30 seconds; repeat the above two-step reaction of denaturation and annealing for a total of 40 times.

[0352] Test results;

[0353] The results were obtained by digital PCR: the specific copy number A of Astragalus membranaceus was 6841.65, the internal reference copy number B was 6536.36, and A / B×100%=100.46%.

[0354] Experimental Example 13:

[0355] The specific process for detecting Astragalus membranaceus using the PCR detection method described above is as follows:

[0356] Synthesize primer assemblies and ligation probe assemblies;

[0357] Table 1: Ligation probe combinations, primer combinations, and control template sequences for the 15th base C of the SNP site at sequence 5'-ACCCATCATGTGTACGCTCCCCATAATATG-3'

[0358] name Serial Number Nucleic acid sequence Forward primer FP SEQ ID NO.44 5'-GGGTTCGCTAAGGGTTGTGA-3' Detection probe SEQ ID NO.45 5'FAM-ACTCTCCATCTGCGGTCT-MGB-3'

[0359] The other primers and probes are the same as in Experiment 1.

[0360] DNA extraction was performed using the same method as step (ii) of Experiment 1.

[0361] Probe ligation and PCR amplification;

[0362] The probe-connection reaction was the same as in Experiment 1.

[0363] PCR reaction system: Take 5 μL of each ligation product, 0.1 μL of Accurate Taq HSDNA polymerase (5 U / μL) (Hunan Aike Rui Biotechnology Co., Ltd., model AG11206), 2 μL of 10×Taq PCR Buffer, 1 μL of 50 mM MgCl2, 0.4 μL of 10 mM dNTP Mix, 9.1 μL of ddH2O, and 0.8 μL each of the forward primer FP (10 μM) shown in SEQ ID NO.44, the reverse primer RP (10 μM) shown in SEQ ID NO.5, and the detection probe (10 μM) shown in SEQ ID NO.45.

[0364] Perform PCR amplification according to the following procedure: pre-denaturation at 95℃ for 5 minutes; denaturation at 95℃ for 10 seconds; annealing and extension at 60℃ for 30 seconds; repeat the above two-step reaction of denaturation and annealing for a total of 40 times.

[0365] (4) Test results

[0366] The results of the quantitative real-time PCR test were as follows: the Ct value of Astragalus membranaceus slices was 22.175, the Ct value of Zhenqi Fuzheng granules was 24.677, and the Ct value of Guhan Yangshengjing was 28.247, indicating that Astragalus DNA could be detected in all three samples.

[0367] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. 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 PCR detection method, characterized in that, Includes the following steps: Step 1: Design ligation probe and primer combinations targeting the known mutation site or its complementary sequence; The known mutation site is set to at least one SNP site; When there is only one SNP site, the ligation probe combination consists of a left probe LLP and a right probe RLP, and the primer combination consists of a forward primer FP and a reverse primer RP. When there are two or more SNP sites, the ligation probe combination consists of a left probe LLP, a middle probe MLP and a right probe RLP, and the primer combination consists of a forward primer FP and two or more reverse primers RP. The left probe LLP has two parts, the left extension sequence X and the hybridization sequence M1, from the 5' end to the 3' end. The hybridization sequence M1 of the left probe is the same as the upstream sequence adjacent to the SNP1 site. The 5' to 3' ends of the intermediate probe MLP1 are the hybridization sequence N1, the linking sequence Z, and the hybridization sequence M2, respectively. The hybridization sequence N1 is the same as the downstream sequence adjacent to the SNP1 site, and the hybridization sequence M2 is the same as the upstream sequence adjacent to the SNP2 site. The SNP1 site is located at the 3' end of the left probe hybridization sequence M1 or the 5' end of the intermediate probe hybridization sequence N1. The design of other intermediate probes follows the same pattern. The hybridization sequence N is the same as the downstream sequence adjacent to the previous SNP site, and the hybridization sequence M is the same as the upstream sequence adjacent to the next SNP site. The right probe RLP has two parts, the hybridization sequence Nn and the right extension sequence Y, from the 5' end to the 3' end. The hybridization sequence Nn of the right probe is the same as the downstream sequence adjacent to the SNPn site. The SNPn site is located at the 3' end of the hybridization sequence Mn of the intermediate probe or the 5' end of the hybridization sequence Nn of the right probe. All hybridization sequences are 5-18 nucleotides in length; The 5' ends of all intermediate probes MLP and right probe RLP were phosphorylated. The forward primer FP has two parts, the extended sequence E and the sequence XM, from its 5' end to its 3' end, or it may only have the sequence XM; the sequence XM is partially or completely identical to the left probe LLP. The reverse primer RPn has two parts, the extension sequence Fc and the complementary sequence YcNc, from the 5' end to the 3' end, or only the complementary sequence YcNc. The complementary sequence YcNc is complementary to part or all of the sequence of the right probe RLP. Simultaneously, for each intermediate probe, a reverse primer is synthesized, namely RP1, RP2 to RPn-1, whose sequence is complementary to part or all of the sequence of the intermediate probe; Step 2: Extract nucleic acid from the sample to be tested, and react it with the ligation probe combination, primer combination, DNA polymerase, and DNA ligase to obtain PCR products; Step 3: Detect the PCR products.

2. The PCR detection method according to claim 1, characterized in that, The specific process for obtaining PCR products in step two is as follows: S2.11 Extract nucleic acid from the sample to be tested, add ligation probe combination and DNA ligase, and prepare ligation reaction system; S2.

12. Heating at 90-98℃ for at least 5 seconds to denature, followed by a linkage reaction for several minutes; repeating the heating denaturation and linkage reaction one to several times, or not repeating, to obtain the linkage product; S2.

13. Take the ligation product, add the primer combination and DNA polymerase, prepare the reaction system, perform PCR reaction, and obtain the PCR product.

3. The PCR detection method according to claim 1, characterized in that, The specific process for obtaining PCR products in step two is as follows: S2.21 Extract nucleic acid from the sample to be tested, add the ligation probe assembly, heat at 90-98℃ for no less than 5 seconds, and then cool. S2.

22. Add primer combination, DNA ligase, and DNA polymerase to prepare the reaction system. After the ligation reaction takes several minutes, heat at no less than 90°C for at least 5 seconds, and then continue the PCR reaction to obtain the PCR product.

4. The PCR detection method according to claim 2, characterized in that, For SNPs with two or more mutation sites, the parameters for step two during the PCR reaction are set as follows: S2.

13. Take the ligation product, add the forward primer, reverse primer RP1 and DNA polymerase to prepare PCR reaction system 1; take the ligation product, add the intermediate probe MLP1, reverse primer RP2 and DNA polymerase to prepare PCR reaction system 2; and so on up to the intermediate probe MLPn-1 and reverse primer RPn and DNA polymerase to prepare PCR reaction system n; after each reaction system is PCR reacted separately, the PCR reaction solutions are combined and the PCR reaction is continued to obtain the PCR product.

5. The PCR detection method according to any one of claims 1-4, characterized in that, The melting temperature T of the left probe LLP and the right probe RLP mentioned in step one m Not lower than 65℃; The melting temperature T of the portion of sequence XM in the forward primer FP that is identical to the portion of sequence LLP in the left probe. m Not lower than 65℃; The melting temperature T of the complementary sequence YcNc in the reverse primer RP and the complementary portion of the right probe RLP sequence. m No lower than 65℃.

6. The PCR detection method according to claim 5, characterized in that, In step two, when performing the PCR reaction, the reaction is first cycled at a lower annealing temperature at least once, and then cycled at an annealing temperature at a higher temperature of 3°C or more at least once.

7. The PCR detection method according to claim 1, characterized in that, The mutation sites are two or more SNP sites; in step one, for each SNP site, a corresponding linker template LMB is designed and synthesized, whose sequence is complementary to the target sequence containing the SNP site. Step two involves adding 1 to n-1 linker templates (LMBs) to the linker reaction system.

8. The PCR detection method according to claim 1, characterized in that, In step three, the PCR products are detected by electrophoresis. The electrophoresis method is set to any one of agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis.

9. The PCR detection method according to claim 1, characterized in that, Step two involves adding a fluorescent dye to the PCR reaction system, wherein the fluorescent dye is any one of SYBR Green I, EvaGreen, LC Green, or SolisGreen; Step three involves detecting the PCR products using the fluorescence signal as an indicator.

10. The PCR detection method according to claim 1, characterized in that, In step one, one of the forward primers FP or the reverse primer RP is used. During the design and synthesis, a fluorescent group and a quenching group are also attached. In step three, the PCR product is detected using the fluorescence signal as an indicator.

11. The PCR detection method according to claim 1, characterized in that, In step one, the forward primer FP is designed and synthesized with a fluorescent group attached to its 5' end; at the same time, a quenching primer QP is synthesized, with a quenching group attached to its 3' end, and its sequence is complementary to the 5' end of the forward primer FP. In step two, the quenching primer QP is added to the PCR reaction system. In step three, the PCR product is detected using fluorescence signal as an indicator.

12. The PCR detection method according to claim 1, characterized in that, Step one also includes designing and synthesizing a TaqMan fluorescent probe, the sequence of which is partially the same as the left probe LLP and is located downstream of the forward primer. The TaqMan fluorescent probe is connected to a fluorescent group and a quenching group. Step two involves adding the TaqMan fluorescent probe to the PCR reaction system. Step three involves detecting the PCR product using the fluorescence signal as an indicator.

13. The PCR detection method according to claim 1, characterized in that, The primer combination described in step one also includes the tag primer TagEXc and the detection template MB; The 5' to 3' ends of the tag primer TagEXc are the tag sequence Tag and the sequence EXc, respectively. The sequence EXc is partially or completely complementary to the extended sequence EX of the forward primer FP. The 5' to 3' ends of the detection template MB are respectively the probe sequence T and the extension sequence G. The probe sequence T is connected to a fluorescent group and a quenching group, and the extension sequence G contains a sequence complementary to the tag sequence Tag. Step two involves adding the tag primer TagEXc and the detection template MB to the PCR reaction system. Step three involves detecting the PCR product using fluorescence signals as an indicator.

14. A kit comprising the ligation probe combination and primer combination used in the PCR detection method according to any one of claims 1-13.

15. The PCR detection method according to any one of claims 1-13 is used for detecting mutation sites.

16. The use according to claim 15, characterized in that, Includes the following steps: Step 1: Select specific SNP sites and perform fluorescence detection using the PCR detection method described in any one of claims 9-13 to obtain the copy number A; Step 2: Select a conserved sequence as an internal reference sequence, take any base site within the internal reference sequence as a SNP, and perform fluorescence detection using the PCR detection method described in any one of claims 9-13 to obtain the copy number B of the internal reference sequence; Step 3: Calculate the ratio R of A / B.

17. The use according to claim 15 or 16, characterized in that, The specific SNP site includes any one of the following: (1) The SNP site is the 15th base C of the sequence 5'-ACCCATCATGTGTACGCTCCCCATAATATG-3', which is used for the detection of products containing Astragalus membranaceus and / or Astragalus membranaceus DNA. (2) The SNP site is the 15th base G in the sequence 5'-GAACCCTCAACTCCGTCCGCCTTTGTTGGG-3', which is used for the detection of products containing Poria cocos DNA. (3) The SNP site is one of the 16th base G and the 20th base C in the sequence 5'-TAACAATACCGGGCTGATTCAGTCTGGTAATTGG-3', which is used for the detection of products containing ginseng DNA; (4) The SNP site is the 16th base T in the sequence 5'-CCAACCCATCACTCCTTTGCGGGAGTCGAG-3', which is used for the detection of products containing American ginseng DNA; (5) The SNP site is the 15th base A in the sequence 5'-ACGACCCGCGAACAAGTTACAATACCGGGT-3', which is used for the detection of products containing Panax notoginseng DNA. (6) The SNP site is the 16th base C of the sequence 5'-TTCAGTGCGCCCCCGCCGGCCCGGAGACGG-3', which is used for the detection of products containing licorice DNA. (7) The SNP site is one of the 13th base C, 14th base A, or 15th base A in the sequence 5'-ACCGTTGCCCGACAACAATTGCCTCGCGAT-3', and is used for the detection of DNA products containing Glycyrrhiza glabra and / or Glycyrrhiza inflata. (8) The SNP site is the 15th base A in the sequence 5'-GAACATGTTTAACCACATCGGGACCGTGGT-3', which is used for the detection of products containing Rehmannia glutinosa DNA; (9) The SNP site is the 15th base G in the sequence 5'-TTGAATTGTACGTCGGCATCCCGTTAGCGG-3', which is used for the detection of products containing Angelica sinensis DNA; (10) The SNP site is the 15th base T in the sequence 5'-CGGGCATCGGCGTCTTTCCAAAATACAACG-3', which is used for the detection of products containing Angelica dahurica DNA; (11) The SNP site is one of the 16th base C and the 17th base A in the sequence 5'-CTGTCGTGCGTGCCGCAGTCGCCAGCGCGA-3', which is used for the detection of products containing Cornus officinalis DNA; (12) The SNP site is one of the 16th base C and the 17th base C of the sequence 5'-GTGACCACCTGTGGGCCTTCCCTTTGGCAT-3', which is used for the detection of products containing Codonopsis pilosula DNA; (13) The SNP site is the 16th base T in the sequence 5'-TAATGGCCCTCGTATTGAGCCGTGCGTCGC-3', which is used for the detection of products containing Atractylodes macrocephala DNA. (14) The SNP site is one of the 16th base C and the 17th base A in the sequence 5'-GACTCCCGACGATCGCACCGTGGGGCGGCG-3', which is used for the detection of products containing Pinellia ternata DNA; (15) The SNP site is one of the 16th base G and the 19th base A in the sequence 5'-GGTGTGTGTCTGGGGGTTAAGGCGTGTCTTGA-3', which is used for the detection of products containing Forsythia DNA; (16) The SNP site is the 15th base A in the sequence 5'-CCTCGTGCGGGCCCATCCTGGCCCATTAAC-3', which is used for the detection of products containing Sophora flavescens DNA. (17) The SNP site is one of the 15th T and 16th T bases in the sequence 5'-CGAGGCCTTTTAGGTTGAGGGCACATCTGCT-3', which is used for the detection of products containing Houttuynia cordata DNA; (18) The SNP site is one of the 15th base G or the 16th base A in the sequence 5'-CCGAAAACAGAGACGACGGCAACGGACGTC-3', which is used for the detection of products containing Eucommia ulmoides DNA; (19) The SNP site is the 16th base T in the sequence 5'-GAAAACCAAAAGAGATCGTCCCCCCTCCGT-3', which is used for the detection of products containing Scutellaria baicalensis DNA; (20) The SNP site is one of the 15th base A and the 16th base G in the sequence 5'-GCCCCCGTTGCCCCAGCTTTGGGATGCGCG-3', which is used for the detection of products containing peony DNA; (21) The SNP site is the 15th base C of the sequence 5'-CGTGAGCCTCTCCTCCATCCCATGTCCGGT-3', which is used to detect products containing Paeonia lactiflora DNA. (22) The SNP site is the 15th base G in the sequence 5'-AATGCTCGGGCTGAGGGAAGGCGTGAGCCT-3', which is used for the detection of products containing peony DNA. (23) The SNP site is the 15th base T in the sequence 5'-AGTATAAACCCAATTCACTGGATCCTTTGC-3', which is used for the detection of products containing Alisma plantago-aquatica DNA; (24) The SNP site is the 15th base G in the sequence 5'-CTCGCGTGCTGTCGGGCGCCAAGGCGTCGT-3', which is used for the detection of products containing Scutellaria barbata DNA. (25) The SNP site is the 15th base G in the sequence 5'-TTATCGTGAATTCTGCGATCATTACATATA-3', which is used for the detection of products containing yam DNA; (26) The SNP site is one of the 13th base T and the 16th base C in the sequence 5'-CTCACTAAACACTCCCGAATTTCCAATATC-3', which is used for the detection of products containing donkey DNA; (27) The SNP site is the 15th base G in the sequence 5'-CCGACTCACTATCAGCACCCCTACTAGTTC-3', which is used for the detection of products containing pig DNA. (28) The SNP site is the 15th base T in the sequence 5'-GGAATCCCATCCGATATGGACAAAATCCCA-3', which is used for the detection of products containing horse DNA; (29) The SNP site is the 15th base T in the sequence 5'-TTAGAAAGCCAAATTTCAGGATACTGTTCT-3', which is used to detect products containing cattle or zebu DNA. (30) The SNP site is the 15th base A of the sequence 5'-TTATTCTACCCACCATTATAGCAATCACAG-3', which is used for the detection of products containing sika deer DNA. (31) The SNP site is the 15th base G in the sequence 5'-TGATGATATGGACGGACAGACGCCAACACAGCAG-3', which is used for the detection of products containing sheep DNA. (32) The SNP site is the 15th base C of the sequence 5'-TATCCGCTCTCCTACTAACATCCGGCCTCA-3', which is used for the detection of products containing goat DNA; (33) The SNP site is the 15th base C of the sequence 5'-TACTATCTGTCCGACAAAGCGGTCTCAACTC-3', which is used to detect products containing Lonicera grahami DNA. (34) The SNP site is the 15th base T in the sequence 5'-AAATGAGGATGCAGTGTCGGGAATGGTCGG-3', which is used for the detection of products containing Lonicera japonica DNA from South China. (35) The SNP site is the 16th base T in the sequence 5'-CGTGTACAAATGAACTTCTTTGAGTAAGGAATC-3', which is used to detect products containing Lonicera japonica DNA. (36) The SNP site is the 15th base A of the sequence 5'-CGGGTCCTGATTGTATTTTGGGCTTTCATTT-3', which is used for the detection of products containing honeysuckle DNA. (37) The SNP site is the 15th base A in the sequence 5'-GCGCACGTCCTGCGATGGGTTGGCTCTCGTG-3', which is used for the detection of products containing Codonopsis pilosula or Codonopsis pilosula DNA. (38) The SNP site is one of the 15th base C and the 16th base A in the sequence 5'-CCTGAGCGATGGTGCAGGCTCTCGTGACCCTG-3', which is used for the detection of products containing Platycodon grandiflorus DNA; (39) The SNP site is one of the 16th base T, 17th base T, 18th base C, or 19th base T in the sequence 5'-GGGATTACCTACTCTTTCTTAACGGTCAAAGCGA-3', and is used for the detection of products containing Bupleurum chinense DNA. (40) The SNP site is the 16th base T in the sequence 5'-TACTCTGTGAGCAACTGCGACCCTTTGGCGC-3', which is used for the detection of products containing Bupleurum chinense DNA. (41) The SNP site is one of the 16th base T and the 17th base A in the sequence 5'-GGTGGAAGGCACTACTAACCTCTTGCCATCTTG-3', which is used to detect products containing Bupleurum chinense DNA; (42) The SNP site is the 16th base C of the sequence 5'-CCGGCTCAAGTAGGTCCACCAACTAAATAAAG-3', which is used for the detection of products containing Danshen DNA; (43) The SNP site is one of the 16th base C and the 17th base A in the sequence 5'-GGGGATGCGTTCCATCAGGGGCGGAGACTGGTC-3', which is used for the detection of products containing Costus Root DNA; (44) The SNP site is the 16th base A in the sequence 5'-ACTTTAAGACGGCCTAGTGTCATGTTGCCCC-3', which is used for the detection of products containing *Inula japonica* DNA. (45) The SNP site is one of the 15th base T and the 16th base G in the sequence 5'-TGGTCTCCCGTGCCTGCGGTGTGGTTGGCC-3', which is used for the detection of products containing Sichuan costus DNA; (46) The SNP site is the 15th base A in the sequence 5'-TCGGGTGCCCATCGACCGACGAAACCAACC-3', which is used for the detection of products containing Notopterygium incisum DNA; (47) The SNP site is the 15th base T in the sequence 5'-GTGCCTTGCGGCGCTGCTGGCCCAAAAGCG-3', which is used for the detection of products containing Notopterygium incisum DNA. (48) The SNP site is the 15th base T of the sequence 5'-CTACTTGGCCCCATTTTGGTGGCCGACTGA-3', which is used for the detection of products containing Amomum villosum DNA. (49) The SNP site is the 16th base C of the sequence 5'-TTTATTTCCAATTCACTTTCAATCTAAAATAGA-3', which is used for the detection of products containing safflower DNA. (50) The SNP site is one of the 16th base C and the 19th base G in the sequence 5'-CGCCAAGGAACACTTCTTGGAAACGCCGTCGCGGCC-3', which is used for the detection of products containing saffron DNA; (51) The SNP site is the 15th base G in the sequence 5'-GCCGCGCGGTGCAAGCCCGTGGGACCATAC-3', which is used for the detection of products containing cinnamon DNA. (52) The SNP site is one of the 16th T and 17th T bases in the sequence 5'-CCCCCAGGGCTGTCCTTGGACGGCGCCCAC-3', which is used for the detection of products containing Corydalis DNA; (53) The SNP site is one of the 15th base G or the 18th base C in the sequence 5'-GATAGTACCCATTTGGGCAACGTCCAGTGCCAATG-3', which is used for the detection of products containing Aristolochia DNA; (54) The SNP site is the 18th base G in the sequence 5'-CAAGGATTTGGAATTACGTGTATGCCATTCTCATC-3', which is used for the detection of products containing Asarum DNA; (55) The SNP site is one of the 19th base T and the 20th base G in the sequence 5'-CGCCAGTCTTAGTGTATATGAGTAGGAGTTATTGAAAAAT-3', which is used for the detection of products containing Gelsemium DNA; (56) The SNP site is the 16th base T in the sequence 5'-TAAGACCCCGGTCTTTTTGTATAGGAGTTATTGAAA-3', which is used for the detection of products containing Strychnos nucifera DNA. (57) The SNP site is one of the 15th base T and the 16th base C in the sequence 5'-ATCCTCCAAAGACATCAAGACGCGTCGTCCTC-3', which is used for the detection of products containing Aconitum DNA; (58) The SNP site is the 16th base C of the sequence 5'-CCATGGATGGGAACTCATGATTGGCTCTGTCT-3', which is used for the detection of products containing Corydalis DNA; (59) The SNP site is the 15th base T in the sequence 5'-TGTGGATCGGCCTTTGGTGTGATAATTGTCTA-3', which is used for the detection of products containing Poria cocos DNA. (60) The SNP site is the 16th base C of the sequence 5'-GAAAAGACTAGTTGACAAGGCTTCTATGTTC-3', which is used for the detection of products containing shiitake mushroom DNA; (61) The SNP site is the 15th base T in the sequence 5'-AGGCATGTGCACGCTCTGCTCATCCACTCT-3', which is used for the detection of products containing Yunzhi DNA. (62) The SNP site is one of the 14th base T and the 15th base G in the sequence 5'-TTCTAACGGTCTCTGTATGGAGACAAAGCT-3', which is used for the detection of products containing Ganoderma lucidum DNA; (63) The SNP site is the 16th base T in the sequence 5'-CAAGAACACTAATGATCAATACCCGAAAAACC-3', which is used for the detection of products containing red deer DNA. (64) The SNP site is the 16th base G in the sequence 5'-ACAAGGCATCCCCCTGTCATCAGGCCTAATCC-3', which is used for the detection of products containing deer DNA.

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