Probe primer, application of probe primer in lumbricus identification and PCR (Polymerase Chain Reaction) detection method

Abstract

The invention belongs to the technical field of medicines, relates to a medicine identification and detection method, and in particular relates to a probe primer, application of the probe primer to earthworm identification and a PCR (Polymerase Chain Reaction) detection method. The probe primer is composed of sequences as shown in SEQ ID NO. 1 to 8 and SEQ ID NO. 9 to 17; wherein the probe primer is used for realizing specific detection and quantitative detection of lumbricus through a fluorescent quantitative PCR detection method (including real-time fluorescent quantitative PCR and digital PCR). The detection method provided by the invention can effectively detect the lumbricus, including newly found lumbricus subgroups, is strong in specificity and high in sensitivity, can be used for qualitative or quantitative detection of the lumbricus in traditional Chinese medicines in forms of medicinal materials, decoction pieces, formula granules, decoction, prescription preparations and the like, and has relatively strong innovativeness and good application value.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a drug identification and detection method, in particular to a probe primer and its use in identifying Lumbricus radiatus and a PCR detection method. Background Art

[0002] Earthworms are a commonly used animal medicine in clinical practice, known for their heat-clearing and antispasmodic effects, calming the liver and relieving wind, promoting blood circulation and activating collaterals, and providing relief from asthma and diuresis. Modern pharmacological research has shown that earthworms possess multiple pharmacological effects, including anticoagulant, thrombolytic, antihypertensive, and antiasthmatic properties. The 2020 edition of the Chinese Pharmacopoeia stipulates that earthworms are derived from the dried bodies of the earthworms Pheretima aspergillum (E. Perrier), Pheretima vulgaris Chen (Metaphire vulgaris), Pheretima guillelmi (Michaelsen), or Pheretima pectinifera Michaelsen, members of the family Metaphireidae. The former is commonly known as "Guangdilong," while the latter three are commonly known as "Hudilong." Guangdilong is primarily produced in Guangdong, Guangxi, Fujian, and other regions. The quality standards for formulated granules explicitly require that the raw materials used be derived from Guangdilong, resulting in significant market demand. The latest research results (Acta Pharm Sin B. 2023Apr; 13(4): 1755-1770) show that there is significant genetic differentiation within the species of P. glabra, which can be divided into two different subgroups.

[0003] my country boasts a rich variety of earthworms. To date, Chinese and foreign scholars have documented 388 species (including subspecies) of terrestrial earthworms in my country. The origins of commercially available earthworms in China are unclear, with sources originating from at least 34 species. Several species of the same genus, including those of the Lumbricidae family, are also used medicinally in various locations. These include the non-pharmacopoeial earthworms Metaphire magna, Amynthas carnosus, and Amynthas obscuritoporus. Furthermore, with the increasing demand for earthworms in recent years, domestic production has outstripped demand, leading to increasing imports from abroad and further complicating their sourcing. Earthworms lose significant characteristics after processing, and their internal components are susceptible to changes during processing, processing, and storage, making authenticity difficult to distinguish. Furthermore, the prevalence of counterfeit and counterfeit products with similar morphologies and homonyms makes identification extremely challenging. Currently, commonly used methods for identifying earthworms include traditional morphological identification, microscopic identification, physical and chemical analysis, and molecular biology. However, these methods are often limited in their application due to lack of objectivity, poor specificity, low sensitivity, or weak anti-interference capabilities. These methods are particularly inapplicable to highly processed samples, such as traditional Chinese medicines or formulated granules. Therefore, it is necessary to establish a more effective and specific identification method for Earthworms.

[0004] The TaqMan probe method generates a fluorescent signal by adding a probe that specifically binds to the template to the reaction system, allowing for real-time monitoring and analysis of PCR amplification products. This method is both qualitative and quantitative. Compared to conventional PCR, it eliminates the need for steps like agarose gel electrophoresis, EB, or GoldView staining, is less time-consuming, and is less likely to cause contamination in the gene amplification laboratory. It also boasts higher specificity and sensitivity, and is rapid and accurate. By adding probes designed for different templates to the same reaction system, multiple fluorescence detections can be performed within the same reaction system, allowing for simultaneous detection of one or more specific targets in a mixed sample, greatly improving detection efficiency.

[0005] Digital PCR (dPCR) is the third generation of PCR technology following real-time fluorescence quantitative PCR technology. It is a brand-new method for nucleic acid detection and quantification. In dPCR, the sample is first divided into many small volumes (in microwells, chambers or droplets). Each independent parallel micro-reaction unit contains an average of only one copy or no target DNA molecule. After the PCR is completed, the number of positive droplets / total droplets is counted to achieve absolute counting of target nucleic acid molecules based on the Poisson distribution principle. dPCR can directly detect the copy number of the target sequence without relying on standard curves and reference samples. The detection limit can reach single copy. It has better sensitivity, specificity and accuracy than traditional qPCR, has strong anti-interference ability against inhibitors, and is not affected by the reaction amplification efficiency. It is particularly suitable for absolute quantitative detection of nucleic acids in complex matrix samples. It has been widely used in medicine, biology, food and other fields. In addition, dPCR has demonstrated its unique technical advantages and application prospects in the detection of traditional Chinese medicines. It not only provides technical support for the rapid identification of traditional Chinese medicine raw materials, but can also be used for quality control of the production chain of traditional Chinese patent medicines. By developing single and multiple dPCR detection methods, adulterants in highly processed products can be identified. For example, when analyzing the raw materials and finished preparations of traditional Chinese medicines such as Akebia, Biejia, and Notopterygium wilfordii, dPCR technology shows good specificity, sensitivity, and practicality.

[0006] The discovery of a new subpopulation of Dilong guangdilugens suggests that the development of DNA identification methods must fully consider intraspecific variation within Dilong guangdilugens. COI sequence analysis reveals that the new subpopulation shares numerous differential sites with the classic Dilong guangdilugens. Previously established DNA identification methods, without accounting for subpopulation variation, are likely to miss subpopulations and produce false-negative results. Therefore, it is necessary to establish a specific identification method for Dilong guangdilugens based on the genetic information of the newly discovered subpopulation to ensure accurate identification. Summary of the Invention

[0007] The purpose of the present invention is to provide a probe primer and its use in identifying P. truncatum and a PCR detection method.

[0008] The detection method established by the present invention can effectively detect Dilong guangxiong and distinguish its mixed and counterfeit products. It has strong specificity and high sensitivity. It can be used for the qualitative or quantitative detection of Dilong guangxiong in traditional Chinese medicines in the form of medicinal materials, decoction pieces, and formula preparations. It has good practical value and application prospects.

[0009] In order to achieve the above objectives, one of the objectives of the present invention is to provide a probe primer set, which consists of a specific primer pair and a specific probe; the specific primer pair includes: upstream primer GDL_F6_H, downstream primer GDL_R6_H, upstream primer GDL_F7_H, upstream primer GDL_F6_L, upstream primer GDL_F7_L, downstream primer GDL_R6_L, and the specific probe includes: GDL_P6_H and GDL_P6_L.

[0010] Among them, the nucleic acid sequence of GDL_P6_H is shown as SEQ ID NO.1, the nucleic acid sequence of GDL_F6_H is shown as SEQ ID NO.2, the nucleic acid sequence of GDL_R6_H is shown as SEQ ID NO.3, the nucleic acid sequence of GDL_F7_H is shown as SEQ ID NO.4, the nucleic acid sequence of GDL_P6_L is shown as SEQ ID NO.5, the nucleic acid sequence of GDL_F6_L is shown as SEQ ID NO.6, the nucleic acid sequence of GDL_F7_L is shown as SEQ ID NO.7, and the nucleic acid sequence of GDL_R6_L is shown as SEQ ID NO.8.

[0011] Preferably, a universal primer pair and a universal probe are also included. The universal primer pair includes: upstream primer DLTY_F8, downstream primer DLTY_R8, upstream primer DLTY_F9, downstream primer DLTY_R9, upstream primer DLTY_F10, downstream primer DLTY_R10, and the universal probe includes: DLTY_P8, DLTY_P9, DLTY_P10.

[0012] Among them, the nucleic acid sequence of DLTY_P8 is shown as SEQ ID NO.9, the nucleic acid sequence of DLTY_F8 is shown as SEQ ID NO.10, the nucleic acid sequence of DLTY_R8 is shown as SEQ ID NO.11, the nucleic acid sequence of DLTY_P9 is shown as SEQ ID NO.12, the nucleic acid sequence of DLTY_F9 is shown as SEQ ID NO.13, the nucleic acid sequence of DLTY_R9 is shown as SEQ ID NO.14, the nucleic acid sequence of DLTY_P10 is shown as SEQ ID NO.15, the nucleic acid sequence of DLTY_F10 is shown as SEQ ID NO.16, and the nucleic acid sequence of DLTY_R10 is shown as SEQ ID NO.17.

[0013] A second object of the present invention is to provide the use of the above-mentioned probe primer set in any one of the following A1)-A6):

[0014] A1) Identify or assist in the identification of Lumbricus radiatus;

[0015] A2) preparing products for identification or assisting in the identification of P.

[0016] A3) Differentiate or assist in differentiating Lumbricus radiatus from other medicinal earthworms;

[0017] A4) preparing a product that distinguishes or assists in distinguishing Lumbricus radiatus from other medicinal earthworms;

[0018] A5) Determining whether the sample to be tested contains Lugurus radix;

[0019] A6) preparing a product for identifying whether a sample to be tested contains P. radix serrata;

[0020] A7) Identification or auxiliary identification to distinguish Luteolin and Luteolin easily confused products;

[0021] A8) Prepare products for identification or auxiliary identification to distinguish Luteolin and Luteolin easily confused products.

[0022] Preferably, the sample to be tested is at least one of medicinal materials, decoction pieces, prescription preparations, Chinese medicine granules, decoctions, and freeze-dried extract powders.

[0023] Preferably, the products that are easily confused with Guangdilong are at least one of the common earthworm, William earthworm, comb-blind ring earthworm, Baoning earthworm, Earthworm genus, Coelophysis family, Earthworm genus, Zhili earthworm, fleshy Earthworm, dark hole Earthworm, and California earthworm.

[0024] The third object of the present invention is to provide a reagent or kit containing the above-mentioned probe primer set, wherein the function of the reagent or kit is any one of the following B1)-B6)

[0025] B1) Identify or assist in the identification of Lumbricus radiatus;

[0026] B2) preparing products for identification or assisting in the identification of P.

[0027] B3) Differentiate or assist in differentiating Lumbricus radiatus from other medicinal earthworms;

[0028] B4) preparing products that distinguish or assist in distinguishing Lumbricus radiatus from other earthworms for medicinal use;

[0029] B5) Identifying whether the sample to be tested contains Lugurus radix;

[0030] B6) preparing a product for identifying whether the sample to be tested contains P. radix serrata;

[0031] B7) Identify or assist in identification to distinguish Luteolin and Luteolin easily confused products;

[0032] B8) Prepare products for identification or auxiliary identification to distinguish Luteolin and Luteolin easily confused products.

[0033] Preferably, the sample to be tested is at least one of medicinal materials, decoction pieces, prescription preparations, Chinese medicine granules, decoctions, and freeze-dried extract powders.

[0034] Preferably, the products that are easily confused with Guangdilong are at least one of the common earthworm, William earthworm, comb-blind ring earthworm, Baoning earthworm, Earthworm genus, Coelophysis family, Earthworm genus, Zhili earthworm, fleshy Earthworm, dark hole Earthworm, and California earthworm.

[0035] The fourth object of the present invention is to provide a method for detecting Dilong dasyphylla, comprising the following steps: using the DNA of the sample to be tested as a template and amplifying using the above-mentioned probe primer set; if there is an amplification product, the sample to be tested is Dilong dasyphylla or contains Dilong dasyphylla; if there is no amplification product, the sample to be tested is not Dilong dasyphylla or does not contain Dilong dasyphylla.

[0036] The specific steps are as follows: the sample to be tested is processed to extract DNA; the above-mentioned probe primer set is used and the reaction system and reaction conditions are set to perform PCR amplification; if there is an amplification product, the sample to be tested is Guangdilong or contains Guangdilong; if there is no amplification product, the sample to be tested is not Guangdilong or does not contain Guangdilong.

[0037] The fifth object of the present invention is to provide a method for detecting Lu Lu or Lu Lu's confounding products, comprising the following steps: using the DNA of the sample to be tested as a template, and adopting the above-mentioned probe primer set for amplification; if the universal primer pair and the universal probe, and the specific primer pair and the specific probe do not produce an amplification product, then there is no Lu Lu or Lu Lu confounding products in the sample to be tested; if the universal primer pair and the universal probe produce an amplification product, and the specific primer pair and the specific probe do not produce an amplification product, then the sample to be tested is other earthworm species other than Lu Lu or contains other earthworm species other than Lu Lu; if the universal primer pair and the universal probe, and the specific primer pair and the specific probe all produce an amplification product, then the sample to be tested is Lu Lu or contains Lu Lu.

[0038] Preferably, the detection method is a fluorescent quantitative PCR detection method or a digital PCR detection method.

[0039] Preferably, the lower limit of detection concentration of the fluorescent quantitative PCR detection method is 0.0001 ng / μL.

[0040] Preferably, the lower limit of detection concentration of the digital PCR detection method is 0.00001 ng / μL.

[0041] Preferably, in the fluorescent quantitative PCR detection method, the amplification reaction system includes, by volume: 10 parts of fluorescent PCR reaction mixture, 0.3 parts of upstream primer, 0.3 parts of downstream primer, 0.3 parts of probe, 1.1 parts of template DNA, and ultrapure water is added to the total volume of the system to 20 parts, wherein the concentrations of upstream primer and downstream primer are 20 μmol / L, and the concentration of probe is 10 μmol / L.

[0042] Preferably, in the fluorescent quantitative PCR detection method, the amplification reaction conditions are as follows: 95° C., 30 seconds; 95° C., 5 seconds; 63° C., 15 seconds, for a total of 40 cycles.

[0043] Preferably, in the fluorescent quantitative PCR detection method, the above-mentioned probe primer set is used for amplification. If amplification is possible, relative quantitative or absolute quantitative detection can also be performed.

[0044] More preferably, in the relative quantitative detection process, a standard is used as a reference, and the difference in CT value between the standard and the sample to be tested is expressed as ΔCT. The proportion of the target DNA in the sample to be tested can be expressed as: sample to be tested / standard % = 2 -△CT绝对值 ×100%.

[0045] Theoretically, when the absolute ΔCT value is 4, the adulteration rate is 6.25%, and when the absolute ΔCT value is 5, the adulteration rate is 3.125%. For details, see Table 12 below. However, in actual testing, samples with a 5% adulteration rate have an absolute ΔCT value between 4 and 5. This value can be adjusted to control the stringency of the test standard. A larger value lowers the detection limit and sets a more stringent standard.

[0046] More preferably, during the absolute quantitative detection process, a standard is used as a reference, the logarithm of the DNA concentration is used as the horizontal axis, and the CT value is used as the vertical axis to draw an absolute quantitative standard curve, and the DNA concentration of Luteolin contained in the sample to be tested is calculated based on the absolute quantitative standard curve and the CT value obtained by amplification of the sample to be tested.

[0047] Preferably, in the digital PCR detection method, the single-channel amplification reaction system includes, by volume: 10 parts of fluorescent PCR reaction mixture, 0.22 parts of ROX Reference Dye II, 1.1 parts of upstream primer, 1.1 parts of downstream primer, 0.55 parts of probe, 4.4 parts of template DNA, and ultrapure water to a total volume of 22 parts of the system, wherein the concentrations of the upstream primer and the downstream primer are 20 μmol / L, and the concentration of the probe is 10 μmol / L.

[0048] Preferably, in the digital PCR detection method, the dual-label amplification reaction system includes, by volume, 10 parts of fluorescent PCR reaction mixture, 0.22 parts of ROX Reference Dye II, 1.32 parts of upstream primer, 1.32 parts of downstream primer, 0.33 parts of probe, 4.4 parts of template DNA, and ultrapure water to a total volume of 22 parts of the system, wherein the concentrations of the upstream primer, downstream primer, and probe are all 20 μmol / L.

[0049] Preferably, in the digital PCR detection method, the amplification reaction conditions are as follows: 95°C, 30 seconds; 95°C, 5 seconds; 63°C, 15 seconds, 45 cycles.

[0050] Preferably, in the digital PCR detection method, the above-mentioned probe primer set is used for amplification. If amplification is possible, absolute quantitative or relative quantitative detection can also be performed.

[0051] More preferably, in the absolute quantitative detection process, a standard curve is drawn with DNA concentration as the horizontal coordinate and copy number as the vertical coordinate; based on the standard curve, the DNA concentration of Lu Lu in the sample is calculated according to the detected copy number value; the mass of Lu Lu standard (such as medicinal materials or formula granules) is used as the horizontal coordinate and the DNA concentration is used as the vertical coordinate to draw a standard curve; based on the standard curve, the mass of Lu Lu in the sample is calculated according to the DNA concentration;

[0052] More preferably, in the relative quantitative detection process, a standard can be used as a reference, and the copy number of the universal primer pair and universal probe in the standard is expressed as Cp S1 Indicates that the target fragment copy number is Cp S2 The number of copies of universal primer pairs and universal probes in the test sample is expressed as Cp T1 Indicates that the target fragment copy number is Cp T2 If , the proportion of target DNA in the test sample is calculated according to the following formula:

[0053] Test sample / standard product%=(Cp T2 ÷Cp T1 )÷(Cp S2 ÷Cp S1 )×100%.

[0054] Compared with the existing technology, the present invention has the following beneficial effects:

[0055] The present invention designs a probe primer set and establishes a dedicated detection method for Luteolin based on the TaqMan probe fluorescence quantitative PCR technology. The method can distinguish Luteolin from common easily confused products, such as the common earthworm and the Baoning cavity earthworm. The detection threshold limit is reasonably set, and the method is suitable for the qualitative or quantitative detection of Luteolin in medicinal materials, decoction pieces, and prescription preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a diagram showing the specificity of the real-time fluorescence quantitative PCR detection method for Dilong chinensis.

[0057] Figure 2 This is a graph showing the fluorescence quantitative PCR amplification efficiency of the Lumbricus macrocarpa specific probe primer set and the earthworm universal probe primer set.

[0058] Figure 3Figure 2 shows the detection limit of the real-time fluorescence quantitative PCR assay for Luteolin. S1 indicates a DNA template amount of 10 ng / μL, S2 indicates a DNA template amount of 1 ng / μL, S3 indicates a DNA template amount of 0.1 ng / μL, S4 indicates a DNA template amount of 0.01 ng / μL, S5 indicates a DNA template amount of 0.001 ng / μL, S6 indicates a DNA template amount of 0.0001 ng / μL, and S7 indicates a DNA template amount of 0.00001 ng / μL. NTC indicates a no-template negative control.

[0059] Figure 4 This is a diagram showing the specificity of the digital PCR detection method for Dilong chinensis.

[0060] Figure 5 This is a graph investigating the detection limit of the digital PCR detection method for Dilong chinensis.

[0061] Figure 6 This is the absolute quantitative standard curve of the digital PCR detection method for Dilong chinensis. DETAILED DESCRIPTION

[0062] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0063] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0064] The instruments and reagents used in this application are as follows: Real-time fluorescence quantitative PCR instrument (Analytikjena qTOWER 3 G, Roche Pangaea fast fluorescence quantitative PCR system), digital PCR instrument (Sniper DQ24), analytical balance (Mettler AB135-S), water purification instrument (Millipore), ball mill (MM400, Retsch), NanoDrop One ultra-micro spectrophotometer (Thermo Fisher Scientific), Qubit4.0 fluorescence quantitative instrument (Thermo Fisher Scientific), desktop high-speed refrigerated centrifuge (Eppendorf Centrifuge 5427R), micro centrifuge (IKAmini GS025), mixer (Eppendorf C), vortex shaker (Scientific Industries Vortex-Genie2), genomic DNA extraction kit DNeasymericon Food Kit (Qiagen), real-time fluorescence quantitative PCR premix Probe qPCR Mix (2×) (RR391A, TaKaRa).

[0065] Example 1

[0066] A total of 48 batches of accurately sourced Guangdilong original samples, Guangdilong reference medicinal materials and commercial samples of earthworms were collected, 6 batches of commercially available formula preparations were collected, 3 batches of formula preparations and 4 batches of freeze-dried powder of earthworm standard decoctions were prepared (see Tables 1 and 2 for details), and the COI sequences of earthworms and their adulterants are shown in Table 3.

[0067] Table 1 Earthworm medicinal material sample information

[0068]

[0069]

[0070]

[0071] Table 2 Sample information of homemade standard decoction freeze-dried powder and formula preparation of Guangdilong (Pheretima ginseng)

[0072] Serial number Sample number Product Name source 1 XHLW01 Xiaohuoluowan Enterprise 1 2 XHLW04 Xiaohuoluowan Enterprise 2 3 XHLW05 Xiaohuoluowan Enterprise 3 4 XHLW06 Xiaohuoluowan Enterprise 4 5 XHLW07 Xiaohuoluowan Enterprise 5 6 MLTKL Mailuotong Granules Enterprise 6 7 XHLW GDL1 Laboratory-made Xiao Huo Luo Wan (containing Guang Di Long) China Food and Drug Inspection Institute 8 XHLW HDL Laboratory-made Xiao Huo Luo Wan (containing Hu Di Long) China Food and Drug Inspection Institute 9 XHLW NTC Laboratory-made Xiao Huo Luo Wan (without earthworms) China Food and Drug Inspection Institute 10 DGF GDL1 Laboratory-made freeze-dried powder of Dilong China Food and Drug Inspection Institute 11 DGF GDL4 Laboratory-made freeze-dried powder of Dilong China Food and Drug Inspection Institute 12 DGF GDL5 Laboratory-made freeze-dried powder of Dilong China Food and Drug Inspection Institute 13 DGF HDL4 Laboratory-made lyophilized powder of Hudilong China Food and Drug Inspection Institute

[0073] Table 3 COI sequences of earthworms and their adulterants downloaded from NCBI database

[0074]

[0075]

[0076] Example 2

[0077] 1. Sample processing and DNA extraction

[0078] The medicinal material test sample was ground into extremely fine powder, and 15 mg was accurately weighed. Samples were taken according to the content of 15 mg of earthworm for the prepared preparation, and DNA was extracted using the DNeasymericon Food Kit (see the kit instructions for specific steps).

[0079] 2. Establishment of Fluorescence Quantitative PCR Detection Method

[0080] 2.1. Probe and Primer Design and Screening

[0081] Based on the DNA sequence analysis and alignment of COI, 16S rRNA and 12S rRNA regions, Lumbricus-specific probe primers and Lumbricus universal probe primers were designed in the conserved and interspecific regions (Table 4).

[0082] Table 4 Probe and primer sequence information

[0083]

[0084]

[0085] In Table 4, GDL_P6_H, GDL_P6_L, DLTY_P8, DLTY_P9, and DLTY_P10 are probes, VIC and FAM are the fluorescent groups used in this experiment, which can also be replaced by other suitable fluorescent groups; GDL_F6_H, GDL_F7_H, GDL_F6_L, GDL_F7_L, DLTY_F8, DLTY_F9, and DLTY_F10 are upstream primers, and GDL_R6_H, GDL_R6_L, DLTY_R8, DLTY_R9, and DLTY_R10 are downstream primers.

[0086] 2.2 Fluorescence quantitative PCR reaction system and reaction conditions

[0087] The reaction system is as follows: the volume is 20 μL, including 10 μL of fluorescent PCR reaction mixture (2×), 0.3 μL each of upstream and downstream primers (20 μmol / L), probe (10 μmol / L), 1.1 μL of template DNA, and autoclaved ultrapure water to make up to 20 μL.

[0088] Prepare the reaction solutions for the test samples and positive controls according to the above system. A blank control is a reaction system containing no template DNA, with an equal volume of autoclaved ultrapure water replacing the template DNA. Three replicates are performed for each sample and control, and the CT value is the average of these three replicates.

[0089] Reaction conditions: 95°C, 30 seconds; 95°C, 5 seconds; 63°C, 15 seconds, 40 cycles.

[0090] 2.3. Examining the specificity of probe primers

[0091] The probe primers designed for Lupinus guangdilong were all positive when detecting the original species of Lupinus guangdilong; while the mixed and counterfeit products were all negative (see Table 5, Figure 1 shown).

[0092] Table 5 Investigation of the specificity of probe primers

[0093]

[0094]

[0095] 2.4. Examining the applicability of probe primers

[0096] The probe primers were tested on six batches of radix guangdilong, three batches of radix hudilong, and four batches of radix baoningensis. All earthworm samples tested positive with the universal probe primer (reference gene). The radix guangdilong-specific probe primer (target gene) tested positive for all radix guangdilong samples, while negative for radix hudilong and radix baoningensis (see Table 6).

[0097] Table 6 Applicability of probe primers

[0098] name serial number Internal reference gene CT±SD Target gene CT±SD Pheretima quinata GDL A1 18.11±0.06 18.87±0.14 Pheretima quinata GDL A3 17.60±0.08 18.39±0.18 Pheretima quinata GDL A4 19.79±0.12 20.58±0.08 Pheretima quinata GDL A5 18.63±0.08 19.61±0.09 Pheretima quinata GDL A7 18.58±0.11 19.54±0.10 Pheretima quinata GDL A8 18.38±0.10 19.36±0.07 Baoning earthworm MM2 18.12±0.05 NA Baoning earthworm MM3 19.14±0.08 NA Baoning earthworm MM4 21.43±0.03 NA Baoning earthworm MM5 19.34±0.02 NA William's worm HDL 842 18.82±0.06 NA Common earthworm HDL 851 19.15±0.09 NA William's worm HDL 852 18.01±0.11 NA

[0099] 2.5 Amplification efficiency

[0100] The DNA solution of P. truncatum was diluted 10-fold to obtain 7 DNA solutions with different concentrations and tested. The standard curve was drawn with the logarithm of DNA concentration as the abscissa and the CT value as the ordinate. The amplification efficiency was calculated according to the formula: Amplification efficiency (%) = [10( -1 / Slope)-1]×100%, the amplification efficiency of the specific probe primer was calculated to be 100.16%, and the amplification efficiency of the universal probe primer was 95.56% (e.g. Figure 2 shown).

[0101] 2.6 Detection Limit

[0102] The DNA solution of Dilong dasyphylla was diluted 10-fold in a gradient, and the original concentration was 10 ng / μL. The DNA concentration was still detectable at 0.0001 ng / μL (e.g. Figure 3 ) as shown.

[0103] 2.7 Repeatability

[0104] Prepare 5 concentration gradients of standard samples, 10-fold serial dilution, 6 replicates for each concentration, and calculate the coefficient of variation (CV) between experiments to establish a standard curve. Use the two standard deviation principle to compare the slope, correlation coefficient, and y-intercept (see Tables 7 and 8).

[0105] Table 7 Repeatability study of quantitative detection method of Dilong

[0106] Sample concentration (ng / μL) CT value CV (%) 1 22.79 1.23 0.1 26.27 1.60 0.01 29.74 0.58 0.001 32.99 1.19 0.0001 35.94 0.10

[0107] Table 8 Repeatability study of quantitative detection method of Dilong

[0108] Slope y-intercept <![CDATA[R 2 ]]> Amplification efficiency (%) Day 1 -3.318 23.06 0.9962 100.16 the next day -3.340 22.68 0.9984 99.25 mean -3.329 22.87 - 99.71 Standard deviation (SD) 0.02 0.27 - 0.64 Mean±2SD 3.329±0.04 22.87±0.54 - 99.71±1.28

[0109] 2.8 Precision

[0110] In the same experiment, 6 parallel groups were set up for the same sample. The standard error (CT) calculated based on the CT value of each group was 0.32, and the coefficient of variation (CV) was 1.35% (see Table 9).

[0111] Table 9 Investigation on the precision of quantitative detection method of P.

[0112]

[0113]

[0114] 2.9 Accuracy

[0115] 2.9.1 DNA solution mixing

[0116] P. guangxiong and terrestris DNA solutions were mixed at ratios of 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, and 1.5625% of the theoretical value and tested. The adulteration ratio was calculated using the CT difference method and a standard curve. The percentage of the measured value to the theoretical value was used as the recovery rate. The recovery rates for quantitative adulteration of P. guangxiong ranged from 88.68% to 112.12% (see Table 10).

[0117] Table 10 Quantitative detection of adulteration of Dilong chinensis

[0118]

[0119] 2.9.2 Sample powder mixing

[0120] Powders of Dilong dasyphylla and Dilong huenensis were mixed at ratios of 100%, 75%, 50%, 10%, 5%, and 1% of the theoretical value and tested. The adulteration ratio was calculated by the CT difference method, and the percentage of the measured value to the theoretical value was used as the recovery rate. The recovery rate of the quantitative adulteration test of Dilong dasyphylla ranged from 84.49% to 113.65% (see Table 11).

[0121] Table 11 Quantitative detection of adulteration of Dilong chinensis

[0122]

[0123]

[0124] 2.10 Durability

[0125] The fluorescence quantitative PCR instruments of different manufacturers were used to test the durability of the test method. Four concentrations of samples were taken and Roche and Analytikjena qTOWER 3The results of the test on samples of different concentrations using two instruments showed that the RSDs were all below 4% (see Table 12), indicating that the method has good durability.

[0126] Table 12 Detection results of different fluorescence quantitative PCR instruments

[0127]

[0128] 2.11. Positive Detection Determination and Quantitative Calculation Method

[0129] 1) For qualitative testing, set a reasonable △CT value as the reporting threshold depending on different situations.

[0130] 2) For relative quantitative detection, a standard (such as Dilong medicinal powder, DNA solution / dry powder, etc.) is required as a reference. Given that the polymerase chain reaction is an exponential amplification, the number of amplification cycles when the threshold is reached is the CT value; the difference in CT values ​​between the standard (S) and the test sample (T) is expressed as ΔCT. The proportion of target DNA in the test sample can be expressed as: test sample (T) / standard (S) % = 2 -△CT绝对值 ×100%.

[0131] Theoretically, when the absolute value of △CT is 4, the adulteration ratio is 6.25%, and when the absolute value of △CT is 5, the adulteration ratio is 3.125%. See Table 13 for details.

[0132] Table 13 Absolute values ​​of △CT corresponding to different adulteration ratios

[0133]

[0134]

[0135] However, in actual testing, a sample with a 5% adulteration ratio has an absolute ΔCT value between 4 and 5. This value can be adjusted to control the stringency of the test standard: a larger value lowers the detection limit and creates a more stringent standard.

[0136] 3) For absolute quantitative testing, a standard (e.g., Dilong herbal powder, DNA solution / dry powder, etc.) is required as a reference. Given that polymerase chain reaction (PCR) amplification is exponential, the number of amplification cycles required to reach the threshold is the CT value. An absolute quantitative standard curve is drawn with the logarithm of DNA concentration as the abscissa and the CT value as the ordinate. qPCR is performed on the test sample, and the concentration of Dilong DNA in the sample is calculated based on the CT value generated by the standard curve.

[0137] 2.12. Testing of commercially available medicinal materials, prepared prescriptions, homemade earthworm standard decoction freeze-dried powder, and homemade prescriptions

[0138] qPCR detection was performed on 9 batches of commercially available tertiary lumbar herbal medicine, 11 batches of non-tertiary lumbar samples, 4 batches of homemade lyophilized powder of standard tertiary lumbar decoction, 2 batches of homemade Xiaohuoluo pills, 1 batch of commercially available Mailuotong granules, and 5 batches of commercially available Xiaohuoluo pills (from Tables 1 and 2). See Tables 14 and 15 for details.

[0139] Among the 9 batches of commercially available Lupin medicinal materials, Lupin-specific signals were detected in 5 batches, and sequencing confirmed that they were all Lupins.

[0140] The target genes of 4 batches of imported commercially available Dilong medicinal materials and 10 batches of non-Dilong medicinal materials had no CT values, and sequencing confirmed that they were not Pheretima ginseng.

[0141] The target gene CT values ​​for three batches of homemade lyophilized powder of standard decoction of Dilong dasyphylla ranged from 24.82 to 27.04. The target gene CT value for homemade Xiaohuoluo pills (containing Dilong dasyphylla) was 13.48, the target gene CT value for commercially available Xiaohuoluo pills ranged from 16.09 to 35.28, and the target gene CT value for commercially available Mailuotong granules was 33.56.

[0142] Table 14 Detection of commercially available earthworm medicinal materials

[0143]

[0144]

[0145] Table 15 Testing of commercially available prescriptions, homemade earthworm standard decoction freeze-dried powder, and homemade prescriptions

[0146]

[0147]

[0148] In summary, the present invention has established a quantitative detection method for Guangdilong based on TaqMan probe fluorescence quantitative PCR technology, which can be distinguished from the three bases of Shanghai Dilong and the common Dilong easily confused products; and the detection fragment is short (<100bp), the amplification efficiency is good, the sensitivity is high, and it has good repeatability and durability. It is suitable for various forms of traditional Chinese medicine such as dried medicinal materials with severe DNA degradation, processed decoction pieces, formula granules, decoctions, and prescription preparations. Qualitative or quantitative detection of Guangdilong can be performed according to the detection requirements, and the quantitative results are highly accurate. At the same time, the invention also gives a principled suggestion for reasonably setting the reporting threshold in combination with the characteristics of traditional Chinese medicine.

[0149] Example 3

[0150] 3.1 Digital PCR (dPCR) reaction system and reaction conditions

[0151] Digital PCR detection was performed using the probe primers shown in Table 4.

[0152] Single-channel reaction system: The volume is 22 μL, including 10 μL of fluorescent PCR reaction mixture (2×), 0.22 μL of ROX Reference Dye II (50×), 1.1 μL each of upstream and downstream primers (20 μmol / L), 0.55 μL of probe (10 μmol / L), 4.4 μL of template DNA, and the volume is made up to 22 μL with high-pressure sterilized ultrapure water.

[0153] Prepare reaction solutions for the test samples and positive controls according to the above system. A blank control consists of a reaction system containing no template DNA and replacing the template DNA with an equal volume of autoclaved ultrapure water. Three replicates were performed for each sample and control, and the copy number was calculated as the average of the three replicates.

[0154] Double-label reaction system: The volume is 22 μL, including 10 μL of fluorescent PCR reaction mixture (2×), 0.22 μL of ROX Reference Dye II (50×), 1.32 μL each of upstream and downstream primers (20 μmol / L), 0.33 μL of probe (20 μmol / L), 4.4 μL of template DNA, and the volume is made up to 22 μL with autoclaved ultrapure water.

[0155] Prepare reaction solutions for the test samples and positive controls according to the above system. A blank control consists of a reaction system containing no template DNA and replacing the template DNA with an equal volume of autoclaved ultrapure water. Three replicates were performed for each sample and control, and the copy number was calculated as the average of the three replicates.

[0156] Reaction conditions: 95°C, 30 seconds; 95°C, 5 seconds; 63°C, 15 seconds, 45 cycles.

[0157] 3.2. Examining the specificity of probe primers

[0158] The probe primers designed for Lupinus guangdilong were all positive when detecting the original species of Lupinus guangdilong; while the mixed and counterfeit products were all negative (such as Figure 4 shown).

[0159] 3.3 Sensitivity

[0160] The DNA solution of the Luteinosa spp. sample was diluted 10-fold in a gradient, and the original concentration was 0.01 ng / μL. The DNA concentration was still detectable at 0.00001 ng / μL (e.g. Figure 5 shown).

[0161] 3.4 Accuracy

[0162] 3.4.1 Absolute Quantification Method Using Standard Curve

[0163] DNA was extracted from Lumbricus spp. samples and diluted to 0.01 ng / μL. The DNA solution was used as the standard to draw the standard curve for absolute quantitative PCR detection.

[0164] The absolute quantitative standard curve was drawn with DNA concentration as the horizontal axis and copy number as the vertical axis. The standard curve equation was: y = 27989x - 0.8832, and the correlation coefficient R 2 =0.9996 (as Figure 6 This shows that the detection results have good correlation and this standard curve can be used for subsequent experiments.

[0165] The samples to be tested were subjected to dPCR detection, and the DNA concentration of Lugulus dasyphylla in the samples was calculated based on the standard curve and the copy number generated by the detection.

[0166] A 0.1 ng / μL solution of Pteris annuli was mixed with a solution of Earthworm DNA from Pteris bonyeongensis at ratios of 100%, 50%, 10%, 5%, 3%, 1%, and 0. The mixture ratio was calculated by substituting the mixture ratio into the standard curve. The recovery rate was calculated as the percentage of the measured value to the theoretical value to evaluate the accuracy of the test method. The recovery rates for the quantitative detection of adulteration of Pteris bonyeongensis ranged from 90.54% to 109.81% (see Table 16).

[0167] Table 16 Recovery calculated by absolute quantitative method

[0168] Theoretical value (ng / μL) Detection value (ng / μL) Recovery rate 0.1 0.10981 109.81％ 0.05 0.05481 109.62％ 0.01 0.01051 105.10％ 0.005 0.00486 97.27％ 0.003 0.00272 90.54％ 0.001 0.00102 101.83％ 0 - -

[0169] 3.4.2 Relative Quantification Method by Ratio of Test Sample to Standard

[0170] A 0.1 ng / μL solution of P. annuli DNA was used as the standard. Test samples were prepared by mixing 0.1 ng / μL of P. annuli DNA with that of E. bornyingii DNA in ratios of 100%, 50%, 10%, 5%, 3%, 1%, and 0. The test ratio was calculated by comparing the test sample to the standard. The recovery rate, calculated as the percentage of the test value to the theoretical value, was used to evaluate the accuracy of the test method. The recovery rates for quantitative detection of adulteration in P. rubrum ranged from 81.51% to 99.79% (see Table 17).

[0171] Table 17 Recovery calculated by relative quantitative method

[0172] Theoretical value (mixing ratio) Detection value (mixing ratio) Recovery rate 100％ - - 50％ 49.90％ 99.79％ 10％ 9.54％ 95.45％ 5％ 4.40％ 88.02％ 3％ 2.45％ 81.51％ 1％ 0.90％ 89.88％ 0％ - -

[0173] 3.5 Quantitative calculation method

[0174] 1) For absolute quantitative testing using a standard curve, a standard substance (e.g., Dilong herbal powder, DNA solution / dry powder, etc.) can be used as a reference. An absolute quantitative standard curve can be drawn using DNA concentration as the horizontal axis and copy number as the vertical axis. dPCR is then performed on the test sample. Based on the standard curve, the copy number value generated by the test is used to calculate the concentration of Dilong DNA in the sample.

[0175] 2) In the relative quantitative detection process, the standard is used as a reference, and the copy number of the internal reference fragment in the standard is expressed as Cp S1 Indicates that the target fragment copy number is Cp S2 Indicates that the copy number of the internal reference fragment in the test sample is Cp T1 Indicates that the target fragment copy number is Cp T2 The proportion of target DNA in the test sample can be expressed as: test sample (T) / standard sample (S)%=[Cp(T2) / Cp(T1)] / [Cp(S2) / Cp(S1)]×100%.

[0176] 3.6 Testing of commercially available and homemade formulas

[0177] Digital PCR was performed on three batches of homemade Xiaohuoluowan and five batches of commercially available Xiaohuoluowan (from Table 2). The control medicinal material Dilong was used as a standard to calibrate the content of Dilong in the test samples.

[0178] The percentage of radix luteus was calculated using the relative quantification method using the ratio of the target gene to the reference gene. The content of radix luteus in the homemade Xiaohuoluo Pills (containing radix luteus) was 100.70%. Only the reference gene was positive for the homemade Xiaohuoluo Pills (containing radix luteus). The homemade Xiaohuoluo Pills (containing radix luteus) and the blank control showed no positive droplets. The commercially available samples XHLW01, XHLW04, XHLW05, XHLW06, and XHLW07 contained 0.81% radix luteus, 2.79% radix luteus, 45.81% radix luteus, 0.01% radix luteus, and 64.21% radix luteus (see Table 18).

[0179] Table 18 dPCR detection of commercially available Xiaohuoluo pills and homemade Xiaohuoluo pills

[0180]

[0181] In summary, the present invention establishes a quantitative detection method for Dilong dasyphylla based on digital PCR technology. By distributing nucleic acid samples into a large number of independent, parallel microreaction units, the independent amplification and visualization of individual template molecules is achieved, directly achieving absolute quantitative determination of nucleic acid molecules. Compared with qPCR, dPCR not only eliminates the reliance on standard curves and Ct values, but also reduces the interference of inhibitors on individual reaction units, thereby achieving significant improvements in sensitivity, interference resistance, and quantitative accuracy.

[0182] Compared with the qPCR method, the digital PCR detection method used in the present invention has the following advantages:

[0183] (1) High temperature and high pressure processes in the production of traditional Chinese medicines often lead to severe DNA fragmentation. Traditional qPCR is difficult to accurately quantify because it cannot effectively distinguish between background noise and real signals. However, dPCR uses droplet segmentation, and even if the target DNA concentration is as low as 0.00001 ng / μL, it can still achieve reliable detection through statistical independent positive signals.

[0184] (2) Polysaccharides, polyphenols and other substances commonly present in Chinese medicinal materials can easily inhibit the amplification efficiency of qPCR, resulting in false negatives or quantitative deviations. However, dPCR significantly improves its tolerance in complex matrices due to the segmentation and dilution of the reaction system. It can still maintain high specificity in compound preparations containing multiple medicinal materials, and its detection limit is reduced by at least 1-2 orders of magnitude compared with qPCR.

[0185] The technology of the present invention not only inherits the specific advantages of the qPCR identification method, but also demonstrates unique advantages in the analysis of complex traditional Chinese medicine samples due to its absolute quantification, high sensitivity and anti-interference ability, making up for the limitations of qPCR and providing a more reliable technical path for accurate identification and quality control.

[0186] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A probe primer set, characterized in that: It consists of a specific primer pair and a specific probe; Specific primer pairs include: GDL_F6_H, GDL_R6_H, GDL_F7_H, GDL_F6_L, GDL_F7_L, GDL_R6_L, and specific probes include: GDL_P6_H and GDL_P6_L; The nucleic acid sequence of GDL_P6_H is shown in SEQ ID NO.1, the nucleic acid sequence of GDL_F6_H is shown in SEQ ID NO.2, the nucleic acid sequence of GDL_R6_H is shown in SEQ ID NO.3, the nucleic acid sequence of GDL_F7_H is shown in SEQ ID NO.4, the nucleic acid sequence of GDL_P6_L is shown in SEQ ID NO.5, the nucleic acid sequence of GDL_F6_L is shown in SEQ ID NO.6, the nucleic acid sequence of GDL_F7_L is shown in SEQ ID NO.7, and the nucleic acid sequence of GDL_R6_L is shown in SEQ ID NO.

8.

2. The probe primer set according to claim 1, characterized in that Also included are universal primer pairs and universal probes. Universal primer pairs include: DLTY_F8, DLTY_R8, DLTY_F9, DLTY_R9, DLTY_F10, DLTY_R10; universal probes include: DLTY_P8, DLTY_P9, DLTY_P10; The nucleic acid sequence of DLTY_P8 is shown in SEQ ID NO.9, the nucleic acid sequence of DLTY_F8 is shown in SEQ ID NO.10, the nucleic acid sequence of DLTY_R8 is shown in SEQ ID NO.11, the nucleic acid sequence of DLTY_P9 is shown in SEQ ID NO.12, the nucleic acid sequence of DLTY_F9 is shown in SEQ ID NO.13, the nucleic acid sequence of DLTY_R9 is shown in SEQ ID NO.14, the nucleic acid sequence of DLTY_P10 is shown in SEQ ID NO.15, the nucleic acid sequence of DLTY_F10 is shown in SEQ ID NO.16, and the nucleic acid sequence of DLTY_R10 is shown in SEQ ID NO.

17.

3. Use of the probe primer set according to claim 1 or 2, or a reagent or kit containing the probe primer set according to claim 1 or 2, in any one of the following A1) to A6): A1) Identify or assist in the identification of Lumbricus radiatus; A2) preparing products for identification or assisting in the identification of P. A3) Differentiate or assist in differentiating Lumbricus radiatus from other medicinal earthworms; A4) preparing a product that distinguishes or assists in distinguishing Lumbricus radiatus from other medicinal earthworms; A5) Identifying whether the sample to be tested contains Lugurus radix; A6) preparing a product for identifying whether a sample to be tested contains P. radix serrata; A7) Identification or auxiliary identification to distinguish Luteolin and Luteolin easily confused products; A8) Prepare products for identification or auxiliary identification to distinguish Luteolin and Luteolin easily confused products.

4. The application according to claim 3, characterized in that The sample to be tested is at least one of medicinal materials, decoction pieces, prescription preparations, Chinese medicine granules, decoctions, and freeze-dried powders of extracts.

5. The application according to claim 3, characterized in that: The most easily confused products of Guangdilong are at least one of the following: common earthworm, William earthworm, comb-blind ring earthworm, Baoning earthworm, Earthworm genus, Coelophysis family, Earthworm genus, Zhili earthworm, fleshy Earthworm, dark hole Earthworm, and California earthworm.

6. A method for detecting P. dasyphylla, characterized in that: The method comprises the following steps: using the DNA of the sample to be tested as a template and adopting the probe primer set of claim 1 to perform amplification; if there is an amplification product, the sample to be tested is Lumbricus spp. or contains Lumbricus spp.; if there is no amplification product, the sample to be tested is not Lumbricus spp. or does not contain Lumbricus spp.

7. A method for detecting Luteolin or Luteolin easily confused products, characterized in that: The method comprises the following steps: using the sample DNA to be tested as a template and using the probe primer set according to claims 1 and 2 for amplification; If neither the universal primer pair and universal probe nor the specific primer pair and specific probe produce amplification products, then there is no Lumbricus radiatus or Lumbricus radiatus contaminants in the sample to be tested; If the universal primer pair and universal probe produce an amplification product, but the specific primer pair and specific probe do not produce an amplification product, then the sample to be tested is an earthworm species other than Lumbricus guangxiensis or contains an earthworm species other than Lumbricus guangxiensis; If the universal primer pair and universal probe, and the specific primer pair and specific probe all produce amplification products, then the sample to be tested is Lumbricus guangxiong or contains Lumbricus guangxiong.

8. The detection method according to claim 6 or 7, characterized in that: The detection method is a fluorescent quantitative PCR detection method or a digital PCR detection method; The lower limit of detection of the fluorescence quantitative PCR detection method is 0.0001 ng / μL, and the lower limit of detection of the digital PCR detection method is 0.00001 ng / μL.

9. The detection method according to claim 6 or 7, characterized in that: In the fluorescence quantitative PCR detection method, the amplification reaction system includes, by volume, 10 parts of fluorescent PCR reaction mixture, 0.3 parts of upstream primer, 0.3 parts of downstream primer, 0.3 parts of probe, and 1.1 parts of template DNA. Ultrapure water is added to a total volume of 20 parts of the system. The concentrations of upstream primer and downstream primer are 20 μmol / L, and the concentration of probe is 10 μmol / L. The amplification reaction conditions are as follows: 95°C, 30 seconds; 95°C, 5 seconds; 63°C, 15 seconds, for a total of 40 cycles. Preferably, in the fluorescent quantitative PCR detection method, the probe primer set according to claim 1 or 2 is used for amplification, and if amplification is possible, relative quantitative or absolute quantitative detection can also be performed; In the relative quantitative detection process, the standard is used as a reference, and the difference in CT value between the standard and the sample to be tested is expressed as ΔCT. The proportion of the target DNA in the sample to be tested can be expressed as: sample to be tested / standard % = 2 -△CT绝对值 ×100%; During the absolute quantitative detection process, a standard is used as a reference, the logarithm of the DNA concentration is used as the horizontal axis, and the CT value is used as the vertical axis to draw an absolute quantitative standard curve. The DNA concentration of Luteolin contained in the sample to be tested is calculated based on the absolute quantitative standard curve and the CT value obtained by amplification of the sample to be tested.

10. The detection method according to claim 6 or 7, characterized in that: In the digital PCR detection method, The single-channel amplification reaction system consists of the following by volume: 10 parts fluorescent PCR reaction mix, 0.22 parts ROX Reference Dye II, 1.1 parts upstream primer, 1.1 parts downstream primer, 0.55 parts probe, 4.4 parts template DNA, and ultrapure water to a total volume of 22 parts. The concentrations of the upstream and downstream primers are 20 μmol / L, and the probe concentration is 10 μmol / L. The dual-label amplification reaction system consists of the following by volume: 10 parts fluorescent PCR reaction mix, 0.22 parts ROX Reference Dye II, 1.32 parts upstream primer, 1.32 parts downstream primer, 0.33 parts probe, 4.4 parts template DNA, and ultrapure water to a total volume of 22 parts. The concentrations of the upstream primer, downstream primer, and probe are all 20 μmol / L. The amplification reaction conditions were as follows: 95°C, 30 seconds; 95°C, 5 seconds; 63°C, 15 seconds, for 45 cycles. Preferably, in the digital PCR detection method, the probe primer set according to claim 1 or 2 is used for amplification, and if amplification is possible, absolute quantitative or relative quantitative detection can also be performed; During the absolute quantitative detection process, a standard curve is drawn with DNA concentration as the horizontal coordinate and copy number as the vertical coordinate; based on the standard curve, the DNA concentration of Lu Lu in the sample is calculated according to the detected copy number value; the mass of Lu Lu standard (such as medicinal materials or formula granules) is used as the horizontal coordinate and the DNA concentration is used as the vertical coordinate to draw a standard curve; based on the standard curve, the mass of Lu Lu in the sample is calculated according to the DNA concentration; In the relative quantitative detection process, the standard was used as a reference, and the copy number of the universal primer pair and universal probe in the standard was expressed as Cp S1 Indicates that the target fragment copy number is Cp S2 The number of copies of universal primer pairs and universal probes in the test sample is expressed as Cp T1 Indicates that the target fragment copy number is Cp T2 If , the proportion of target DNA in the test sample is calculated according to the following formula: Test sample / standard product%=(Cp T2 ÷Cp T1 )÷(Cp S2 ÷Cp S1 )×100%.

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