Pcr primer pair and probe for identifying adulteration of sinomenium acutum in north radix sophorae tonkinensis and application thereof

Abstract

This invention belongs to the field of pharmaceutical testing technology, specifically relating to PCR primer pairs and probes for identifying adulteration of *Sinomenium acutum* in *Radix Sophorae Flavescentis* and their applications. The sequences of the PCR primer pairs are shown in SEQ ID NO: 2 and SEQ ID NO: 3, and the sequence of the probe is shown in SEQ ID NO: 1. The method for identifying adulteration of *Sinomenium acutum* in *Radix Sophorae Flavescentis* constructed based on the PCR primer pairs and probes provided by this invention has strong specificity for *Sinomenium acutum*, can distinguish it from common easily confused varieties, such as *Sinomenium acutum*, and has a reasonably set detection threshold limit, making it suitable for qualitative or quantitative detection of *Sinomenium acutum* in medicinal materials, processed medicinal slices, and compound preparations.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical testing technology, specifically relating to PCR primer pairs and probes for identifying adulteration of Sophora flavescens in Sophora tonkinensis and their applications. Background Technology

[0002] The 2020 edition of the Pharmacopoeia of the People's Republic of China (hereinafter referred to as the "Chinese Pharmacopoeia") lists *Menispermum dauricum* DC., a plant in the Menispermaceae family, as the dried rhizome. It is a deciduous vine, mainly found in the wild, primarily in Northeast China, North China, and Shaanxi Province. *Menispermum dauricum* is bitter and cold in nature; it is slightly toxic; it has the effects of clearing heat and detoxifying, dispelling wind and relieving pain; clinically, it is used for sore throat, dysentery due to heat toxicity, and rheumatic pain. Clinically, single-ingredient preparations such as tablets and aerosols have been developed for the treatment of oral and other inflammations; compound preparations are used for clearing heat and reducing inflammation, antibacterial effects, and immune regulation, with significant clinical efficacy.

[0003] With the increasing demand for medicinal use of *Sinomenium acutum*, it is easily confused with closely related vine species, such as *Sinomenium acutum* (Thunb.) Rehd. et Wils. and *Sinomenium acutum* (Thunb.) Rehd. et Wils. var. *cinereum* Rehd. et Wils., both belonging to the Menispermaceae family. These species are closely related and have similar appearances, which can easily lead to confusion during distribution and application, posing a higher risk to clinical medication safety.

[0004] Therefore, it is necessary to develop a method for adulterating Sophora flavescens with Chinese ginseng. Summary of the Invention

[0005] Based on this, the present invention analyzes the ITS sequences of Sophora flavescens and its easily confused varieties, designs specific probes and primers, and establishes a fluorescence quantitative PCR method for the specific detection of Sophora flavescens.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0007] The present invention provides a PCR primer pair and probe for identifying adulteration of *Sinomenium acutum* in *Sophora bean* root. The sequences of the PCR primer pair are shown in SEQ ID NO: 2 and SEQ ID NO: 3, and the sequence of the probe is shown in SEQ ID NO: 1.

[0008] Another aspect of the present invention provides a detection product for identifying adulteration of Sophora flavescens in Sophora tonkinensis, which includes the PCR primer pair and probe of the present invention.

[0009] Preferably, the above-mentioned testing product is a testing reagent or a testing kit.

[0010] Preferably, the above-mentioned detection products also include one or more of the following: PCR reaction mixture, autoclaved ultrapure water, standard or internal control probe.

[0011] In another aspect, the present invention provides a method for identifying adulteration of Sophora flavescens in Sophora tonkinensis, the method comprising: performing PCR amplification and detection on the DNA of the sample to be tested using the PCR primer pair and probe of the present invention or the detection product of the present invention.

[0012] Preferably, in the above method, the PCR amplification program is: 95℃, 30 seconds; 95℃, 5 seconds; 60℃, 15 seconds, 45 cycles.

[0013] Preferably, in the above method,

[0014] For qualitative testing, a CT value <30 is considered a positive result; and / or

[0015] The detection method is semi-quantitative. A standard is set as a reference, and the number of amplification rounds at which the threshold is reached is called the CT value. The difference between the CT values ​​of the standard (S) and the test sample (T) is represented by ΔCT. 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%.

[0016] In another aspect, the present invention provides the application of the PCR primer pair and probe of the present invention or the detection product of the present invention in identifying adulteration of Sophora flavescens in Sophora tonkinensis.

[0017] Preferably, in the above applications, the detection product includes detection reagents, detection kits, or microfluidic chips.

[0018] Preferably, in the above applications, the Sophora flavescens products include compound preparations of Sinensis spp., tablets, granules, or capsules.

[0019] The beneficial effects of this invention include: the method for identifying adulteration of Sinomenium acutum in Sophora flavescens based on the PCR primer pairs and probes provided by this invention has strong specificity for Sinomenium acutum and can be distinguished from common easily confused varieties, such as Sinomenium acutum. The detection threshold limit is reasonably set and it is suitable for qualitative or quantitative detection of Sinomenium acutum in medicinal materials, decoction pieces, and compound preparations. Attached Figure Description

[0020] Figure 1 The screening results for different primer-probe sets;

[0021] Figure 2 This is a specific amplification curve for Sophora flavescens.

[0022] Figure 3 The limit of detection is set for the qPCR detection method of Sophora flavescens. Detailed Implementation

[0023] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0024] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0025] This invention provides a PCR primer pair and probe for identifying adulteration of *Sinomenium acutum* in *Radix Sophorae Flavescentis*. The sequences of the PCR primer pair are shown in SEQ ID NO: 2 and SEQ ID NO: 3, and the sequence of the probe is shown in SEQ ID NO: 1.

[0026] It should be noted that this invention, through analysis of the ITS sequences of *Sophora tonkinensis* and its easily confused varieties, designed specific probes and primers to establish a fluorescence quantitative PCR method for the specific detection of *Sophora tonkinensis*. Furthermore, the internal transcribed spacer (ITS) sequence is a non-coding sequence found in eukaryotic ribosomal RNA (rRNA) genes, located between the 18S and 28S ribosomal RNA. It is widely used in species identification, providing an objective and accurate method to solve the difficult problems of mixed use and adulteration of Chinese medicinal materials, and is conducive to the standardization of Chinese medicine identification.

[0027] This invention also provides a detection product for identifying adulteration of Sophora flavescens in Sophora tonkinensis, which includes the PCR primer pair and probe of this invention.

[0028] It should be noted that the PCR primer pairs and probes in this invention can be prepared into detection products for identifying adulteration of *Sinomenium acutum* in *Radix Sophorae Flavescentis*. The form of the detection product is known in the art, such as a detection reagent or detection kit.

[0029] In some specific examples, the aforementioned testing products also include one or more of the following: PCR reaction mixture, autoclaved ultrapure water, standards, or internal control probes.

[0030] It should be noted that the detection products in this invention also include other auxiliary detection reagents, such as one or more of PCR reaction mixtures, autoclaved ultrapure water, standards, or internal control probes.

[0031] This invention also provides a method for identifying adulteration of Sophora flavescens in Sophora tonkinensis, the method comprising: performing PCR amplification and detection on the DNA of the sample to be tested using the PCR primer pair and probe of this invention or the detection product of this invention.

[0032] In some specific examples, the PCR amplification program in the above method is: 95℃, 30 seconds; 95℃, 5 seconds; 60℃, 15 seconds, 45 cycles.

[0033] In some specific examples, the above method,

[0034] For qualitative testing, a CT value <30 is considered a positive result; and / or

[0035] The detection method is semi-quantitative. A standard is set as a reference, and the number of amplification rounds at which the threshold is reached is called the CT value. The difference between the CT values ​​of the standard (S) and the test sample (T) is represented by ΔCT. 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%.

[0036] It should be noted that the PCR primer pairs and probes provided in this invention can be used to identify adulteration with *Sinomenium acutum* in *Sophora flavescens*. The constructed identification method can effectively detect *Sophora flavescens* and distinguish it from the closely related and easily confused *Sinomenium acutum*. It exhibits high specificity and sensitivity, and can be used for qualitative or quantitative detection of *Sophora flavescens* in various forms of traditional Chinese medicine, including medicinal materials, processed slices, and compound preparations. It has good practical value and application prospects. Furthermore, qualitative, semi-quantitative, or quantitative detection can be performed according to different detection needs using the above method.

[0037] This invention also provides an application of the PCR primer pair and probe of this invention or the detection product of this invention in identifying adulteration of Sophora flavescens in Sophora tonkinensis.

[0038] In some specific examples, the aforementioned testing products include testing reagents, testing kits, or microfluidic chips.

[0039] In some specific examples, the above applications include Sophora flavescens products such as compound preparations, tablets, granules, or capsules.

[0040] It should be noted that the PCR primer pairs and probes in this invention can be used to identify adulteration with Sinomenium acutum in raw materials of Sophora flavescens and its related products. The related products are known in the art, such as compound preparations of Sophora flavescens, tablets, granules or capsules.

[0041] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0042] The instruments used in the following examples are as follows: Real-time quantitative PCR instrument (qPCR-Analytik Jenaq TOW ER) 3 G,Roche Analytical balance (Mettler AB135-S), pure water system (Millipore), ball mill (M400, Retsch), NanoDrop One ultra-micro spectrophotometer (Thermo Fisher Scientific), benchtop high-speed refrigerated centrifuge (Eppendorf Centrifuge 5427R), micro centrifuge (IKA mini G S025), mixer (Eppendorf) C), Vortex oscillator (Scientific Industries Vortex-Genie2). sy mericon Food Kit (Qiagen), Probe qPCR Mix (2x) (RR392, RR391, TaKaRa); Go T Probe qPCR Mix (A610A, PROMEGA); HQ Probe qPCR Mix (ZF601, Beijing Zhuangmeng International Biotechnology Co., Ltd.).

[0043] In the following example, the sample processing and DNA extraction methods are as follows: The test sample is ground into a very fine powder, 20 mg is accurately weighed, and DNA is extracted using the Plant Genome Extraction Kit (DNeasy mericon Food Kit (69514, Qiagen)) (see the kit instructions for specific steps).

[0044] In the following examples, the samples were collected in more than 28 batches, including original samples of *Sinomenium acutum* and *Sinomenium pubescens*, reference medicinal materials of *Sinomenium acutum*, commercially available samples of *Sinomenium acutum*, reference medicinal materials of *Sophora benjamina*, and commercially available samples (Table 1).

[0045] Table 1 Sample Information Table

[0046]

[0047]

[0048] I. Establishment of a Real-Time PCR Detection Method

[0049] (I) Probe Primer Design and Screening

[0050] Based on DNA sequence analysis and alignment of the ITS region, probe primers were designed in conserved regions within species and regions with differences between species. More than two sets of probe primers were designed for each target species (Table 2).

[0051] Table 2 Probe Primer Sequence Information

[0052]

[0053] (II) PCR reaction system and reaction conditions

[0054] Reaction system: The volume is 20 μL, including 10 μL of fluorescent PCR reaction mixture (2×) (using RR392 premixed solution, containing PCR enzyme, dNTP Mixture, Mg2+, Ti RNase H), 0.6 μL each of forward and reverse primers (20 μmol / L) and probe (10 μmol / L), 1.1 μL of template DNA, and autoclaved ultrapure water to a total volume of 7.1 μL; 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 to replace the template DNA; both samples and controls are set in triplicate, and the CT value is taken as the average of the three replicates as the final result; reaction conditions: 95℃, 30 seconds; 95℃, 5 seconds; 60℃, 15 seconds, 45 cycles.

[0055] (III) Primer and probe screening

[0056] Following the PCR reaction system and conditions constructed above, different probe and primer sequences from Table 2 were used to detect BDG-1 and QFT-1 DNA, respectively. The results are as follows: Figure 1 As shown, the results indicate that the Group a primer and probe set elicited a line for Sophora flavescens (BDG-1) but not for Stellaria dichotoma (QFT-1), indicating that the Group a primer and probe set has strong specificity for Sophora flavescens.

[0057] II. Methodological Examination

[0058] (I) Examination of Specificity

[0059] The specificity of the selected probe primers (BDG-1_P, BDG-1_F, BDG-1_R in Table 1) was investigated. The probe primers designed for *Radix Sophorae Flavescentis* were all positive when detecting the original species of *Radix Sophorae Flavescentis*; the closely related and easily confused species *Sinomenium acutum* was negative (Table 3). Figure 2 ).

[0060] Table 3. Investigation of probe primer specificity

[0061]

[0062] (II) Applicability

[0063] The probe primers obtained from the screening were used to test the identifiable origins of *Sinomenium acutum* and *Sophora benjamina*. All *Sinomenium acutum* samples showed positive results, while *Sophora benjamina* samples showed negative results (Table 4).

[0064] Table 4. Sample Detection Results for Gene Identification

[0065]

[0066] (III) Repeatability

[0067] The selected probe primers were used to perform three repeated tests on the original sample of *Sinomenium acutum*. All results were positive (Table 5).

[0068] Table 5 Results of three replicate detections using the *Sinomenium acutum* probe primers.

[0069] Parallel groups CT mean 1 16.64 2 16.09 3 16.12 SD 0.3

[0070] (iv) Precision

[0071] In the same experiment, three parallel groups were set up for the same sample. The standard error (SD) was calculated based on the CT value of each group, and it was 0.1 for Qingfengteng (Table 6).

[0072] Table 6. Precision evaluation of the detection method for *Sinomenium acutum*

[0073]

[0074]

[0075] (v) Recovery rate

[0076] This invention uses screened probe primers to detect adulteration with *Sophora flavescens* and the easily confused *Sinomenium acutum* at ratios of 5% and 50% (powder mass ratio, where 5% and 50% represent 2.5 mg *Sinomenium acutum* + 45 mg *Sophora flavescens* and 25 mg *Sinomenium acutum* + 25 mg *Sophora flavescens*, respectively). The adulteration ratio is calculated using the CT difference method, and the percentage of the calculated value to the expected value is used as the recovery rate. The recovery rate for quantitative detection of adulteration with *Sinomenium acutum* ranges from 81.79% to 102.38%, as detailed in Table 7.

[0077] Table 7. Quantitative Detection of Adulteration with Qingfengteng

[0078] Expected value CT mean Difference in CT values Blending ratio (test value) Recovery rate 50％ 17.12 1.03 48.97％ 97.94％ 100％ 16.09 / / /

[0079] (vi) Limit of detection

[0080] The study investigated the effects of serial 10-fold dilutions of the original *Sinomenium acutum* DNA solution with sterile water, yielding five different concentrations: 1.16 ng / μL, 0.166 ng / μL, 0.0166 ng / μL, 0.00166 ng / μL, and 0.000166 ng / μL. DNA was still detectable at a concentration as low as 0.0001 ng / μL. Figure 3 ).

[0081] (vii) Durability

[0082] In the following examples, the RR391 and RR392 premixes contain PCR enzyme, dNTP mixture, and Mg. 2+ TiRNaseH; A610A premix contains GoTaq(R) hot-start polymerase, MgCl2, dNTPs, BRYT Green(R), and proprietary reaction buffer; ZF601Y premix contains Dye ROXA, Dye ROXB, and ddH2O.

[0083] (1) Investigation of different premixed solutions for quantitative PCR (probe method)

[0084] Using RR391 and RR392 (Probe qPCR Mix, 2X), and A610A (Go) respectively PCR amplification experiments were conducted using Probe qPCR MMx, 2X, PROMEGA and ZF601 (2xHQ Probe qPCR Mix, Beijing Zhuangmeng International Biotechnology Co., Ltd.). Due to differences in the types and activities of DNA polymerases from different manufacturers, the Ct values ​​generated by the PCR reaction were different, but all of them could detect CT values, indicating that the method has good applicability (Table 8).

[0085] Table 8. Evaluation of different premixed solutions for quantitative PCR (probe method)

[0086] Different reagents CT mean RR391 18.99 RR392 19.61 A610A 23.02 ZF601 24.46

[0087] (2) Investigation of different models of PCR instruments

[0088] Different manufacturers' real-time PCR instruments were used to test the method's tolerance. 20 mg of *Sinomenium acutum* sample was analyzed using Roche... and Analytikjena qTOWER 3 The G-type PCR instrument could obtain the expected identification results, with CT values ​​of 19.22 and 20.36, respectively, indicating that the method has strong applicability (Table 9).

[0089] Table 9. Detection results of different real-time PCR instruments

[0090]

[0091] (VIII) Methods for Determining and Quantifying Positive Detection

[0092] 1) For qualitative detection, the CT value threshold can be set at 30, and CT < 30 is considered as detected.

[0093] 2) For semi-quantitative detection, a standard (such as *Sinomenium acutum* 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%. Theoretically, when the absolute value of ΔCT is 4, the adulteration rate is 6.25%; when the absolute value of ΔCT is 5, the adulteration rate is 3.125%, as detailed in Table 10. In actual testing, samples with an adulteration rate of 5% have an absolute value of ΔCT between 4 and 5. Given that medicinal materials are allowed to contain impurities up to 3%, and considering the possibility of errors in testing, an absolute value of ΔCT ≤ 4 is usually used as the criterion for detection. 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.

[0094] Table 10 Absolute values ​​of ΔCT corresponding to different adulteration ratios

[0095] △CT adulteration ratio 1 50％ 2 25％ 3 12.5％ 4 6.25％ 5 3.13％ 6 1.56％

[0096] III. Sample Testing

[0097] The detection method constructed above could detect *Smilax china* (QFT1–QFT7 and QFT10–QFT18) and *Polygonum multiflorum* (BDG1–BDG7) in commercially available samples, with CT values ​​less than 30 in all samples. *Smilax china* samples did not show any detection (see Table 11). Specific signals were detected in all 7 batches of commercially available *Polygonum multiflorum* samples, and sequencing confirmed they were all *Pueraria lobata*. The target gene in all 16 batches of *Smilax china* samples showed no CT value, and sequencing confirmed they were all *Smilax china*.

[0098] Table 11 Market Sample Testing Results

[0099]

[0100]

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A PCR primer pair and probe for identifying adulteration with *Sinomenium acutum* in *Radix Sophorae Flavescentis*, characterized in that, The sequences of the PCR primer pairs are shown in SEQ ID NO: 2 and SEQ ID NO: 3, and the sequence of the probe is shown in SEQ ID NO:

1.

2. A detection product for identifying adulteration of Sinicanoctis sinica in S. sinica, characterized by, Includes the PCR primer pair and probe as described in claim 1.

3. The test product according to claim 2, characterized in that The product being tested is a testing reagent or testing kit.

4. The detection product according to claim 2 or 3, characterized in that, The testing products also include one or more of the following: PCR reaction mixture, autoclaved ultrapure water, standards or internal control probes.

5. A method for identifying adulteration of *Sinomenium acutum* in *Radix Sophorae Flavescentis*, characterized in that... The methods include: The DNA of the sample to be tested is amplified and detected by PCR using the PCR primer pair and probe of claim 1 or the detection product of any one of claims 2 to 4.

6. The method of claim 5, wherein, The PCR amplification program was: 95℃ for 30 seconds; 95℃ for 5 seconds; 60℃, 15 seconds, 45 cycles.

7. The method according to claim 5 or 6, characterized in that, For qualitative testing, a CT value <30 is considered a positive result; and / or The detection method is semi-quantitative. A standard is set as a reference, and the number of amplification rounds at which the threshold is reached is called the CT value. The difference between the CT values ​​of the standard (S) and the test sample (T) is represented by ΔCT. The proportion of the target DNA in the test sample is then expressed as: Test sample (T) / Standard (S)% = 2 -△CT绝对值 ×100%.

8. The use of the PCR primer pair and probe of claim 1 or the detection product of any one of claims 2 to 4 in identifying adulteration of Sophora flavescens in Sophora tonkinensis products.

9. Use according to claim 8, characterized in that, Testing products include testing reagents, testing kits, or microfluidic chips.

10. Use according to claim 8 or 9, characterized in that, Products made from Sophora flavescens include compound preparations of Sophora flavescens.

11. Use according to claim 8 or 9, characterized in that, Products containing Sophora flavescens include tablets, granules, or capsules.

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