A method for identifying adulteration in Polygonatum sibiricum based on real-time quantitative PCR technology

Abstract

This invention discloses a method for identifying adulteration in Polygonatum sibiricum based on quantitative real-time PCR (qPCR), belonging to the field of pharmaceutical testing technology. This invention provides a primer pair and probe for identifying adulteration in Polygonatum sibiricum based on qPCR. It also provides a method for identifying adulteration in Polygonatum sibiricum, which includes the following steps: first, using the primer pair and probe, PCR amplification is performed on the test sample and Polygonatum sibiricum standard, respectively, to obtain amplification product A and amplification product B; then, identification is performed based on the presence or absence of fluorescence logarithmic growth in amplification product A, and the CT values ​​of amplification products A and B. This invention establishes a rapid nucleic acid detection PCR primer, probe, and method for identifying adulteration in Hubei Polygonatum sibiricum. This method is simple to operate, requiring only one reaction for identification, and features fast detection speed, high accuracy, high specificity, and good repeatability. It can perform accurate and rapid analysis, which is beneficial for its widespread application in enterprise testing practices.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical testing technology, and in particular to a method for identifying adulteration of Polygonatum odoratum based on quantitative real-time PCR technology. Background Technology

[0002] Polygonatum is a medicinal and edible resource in traditional Chinese medicine. It not only enhances the body's immunity but also has effects such as lowering blood pressure, blood sugar, and cholesterol, as well as anti-tumor, antibacterial, and antiviral properties. It also has broad development prospects in the research and development of new drugs and health products. my country has diverse Polygonatum germplasm resources, with more than 30 species of the genus Polygonatum found in my country.

[0003] Historical herbal texts record that medicinal Polygonatum comes from the rhizomes of various plants in the Polygonatum genus, including *Polygonatum zanlanscianense* Pamp. Its rhizome, as one source of the traditional Chinese medicine Polygonatum, has the effects of moistening the lungs and nourishing yin, tonifying the spleen and kidneys, and can be used to treat various diseases such as physical weakness, palpitations, shortness of breath, dry cough due to lung dryness, and diabetes. However, the Chinese Pharmacopoeia Commission explicitly stipulates that only three sources of Polygonatum are used medicinally: *Polygonatum sibiricum* Red, *Polygonatum cyrtonema* Hua, and *Polygonatum ingianum* Coll. et Hemsl. *Polygonatum zanlanscianense* Pamp., a commonly used folk medicine, is not included in these categories. Therefore, the phenomenon of confusing the dried rhizomes of *Polygonatum zanlanscianense* with Polygonatum in the market is quite common.

[0004] People usually classify Polygonatum plants based on their morphological characteristics. However, due to factors such as growing environment, climate, soil nutrients, and growth period, even the same species of Polygonatum can have different morphological characteristics when grown in different regions, so morphological identification methods often lack accuracy. Furthermore, the tubers of Polygonatum, as its main medicinal and edible material, are all nodular or beaded swellings, with extremely similar microscopic characteristics and highly similar physicochemical components. Therefore, traditional identification methods are easily affected by human or environmental factors and are difficult to use for identification.

[0005] Current technologies for quality control of Polygonatum sibiricum primarily rely on chemical component identification, but this method is only qualitative. Adulteration of Polygonatum sibiricum is rampant in the market, and adulteration methods are not entirely substitutes; rather, small amounts of Hubei Polygonatum sibiricum or other types of Polygonatum sibiricum are added to the raw material. Current technologies still cannot solve the problem of quantitative detection of this type of adulteration.

[0006] Given the limitations of morphological identification in the *Polygonatum* genus, molecular identification techniques have been used for phylogenetic studies, germplasm resource identification, and genetic diversity analysis of these plants. Real-time quantitative PCR (qPCR) is recognized as an important method for gene expression level analysis due to its high efficiency, high sensitivity, and good repeatability. qPCR technology has been widely applied in the biomedical field, particularly in testing and inspection, where numerous relevant standards exist. However, no standards have yet been published for the application of qPCR in the testing of traditional Chinese medicine.

[0007] The widespread and serious adulteration of Polygonatum sibiricum disrupts the fair competition of the market, significantly impacts the safety and efficacy of its use, and ultimately harms consumers' economic and health interests. Therefore, establishing relevant adulteration detection methods using quantitative real-time PCR technology is of great significance. Summary of the Invention

[0008] The purpose of this invention is to provide a method for identifying adulteration of Polygonatum odoratum based on quantitative real-time PCR (qPCR). To achieve quantitative detection of Polygonatum odoratum adulterated in the original Polygonatum odoratum, a qPCR detection system is used to detect and semi-quantitatively measure the copy number of Polygonatum odoratum in the template DNA. A rapid nucleic acid detection PCR primer, probe, and method for identifying adulteration with Polygonatum odoratum from Hubei Province are established.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] Technical Solution 1: A primer pair and probe for identifying adulteration of Polygonatum odoratum based on real-time PCR technology, wherein the primer pair contains an upstream primer 5'-CAGAGGCAAAGGACTAAG-3' and a downstream primer 5'-TCCCTATTGATACCGATTTG-3'; and the probe is 5'-ATTTTGATAACACATGGC-3'.

[0011] Furthermore, the probe is labeled with a fluorescent gene at its 5' end and a quencher group at its 3' end; the fluorescent gene is FAM and the quencher group is BHQ-1.

[0012] Technical Solution 2: A method for identifying adulteration of Polygonatum odoratum based on real-time fluorescence PCR technology, comprising the following steps: (1) using DNA from the sample to be tested and Polygonatum odoratum standard as templates, respectively, PCR amplification is performed using the primer pair and probe to obtain amplification product A and amplification product B; (2) whether Hubei Polygonatum odoratum is added to the original Polygonatum odoratum based on whether there is a fluorescence logarithmic growth in amplification product A and the CT values ​​of amplification product A and amplification product B.

[0013] Further, in step (1), the PCR amplification reaction program is: 94℃ / 3min; 94℃ / 10sec, 58℃ / 20sec, 72℃ / 30sec, 40 cycles.

[0014] Further, in step (1), the PCR amplification reaction system includes the following components: 10 μL Premix Ex-Taq, 0.4 μL upstream primer, 0.4 μL downstream primer, 0.4 μL probe, and 2 μL template, with water added to 20 μL.

[0015] Further, in step (2), the identification method is as follows: if the amplification product A has no fluorescence log growth, or the absolute value of the difference between the CT values ​​of the amplification product A and the amplification product B is >3.2, then it is identified as not being doped with Hubei Polygonatum; if the amplification product A has fluorescence log growth, and the absolute value of the difference between the CT values ​​of the amplification product A and the amplification product B is ≤3.2, then it is identified as being doped with Hubei Polygonatum.

[0016] Technical Solution 3: The application of the primer pair and probe described above in the identification of adulteration of Polygonatum odoratum.

[0017] The present invention discloses the following technical effects:

[0018] To achieve quantitative detection of *Polygonatum hupehensis* adulterated in *Polygonatum hupehensis* var. *hupehensis*, a real-time PCR system was used to detect and semi-quantitatively measure the copy number of *Polygonatum hupehensis* in the template DNA. This invention establishes a rapid nucleic acid detection PCR primer, probe, and method for identifying adulteration with *Polygonatum hupehensis*. This method is simple to operate, requiring only one reaction for identification. It is fast, accurate, specific, and reproducible, and can perform accurate and rapid analysis, which is beneficial for its widespread application in enterprise testing practices. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example 1

[0025] 1.1 Extraction of genomic DNA from *Polygonatum sibiricum* and its precursor samples from Hubei Province

[0026] (1) Take 0.1g of each of the three original plants (Polygonatum sibiricum Red, Polygonatum cyrtonema Hua, and Polygonatum ingianum Coll. et Hemsl) with accurate origins. Wash them sequentially with 1ml of 75% ethanol and 1ml of sterile ultrapure water, dry the surface moisture, and grind them into a fine powder in a ball mill (or liquid nitrogen grinding). Take 20mg and place it in a 1.5ml centrifuge tube. Use the plant genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. to extract nucleic acid and obtain the DNA template.

[0027] (2) Fluorescent PCR amplification reaction: The DNA template obtained above was added to the PCR reaction system for fluorescent PCR amplification reaction and CT value analysis. The total volume of the PCR reaction system was 20 μl, specifically: 10 μL Premix Ex-Taq, 0.4 μL upstream primer, 0.4 μL downstream primer, 0.4 μL probe, 2 μL template, and water was added to 20 μL. The PCR amplification reaction program was: 94℃ / 3 min, 94℃ / 10 ​​sec, 58℃ / 20 sec, 72℃ / 30 sec, with the 94℃ / 10 ​​sec to 72℃ / 30 sec cycle repeated 40 times.

[0028] (3) CT value analysis: PCR amplification products were analyzed using a Bio-Rad CFX96 analyzer.

[0029] The analysis results show that the probes and primers used in the experiment were tested on the leaves and rhizomes of Polygonatum hupehensis. The sampling details of the leaves and rhizomes are shown in Table 1.

[0030] Table 1

[0031]

[0032] All samples yielded positive results, while the original species of Polygonatum (Polygonatum sibiricum), Polygonatum cyrtonema, and Polygonatum kingianum all yielded negative results. Based on the amplification curves, it can be confirmed that the primers and probes of this invention can be used for the identification of adulteration in Hubei Polygonatum.

[0033] The base sequences of the primer pairs are as follows: Polygonatum odoratum - upstream primer: 5'-CAGAGGCAAAGGACTAAG-3'; Polygonatum odoratum - downstream primer: 5'-TCCCTATTGATACCGATTTG-3'; the base sequence of probe p is: 5'-attttGatAacAcaTggc-3'. The base sequence of the Polygonatum odoratum - upstream primer is shown in SEQ ID NO: 1; the base sequence of the Polygonatum odoratum - downstream primer is shown in SEQ ID NO: 2; and the base sequence of probe p is shown in SEQ ID NO: 3.

[0034] Example 2

[0035] Establishment of a method for identifying adulterated Polygonatum sibiricum from Hubei: Using the primers and probes obtained in Example 1, quantitative analysis of adulterated Polygonatum sibiricum from Hubei was performed, including the following steps:

[0036] (1) DNA extraction: Take the above-mentioned fine powders of three original samples of Polygonatum sibiricum Red, Polygonatum cyrtonema Hua, and Polygonatum ingianum Coll. et Hemsl. from Hubei, and accurately weigh 0.1g according to the proportions in Table 2, and mix them evenly. Take 20mg and place it in a 1.5ml centrifuge tube. Use the plant genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. to extract nucleic acid and obtain DNA template.

[0037] Table 2

[0038]

[0039] (2) Fluorescent PCR amplification reaction: The DNA template obtained above was added to the PCR reaction system for fluorescent PCR amplification reaction and CT value analysis. The total volume of the PCR reaction system was 20 μl, specifically consisting of 10 μL Premix Ex-Taq, 0.4 μL upstream primer, 0.4 μL downstream primer, 0.4 μL probe, and 2 μL template, with water added to a final volume of 20 μL. The PCR amplification reaction program was as follows: 94℃ / 3 min, 94℃ / 10 ​​sec, 58℃ / 20 sec, 72℃ / 30 sec, with the 94℃ / 10 ​​sec to 72℃ / 30 sec cycle repeated 40 times.

[0040] (3) CT value analysis: PCR amplification products were analyzed using a Bio-Rad CFX96 analyzer. The CT values ​​of samples with different adulteration ratios were analyzed, with 100% adulterated Hubei Polygonatum as a positive control. The relationship between the absolute value of the difference in CT values ​​(ΔCT absolute value) between samples with different adulteration ratios and positive samples was analyzed and the adulteration ratio was analyzed.

[0041] Given that polymerase chain reaction (PCR) exhibits exponential amplification, the number of amplification rounds required to reach the threshold is denoted by 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 target DNA in the test sample can be expressed as: [Test sample (T) / Standard (S)]% = 2 -△CT绝对值 ×100%.

[0042] Based on the above formula, the calculated and measured values ​​of the proportion of DNA in Hubei Polygonatum were calculated under different adulteration ratios. The calculation results are shown in Table 3. Table 3 shows the relationship between the actual adulteration ratio of Hubei Polygonatum and the absolute value of ΔCT.

[0043] Table 3

[0044]

[0045] In actual testing, given that medicinal materials are allowed to contain no more than 3% impurities, and taking into full account the error in the testing, when the actual adulteration is 10%, the ΔCT is 3.2. Therefore, the absolute value of ΔCT ≤ 3.2 is used as the basis for detection.

[0046] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer pair and probe for identifying adulteration of *Polygonatum sibiricum* from Hubei Province based on quantitative real-time PCR technology, characterized in that, The sequences of the primer pairs and probes are shown below: The upstream primer is shown in SEQ ID NO:1; The downstream primer is shown in SEQ ID NO:2; The probe is shown in SEQ ID NO:3; The probe is labeled with a fluorescent gene at its 5' end and a quencher group at its 3' end; the fluorescent gene is FAM and the quencher group is BHQ-1.

2. A method for identifying adulteration of *Polygonatum sibiricum* from Hubei Province based on quantitative real-time PCR technology, characterized in that... Includes the following steps: (1) Using the DNA of the sample to be tested and the Polygonatum standard as templates, PCR amplification was performed using the primer pair and probe described in claim 1 to obtain the amplification product A of the sample to be tested and the amplification product B of the Polygonatum standard, respectively. (2) The presence or absence of fluorescence log growth in the amplification product A of the test sample, and the CT values ​​of the amplification product A of the test sample and the amplification product B of the Polygonatum standard are used to identify whether Hubei Polygonatum is mixed in the Polygonatum sibiricum source. In step (2), the identification method is as follows: if the amplification product A of the sample to be tested has no fluorescence log growth, or the absolute value of the difference between the amplification product A of the sample to be tested and the amplification product B of the Polygonatum standard is >3.2, then it is identified as not being adulterated with Hubei Polygonatum. If the amplification product A of the test sample shows a logarithmic increase in fluorescence, and the absolute difference in CT values ​​between the amplification product A of the test sample and the amplification product B of the Polygonatum standard is ≤3.2, then it is identified as being adulterated with Hubei Polygonatum.

3. The method for identifying adulteration of Hubei Polygonatum as described in claim 2, characterized in that, In step (1), the PCR amplification reaction program is: 94℃ / 3min; 94℃ / 10sec, 58℃ / 20sec, 72℃ / 30sec, 40 cycles.

4. The method for identifying adulteration of Hubei Polygonatum as described in claim 2, characterized in that, In step (1), the PCR amplification reaction system includes the following components: 10 μL of Premix Ex-Taq, 0.4 μL of upstream primer, 0.4 μL of downstream primer, 0.4 μL of probe and 2 μL of template, with water added to 20 μL.

5. The application of the primer pair and probe as described in claim 1 in identifying adulteration of Polygonatum sibiricum in Hubei.

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