Digital PCR (Polymerase Chain Reaction) detection method and kit for detecting ginseng contained in product

Abstract

The invention belongs to the field of quality detection of traditional Chinese medicines, and particularly relates to a digital PCR (Polymerase Chain Reaction) detection method and a kit for detecting ginseng contained in a product. The invention discloses a novel specific SNP (Single Nucleotide Polymorphism) site capable of being used for ginseng detection, wherein the SNP site is that a 5896th basic group of NO.KM036296.1 in Genebank is G. Primers and / or probes are designed according to the specific SNP sites, a probe method or a fluorescent dye method reaction system is configured, a digital PCR method is adopted for detection, the copy number A of a specific DNA sequence representing ginseng is obtained, meanwhile, the copy number B of an internal reference sequence is obtained, and the internal reference sequence calculation result is obtained by calculating the ratio of A to B and used for quality control of ginseng related products. The method provided by the invention can be used for ginseng medicinal materials, ginseng decoction pieces, ginseng formula granules, ginseng extracts, traditional Chinese medicine preparations containing ginseng, foods containing ginseng, health foods, beverages and other products, and can also be used for detecting ginseng leaves, ginseng fruits and the like.

Description

Technical Field

[0001] The invention belongs to the field of quality detection of traditional Chinese medicines, and in particular relates to a digital PCR detection method and a kit for detecting ginseng contained in a product. Background Art

[0002] Ginseng (Latin name: Panax ginseng CAMey.) is a perennial herbaceous plant of the genus Panax in the Araliaceae family and is a famous precious medicinal material. Due to its high price, ginseng is often impersonated or adulterated by unscrupulous merchants with other raw materials. Traditional identification methods (character identification, microscopic identification, physical and chemical identification, etc.) all have certain limitations. In many cases, it is difficult to achieve the requirements for accurate identification of the authenticity of medicinal materials, and the quality control of Chinese medicine preparations is even more difficult. Molecular identification is the detection of genetic material DNA, which can accurately identify the origin of medicinal materials and is one of the effective means of medicinal material quality control. At present, there have been some reports on ginseng molecular identification methods, but they are mainly used for molecular identification of ginseng medicinal materials or powders. Ginseng extraction and processing products (such as formula granules, extracts, compound preparations containing ginseng, etc.) are difficult to detect using molecular identification methods due to the serious degradation of DNA during the extraction process and the low DNA content in the products.

[0003] To molecularly identify ginseng, we must first find specific DNA fragments or SNP sites that can represent the ginseng genome. The specific SNP sites reported for ginseng are G and C at the 496th and 500th bases of Genebank NO.KM036296.1, respectively, located in the 18SRNA gene. The current molecular identification method for ginseng mainly uses software to design primers and probes for this SNP site. The forward primer is 5'-CACGGGGAGGTAGTGACAATA-3', the reverse primer is 5'AGACTTGCCCTCCAATGGAT-3', and the probe is FAM-CGGGCTGATTCAGTCT-MGB (patent application number: 201510479788.3). The 5' of the probe is 11 bp away from the 3' of the forward primer. The DNA fragment required for detection is relatively long, and the amplified product is 107 bp, which is not suitable for the detection of severely degraded DNA products. Summary of the invention

[0004] The present invention aims to solve the above-mentioned problem and proposes a digital PCR detection method and a kit for detecting ginseng contained in products with severely degraded DNA.

[0005] In order to achieve the above object, the technical solution provided by the present invention is:

[0006] In a first aspect, the present invention has discovered a specific SNP site for ginseng detection, wherein the SNP site is the 5896th base of NO.KM036296.1 in Genebank, which is G.

[0007] In the second aspect, the present invention designs probes according to one or two or three SNP sites in Genebank NO.KM036296.1 for ginseng detection, wherein the 5896th base is G, the 496th base is G and the 500th base is C as follows:

[0008] The probe is a fluorescent probe, and its nucleotide sequence is:

[0009] 5'-CTGATTCAGTCTGGTAATT-3'(SEQ ID NO1)

[0010] 5'-TCTGGTAATTGGAATGAG-3'(SEQ ID NO2)

[0011] 5'-ATGCCCGAGTGAAAGTGAC-3'(SEQ ID NO3)

[0012] 5'-TGAAAGTGACAAAGTCGC-3'(SEQ ID NO4)

[0013] 5'-AGACTGAATCAGCCCGGTAT-3'(SEQ ID NO5)

[0014] 5'-CTGAATCAGCCCGGTATTG-3'(SEQ ID NO6)

[0015] 5'-CCCGGTATTGTTATTTAT-3'(SEQ ID NO7)

[0016] 5'-TCACTCGGGCATTAGTATC-3'(SEQ ID NO8)

[0017] 5'-ATTAGTATCGGTGAACCTC-3'(SEQ ID NO9)

[0018] Further, according to one, two or three SNP sites among the specific SNP sites for ginseng detection (base 5896 is G, base 496 is G and base 500 is C), primers are designed as follows:

[0019] The nucleotide sequence of the forward primer is:

[0020] 5'-GACAATAAATAACAATACCGG-3'(SEQ ID NO10)

[0021] 5'-TAACAATACCGGGCTGATAC-3'(SEQ ID NO11)

[0022] 5'-CGTCGAGGTTCACCGATACT-3'(SEQ ID NO12)

[0023] 5'-TTCACCGATACTAATGCCGG-3'(SEQ ID NO13)

[0024] 5'-TTGTACTCATTCCAATTAC-3'(SEQ ID NO14)

[0025] 5'-GTACTCATTCCAATTACCAG-3'(SEQ ID NO15)

[0026] 5'-TCCAATTACCAGACTGAACC-3'(SEQ ID NO16)

[0027] 5'-CAGTCGGCGACTTTGTCACT-3'(SEQ ID NO17)

[0028] 5'-CGACTTTGTCACTTTCACCC-3'(SEQ ID NO18)

[0029] The nucleotide sequence of the reverse primer is:

[0030] 5'-CTCATTCCAATTACCAGACT-3'(SEQ ID NO19)

[0031] 5'-GCGACTTTGTCACTTTCACT-3'(SEQ ID NO20)

[0032] 5'-ATAAATAACAATACCGGGCT-3'(SEQ ID NO21)

[0033] 5'-GTTCACCGATACTAATGCC-3'(SEQ ID NO22)

[0034] In a third aspect, the present invention provides a digital PCR detection method for detecting ginseng in a product, comprising the following steps:

[0035] Step 1: Extract DNA from the product to be tested as the detection DNA.

[0036] Step 2: Design primers or primers or primers and probes according to one or two or three SNP sites in Genebank NO.KM036296.1 for ginseng detection, in which the 5896th base is G, the 496th base is G and the 500th base is C.

[0037] Step 3: Take the detection DNA in step 1, add the primers or primers and probes designed in step 2, configure the reaction system, and use the digital PCR method for detection to obtain the copy number A of the specific DNA sequence representing ginseng.

[0038] Step 4: Select the conserved sequence in NO.KM036296.1 in Genebank as the internal reference sequence, and design PCR primers, or primers and probes that can detect this internal reference sequence, so that the length of the amplified fragment is the same or similar to the length of the fragment amplified in step 3, and the length difference does not exceed 5bp.

[0039] Step 5: Take the detection DNA in step 1, the PCR primers designed in step 4, or the primers and probes, add them into the configured reaction system, and use the digital PCR method for detection to obtain the copy number B of the internal reference sequence.

[0040] Step 6: Calculate the A / B ratio to obtain the result calculated with the internal reference sequence for product quality control.

[0041] The internal reference sequence is a conserved sequence common to most species, a nucleotide fragment in a DNA molecule that remains essentially unchanged during evolution.

[0042] Furthermore, the reaction system configured in step 3 and step 5 includes a probe method or fluorescent dye method reaction system, the probe method reaction system includes a combination of a forward primer and a fluorescent probe, and a reverse primer; the fluorescent dye method reaction system includes a forward and reverse primer pair, and a fluorescent dye.

[0043] The forward primer and fluorescent probe included in the probe reaction system are one of the following combinations:

[0044] The nucleotide sequences of the combination 1 are: forward primer (SEQ ID NO10) and probe (SEQ ID NO1);

[0045] The nucleotide sequence of combination 2 is: forward primer (SEQ ID NO11) and probe (SEQ ID NO2);

[0046] The nucleotide sequence of combination 3 is: forward primer (SEQ ID NO12) and probe (SEQ ID NO3);

[0047] The nucleotide sequence of combination 4 is: forward primer (SEQ ID NO13) and probe (SEQ ID NO4);

[0048] The nucleotide sequence of combination 5 is: forward primer (SEQ ID NO14) and probe (SEQ ID NO5);

[0049] The nucleotide sequence of combination 6 is: forward primer (SEQ ID NO15) and probe (SEQ ID NO6);

[0050] The nucleotide sequence of combination 7 is: forward primer (SEQ ID NO16) and probe (SEQ ID NO7);

[0051] The nucleotide sequence of the combination 8 is: forward primer (SEQ ID NO17) and probe (SEQ ID NO8);

[0052] The nucleotide sequence of the combination 9 is: forward primer (SEQ ID NO18) and probe (SEQ ID NO9);

[0053] The primer pair included in the fluorescent dye method system is one of the following:

[0054] Primer pair 1: forward primer (SEQ ID NO11) and reverse primer (SEQ ID NO19);

[0055] Primer pair 2: forward primer (SEQ ID NO 13) and reverse primer (SEQ ID NO 20);

[0056] Primer pair 3: forward primer (SEQ ID NO 16) and reverse primer (SEQ ID NO 21);

[0057] Primer pair 4: forward primer (SEQ ID NO: 18) and reverse primer (SEQ ID NO: 22).

[0058] Furthermore, in step 4: the length of the amplified fragment is made the same or similar to the length of the amplified fragment in step 3, and the length difference does not exceed 3 bp, preferably does not exceed 1 bp.

[0059] Furthermore, the fluorescent probe sequence in the probe method reaction system keeps the 5' end sequence unchanged, deletes 1 to several bases from the 3' end, shortens the probe length to 10 to 19 bp, preferably shortens the probe length to 13 to 15 bp, and the bases in the probe sequence are modified with 1 to several locked nucleic acids (LNA) or peptide nucleic acids (PNA);

[0060] The forward primer and / or reverse primer sequence in the fluorescent dye method or probe method reaction system keeps the 3' end sequence unchanged, deletes 1 to several bases from the 5' end, shortens the primer length to 12 to 19 bp, preferably shortens the primer length to 15 to 18 bp, and the bases in the primer sequence can be modified with 1 to several locked nucleic acids (LNA) or peptide nucleic acids (PNA).

[0061] Furthermore, the step 1 is: extracting DNA from the product to be detected, then using an enzyme to add a DNA linker to the 3' end and / or 5' end of the double-stranded or single-stranded DNA as the detection DNA, and designing a reverse primer using the DNA linker sequence as a template.

[0062] Furthermore, the step 1 is: extracting DNA from the product to be detected, breaking the DNA into fragments of 20 to 200 bp in size, preferably breaking it into fragments of 30 to 100 bp in size, and more preferably breaking it into fragments of 40 to 60 bp in size, and then using an enzyme to add a DNA linker to the 3' end and / or 5' end of the double-stranded or single-stranded DNA as the detection DNA, and designing a reverse primer using the DNA linker sequence as a template.

[0063] In a fourth aspect, the above-mentioned digital PCR detection method for detecting ginseng in products is used in the preparation of a kit for detecting ginseng.

[0064] The present invention has the following beneficial effects:

[0065] (1) The DNA fragment required for the detection of the present invention is very short, and is suitable for the detection of deep-processed products that have been extracted and dried for a long time at high temperature. The DNA template required for the dye method is as short as 30 to 40 bp, and the DNA template required for the probe method does not exceed 65 bp. If the DNA linker method is used, the DNA template required for the probe method can be as short as 40 bp. If the length of the primer and probe is shortened, the required DNA template can be shortened to 22 to 28 bp. This is not available in the methods currently reported in the literature.

[0066] (2) The present invention adopts digital PCR for detection. Digital PCR technology performs amplification independently in thousands to tens of thousands of droplets, which reduces the interference of background and inhibitors on the PCR reaction and greatly improves the sensitivity of detection. Even trace amounts of DNA can be detected. It is suitable for the detection of ginseng deep-processing products with only trace amounts of DNA residues.

[0067] (3) The present invention selects an internal reference sequence as a reference. The length of the internal reference sequence detection fragment is the same as or similar to the length of the ginseng-specific fragment. This can offset the difference caused by DNA degradation during the processing. For products with fixed feed amount and fixed production process, the copy number ratio is also relatively fixed and can be used for product quality control.

[0068] (4) The detection method of the present invention can be used for the detection of ginseng medicinal materials, ginseng decoction pieces, ginseng formula granules, ginseng extracts, ginseng-containing Chinese medicine preparations, ginseng-containing foods, health foods, beverages and other products, and can also be used for the detection of ginseng leaves, ginseng fruits, etc.

[0069] (5) The detection tools used in the detection method of the present invention, such as primers, probes, etc., can be further developed into a kit for detecting ginseng products. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 The 5896th base of the specific SNP site detected for ginseng in Genebank No. KM036296.1 is G;

[0071] Figure 2 The 496th and 500th bases of the specific SNP site NO.KM036296.1 in Genebank for ginseng detection are G and C respectively. DETAILED DESCRIPTION

[0072] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0073] The research team found in the previous study that the probe designed in patent application number: 201510479788.3 is 11 bp away from the 3' of the forward primer. The DNA fragment required for detection is relatively long, and the amplified product is 107 bp, which is not suitable for the detection of severely degraded DNA products.

[0074] The present invention discovered a new specific SNP site for ginseng detection: the 5896th base of NO.KM036296.1 in Genebank is G. Figure 1 shown.

[0075] like Figure 1-2 As shown, according to one or two or three SNP sites in Genebank NO.KM036296.1, in which the 5896th base is G, the 496th base is G and the 500th base is C, primers or primers and probes (i.e., fluorescent probes) are designed as follows:

[0076] The nucleotide sequence of the fluorescent probe is:

[0077] 5'-CTGATTCAGTCTGGTAATT-3'(SEQ ID NO1)

[0078] 5'-TCTGGTAATTGGAATGAG-3'(SEQ ID NO2)

[0079] 5'-ATGCCCGAGTGAAAGTGAC-3'(SEQ ID NO3)

[0080] 5'-TGAAAGTGACAAAGTCGC-3'(SEQ ID NO4)

[0081] 5'-AGACTGAATCAGCCCGGTAT-3'(SEQ ID NO5)

[0082] 5'-CTGAATCAGCCCGGTATTG-3'(SEQ ID NO6)

[0083] 5'-CCCGGTATTGTTATTTAT-3'(SEQ ID NO7)

[0084] 5'-TCACTCGGGCATTAGTATTC-3'(SEQ ID NO8)

[0085] 5'-ATTAGTATCGGTGAACCTC-3'(SEQ ID NO9)

[0086] Read more:

[0087] 5'-GACAATAAATACAATACCGG-3'(SEQ ID NO10)

[0088] 5'-TAACAATACCGGGCTGATAC-3'(SEQ ID NO11)

[0089] 5'-CGTCGAGGTTCACCGATACT-3'(SEQ ID NO12)

[0090] 5'-TTCACCGATACTAATGCCGG-3'(SEQ ID NO13)

[0091] 5'-TTGTACTCATTCCAATTAC-3'(SEQ ID NO14)

[0092] 5'-GTACTCATTCCAATTACCAG-3'(SEQ ID NO15)

[0093] 5'-TCCAATTACCAGACTGAACC-3'(SEQ ID NO16)

[0094] 5'-CAGTCGGCGACTTTGTCACT-3'(SEQ ID NO17)

[0095] 5'-CGACTTTGTCACTTTCACCC-3'(SEQ ID NO18)

[0096] The nucleotide sequence of the reverse primer is:

[0097] 5'-CTCATTCCAATTACCAGACT-3'(SEQ ID NO19)

[0098] 5'-GCGACTTTGTCACTTTCACT-3'(SEQ ID NO20)

[0099] 5'-ATAAATAACAATACCGGGCT-3'(SEQ ID NO21)

[0100] 5'-GTTCACCGATACTAATGCC-3'(SEQ ID NO22)

[0101] Note: The "primers or primers and probes" mentioned above refer to the differences between the fluorescent dye method reaction system and the probe method reaction system, that is, the fluorescent dye method reaction system requires the addition of forward and reverse primers and fluorescent dyes (no fluorescent probes are needed), while the probe method requires the addition of forward and reverse primers and probes. The "primers" before "or" refer to the reaction system for the dye method, and the "primers and probes" after "or" refer to the reaction system for the probe method. Similarly, the "PCR primers, or primers and probes" described in the claims have the same meaning.

[0102] When designing primers and probes in the present invention, the forward primer sequence of the dye method is designed to be adjacent to the reverse primer sequence, and the forward primer and reaction primer sequences of the probe method are designed to be adjacent to the fluorescent probe sequence. Combined with the base modification of locked nucleic acid (LNA) or peptide nucleic acid (PNA), the length of the primers and probes themselves is also shorter, so that the DNA template required for detection is shorter, only 30-65bp is required, and it is suitable for the analysis of ginseng preparation products with severe DNA degradation. The primers and probes used in the prior art require relatively long DNA templates for detection, and can usually only be used for the detection of ginseng medicinal materials, and are difficult to be used for the detection of ginseng components in traditional Chinese medicine preparations.

[0103] The primers and probes designed by the present invention are obtained by artificial design because the DNA template to be detected is short and it is difficult to design them using conventional primer design software. In addition, when designing the primers or probes of the present invention, mismatch design is also performed to improve the specificity of SNP site detection and eliminate nonspecific interference. Combined with the optimization of digital PCR detection conditions, the detection of ginseng DNA in ginseng-containing traditional Chinese medicine preparation samples can be realized.

[0104] Example 1

[0105] Testing of Ginseng Extract

[0106] (1) DNA extraction

[0107] Take 200 mg of ginseng extract, place it in a 1.5 ml centrifuge tube, add 1 ml of water to dissolve, heat at 56°C for 15 min, and centrifuge. Use a DNA extraction kit (magnetic bead method) to extract DNA according to the instructions, and store it at -20°C for later use.

[0108] (2) Primers

[0109] Specific primer pairs for detecting ginseng:

[0110] Forward primer: 5′-TAACAATACCGGGCTGATAC-3′ (SEQ ID NO 11).

[0111] Reverse primer 5'-CTCATTCCAATTACCAGACT-3' (SEQ ID NO 19)

[0112] The length of the fragment amplified using the above-mentioned ginseng-specific primers is 40 bp.

[0113] Internal reference sequence primer pair

[0114] Forward primer 5'-CAGGGATAACTGGCTTGTGG-3'

[0115] Reverse primer 5′-TCGCTATGAACGCTTGGCTG-3′

[0116] The fragment length amplified using the above-mentioned internal reference sequence primers is 40 bp.

[0117] (3) Digital PCR detection conditions

[0118] Using the sample DNA as a template, the ginseng or internal reference sequence primer pair, Evagreen dye, and ddPCR Mix in the above step (2) were added to prepare the reaction system, and the detection was performed using a droplet digital PCR instrument (Beijing Xinyi Biotechnology Co., Ltd.) according to the instrument operating procedures.

[0119] The PCR reaction program was: 95°C for 10 min; 95°C for 15 s, 58°C for 30 s, 40 amplifications; 98°C for 10 min

[0120] (4) Test results

[0121] The copy number of ginseng is 2601.5, the copy number of the internal reference sequence is 2516.4, and the ratio is 103.4%

[0122] Example 2

[0123] Testing of Ginseng Formula Granules

[0124] (1) DNA extraction: Same as Example 1.

[0125] (2) Primers

[0126] Specific primer pairs for detecting ginseng:

[0127] The forward primer was 5'-ATACCGGGCTGATAC-3', and the 11th and 15th bases were modified with LNA.

[0128] The reverse primer was 5'-TCCAATTACCAGACT-3', and the 11th and 15th bases were modified with LNA.

[0129] The length of the fragment amplified using the above-mentioned ginseng-specific primers is 30 bp.

[0130] Internal reference sequence primer pair

[0131] The forward primer was 5'-ATAACTGGCTTGTGG-3', and the 11th and 15th bases were modified with LNA.

[0132] The reverse primer was 5'-ATGAACGCTTGGCTG-3', and the 11th and 15th bases were modified with LNA.

[0133] The length of the fragment amplified using the above-mentioned internal reference sequence primers is 30 bp.

[0134] (3) Digital PCR detection conditions: same as in Example 1.

[0135] (4) Test results

[0136] The copy number of ginseng is 1926.6, the copy number of the internal reference sequence is 1887.4, and the ratio is 102.1%

[0137] Example 3

[0138] Testing of a Chinese medicine compound preparation (pills made from 11 herbs including ginseng and Atractylodes macrocephala)

[0139] (1) DNA extraction: Take 5 g of sample, grind it, add 100 ml of water and let it stand for 30 minutes to dissolve, filter, take the filter residue and use a DNA extraction kit (magnetic bead method) to extract DNA according to the instructions, and store it at -20°C for later use.

[0140] (2) Primers and probes

[0141] The specific forward primer sequence for detecting ginseng is 5'-GACAATAAATAACAATACCGG-3' (SEQ ID NO 10)

[0142] The reverse primer sequence is 5'-GATTTAGATTGTACTCATTC-3'

[0143] The MGB probe sequence is FAM-CTGATTCAGTCTGGTAATT-BHQ1 (SEQ ID NO 1)

[0144] The length of the fragment amplified using the above-mentioned specific primers is 62 bp.

[0145] The forward primer sequence for detecting the internal reference sequence is 5'-AAGGTAGCCAAATGCCTCGT-3'

[0146] The reverse primer sequence is 5'-GGGAATCTCGTTAATCCATT-3'

[0147] The MGB probe sequence is VIC-ATCTAATTAGTGACGCGCAT-BHQ1

[0148] The length of the fragment amplified using the above primers is 62 bp.

[0149] (3) Digital PCR detection conditions

[0150] The sample DNA was used as a template, and the primers and probes in the above step (2) were added to prepare a reaction system. Other conditions were the same as those in Example 1.

[0151] 4. Test results

[0152] The copy number of ginseng is 3541.5, the copy number of the internal reference sequence is 20548.7, and the ratio is 17.23%

[0153] Example 4

[0154] Testing of a Chinese medicine compound preparation (an oral solution made from 11 herbs including ginseng and astragalus)

[0155] (1) DNA extraction: Take 5 ml of sample and use a DNA extraction kit (magnetic bead method) to extract DNA according to the instructions and store at -20°C for later use.

[0156] (2) Adding DNA adapter

[0157] The DNA extracted in step (1) was purified using the Adaptase Module kit according to the instructions.

[0158] (3) Primers and probes

[0159] The primers and probes for detecting ginseng are

[0160] Forward primer 5'-AAATAACAATACCGG-3', where bases 3, 7, and 10 are modified with LNA

[0161] MGB probe FAM-CTGATTCAGTCTG-BHQ1, in which the 3rd, 7th, and 10th bases are modified with LNA

[0162] Reverse primer: 5'ACTGGAGTTCAGACGTGTGC-3'

[0163] Primers and probes for detecting internal reference sequences

[0164] Forward primer 5'-AGCCAAATGCCTCGT-3', in which the 3rd, 7th, and 10th bases are modified with LNA

[0165] MGB probe: VIC-ATCTAATTAGTGA-BHQ1, where the 3rd, 7th, and 10th bases are modified with LNA

[0166] Reverse primer: 5'ACTGGAGTTCAGACGTGTGC-3'

[0167] (4) Digital PCR detection conditions, same as in Example 3

[0168] The copy number of ginseng is 1532.1, the copy number of the internal reference sequence is 8452.6, and the ratio is 18.13%.

[0169] Example 5

[0170] Ginseng medicinal material testing

[0171] (1) DNA extraction: 200 mg of ginseng powder was taken and DNA was extracted using a commercial DNA extraction kit. The resulting DNA solution was ultrasonically treated for 60 seconds, and then a DNA adapter was added according to the instructions of the Adaptase Module kit.

[0172] (2) Primers, probes, and detection methods are the same as in Example 4

[0173] The copy number of ginseng is 34545.7, the copy number of the internal reference sequence is 35412.1, and the ratio is 97.55%.

[0174] Example 6

[0175] Testing of Ginseng Extract

[0176] The specific primers used to detect ginseng are shown in the table below, and other conditions are the same as in Example 1. The detection results are shown in Table 1.

[0177] Table 1

[0178]

[0179] Example 7

[0180] Testing of a Chinese medicine compound preparation (pills made from 11 herbs including ginseng and Atractylodes macrocephala)

[0181] The specific primers and probes used to detect ginseng are shown in Table 2, and the reverse primer and other conditions are the same as those in Example 4. The detection results are shown in Table 2.

[0182] Table 2

[0183]

[0184]

[0185] Example 8

[0186] Testing of a Chinese medicine compound preparation (an oral solution made from 11 herbs including ginseng and astragalus)

[0187] The specific primers and probes used to detect ginseng are shown in Table 3, and the reverse primer and other conditions are the same as those in Example 4. The detection results are shown in Table 3.

[0188] Table 3

[0189]

[0190]

[0191] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the parameters for implementation. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A specific SNP site for ginseng detection, wherein the SNP site is the 5896th base of NO.KM036296.1 in Genebank, which is G.

2. A method for designing primers or primers and probes based on one or two or three SNP sites in Genebank NO. KM036296.1 for specific SNP sites for ginseng detection, wherein the 5896th base is G, the 496th base is G and the 500th base is C, for the detection of ginseng, wherein the primers or primers and probes are designed as follows: The probe is a fluorescent probe, and its nucleotide sequence is: 5'-CTGATTCAGTCTGGTAATT-3'(SEQ ID NO1) 5'-TCTGGTAATTGGAATGAG-3'(SEQ ID NO2) 5'-ATGCCCGAGTGAAAGTGAC-3'(SEQ ID NO3) 5'-TGAAAGTGACAAAGTCGC-3'(SEQ ID NO4) 5'-AGACTGAATCAGCCCGGTAT-3'(SEQ ID NO5) 5'-CTGAATCAGCCCGGTATTG-3'(SEQ ID NO6) 5'-CCCGGTATTGTTATTTAT-3'(SEQ ID NO7) 5'-TCACTCGGGCATTAGTATC-3'(SEQ ID NO8) 5'-ATTAGTATCGGTGAACCTC-3'(SEQ ID NO9) The nucleotide sequence of the forward primer is: 5'-GACAATAAATAACAATACCGG-3'(SEQ ID NO10) 5'-TAACAATACCGGGCTGATAC-3'(SEQ ID NO11) 5'-CGTCGAGGTTCACCGATACT-3'(SEQ ID NO12) 5'-TTCACCGATACTAATGCCGG-3'(SEQ ID NO13) 5'-TTGTACTCATTCCAATTAC-3'(SEQ ID NO14) 5'-GTACTCATTCCAATTACCAG-3'(SEQ ID NO15) 5'-TCCAATTACCAGACTGAACC-3'(SEQ ID NO16) 5'-CAGTCGGCGACTTTGTCACT-3'(SEQ ID NO17) 5'-CGACTTTGTCACTTTCACCC-3'(SEQ ID NO18) The nucleotide sequence of the reverse primer is: 5'-CTCATTCCAATTACCAGACT-3'(SEQ ID NO19) 5'-GCGACTTTGTCACTTTCACT-3'(SEQ ID NO20) 5'-ATAAATAACAATACCGGGCT-3'(SEQ ID NO21) 5'-GTTCACCGATACTAATGCC-3'(SEQ ID NO22) 3. A digital PCR detection method for detecting ginseng in products, characterized in that: The steps include: Step 1: Extract DNA from the product to be tested as the detection DNA. Step 2: Design primers or primers and probes according to one, two or three SNP sites in Genebank NO. KM036296.1, in which the 5896th base is G, the 496th base is G and the 500th base is C. Step 3: Take the detection DNA in step 1, add the primers or primers and probes designed in step 2, configure the reaction system, and use the digital PCR method for detection to obtain the copy number A of the specific DNA sequence representing ginseng. Step 4: Select the conserved sequence in NO.KM036296.1 in Genebank as the internal reference sequence, and design PCR primers, or primers and probes that can detect this internal reference sequence, so that the length of the amplified fragment is the same or similar to the length of the fragment amplified in step 3, and the length difference does not exceed 5bp. Step 5: Take the detection DNA in step 1, the PCR primers designed in step 4, or the primers and probes, add them into the configured reaction system, and use the digital PCR method for detection to obtain the copy number B of the internal reference sequence. Step 6: Calculate the A / B ratio to obtain the result calculated with the internal reference sequence for product quality control.

4. A digital PCR detection method for detecting ginseng in products according to claim 3, characterized in that: The reaction system configured in step 3 and step 5 includes a probe method or fluorescent dye method reaction system. The probe method reaction system includes a combination of a forward primer and a fluorescent probe, and a reverse primer; the fluorescent dye method reaction system includes a forward and reverse primer pair, and a fluorescent dye.

5. A digital PCR detection method for detecting ginseng in products according to claim 4, characterized in that: The forward primer and fluorescent probe included in the probe reaction system are one of the following combinations: The nucleotide sequences of the combination 1 are: forward primer (SEQ ID NO10) and probe (SEQ ID NO1); The nucleotide sequence of the combination 2 is: forward primer (SEQ ID NO11) and probe (SEQ ID NO2); The nucleotide sequence of combination 3 is: forward primer (SEQ ID NO12) and probe (SEQ ID NO3); The nucleotide sequence of combination 4 is: forward primer (SEQ ID NO13) and probe (SEQ ID NO4); The nucleotide sequence of combination 5 is: forward primer (SEQ ID NO14) and probe (SEQ ID NO5); The nucleotide sequence of combination 6 is: forward primer (SEQ ID NO15) and probe (SEQ ID NO6); The nucleotide sequence of combination 7 is: forward primer (SEQ ID NO16) and probe (SEQ ID NO7); The nucleotide sequence of the combination 8 is: forward primer (SEQ ID NO17) and probe (SEQ ID NO8); The nucleotide sequence of the combination 9 is: forward primer (SEQ ID NO18) and probe (SEQ ID NO9); The primer pair included in the fluorescent dye method system is one of the following: Primer pair 1: forward primer (SEQ ID NO11) and reverse primer (SEQ ID NO19); Primer pair 2: forward primer (SEQ ID NO 13) and reverse primer (SEQ ID NO 20); Primer pair 3: forward primer (SEQ ID NO 16) and reverse primer (SEQ ID NO 21); Primer pair 4: forward primer (SEQ ID NO: 18) and reverse primer (SEQ ID NO: 22).

6. A digital PCR detection method for detecting ginseng in products according to claim 3, characterized in that: In step 4: the length of the amplified fragment is made the same or similar to the length of the fragment amplified in step 3, and the length difference does not exceed 3 bp, preferably does not exceed 1 bp.

7. A digital PCR detection method for detecting ginseng in a product according to any one of claims 3 to 6, characterized in that: The fluorescent probe sequence in the probe method reaction system keeps the 5' end sequence unchanged, deletes 1 to several bases from the 3' end, shortens the probe length to 10 to 19 bp, preferably shortens the probe length to 13 to 15 bp, and the bases in the probe sequence are modified with 1 to several locked nucleic acids (LNA) or peptide nucleic acids (PNA); The forward primer and / or reverse primer sequence in the fluorescent dye method or probe method reaction system keeps the 3' end sequence unchanged, deletes 1 to several bases from the 5' end, shortens the primer length to 12 to 19 bp, preferably shortens the primer length to 15 to 18 bp, and the bases in the primer sequence can be modified with 1 to several locked nucleic acids (LNA) or peptide nucleic acids (PNA).

8. A digital PCR detection method for detecting ginseng in a product according to any one of claims 3 to 7, characterized in that: The step 1 is: extracting DNA from the product to be detected, then using an enzyme to add a DNA linker to the 3' end and / or 5' end of the double-stranded or single-stranded DNA as the detection DNA, and designing a reverse primer using the DNA linker sequence as a template.

9. A digital PCR detection method for detecting ginseng in a product according to any one of claims 3 to 7, characterized in that: The step 1 is as follows: extracting DNA from the product to be detected, breaking the DNA into fragments of 20 to 200 bp in size, preferably breaking the DNA into fragments of 30 to 100 bp in size, and more preferably breaking the DNA into fragments of 40 to 60 bp in size, and then using an enzyme to add a DNA linker to the 3' end and / or 5' end of the double-stranded or single-stranded DNA as the detection DNA, and designing a reverse primer using the DNA linker sequence as a template.

10. Use of the method according to any one of claims 1 to 9 in preparing a kit for detecting ginseng.

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