Probe primers and uses thereof for identification of radix pulmonalis cum radicibus and PCR detection method

Abstract

This invention belongs to the field of pharmaceutical technology and relates to drug identification and detection methods, specifically to probe primers and their applications in the identification of *Pheretima aspergillum* and PCR detection methods. The probe primers consist of the sequences shown in SEQ ID NO. 1-8 and SEQ ID NO. 9-17; these probe primers are used for the specific and quantitative detection of *Pheretima aspergillum* using quantitative real-time PCR methods (including real-time quantitative PCR and digital PCR). The detection method provided by this invention can effectively detect *Pheretima aspergillum*, including newly discovered subgroups, exhibiting strong specificity and high sensitivity. It can be used for the qualitative or quantitative detection of *Pheretima aspergillum* in various forms of traditional Chinese medicine, including raw materials, processed slices, granules, decoctions, and compound preparations, demonstrating strong innovation and good application value.

Description

Probe primers and their applications in the identification of earthworms and PCR detection methods Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to drug identification and detection methods, specifically to probe primers and their use in the identification of earthworms and PCR detection methods. Background Technology

[0002] Earthworm is a commonly used animal-based medicine in clinical practice, possessing the effects of clearing heat and relieving spasms, calming the liver and extinguishing wind, promoting blood circulation and relieving asthma and promoting diuresis. Modern pharmacological studies have shown that earthworm has various pharmacological effects such as anticoagulation, thrombolysis, lowering blood pressure, and relieving asthma. The 2020 edition of the Chinese Pharmacopoeia specifies that it is derived from the dried bodies of the following species: *Pheretima aspergillum* (E. Perrier), *Pheretima vulgaris* Chen (also known as *Metaphire vulgaris*), *Pheretima guillelmi* (Michaelsen) (also known as *Metaphire guillelmi*), or *Pheretima pectinifera* Michaelsen. The former is commonly known as "Guangdong earthworm," and the latter three are commonly known as "Shanghai earthworm." Guangdong earthworm is mainly produced in Guangdong, Guangxi, and Fujian provinces. The quality standards for formulated granules explicitly require that the raw materials used be from Guangdong earthworm, therefore, the current market demand is relatively large. The latest research results (Acta Pharm SinB.2023Apr;13(4):1755-1770) show that there is significant genetic differentiation within the species of *Gnaphalium guangdianense*, which can be divided into two different subgroups.

[0003] my country boasts a rich diversity of earthworm species. To date, scholars from both China and abroad have recorded 388 species (including subspecies) of terrestrial earthworms in China. The origins of commercially available earthworm medicinal materials in my country are inconsistent, originating from at least 34 different species. Several other earthworms within the same genus and from the Eisenia genus of the Lumbriaceae family are also used medicinally in various regions, including non-pharmacopoeia species such as *Metaphire magna*, *Amynthas carnosus*, and *Amynthas obscuritoporus*. Furthermore, in recent years, with the increasing demand for earthworm medicinal materials and domestic production falling short of supply, the importation of earthworms from abroad has become increasingly common, further complicating their sourcing. The characteristics of earthworms are significantly diminished after processing, and their internal components are easily altered by processing, storage, and other factors, making it difficult to distinguish genuine from counterfeit products. In addition, the prevalence of adulterants with similar morphologies and the presence of different species with the same name further complicates the identification process. Currently, commonly used methods for identifying earthworms include traditional morphological identification, microscopic identification, physicochemical analysis, and molecular biological methods. However, these methods are often limited in application due to insufficient objectivity, poor specificity, low sensitivity, or weak resistance to interference, especially for highly processed samples such as traditional Chinese medicine preparations or granules. Therefore, it is necessary to establish a more effective method for identifying earthworms with broad specificity.

[0004] TaqMan probe assays monitor and analyze PCR amplification products in real time by adding probes that specifically bind to the template into the reaction system to generate fluorescence signals, enabling both qualitative and quantitative analysis. Compared to conventional PCR, it eliminates the need for steps such as agarose gel electrophoresis, EB or GoldView staining, is less time-consuming, and is less likely to cause contamination in gene amplification laboratories. It offers higher specificity and sensitivity, is rapid and accurate, and allows for multiplex fluorescence detection within the same reaction system by adding probes designed with different templates. This enables simultaneous detection of one or more specific targets in mixed samples, significantly improving detection efficiency.

[0005] Digital PCR (dPCR) is the third generation of PCR technology following real-time quantitative PCR (qPCR), and it is a novel 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 microreaction unit contains, on average, only one copy or no target DNA molecule. After PCR, the absolute count of target nucleic acid molecules is achieved by counting the positive droplets / total droplets, based on the Poisson distribution principle. dPCR can directly detect the copy number of the target sequence without relying on a standard curve or reference sample, with a detection limit down to single copy. It has superior sensitivity, specificity, and accuracy compared to traditional qPCR, exhibits strong resistance to inhibitors, and is unaffected by reaction amplification efficiency. It is particularly suitable for the absolute quantitative detection of nucleic acids in samples with complex matrices and is currently widely used in medicine, biology, food, and other fields. Furthermore, dPCR has demonstrated unique technical advantages and application prospects in the detection of Chinese medicinal materials. It not only provides technical support for the rapid identification of raw materials of Chinese medicinal materials, but can also be used for quality control in the production chain of prepared Chinese medicines. By developing single and multiplex dPCR detection methods, adulterants in highly processed products can be identified. For example, when analyzing raw materials and prepared formulations of Chinese medicinal materials such as Akebia quinata, turtle shell, and Notopterygium incisum, dPCR technology has shown good specificity, sensitivity and practicality.

[0006] The discovery of a new subpopulation of *Gnaphalium guangyuanensis* suggests that the establishment of DNA identification methods must fully consider the intraspecific variation within *Gnaphalium guangyuanensis*. COI sequence analysis reveals numerous differing sites between the new subpopulation and the classic *Gnaphalium guangyuanensis*. Previous DNA identification methods, without considering subpopulation variation, are likely to miss subpopulations, leading to false negative results. Therefore, it is necessary to establish a specific identification method for *Gnaphalium guangyuanensis* based on the genetic information of the newly discovered subpopulation to ensure the accuracy of *Gnaphalium guangyuanensis* identification. Summary of the Invention

[0007] The purpose of this invention is to provide probe primers and their applications in the identification of *Pheretima aspergillum* and PCR detection methods.

[0008] The detection method established in this invention can effectively detect *Pheretima aspergillum* and distinguish it from adulterants. It has strong specificity and high sensitivity, and can be used for the qualitative or quantitative detection of *Pheretima aspergillum* in Chinese medicine in the form of medicinal materials, decoction pieces, and compound preparations. It has good practical value and application prospects.

[0009] To achieve the above objectives, one objective of this invention is to provide a probe primer set, consisting 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, and downstream primer GDL_R6_L, and the specific probe includes: GDL_P6_H and GDL_P6_L.

[0010] The nucleic acid sequences of GDL_P6_H, GDL_F6_H, GDL_R6_H, GDL_F7_H, GDL_P6_L, GDL_F6_L, GDL_F7_L, and GDL_R6_L are shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, respectively.

[0011] Preferably, it also includes universal primer pairs and universal probes. The universal primer pairs include: upstream primer DLTY_F8, downstream primer DLTY_R8, upstream primer DLTY_F9, downstream primer DLTY_R9, upstream primer DLTY_F10, and downstream primer DLTY_R10. The universal probes include: DLTY_P8, DLTY_P9, and DLTY_P10.

[0012] The nucleic acid sequences of DLTY_P8 are shown in SEQ ID NO.9, DLTY_F8 in SEQ ID NO.10, DLTY_R8 in SEQ ID NO.11, DLTY_P9 in SEQ ID NO.12, DLTY_F9 in SEQ ID NO.13, DLTY_R9 in SEQ ID NO.14, DLTY_P10 in SEQ ID NO.15, DLTY_F10 in SEQ ID NO.16, and DLTY_R10 in SEQ ID NO.17.

[0013] The second objective of this invention is to provide the application of the above-mentioned probe primer set in any of the following A1)-A6):

[0014] A1) Identification or auxiliary identification of Guangdilong;

[0015] A2) Prepare products for identification or auxiliary identification of *Gastrodia elata*;

[0016] A3) To differentiate or assist in differentiating Guangdilong from other medicinal earthworms;

[0017] A4) Prepare products to differentiate or assist in differentiating *Pheretima aspergillum* from other medicinal earthworms;

[0018] A5) Identify whether the sample to be tested contains *Gastrodia elata*;

[0019] A6) Prepare a product to identify whether the sample to be tested contains *Gastrodia elata*;

[0020] A7) Identify or assist in the identification of *Gastrodia elata* and easily confused varieties of *Gastrodia elata*;

[0021] A8) Prepare products for identification or auxiliary identification to distinguish between *Diplophora guangdilong* and easily confused *Diplophora guangdilong* products.

[0022] Preferably, the sample to be tested is at least one of the following: medicinal materials, processed medicinal slices, compound preparations, traditional Chinese medicine formula granules, decoctions, and freeze-dried extract powder.

[0023] Preferably, the easily confused species of *Pheretima asiatica* are at least one of the following: *Pheretima spp.*, ... and *Pheretima spp.*.

[0024] A third objective of this invention is to provide a reagent or kit containing the above-mentioned probe primer set, wherein the reagent or kit functions as any one of the following B1)-B6).

[0025] B1) Identification or auxiliary identification of Guangdilong;

[0026] B2) Preparation of products for identification or auxiliary identification of *Gastrodia elata*;

[0027] B3) To differentiate or assist in differentiating Guangdilong from other medicinal earthworms;

[0028] B4) Prepare products to differentiate or assist in differentiating *Pheretima aspergillum* from other medicinal earthworms;

[0029] B5) Identify whether the sample to be tested contains *Gastrodia elata*;

[0030] B6) Prepare products to identify whether the sample to be tested contains *Gastrodia elata*;

[0031] B7) Identify or assist in the identification of *Pteris vittata* and easily confused varieties of *Pteris vittata*;

[0032] B8) Prepare products for identification or auxiliary identification to distinguish between *Diplophora guangdilong* and easily confused *Diplophora guangdilong* products.

[0033] Preferably, the sample to be tested is at least one of the following: medicinal materials, processed medicinal slices, compound preparations, traditional Chinese medicine formula granules, decoctions, and freeze-dried extract powder.

[0034] Preferably, the easily confused species of *Pheretima asiatica* are at least one of the following: *Pheretima spp.*, ... and *Pheretima spp.*.

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

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

[0037] The fifth objective of this invention is to provide a method for detecting *Pheretima aspergillum* or easily confused varieties of *Pheretima aspergillum*, comprising the following steps: using the DNA of the sample to be tested as a template, amplification is performed using the aforementioned probe and primer set; if neither the universal primer pair and universal probe, nor the specific primer pair and specific probe produce amplification products, then the sample to be tested does not contain *Pheretima aspergillum* or easily confused varieties of *Pheretima aspergillum*; if the universal primer pair and universal probe produce amplification products, but the specific primer pair and specific probe do not produce amplification products, then the sample to be tested contains other earthworm species besides *Pheretima aspergillum* or contains other earthworm species besides *Pheretima aspergillum*; if both the universal primer pair and universal probe, and the specific primer pair and specific probe produce amplification products, then the sample to be tested is *Pheretima aspergillum* or contains *Pheretima aspergillum*.

[0038] Preferably, the detection method is a real-time PCR method or a digital PCR method.

[0039] Preferably, the detection limit of the real-time PCR method is 0.0001 ng / μL.

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

[0041] Preferably, in the quantitative real-time PCR detection method, the amplification reaction system comprises, by volume fraction: 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 to make up to a total volume of 20 parts, wherein the concentration of upstream and downstream primers is 20 μmol / L, and the concentration of probe is 10 μmol / L.

[0042] Preferably, in the real-time PCR detection method, the amplification reaction conditions are as follows: 95℃ for 30 seconds; 95℃ for 5 seconds; 63℃ for 15 seconds, for a total of 40 cycles.

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

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

[0045] Theoretically, when the absolute value of ΔCT is 4, the adulteration rate is 6.25%, and when the absolute value of ΔCT is 5, the adulteration rate is 3.125%, as detailed in Table 12 below. However, in actual testing, samples with an adulteration rate of 5% have an absolute value of ΔCT between 4 and 5. The stringency of the testing standard can be controlled by adjusting this value; the larger the value, the lower the detection limit and the stricter the standard.

[0046] More preferably, in the absolute quantitative detection process, a standard is used as a reference, and an absolute quantitative standard curve is plotted with the logarithm of DNA concentration as the abscissa and the CT value as the ordinate. Based on the absolute quantitative standard curve and the CT value obtained from the amplification of the sample to be tested, the concentration of *Pheretima aspergillum* DNA contained in the sample to be tested is calculated.

[0047] Preferably, in the digital PCR detection method, the single-channel amplification reaction system comprises, by volume fraction: 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 make up to a total volume of 22 parts, wherein the concentration of upstream and downstream primers is 20 μmol / L, and the concentration of probe is 10 μmol / L.

[0048] Preferably, in the digital PCR detection method, the double-label amplification reaction system comprises, by volume fraction: 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 make up to a total volume of 22 parts, wherein the concentration of upstream primer, downstream primer and probe is 20 μmol / L.

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

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

[0051] More preferably, in the absolute quantitative detection process, a standard curve is plotted with DNA concentration as the abscissa and copy number as the ordinate; based on the standard curve, the DNA concentration of *Pheretima aspergillum* in the sample is calculated according to the detected copy number; a standard curve is plotted with the mass of *Pheretima aspergillum* standard (such as medicinal material or formula granules) as the abscissa and DNA concentration as the ordinate; based on the standard curve, the mass of *Pheretima aspergillum* 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 represented by Cp. S1 The number of copies of the target fragment is represented by Cp. S2 The copy number of universal primer pairs and universal probes in the test sample is indicated by Cp. T1 The number of copies of the target fragment is represented by Cp. T2 The proportion of the target DNA in the test sample is calculated using the following formula:

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

[0054] Compared with existing technologies, the present invention has the following advantages:

[0055] This invention designs a probe primer set and establishes a specific detection method for *Pheretima asiatica* based on TaqMan probe-based quantitative PCR technology. This method can distinguish *Pheretima asiatica* from common and easily confused varieties, such as *Pheretima asiatica* and *Pheretima baoningensis*. The detection threshold limit is reasonably set, making it suitable for qualitative or quantitative detection of *Pheretima asiatica* in medicinal materials, processed medicinal slices, and compound preparations. Attached Figure Description

[0056] Figure 1 shows the specificity of the real-time fluorescence quantitative PCR detection method for *Pheretima aspergillum*.

[0057] Figure 2 shows the fluorescence quantitative PCR amplification efficiency of the earthworm-specific probe primer set and the earthworm universal probe primer set.

[0058] Figure 3 shows the detection limit of the real-time quantitative PCR method for detecting *Pheretima aspergillum*. S1 represents a DNA template amount of 10 ng / μL, S2 represents a DNA template amount of 1 ng / μL, S3 represents a DNA template amount of 0.1 ng / μL, S4 represents a DNA template amount of 0.01 ng / μL, S5 represents a DNA template amount of 0.001 ng / μL, S6 represents a DNA template amount of 0.0001 ng / μL, S7 represents a DNA template amount of 0.00001 ng / μL, and NTC represents a template-free negative control.

[0059] Figure 4 shows the specificity of the digital PCR detection method for *Pheretima aspergillum*.

[0060] Figure 5 shows the detection limit of the digital PCR detection method for *Pheretima aspergillum*.

[0061] Figure 6 shows the absolute quantitative standard curve of the digital PCR detection method for *Pheretima aspergillum*. Detailed Implementation

[0062] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

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

[0064] The instruments and reagents used in this application are as follows: Real-time quantitative PCR instrument (Analytikjena qTOWER) 3 G, Roche The following equipment was used: Pangaea rapid quantitative PCR system, digital PCR instrument (Sniper DQ24), analytical balance (Mettler AB135-S), pure water system (Millipore), ball mill (MM400, Retsch), NanoDrop One ultra-micro spectrophotometer (Thermo Fisher Scientific), Qubit 4.0 quantitative PCR instrument (Thermo Fisher Scientific), benchtop high-speed refrigerated centrifuge (Eppendorf Centrifuge 5427R), microcentrifuge (IKAmini GS025), and mixer (Eppendorf). C), Vortex oscillator (Scientific Industries Vortex-Genie2), DNeasymericon Food Kit (Qiagen), Probe qPCR Mix (2×) (RR391A, TaKaRa).

[0065] Example 1

[0066] A total of 48 batches of samples were collected, including original samples of *Pheretima aspergillum*, reference medicinal materials of *Pheretima aspergillum*, and commercially available samples of *Pheretima aspergillum*. Six batches of commercially available prepared formulations were collected, and three batches of self-made prepared formulations and four batches of freeze-dried powder of *Pheretima aspergillum* standard decoction were collected (see Tables 1 and 2 for details). The COI sequences of *Pheretima aspergillum* and its adulterants are shown in Table 3.

[0067] Table 1 Information on Earthworm Medicinal Material Samples

[0068]

[0069]

[0070]

[0071] Table 2 Information on Freeze-dried Powder and Compound Preparations of Self-made Standard Decoction of Earthworm (Pheretima aspergillum)

[0072] Serial Number Sample Number Product Name Source 1XHLW01 Xiaohuoluo Pills Manufacturer 12XHLW04 Xiaohuoluo Pills Manufacturer 23XHLW05 Xiaohuoluo Pills Manufacturer 34XHLW06 Xiaohuoluo Pills Manufacturer 45XHLW07 Xiaohuoluo Pills Manufacturer 56MLTKL Mailuotong Granules Manufacturer 67XHLW GDL1 Laboratory-made Xiaohuoluo Pills (containing Guangdilong) China National Institutes for Food and Drug Control 8XHLW HDL Laboratory-made Xiaohuoluo Pills (containing Hudilong) China National Institutes for Food and Drug Control 9XHLW NTC Laboratory-made Xiaohuoluo Pills (excluding Dilong) China National Institutes for Food and Drug Control 10DGF GDL1 Laboratory-made Guangdilong Freeze-dried Powder China National Institutes for Food and Drug Control 11DGF GDL4 Laboratory-made Guangdilong Freeze-dried Powder China National Institutes for Food and Drug Control 12DGF GDL5 Laboratory-made Guangdilong Freeze-dried Powder China National Institutes for Food and Drug Control 13DGF HDL4 Laboratory-made Hudilong Freeze-dried Powder China National Institutes for Food and Drug Control surface

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

[0074]

[0075]

[0076] Example 2

[0077] 1. Sample processing and DNA extraction

[0078] The medicinal material was ground into an extremely fine powder, and 15 mg was accurately weighed. For the prepared medicine, 15 mg of earthworm was taken as the sample. DNA was extracted using the DNeasymericon Food Kit (see the kit instructions for specific steps).

[0079] 2. Establish a real-time PCR detection method

[0080] 2.1 Probe Primer Design and Screening

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

[0082] Table 4. Probe Primer Sequence Information

[0083]

[0084]

[0085] In Table 4, GDL_P6_H, GDL_P6_L, DLTY_P8, DLTY_P9, and DLTY_P10 are probes, and VIC and FAM are the fluorescent groups used in this experiment. They can also be replaced with 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, while GDL_R6_H, GDL_R6_L, DLTY_R8, DLTY_R9, and DLTY_R10 are downstream primers.

[0086] 2.2. Real-time PCR reaction system and reaction conditions

[0087] The reaction mixture is as follows: 20 μL, including 10 μL of fluorescent PCR reaction mixture (2×), 0.3 μL each of upstream and downstream primers (20 μmol / L) and 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. The blank control is a reaction system without template DNA, and an equal volume of autoclaved ultrapure water is used instead of template DNA. Both samples and controls are prepared in triplicate, and the CT value is taken as the average of the three replicates as the final result.

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

[0090] 2.3. Investigate the specificity of the probe primers.

[0091] The probe primers designed for *Pheretima asiatica* were all positive when detecting the original species of *Pheretima asiatica*; while the adulterants were all negative (see Table 5, as shown in Figure 1).

[0092] Table 5. Specificity assessment of probe primers

[0093]

[0094]

[0095] 2.4. Examine the suitability of the probe primers.

[0096] Six batches of *Pheretima guangyuan*, three batches of *Pheretima hulian*, and four batches of *Pheretima baoningensis*, whose original genes were identified, were tested using probe primers. Universal probe primers (internal reference gene) yielded positive results in all earthworm samples. *Pheretima guangyuan*-specific probe primers (target gene) were also used to test all *Pheretima guangyuan* samples, while *Pheretima hulian* and *Pheretima baoningensis* were negative (see Table 6).

[0097] Table 6. Suitability assessment of probe primers

[0098] Name Number Internal Reference Gene CT±SD Target Gene CT±SD *Pheretima asiatica* GDL A1 18.11±0.06 18.87±0.14 *Pheretima asiatica* GDL A3 17.60±0.08 18.39±0.18 *Pheretima asiatica* GDL A4 19.79±0.12 20.58±0.08 *Pheretima asiatica* GDL A5 18.63±0.08 19.61±0.09 *Pheretima asiatica* GDL A7 18.58±0.11 19.54±0.10 *Pheretima asiatica* GDL A8 18.38±0.10 19.36±0.07 Baoning Cordyceps MM2 18.12±0.05NA Baoning Cordyceps MM3 19.14±0.08NA Baoning Cordyceps MM4 21.43±0.03NA Baoning Cordyceps MM5 19.34±0.02NA William Cordyceps HDL 842 18.82±0.06NA Common Cordyceps HDL 851 19.15±0.09NA William Cordyceps HDL 852 18.01±0.11NA surface

[0099] 2.5 Amplification Efficiency

[0100] The *Pheretima aspergillum* DNA solution was serially diluted 10-fold to obtain seven different concentrations of DNA solution, which were then tested. A standard curve was plotted with the logarithm of DNA concentration on the x-axis and the CT value on the y-axis. The amplification efficiency was calculated using the formula: Amplification efficiency (%) = [10( -1 / The amplification efficiency of the specific probe primer was calculated to be 100.16% and that of the universal probe primer was 95.56% (as shown in Figure 2).

[0101] 2.6 Detection Limit

[0102] The DNA solution from *Pheretima aspergillum* was serially diluted 10-fold, with an original concentration of 10 ng / μL. The DNA was still detectable at a concentration of 0.0001 ng / μL (as shown in Figure 3).

[0103] 2.7 Repeatability

[0104] Five concentration gradient standards were prepared, serially diluted 10-fold, with six replicates for each concentration. The coefficient of variation (CV) between experiments was calculated, and a standard curve was established. The slope, correlation coefficient, and y-intercept were compared using the two standard deviations principle (see Tables 7 and 8).

[0105] Table 7 Repeatability Study of Quantitative Detection Method for *Diphtheria guangdilong*

[0106] Sample concentration (ng / μL) CT value CV (%) 122.79 1.23 0.126.27 1.60 0.0129.74 0.58 0.00132.99 1.19 0.000135.94 0.10 surface

[0107] Table 8 Repeatability test of quantitative detection method for *Pheretima aspergillum*

[0108] Slope y-intercept R 2 Amplification efficiency (%) Day 1: -3.318 23.06 0.996 210 0.16 Day 2: -3.340 22.68 0.998 499.25 Mean: -3.329 22.87 -99.71 Standard deviation (SD): 0.02 0.27 -0.64 Mean ± 2 SD: 3.329 ± 0.04 22.87 ± 0.54 -99.71 ± 1.28 surface

[0109] 2.8 Precision

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

[0111] Table 9. Precision evaluation of quantitative detection methods for *Earthworm*.

[0112]

[0113]

[0114] 2.9 Accuracy

[0115] 2.9.1 DNA solution mixing

[0116] DNA solutions of *Pheretima asiatica* and *Pheretima bambusoides* were mixed at theoretical ratios of 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, and 1.5625% and then detected. The adulteration ratio was calculated using CT difference and standard curve methods, and the percentage of the detected value to the theoretical value was used as the recovery rate. The recovery rate for quantitative detection of adulteration in *Pheretima asiatica* ranged from 88.68% to 112.12% (see Table 10).

[0117] Table 10 Quantitative Detection Results of Adulteration with Guangdilong

[0118]

[0119] 2.9.2 Sample Powder Mixing

[0120] Powders of Guangdilong and Hudilong were mixed at theoretical ratios of 100%, 75%, 50%, 10%, 5%, and 1% and then tested. The adulteration ratio was calculated using the CT difference method, and the percentage of the detected value to the theoretical value was used as the recovery rate. The recovery rate of Guangdilong adulteration in quantitative detection ranged from 84.49% to 113.65% (see Table 11).

[0121] Table 11 Quantitative Detection Results of Adulteration of Guangdilong

[0122]

[0123]

[0124] 2.10 Durability

[0125] The robustness of the experimental method was examined by using real-time PCR instruments from different manufacturers. Four concentrations of samples were tested using Roche methods. and Analytikjena qTOWER 3 The G-type PCR instrument was used for detection. The results of the detection of samples with different concentrations using two instruments showed that the RSD was below 4% (see Table 12), indicating that the method has good robustness.

[0126] Table 12 Detection results of different real-time PCR instruments

[0127]

[0128] 2.11. Methods for determining and quantifying positive detection

[0129] 1) For qualitative testing, set a reasonable ΔCT value as the reporting threshold, depending on the specific circumstances.

[0130] 2) For relative quantitative detection, a standard (such as earthworm powder, DNA solution / dry powder, etc.) needs to be set as a reference. Given that polymerase chain reaction (PCR) exhibits exponential amplification, the number of amplification rounds required to reach the threshold is the CT value. The difference between the CT values ​​of the standard (S) and the test sample (T) is represented by ΔCT. Therefore, the proportion of the 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 ΔCT is 4, the doping ratio is 6.25%, and when the absolute value of ΔCT is 5, the doping ratio is 3.125%. See Table 13 for details.

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

[0133]

[0134]

[0135] However, in actual testing, the absolute value of ΔCT for samples adulterated at a rate of 5% is between 4 and 5. The stringency of the testing standard can be controlled by adjusting this value; a higher value results in a lower detection limit and a more stringent standard.

[0136] 3) For absolute quantitative detection, a standard (such as earthworm powder, DNA solution / dry powder, etc.) needs to be set as a reference. Given that polymerase chain reaction (PCR) exhibits exponential amplification, the number of amplification rounds at which the threshold is reached is the CT value. An absolute quantitative standard curve is plotted with the logarithm of DNA concentration on the x-axis and the CT value on the y-axis. The sample to be tested is then subjected to qPCR detection. Based on the standard curve, the concentration of earthworm DNA in the sample is calculated according to the CT value generated by the detection.

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

[0138] Nine batches of commercially available Guangdilong medicinal materials, eleven batches of non-Guangdilong samples, four batches of self-made Guangdilong standard decoction freeze-dried powder, two batches of self-made Xiaohuoluo pills, one batch of commercially available Mailuotong granules, and five batches of commercially available Xiaohuoluo pills (from Tables 1 and 2) were tested by qPCR. See Tables 14 and 15 for details.

[0139] Five out of nine batches of commercially available *Pheretima aspergillum* medicinal materials were found to contain *Pheretima aspergillum*-specific signals, and sequencing confirmed that they were all *Pheretima spp.*

[0140] The target genes of 4 batches of imported commercially available *Pheretima aspergillum* medicinal materials and 10 batches of non-*Pheretima aspergillum* medicinal materials all had no CT values, and sequencing confirmed that none of them were *Pheretima aspergillum*.

[0141] The target gene CT values ​​of three batches of self-made Guangdilong standard decoction freeze-dried powder ranged from 24.82 to 27.04. The target gene CT value of self-made Xiaohuoluo pills (containing Guangdilong) was 13.48, the target gene CT values ​​of commercially available Xiaohuoluo pill samples ranged from 16.09 to 35.28, and the target gene CT value of commercially available Mailuotong granules was 33.56.

[0142] Table 14. Testing of Commercially Available Earthworm Herbs

[0143]

[0144]

[0145] Table 15 Tests of commercially available prepared medicines, homemade earthworm standard decoction freeze-dried powder, and self-made prepared medicines.

[0146]

[0147]

[0148] In summary, this invention establishes a specific quantitative detection method for *Pheretima aspergillum* based on TaqMan probe-based quantitative PCR technology. This method can differentiate *Pheretima aspergillum* from three original *Pheretima aspergillum* species and other commonly confused *Pheretima* varieties. Furthermore, it features a short detection fragment (<100bp), high amplification efficiency, high sensitivity, good repeatability and robustness, and is applicable to various forms of traditional Chinese medicine, including dried medicinal materials with severe DNA degradation, processed medicinal slices, granules, decoctions, and compound preparations. Qualitative or quantitative detection of *Pheretima aspergillum* can be performed according to specific testing requirements, with high accuracy in quantitative results. Additionally, this invention provides principled suggestions for rationally setting reporting thresholds based on 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 probes and 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 autoclaved ultrapure water to make up to 22 μL.

[0153] Prepare the reaction solutions for the test samples and positive controls according to the above system. The blank control is a reaction system without template DNA, and an equal volume of autoclaved ultrapure water is used instead of template DNA. Both samples and controls are prepared in triplicate, and the copy number is taken as the average of the three replicates as the final result.

[0154] Double-label reaction system: The volume is 22 μL, including 10 μL of fluorescent PCR reaction mixture (2×), 0.22 μL of ROX ReferenceDyeⅡ (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 autoclaved ultrapure water to make up to 22 μL.

[0155] Prepare the reaction solutions for the test samples and positive controls according to the above system. The blank control is a reaction system without template DNA, and an equal volume of autoclaved ultrapure water is used instead of template DNA. Both samples and controls are prepared in triplicate, and the copy number is taken as the average of the three replicates as the final result.

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

[0157] 3.2. Investigate the specificity of the probe primers.

[0158] The probe primers designed for *Pheretima asiatica* were all positive when detecting the original species of *Pheretima asiatica*; while the adulterants were all negative (as shown in Figure 4).

[0159] 3.3 Sensitivity

[0160] DNA solutions from *Pheretima aspergillum* samples were serially diluted 10-fold. The original concentration was 0.01 ng / μL, and DNA was still detectable at a concentration of 0.00001 ng / μL (as shown in Figure 5).

[0161] 3.4 Accuracy

[0162] 3.4.1 Absolute Quantification Method of Standard Curve

[0163] DNA was extracted from the earthworm sample and diluted to 0.01 ng / μL. Using this DNA solution as a standard, a standard curve for absolute quantitative PCR detection was plotted.

[0164] An absolute quantification standard curve was plotted with DNA concentration on the x-axis and copy number on the y-axis. The equation of the standard curve is: y = 27989x - 0.8832, and the correlation coefficient R0 is 0. 2 =0.9996 (as shown in Figure 6), proving that the detection results have good correlation. This standard curve can be used for subsequent experiments.

[0165] The samples to be tested were subjected to dPCR detection. Based on the standard curve, the concentration of *Pheretima aspergillum* DNA in the samples was calculated according to the number of copies generated.

[0166] DNA solutions of 0.1 ng / μL *Pheretima asiatica* and *Pheretima bournei* were mixed at ratios of 100%, 50%, 10%, 5%, 3%, 1%, and 0. The detection values ​​were calculated by substituting the mixtures into a standard curve. The percentage ratio of the detected value to the theoretical value was used as the recovery rate to evaluate the accuracy of the detection method. The recovery rate for quantitative detection of adulteration in *Pheretima asiatica* ranged from 90.54% to 109.81% (see Table 16).

[0167] Table 16. Recovery rate calculated using the absolute quantification method.

[0168] Theoretical value (ng / μL) Detected 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 0.0048697.27% 0.003 0.0027290.54% 0.001 0.00102101.83% 0 -- surface

[0169] 3.4.2 Relative Quantitative Method Based on the Ratio of Test Sample to Standard

[0170] A 0.1 ng / μL solution of *Pheretima aspergillum* DNA was used as a standard. 0.1 ng / μL solutions of *Pheretima aspergillum* and *Pheretima baoningensis* DNA were mixed at ratios of 100%, 50%, 10%, 5%, 3%, 1%, and 0 to prepare the test samples. The detection value of the mixture was calculated by comparing the test sample with the standard. The percentage of the detected value to the theoretical value was used as the recovery rate to evaluate the accuracy of the detection method. The recovery rate for quantitative detection of adulteration in *Pheretima aspergillum* ranged from 81.51% to 99.79% (see Table 17).

[0171] Table 17 Recovery rate calculated using the relative quantification method

[0172] Theoretical value (mixing ratio) Detected 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% -- surface

[0173] 3.5 Quantitative Calculation Methods

[0174] 1) For absolute quantitative detection using a standard curve, a standard (such as earthworm powder, DNA solution / dry powder, etc.) can be set as a reference; an absolute quantitative standard curve is plotted with DNA concentration as the x-axis and copy number as the y-axis. The sample to be tested is then subjected to dPCR detection, and based on the standard curve, the concentration of earthworm DNA in the sample is calculated according to the copy number generated by the detection.

[0175] 2) For relative quantitative detection, a standard is used as a reference, and the copy number of the internal reference fragment in the standard is expressed as Cp. S1 The number of copies of the target fragment is represented by Cp. S2 The copy number of the internal reference fragment in the test sample is represented by Cp. T1 The number of copies of the target fragment is represented by Cp. T2 If we express the percentage of target DNA in the test sample, it can be expressed as: Test sample (T) / Standard (S)% = [Cp(T2) / Cp(T1)] / [Cp(S2) / Cp(S1)] × 100%.

[0176] 3.6 Testing of commercially available and homemade formulations

[0177] Digital PCR was performed on 3 batches of self-made Xiaohuoluo pills and 5 batches of commercially available Xiaohuoluo pills (from Table 2). Guangdilong (a type of earthworm) was used as a reference material to correct the proportion of Guangdilong in the test samples.

[0178] The proportion of *Pheretima aspergillum* was calculated using the relative quantification method based on the ratio of the target gene to the internal reference gene: the content of *Pheretima aspergillum* in the self-made Xiaohuoluo Pill (containing *Pheretima aspergillum*) was 100.70%; only the internal reference gene in the self-made Xiaohuoluo Pill (containing *Pheretima aspergillum*) showed positive droplets; and neither the self-made Xiaohuoluo Pill (without *Pheretima aspergillum*) nor the blank control showed positive droplets. The content of *Pheretima aspergillum* in commercially available samples XHLW01 was 0.81%, XHLW04 was 2.79%, XHLW05 was 45.81%, XHLW06 was 0.01%, and XHLW07 was 64.21% (see Table 18).

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

[0180]

[0181] In summary, this invention establishes a specific quantitative detection method for earthworms based on digital PCR technology. By distributing nucleic acid samples into a large number of independent, parallel microreaction units, it achieves independent amplification and visualization of individual template molecules, directly realizing the absolute quantitative determination of nucleic acid molecules. Compared with qPCR, dPCR not only eliminates the dependence on standard curves and Ct values, but also reduces the interference of inhibitors on individual reaction units, thereby achieving significant improvements in sensitivity, anti-interference ability, and quantitative accuracy.

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

[0183] (1) In the production process of traditional Chinese medicine, high temperature and high pressure often lead to severe DNA fragmentation. Traditional qPCR is difficult to accurately quantify because it cannot effectively distinguish between background noise and real signal. However, dPCR can reliably detect DNA even if the target DNA concentration is as low as 0.00001 ng / μL by splitting it with microdroplets.

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

[0185] This invention not only inherits the specific advantages of qPCR identification methods, 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. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A probe primer set, characterized in that, It consists of specific primer pairs and specific probes; the specific primer pairs include: GDL_F6_H, GDL_R6_H, GDL_F7_H, GDL_F6_L, GDL_F7_L, and GDL_R6_L, and the 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. A probe primer set, characterized in that, include: The probe primer set of claim 1 further includes universal primer pairs and universal probes. The universal primer pairs include: DLTY_F8, DLTY_R8, DLTY_F9, DLTY_R9, DLTY_F10, and DLTY_R10. The universal probes include: DLTY_P8, DLTY_P9, and DLTY_P10. The nucleic acid sequences of DLTY_P8, DLTY_F8, DLTY_R8, and DLTY_P9 are shown in SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, and SEQ ID NO.

17.

3. The use of the probe primer set of claim 1 or 2, and the reagent or kit containing the probe primer set of claim 1 or 2, in the identification or auxiliary identification of *Pheretima asiatica*.

4. The use of the probe primer set of claim 1 or 2, or the reagent or kit containing the probe primer set of claim 1 or 2, in the preparation of products for identification or auxiliary identification of *Pheretima asiatica*.

5. A method for detecting *Pheretima asiatica*, characterized in that, The detection method is a real-time PCR method or a digital PCR method; including the following steps: using the DNA of the sample to be tested as a template, amplification is performed using the probe and primer set described in claim 1; if there is an amplification product, the sample to be tested is *Eriocheir chinensis* or contains *Eriocheir chinensis*, and if there is no amplification product, the sample to be tested is not *Eriocheir chinensis* or does not contain *Eriocheir chinensis*.

6. The detection method according to claim 5, characterized in that, In the quantitative real-time PCR detection method, the amplification reaction system includes the following volume fractions: 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 to make up to a total volume of 20 parts. The concentrations of upstream and downstream primers are 20 μmol / L, and the probe concentration is 10 μmol / L. The amplification reaction conditions are as follows: 95℃ for 30 seconds; 95℃ for 5 seconds; 63℃ for 15 seconds, for a total of 40 cycles.

7. The detection method according to claim 5, characterized in that, In the digital PCR detection method, the single-channel amplification reaction system, by volume fraction, includes: 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 make up to a total volume of 22 parts. The concentrations of upstream and downstream primers are 20 μmol / L, and the probe concentration is 10 μmol / L. The double-label amplification reaction system, by volume fraction, includes: 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 make up to a total volume of 22 parts. The concentrations of upstream, downstream primers, and probe are all 20 μmol / L. The amplification reaction conditions are as follows: 95℃ for 30 seconds; 95℃ for 5 seconds; 63℃ for 15 seconds, 45 cycles.

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