Digital PCR (Polymerase Chain Reaction) detection method suitable for bio-based original product

Abstract

The invention belongs to the field of traditional Chinese medicine quality detection, and particularly relates to a technical scheme of a digital PCR detection method of a traditional Chinese medicine whole industrial chain, a detection kit and application. Based on the characteristics of the digital PCR technology, the invention establishes a set of detection technical scheme suitable for quality control of traditional Chinese medicines, and the detection technical scheme can be used for quality control of related products in the whole industry chain of traditional Chinese medicines, including traditional Chinese medicinal materials, traditional Chinese medicinal decoction pieces, traditional Chinese medicinal formula granules, traditional Chinese medicinal preparations and extracts from biological materials. In addition, the method can also be used for food, health food, beverage, cosmetics, agriculture, forestry, animal husbandry, fishery and other products containing bio-based raw materials.

Description

Technical Field

[0001] The present application relates to the technical field of quality inspection of traditional Chinese medicine, and in particular to a digital PCR inspection method applicable to products containing bio-based raw materials. Background Art

[0002] The quality of traditional Chinese medicine is an important prerequisite for ensuring the development of the traditional Chinese medicine industry. Traditional identification methods (property identification, microscopic identification, physical and chemical identification, etc.) have certain limitations. It is often difficult to achieve the requirements for accurate identification of the authenticity of medicinal materials, and the quality control of traditional Chinese medicine preparations is even more difficult. Molecular identification of traditional Chinese medicine is one of the most promising technologies for accurately identifying the origin of medicinal materials, but it is currently difficult to apply to the quality control of traditional Chinese medicine preparations. Digital PCR can achieve absolute quantification of the initial concentration of the sample without the need for calibrators and drawing standard curves, and has the characteristics of high sensitivity, high specificity and high accuracy.

[0003] Bio-origins refer to biological species containing DNA, such as animals, plants, and microorganisms. For products containing bio-origins, such as traditional Chinese medicine preparations, the prescription amount of each medicine is fixed, and the production process is fixed. Therefore, the proportion of DNA of each medicinal material to the total DNA in the sample is basically fixed, that is, the proportion of the number of gene copies representing each medicinal material to the total number of copies is also basically fixed. Based on this, by detecting the copy number A of the biological origin representing the medicinal flavor and the copy number B of the internal reference sequence, the ratio A / B can be obtained for product quality control.

[0004] In the patent "A droplet digital PCR primer, probe, kit and method for quantitative detection of ginseng" (application number 202110195783.3), the purpose of detecting the internal reference gene is to calculate the actual ginseng content in the test sample through a pre-established linear relationship of content, but this method can only be used for the quality detection of single medicinal materials, and it is more difficult to use for samples with severe DNA degradation, such as Chinese medicine formula granules and Chinese medicine preparations.

[0005] Existing technologies such as fluorescent quantitative PCR also use internal reference genes, but their purpose is to correct the differences caused by different initial sample concentrations so that the expression levels of genes in the measured samples can be compared, and they are not used for sample quality detection and control.

[0006] The present invention establishes a set of technical development solutions for the quality control of traditional Chinese medicine, which can be used for the quality control of related products in the entire industrial chain of traditional Chinese medicine, including Chinese medicinal materials, Chinese herbal medicine slices, Chinese herbal medicine formula granules, Chinese herbal medicine preparations, extracts derived from biological materials, and can also be used for primary products and deep-processed products containing biological origins in food, health food, beverages, cosmetics, agriculture, forestry, animal husbandry and fishery, etc. Summary of the invention

[0007] The present invention aims to develop a set of technology development solutions based on digital PCR to solve the problem of quality control of traditional Chinese medicine, which can be used for the quality control and development of detection kits for traditional Chinese medicine and related bio-based raw products.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] In a first aspect, the present invention provides a digital PCR method for detecting biological origins contained in a product, comprising the following steps:

[0010] Step 1: Extract DNA from the product to be tested as the detection DNA;

[0011] Step 2: Select a specific DNA sequence that can represent the biological origin, and design a PCR primer or a PCR primer and a fluorescent probe that can detect the specific DNA sequence;

[0012] Step 3: Select a conservative sequence as an internal reference sequence, design a PCR primer or a PCR primer and a fluorescent probe that can detect the internal reference sequence, and make the length of the amplified DNA fragment the same or similar to the length of the DNA fragment representing the biological origin in step 2, with the length difference not exceeding 5 bp;

[0013] Step 4: Take the detection DNA in step 1, add the PCR primers or PCR primers and fluorescent probes in step 2 and / or step 3, configure the reaction system, and use the digital PCR method for detection to obtain the copy number A representing the biological origin and the copy number B of the internal reference sequence respectively;

[0014] Step 5: Calculate the A / B ratio to obtain the result calculated using the internal reference sequence.

[0015] Wherein, the specific DNA sequence in step 2 is preferably a specific DNA sequence of a ribosomal RNA gene.

[0016] The internal reference sequence in step 3 is preferably a conserved sequence of ribosomal RNA, and more preferably a conserved sequence in eukaryotic ribosomal RNA genes.

[0017] The length difference in step 3 is preferably no more than 3 bp, and more preferably no more than 1 bp.

[0018] In a second aspect, the present invention provides a digital PCR method for detecting biological origins contained in a product, comprising the following steps:

[0019] Step 1: Extract the DNA from the product to be tested, and use 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;

[0020] Step 2: Select a specific DNA sequence that can represent the biological origin, and design PCR primers and fluorescent probes that can detect the specific DNA sequence, wherein the forward primer and / or reverse primer is designed using the DNA linker sequence in step 1 as a template;

[0021] Step 3: Select a conserved sequence as an internal reference sequence, and design a PCR primer or a PCR primer and a fluorescent probe that can detect the internal reference sequence, wherein the forward primer and / or reverse primer is designed using the DNA adapter sequence in step 1 as a template;

[0022] Step 4: Take the detection DNA in step 1, add the PCR primers or PCR primers and fluorescent probes in step 2 and / or step 3, configure the reaction system, and use the digital PCR method for detection to obtain the copy number A representing the biological origin and the copy number B of the internal reference sequence respectively;

[0023] Step 5: Calculate the A / B ratio to obtain the result calculated using the internal reference sequence.

[0024] In step 1, preferably, an enzyme is used to add a DNA linker to the 3' end of the single-stranded DNA.

[0025] In step 2, the specific DNA sequence is preferably a specific DNA sequence of a ribosomal RNA gene.

[0026] In step 2, the PCR primers include a forward primer and a reverse primer, wherein the forward primer and / or the reverse primer are designed using the DNA linker in step 1 as a template.

[0027] The internal reference sequence in step 3 is preferably a conserved sequence of ribosomal RNA, and more preferably a conserved sequence in eukaryotic ribosomal RNA genes.

[0028] The PCR primers in step 3 include a forward primer and a reverse primer, wherein the forward primer and / or the reverse primer are designed using the DNA linker in step 1 as a template.

[0029] In a third aspect, the present invention provides a digital PCR method for detecting biological origins contained in a product, characterized in that it comprises the following steps:

[0030] Step 1: Extract the DNA from the product to be tested, break the DNA into fragments of 20 to 200 bp in size, and use 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;

[0031] Step 2: Select a specific DNA sequence that can represent the biological origin, and design a PCR primer or a PCR primer and a fluorescent probe that can detect the specific DNA sequence, wherein the forward primer and / or reverse primer is designed using the DNA linker sequence in step 1 as a template;

[0032] Step 3: Select a conserved sequence as an internal reference sequence, and design a PCR primer or a PCR primer and a fluorescent probe that can detect the internal reference sequence, wherein the forward primer and / or reverse primer is designed using the DNA adapter sequence in step 1 as a template;

[0033] Step 4: Take the detection DNA in step 1, add the PCR primers or PCR primers and fluorescent probes in step 2 and / or step 3, configure the reaction system, and use the digital PCR method for detection to obtain the copy number A representing the biological origin and the copy number B of the internal reference sequence respectively;

[0034] Step 5: Calculate the A / B ratio to obtain the result calculated using the internal reference sequence.

[0035] In the step 1, the DNA is preferably broken into fragments of 30 to 100 bp in size, and more preferably broken into fragments of 40 to 60 bp in size.

[0036] In step 1, preferably, an enzyme is used to add a DNA linker to the 3' end of the single-stranded DNA.

[0037] In some embodiments, the reaction system configured in step 4 includes a probe method or a fluorescent dye method reaction system, the fluorescent dye method reaction system includes a forward and reverse primer pair, and a fluorescent dye; the probe method reaction system includes a forward primer, a reverse primer and a fluorescent probe, and the fluorescent probe is selected from one of a TaqMan probe, a TaqMan-MGB probe and a molecular beacon probe.

[0038] In some embodiments, the fluorescent probe in step 2 and step 3 is an MGB probe, whose length does not exceed 15 bp, preferably does not exceed 13 bp; and the forward primer sequence is designed to be adjacent to the fluorescent probe sequence, and the distance between the two does not exceed 3 bp, preferably does not exceed 1 bp.

[0039] In some embodiments, the fluorescent probe is a TaqMan probe, a TaqMan-MGB probe or a molecular beacon probe, preferably a TaqMan-MGB probe, the length of the TaqMan-MGB probe is no more than 20 bp, preferably no more than 15 bp, and more preferably no more than 13 bp, and the bases in the probe sequence can be modified with 1 to several locked nucleic acids (LNA) or peptide nucleic acids (PNA). The probe is labeled with a quencher group and different fluorescent groups, and the fluorescent group is preferably one of FAM, TET, HEX, CY3, JOE, VIC, ROX or CY5; the quencher group is preferably one of BHQ1, BHQ2, TAMRA, DABCYL, MGB or Eclipse.

[0040] In a fifth aspect, the present invention provides a digital PCR method for detecting biological origins contained in a product, comprising the following steps:

[0041] Step 1: Extract DNA from the product to be tested as the detection DNA;

[0042] Step 2: Select a specific DNA sequence that can represent the biological origin, design PCR primers that can detect this specific DNA sequence, and make the length of the amplified DNA fragment 30 to 100 bp;

[0043] Step 3: Select a conservative sequence as an internal reference sequence, design PCR primers that can detect this internal reference sequence, and make the length of the amplified DNA fragment the same or similar to the length of the DNA fragment representing the biological origin in step 2, with the length difference not exceeding 5 bp;

[0044] Step 4: Take the detection DNA in step 1, add the primers in step 2 or 3 respectively, then add the fluorescent dye, configure the PCR reaction system, and use the digital PCR method to perform detection to obtain the representative biological base original copy number A and the internal reference sequence copy number B;

[0045] Step 5: Calculate the A / B ratio to obtain the result calculated using the internal reference sequence.

[0046] Wherein, the specific DNA sequence in step 2 is preferably a specific DNA sequence of a ribosomal RNA gene.

[0047] The length in step 2 is preferably 30 to 80 bp, and more preferably 40 to 50 bp.

[0048] The internal reference sequence in step 3 is preferably a conserved sequence of ribosomal RNA, and more preferably a conserved sequence in eukaryotic ribosomal RNA genes.

[0049] The length difference in step 3 is preferably no more than 3 bp, and more preferably no more than 1 bp.

[0050] In some embodiments, when the fluorescent probe method is used, the forward primer sequence in claims 1 to 4 is designed to be adjacent to the fluorescent probe sequence, and the distance between the two does not exceed 3 bp, preferably not more than 1 bp. The reverse primer sequence is designed to be adjacent to the fluorescent probe sequence, and the distance between the two does not exceed 5 bp; preferably not more than 3 bp, and more preferably not more than 1 bp. When the dye method is used, the forward primer sequence in claims 1-5 is designed to be adjacent to the reverse primer sequence, and the distance between the two does not exceed 3 bp. Preferably, it does not exceed 1 bp.

[0051] In some embodiments, the length of the primers in steps 2 and 3 is no more than 21 bp, preferably no more than 18 bp, and more preferably no more than 15 bp.

[0052] In some embodiments, the primers described in steps 2 and 3 are no longer than 21 bp, and the bases in the primer sequence can be modified with one or more locked nucleic acids (LNA) or peptide nucleic acids (PNA) to increase the Tm required for PCR.

[0053] In some embodiments, when the product contains auxiliary materials of biological origin such as starch and / or honey, the aforementioned steps 1-4 remain unchanged. When calculating the result in step 5, it is necessary to determine the copy number C representing the biological origin of the auxiliary material, and calculate the ratio according to A / (B-C) to obtain the result calculated with the internal reference sequence.

[0054] Since biologically derived excipients, such as starch and honey, are often used in Chinese herbal formula granules and compound preparations, these excipients also contain DNA. The amount of excipients added will be adjusted according to the extract yield during the production process, which will cause the measured internal reference sequence copy number B to fluctuate. The specific sequence copy number C representing the excipient can be detected at the same time, and then deducted from the internal reference sequence copy number B and then calculated, that is, A / (B-C), so that the results calculated with the internal reference sequence are more reliable.

[0055] In some embodiments, during the detection, a positive control group and a negative control group can be further set up, wherein the positive control group is a template of a specific DNA sequence that can detect the biological origin, and the negative control group is a DNA template with the same sequence as the positive control group except for the specific base site, and the PCR conditions are adjusted so that the results of the positive control group and the negative control group can be effectively distinguished, and the PCR conditions are used to detect the number of gene sequence copies in the product. Among them, adjusting the PCR conditions includes but is not limited to adjusting by optimizing annealing temperature, number of cycles, fluorescence signal detection threshold and other PCR parameters. By optimizing the detection conditions, the interference of non-specific fragments can be effectively eliminated, so that the copy number of the specific sequence representing the biological origin and the copy number of the internal reference sequence are more accurate.

[0056] The present invention provides a detection tool required for detection, including the aforementioned primers, and / or probes, and / or DNA control templates.

[0057] The present invention also provides a detection kit, comprising the aforementioned detection tool, including primers, and / or probes, and / or DNA control templates.

[0058] Beneficial Effects

[0059] 1. By setting up an internal reference sequence and detecting the copy number A of the specific sequence representing the medicinal flavor and the copy number B of the internal reference sequence, the ratio is calculated as an indicator of product quality control.

[0060] Due to differences in varieties and medicinal parts, the DNA content in different medicinal materials is not the same, and the ratio is not equal to the content of this medicinal material in the preparation. However, for the same medicinal material, although its DNA content will change, the fluctuation range will not be too large in general. Although the ratio is not an absolute fixed value, its range of variation is limited, so it can be used as a quality control indicator. Specifically: for products with a single biological origin, such as Chinese medicinal materials, Chinese herbal medicines, Chinese herbal formula granules, etc., when the specific sequence representing the taste of the medicine and the internal reference sequence are derived from the same gene sequence, the ratio should theoretically be 1:1. When other biological origins are mixed in the product, the ratio will decrease. After establishing the internal reference sequence, the purpose of accurately controlling product quality can be achieved.

[0061] For products such as Chinese herbal compound preparations that contain multiple biological origins, when the formula is fixed and the production process is mature and fixed, the ratio is also relatively fixed and can also be used to control product quality, thereby ensuring that the origin of the medicinal materials used in the product is correct and the amount of materials added is accurate.

[0062] 2. The digital PCR method is used for detection, which reduces the interference of background and inhibitors on the PCR reaction, greatly improves the sensitivity of detection, and can detect trace amounts of DNA. It is suitable for the detection of products such as traditional Chinese medicine preparations that contain trace amounts of DNA, and can effectively eliminate the interference of drug components on the detection. In addition, the gene to be detected is preferably the gene sequence of ribosomal RNA, because the ribosomal RNA gene is a multi-copy gene, and a genome can contain 500 to 40,000 copies, which can greatly improve the sensitivity of detection.

[0063] 3. When the DNA fragments required for detection are very short, such as Chinese medicine, food and other deep-processed products that have undergone long-term high-temperature extraction and drying, the DNA contained in them is severely degraded and the fragments are short. It is also very suitable to add a DNA linker to the 3' end and / or 5' end of the DNA by using an enzyme.

[0064] 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.

[0065] In the description of the present application, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the feature. In the description of the present application, "plurality" means two or more, unless otherwise clearly and specifically defined.

[0066] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0067] In this application, the English abbreviations are:

[0068] DNA: deoxyribonucleic acid

[0069] RNA: ribonucleic acid

[0070] PCR: polymerase chain reaction

[0071] SNP: single nucleotide polymorphism

[0072] bp:base pair

[0073] Tm: melting temperature

[0074] MGB: Minor Groove Binder, minor groove binder of DNA molecule

[0075] “min” means minute; “h” means hour; “g” means gram; “mL” means milliliter. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 : Graph showing the results of digital PCR detection of ginseng-specific sequences in Example 2;

[0077] Figure 2 : Digital PCR detection results of the internal reference sequence in Example 2. DETAILED DESCRIPTION

[0078] In order to enable those skilled in the art to better understand the technical solution of the present application, some non-limiting embodiments are further disclosed below to further explain the present application in detail.

[0079] The reagents used in this application can be purchased from the market or prepared by the methods described in this application.

[0080] In the present application, "min" means minute; "h" means hour; "g" means gram; and "mL" means milliliter.

[0081] Example 1

[0082] Testing of Ginseng Extract

[0083] (1) DNA extraction

[0084] 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.

[0085] (2) Primers

[0086] Specific primer pairs for detecting ginseng:

[0087] Forward primer: 5′-TAACAATACCGGGCTGATAC-3′ (SEQ ID NO 11).

[0088] Reverse primer 5'-CTCATTCCAATTACCAGACT-3' (SEQ ID NO 19)

[0089] The length of the fragment amplified using the above-mentioned ginseng-specific primers was 40 bp.

[0090] Internal reference sequence primer pair

[0091] Forward primer 5'-CAGGGATAACTGGCTTGTGG-3'

[0092] Reverse primer 5′-TCGCTATGAACGCTTGGCTG-3′

[0093] The fragment length amplified using the above-mentioned internal reference sequence primers is 40 bp.

[0094] (3) Digital PCR detection conditions

[0095] 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.

[0096] 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

[0097] (4) Test results

[0098] 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%.

[0099] Example 2

[0100] Testing of Ginseng Formula Granules

[0101] (1) DNA extraction: Same as Example 1.

[0102] (2) Primers

[0103] Specific primer pairs for detecting ginseng:

[0104] The forward primer was 5'-ATACCGGGCTGATAC-3', and the 11th and 15th bases were modified with LNA.

[0105] The reverse primer was 5'-TCCAATTACCAGACT-3', and the 11th and 15th bases were modified with LNA.

[0106] The length of the fragment amplified using the above-mentioned ginseng-specific primers was 30 bp.

[0107] Internal reference sequence primer pair

[0108] The forward primer was 5'-ATAACTGGCTTGTGG-3', and the 11th and 15th bases were modified with LNA.

[0109] The reverse primer was 5'-ATGAACGCTTGGCTG-3', and the 11th and 15th bases were modified with LNA.

[0110] The length of the fragment amplified using the above-mentioned internal reference sequence primers is 30 bp.

[0111] (3) Digital PCR detection conditions: same as in Example 1.

[0112] (4) Test results

[0113] The copy number of ginseng was 1926.6, the copy number of the internal reference sequence was 1887.4, and the ratio was 102.1%.

[0114] Example 3

[0115] Testing of a Chinese medicine compound preparation (pills made from 11 herbs including ginseng and Atractylodes macrocephala)

[0116] (1) DNA extraction:

[0117] Take 5 g of sample, grind it, add 100 ml of water and let it stand for 30 minutes to dissolve, filter it, take the filter residue and use a DNA extraction kit (magnetic bead method) to extract DNA according to the steps in the instruction manual, and store it at -20℃ for later use.

[0118] (2) Primers and probes

[0119] The specific forward primer sequence for detecting ginseng is 5'-GACAATAAATAACAATACCGG-3' (SEQ ID NO 10)

[0120] The reverse primer sequence is 5'-GATTTAGATTGTACTCATTC-3'

[0121] The MGB probe sequence is FAM-CTGATTCAGTCTGGTAATT-BHQ1 (SEQ ID NO 1)

[0122] The length of the fragment amplified using the above-mentioned specific primers is 62 bp.

[0123] The forward primer sequence for detecting the internal reference sequence is 5'-AAGGTAGCCAAATGCCTCGT-3'

[0124] The reverse primer sequence is 5'-GGGAATCTCGTTAATCCATT-3'

[0125] The MGB probe sequence is VIC-ATCTAATTAGTGACGCGCAT-BHQ1

[0126] The length of the fragment amplified using the above primers is 62 bp.

[0127] (3) Digital PCR detection conditions

[0128] 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.

[0129] (4) Test results

[0130] The copy number of ginseng is 3541.5, and the copy number of the internal reference sequence is 20548.7, with a ratio of 17.23%.

[0131] Example 4

[0132] Testing of a Chinese medicine compound preparation (an oral solution made from 11 herbs including ginseng and astragalus)

[0133] (1) DNA extraction:

[0134] Take 5 ml of sample and use DNA extraction kit (magnetic bead method) to extract DNA according to the instructions and store at -20℃ for later use.

[0135] (2) Adding DNA adapter

[0136] The DNA extracted in step (1) was purified using the Adaptase Module kit according to the instructions.

[0137] (3) Primers and probes

[0138] The primers and probes for detecting ginseng are

[0139] Forward primer 5'-AAATAACAATACCGG-3', where bases 3, 7, and 10 are modified with LNA

[0140] MGB probe FAM-CTGATTCAGTCTG-BHQ1, in which the 3rd, 7th, and 10th bases are modified with LNA

[0141] Reverse primer: 5'ACTGGAGTTCAGACGTGTGC-3'

[0142] Primers and probes for detecting internal reference sequences

[0143] The forward primer was 5′-AGCCAAATGCCTCGT-3′, in which bases 3, 7, and 10 were modified with LNA;

[0144] MGB probe: VIC-ATCTAATTAGTGA-BHQ1, in which bases 3, 7, and 10 were modified with LNA;

[0145] Reverse primer: 5'ACTGGAGTTCAGACGTGTGC-3'

[0146] (4) Digital PCR detection conditions, same as in Example 3

[0147] 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%.

[0148] Example 5

[0149] Ginseng medicinal material testing

[0150] (1) DNA extraction:

[0151] 200 mg of ginseng powder was taken and DNA was extracted using a commercial DNA extraction kit. The obtained DNA solution was ultrasonically treated for 60 seconds, and then a DNA adapter was added according to the instructions of the Adaptase Module kit.

[0152] (2) Primers, probes, and detection methods are the same as in Example 4

[0153] 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%.

[0154] Example 6

[0155] Testing of Ginseng Extract

[0156] 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.

[0157] Table 1

[0158] Specific primers for detecting ginseng Ginseng copy number Internal reference sequence copy number ratio Forward primer (SEQ ID NO 13) 2712.2 2698.4 100.5％ Forward primer (SEQ ID NO 16) 3102.4 3204.1 96.83％ Forward primer (SEQ ID NO 18) 2954.5 3024.7 97.68％

[0159] Example 7

[0160] Testing of a Chinese medicine compound preparation (pills made from 11 herbs including ginseng and Atractylodes macrocephala)

[0161] 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.

[0162] Table 2

[0163]

[0164]

[0165] Example 8

[0166] The detection of a Chinese medicine compound preparation (an oral liquid made from 11 medicinal materials including ginseng and astragalus) uses specific primers and probes for detecting ginseng, as 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.

[0167] Table 3

[0168]

[0169]

[0170]

[0171] The method of the present application 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 application to implement and apply the technology of the present application. Those skilled in the art can refer to the content of this article and appropriately improve the process 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 deemed to be included in the present application.

Claims

1. A digital PCR method for detecting biological origins contained in a product, characterized in that: The steps include: Step 1: Extract DNA from the product to be tested as the detection DNA; Step 2: Select a specific DNA sequence that can represent the biological origin, and design a PCR primer or a PCR primer and a fluorescent probe that can detect the specific DNA sequence; Step 3: Select a conservative sequence as an internal reference sequence, design a PCR primer or a PCR primer and a fluorescent probe that can detect the internal reference sequence, and make the length of the amplified DNA fragment the same or similar to the length of the DNA fragment representing the biological origin in step 2, with the length difference not exceeding 5 bp; Step 4: Take the detection DNA in step 1, add the PCR primers or PCR primers and fluorescent probes in step 2 and / or step 3, configure the reaction system, and use the digital PCR method for detection to obtain the copy number A representing the biological origin and the copy number B of the internal reference sequence respectively; Step 5: Calculate the A / B ratio to obtain the result calculated using the internal reference sequence.

2. A digital PCR method for detecting biological origins contained in a product, characterized in that: The steps include: Step 1: Extract the DNA from the product to be tested, and use 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; Step 2: Select a specific DNA sequence that can represent the biological origin, and design a PCR primer or a PCR primer and a fluorescent probe that can detect the specific DNA sequence, wherein the forward primer and / or reverse primer is designed using the DNA linker sequence in step 1 as a template; Step 3: Select a conserved sequence as an internal reference sequence, and design a PCR primer or a PCR primer and a fluorescent probe that can detect the internal reference sequence, wherein the forward primer and / or reverse primer is designed using the DNA adapter sequence in step 1 as a template; Step 4: Take the detection DNA in step 1, add the PCR primers or PCR primers and fluorescent probes in step 2 and / or step 3, configure the reaction system, and use the digital PCR method for detection to obtain the copy number A representing the biological origin and the copy number B of the internal reference sequence respectively; Step 5: Calculate the A / B ratio to obtain the result calculated using the internal reference sequence.

3. A digital PCR method for detecting biological origins contained in a product, characterized in that: The steps include: Step 1: Extract the DNA from the product to be tested, break the DNA into fragments of 20 to 200 bp in size, and use 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; Step 2: Select a specific DNA sequence that can represent the biological origin, and design a PCR primer or a PCR primer and a fluorescent probe that can detect the specific DNA sequence, wherein the forward primer and / or reverse primer is designed using the DNA linker sequence in step 1 as a template; Step 3: Select a conserved sequence as an internal reference sequence, and design a PCR primer or a PCR primer and a fluorescent probe that can detect the internal reference sequence, wherein the forward primer and / or reverse primer is designed using the DNA adapter sequence in step 1 as a template; Step 4: Take the detection DNA in step 1, add the PCR primers or PCR primers and fluorescent probes in step 2 and / or step 3, configure the reaction system, and use the digital PCR method for detection to obtain the copy number A representing the biological origin and the copy number B of the internal reference sequence respectively; Step 5: Calculate the A / B ratio to obtain the result calculated using the internal reference sequence.

4. The method according to any one of claims 1 to 3, characterized in that: The reaction system configured in step 4 includes a probe method or a fluorescent dye method reaction system. The fluorescent dye method reaction system includes a forward and reverse primer pair, and a fluorescent dye; the probe method reaction system includes a forward primer, a reverse primer and a fluorescent probe, and the fluorescent probe is selected from one of a TaqMan probe, a TaqMan-MGB probe or a molecular beacon probe.

5. A digital PCR method for detecting biological origins contained in a product, characterized in that: The steps include: Step 1: Extract DNA from the product to be tested as the detection DNA; Step 2: Select a specific DNA sequence that can represent the biological origin, design PCR primers that can detect this specific DNA sequence, and make the length of the amplified DNA fragment 30 to 100 bp; Step 3: Select a conservative sequence as an internal reference sequence, design PCR primers that can detect this internal reference sequence, and make the length of the amplified DNA fragment the same or similar to the length of the DNA fragment representing the biological origin in step 2, with the length difference not exceeding 5 bp; Step 4: Take the detection DNA in step 1, add the primers in step 2 or 3 respectively, then add the fluorescent dye, configure the PCR reaction system, and use the digital PCR method to perform detection to obtain the representative biological base original copy number A and the internal reference sequence copy number B; Step 5: Calculate the A / B ratio to obtain the result calculated using the internal reference sequence.

6. The method according to any one of claims 1 to 5, characterized in that: When the fluorescent probe method is adopted, the forward primer sequence in claims 1 to 4 is designed to be adjacent to the fluorescent probe sequence, and the distance between the two does not exceed 3bp; the reverse primer sequence is designed to be adjacent to the fluorescent probe sequence, and the distance between the two does not exceed 5bp; when the dye method is adopted, the forward primer sequence in claims 1-5 is designed to be adjacent to the reverse primer sequence, and the distance between the two does not exceed 3bp.

7. The method according to any one of claims 1 to 6, characterized in that: The length of the primers described in steps 2 and 3 does not exceed 21 bp.

8. The method according to any one of claims 1 to 7, characterized in that: When the product contains excipients of biological origin such as starch and / or honey, steps 1-4 remain unchanged. When calculating the result in step 5, it is necessary to determine the copy number C representing the biological origin of the excipient and calculate the ratio according to A / (B-C) to obtain the result calculated using the internal reference sequence.

9. The method according to any one of claims 1 to 8, characterized in that: During the test, a positive control group and a negative control group can be further set up, wherein the positive control group is a template of a specific DNA sequence that can detect the biological origin, and the negative control group is a DNA template whose sequence is the same as that of the positive control group except for the specific base site. The PCR conditions can be adjusted so that the measurement results of the positive control group and the negative control group can be effectively distinguished, and this PCR condition is used to detect the copy number of the gene sequence in the product.

10. A detection kit, characterized in that: A detection tool comprising any one of claims 1 to 9, comprising primers, and / or probes, and / or DNA control templates.

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