Primer pair for identifying honeysuckle and lonicerae flos and application of primer pair

Abstract

The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a primer pair for identifying honeysuckle and lonicerae flos and application of the primer pair. The sequences of the primer pair are as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. According to the present invention, after the PCR amplification is performed on the honeysuckle and the lonicerae flos by using the primer pair, the high resolution melting curve and the gel electrophoresis band of the PCR amplification product are analyzed to discover that the typing effect is good, and the honeysuckle and lonicerae flos samples with different mass ratios can be distinguished so as to achieve the identification of the honeysuckle and the lonicerae flos.

Description

[0001] This patent application is a subsequent application based on the priority of the patent application with application date of July 31, 2025, application number 2025110701190, and patent name "Primer pairs for identifying honeysuckle and Chinese honeysuckle and their applications". Technical Field

[0002] The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a primer pair for identifying honeysuckle and Chinese honeysuckle and application thereof. Background Art

[0003] As a commonly used and bulky Chinese medicinal material, honeysuckle has the effects of clearing away heat and detoxifying, dispersing wind and dissipating heat, and is widely used in clinical Chinese medicine and epidemic prevention and control. However, its market is often mixed with or substituted by honeysuckle. Although the Pharmacopoeia of the People's Republic of China has clearly stated since its 2005 edition that the origin of honeysuckle is only the plant Lonicera japonica, which is distinguished from honeysuckle (origins include Lonicera japonica, Lonicera rubra, etc.), the two are similar in morphology and have some overlapping pharmacological effects. In addition, the production of honeysuckle is low and the price is high, resulting in prominent market commodity confusion, which seriously affects the safety and effectiveness of medication.

[0004] At present, the identification methods of honeysuckle and Chinese honeysuckle are mainly divided into traditional methods and molecular biology methods, but both have significant shortcomings: among them, traditional methods (such as property identification, microscopic identification, physical and chemical identification) rely on empirical judgment or chemical composition analysis, and have low accuracy for deep-processed samples such as powders and preparations, and are easily affected by factors such as origin and growth environment; in addition, although molecular biology methods (such as RAPD, ISSR, DNA barcodes) have improved accuracy, the operation is complicated, the detection cycle is long, and quantitative analysis cannot be achieved, making it difficult to meet the needs of rapid and quantitative detection.

[0005] The high-resolution melting curve (HRM) technology is based on the changes in melting temperature caused by DNA sequence differences. It has the advantages of no need for sequencing, simple operation, high throughput, and low cost. Therefore, it has certain potential in the identification of honeysuckle and Chinese honeysuckle. Summary of the Invention

[0006] Based on this, the present invention, by designing specific primer pairs and combining HRM technology to analyze the high-resolution melting curves of PCR products, can achieve qualitative identification of honeysuckle and Chinese honeysuckle and quantitative detection of mixed samples. It also improves detection efficiency by establishing a standard high-resolution melting curve database. This method overcomes the shortcomings of traditional and existing molecular technologies and provides an innovative solution for market supervision, quality control, and clinical drug safety in traditional Chinese medicine.

[0007] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0008] In one aspect, the present invention provides a primer pair for identifying honeysuckle and Chinese honeysuckle, wherein the primer pair is selected from any one of the following pairs:

[0009] Primer pair 1: the forward primer sequence is shown in SEQ ID NO: 1, and the reverse primer sequence is shown in SEQ ID NO: 8;

[0010] Primer pair 2: the forward primer sequence is shown in SEQ ID NO: 1, and the reverse primer sequence is shown in SEQ ID NO: 9;

[0011] Primer pair 3: the forward primer sequence is shown in SEQ ID NO: 3, and the reverse primer sequence is shown in SEQ ID NO: 8;

[0012] Primer pair 4: the forward primer sequence is shown in SEQ ID NO: 4, and the reverse primer sequence is shown in SEQ ID NO: 8;

[0013] Primer pair 5: The sequence of the forward primer is shown in SEQ ID NO: 4, and the sequence of the reverse primer is shown in SEQ ID NO: 10.

[0014] Another aspect of the present invention provides a detection reagent for identifying honeysuckle and Chinese honeysuckle, which comprises the above primer pair and a fluorescent dye.

[0015] Preferably, the fluorescent dye is selected from EvaGreen and / or LC Green.

[0016] In another aspect, the present invention provides a method for identifying honeysuckle and Chinese honeysuckle, the method comprising:

[0017] Step 1: Obtain a high-resolution melting curve of the sample to be tested;

[0018] Step 2: Compare the high-resolution melting curve of the sample to be tested with the standard high-resolution melting curves in the standard library to determine the standard high-resolution melting curve with the highest similarity;

[0019] Step 3: Determine the mass ratio of honeysuckle and Chinese honeysuckle in the sample to be tested based on the mass ratio of honeysuckle and Chinese honeysuckle corresponding to the standard dissolution curve with the highest similarity;

[0020] Among them, the high-resolution melting curve in the sample to be tested is obtained by amplifying the DNA genome of the test sample with the above-mentioned detection reagent; the standard high-resolution melting curve in the standard library is obtained by amplifying the DNA genome of the standard sample with the above-mentioned detection reagent; the standard sample types include honeysuckle standard samples and / or honeysuckle standard samples.

[0021] Preferably, the high-resolution melting curve of the sample to be tested and the standard high-resolution melting curve in the standard library are both normalized curves.

[0022] Preferably, the above-mentioned standard sample types include honeysuckle standard samples, Chinese honeysuckle standard samples and mixed standard samples of honeysuckle standards and Chinese honeysuckle standards; in the mixed standard samples, the mass proportion of honeysuckle standards is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% and 10% respectively.

[0023] In another aspect, the present invention provides a device for identifying honeysuckle and Chinese honeysuckle, the device comprising:

[0024] A high-resolution melting curve acquisition module is configured to acquire a high-resolution melting curve of a sample to be detected;

[0025] a high-resolution melting curve comparison module configured to traverse the comparison and identify the standard high-resolution melting curve with the highest similarity between the high-resolution melting curve of the sample to be tested and the standard high-resolution melting curve in the standard library;

[0026] The identification module is configured to output the mass ratio result of honeysuckle or Chinese honeysuckle in the sample to be tested based on the mass ratio of honeysuckle or Chinese honeysuckle in the standard sample corresponding to the standard high-resolution melting curve with the highest similarity.

[0027] Among them, the high-resolution melting curve in the sample to be tested is obtained by amplifying the DNA genome of the test sample with the above-mentioned detection reagent; the standard high-resolution melting curve in the standard library is obtained by amplifying the DNA genome of the standard sample with the above-mentioned detection reagent; the standard sample types include honeysuckle standard samples and / or honeysuckle standard samples.

[0028] Another aspect of the present invention provides an electronic device, comprising: a processor, and a memory communicatively connected to the processor;

[0029] Memory stores computer-executable instructions;

[0030] The processor executes the computer-executable instructions stored in the memory to implement the above-mentioned method for identifying honeysuckle and Chinese honeysuckle.

[0031] On the other hand, the present invention provides a computer-readable storage medium, which stores computer-executable instructions. When the computer-executable instructions are executed by a processor, they are used to implement the above-mentioned method for identifying honeysuckle and Chinese honeysuckle.

[0032] In another aspect, the present invention provides an application of the above primers to the above detection reagents in identifying honeysuckle and Chinese honeysuckle.

[0033] The beneficial effects of the present invention include:

[0034] (1) After PCR amplification of honeysuckle and Chinese honeysuckle by the primer pair provided by the present invention, it was found by analyzing the high-resolution melting curve and gel electrophoresis bands of the PCR amplification products that the typing effect was good, and honeysuckle and Chinese honeysuckle samples with different mass ratios could be distinguished, thereby realizing the identification of honeysuckle and Chinese honeysuckle.

[0035] (2) Based on the primer pairs for identifying honeysuckle and Chinese honeysuckle of the present invention, combined with HRM technology to analyze the high-resolution melting curve of PCR products to establish a standard high-resolution melting curve database, qualitative and quantitative detection of honeysuckle and Chinese honeysuckle can be quickly achieved. The detection method is simple and fast, and the detection cycle is short (the total time for PCR amplification and HRM analysis is about 75 minutes). BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The relative position map of primers for PCR-HRM analysis of honeysuckle and Chinese honeysuckle;

[0037] Figure 2 The graph shows the normalized curve results of PCR amplification and HRM analysis using forward primer JYH1F and different reverse primers;

[0038] Figure 3 The graph shows the normalized curve results of PCR amplification and HRM analysis using forward primer JYH2F and different reverse primers;

[0039] Figure 4 The graph shows the normalized curve results of PCR amplification and HRM analysis using forward primer JYH3F and different reverse primers;

[0040] Figure 5 The graph shows the normalized curve results of PCR amplification and HRM analysis using forward primer JYH4F and different reverse primers;

[0041] Figure 6 The graph shows the normalized curve results of PCR amplification and HRM analysis using forward primer JYH5F and different reverse primers;

[0042] Figure 7 Result diagram of PCR amplification curves using different primers;

[0043] Figure 8 Results of the original solution and mixed solution (sequencing peak diagram); A: Honeysuckle 0%; B: Honeysuckle 30%; C: Honeysuckle 50%; D: Honeysuckle 70%; E: Honeysuckle 100%;

[0044] Figure 9The HRM analysis results of 11 different percentages of honeysuckle and Chinese honeysuckle; A: melting peak diagram; B: normalized high-resolution melting curve; C: differentiation curve;

[0045] Figure 10 The results of 50% mixed powder testing (sequencing peak diagram); A: Honeysuckle; B: Mixed powder 1; C: Mixed powder 2; D: Mixed powder 3; E: Honeysuckle;

[0046] Figure 11 Comparison results of normalized high-resolution melting curves between laboratory-mixed honeysuckle powder and the standard database;

[0047] Figure 12 This is a comparison of the normalized high-resolution melting curves of commercially available medicinal materials and the standard database. DETAILED DESCRIPTION

[0048] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0049] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0050] In the present invention, "detection reagents" not only refer to detection reagents in a narrow sense, but also include detection reagents in different product forms, such as test kits, chips and other product forms, collectively referred to as "detection reagents".

[0051] At present, the identification methods of honeysuckle and Chinese honeysuckle are mainly divided into traditional methods and molecular biology methods, but both have significant shortcomings: among them, traditional methods (such as property identification, microscopic identification, physical and chemical identification) rely on empirical judgment or chemical composition analysis, and have low accuracy for deep-processed samples such as powders and preparations, and are easily affected by factors such as origin and growth environment; in addition, although molecular biology methods (such as RAPD, ISSR, DNA barcodes) have improved accuracy, the operation is complicated, the detection cycle is long, and quantitative analysis cannot be achieved, making it difficult to meet the needs of rapid and quantitative detection.

[0052] In a first aspect, an embodiment of the present invention provides a primer pair for identifying honeysuckle and Chinese honeysuckle, wherein the primer pair is selected from any one of the following pairs:

[0053] Primer pair 1: the forward primer sequence is shown in SEQ ID NO: 1, and the reverse primer sequence is shown in SEQ ID NO: 8;

[0054] Primer pair 2: the forward primer sequence is shown in SEQ ID NO: 1, and the reverse primer sequence is shown in SEQ ID NO: 9;

[0055] Primer pair 3: the forward primer sequence is shown in SEQ ID NO: 3, and the reverse primer sequence is shown in SEQ ID NO: 8;

[0056] Primer pair 4: the forward primer sequence is shown in SEQ ID NO: 4, and the reverse primer sequence is shown in SEQ ID NO: 8;

[0057] Primer pair 5: The sequence of the forward primer is shown in SEQ ID NO: 4, and the sequence of the reverse primer is shown in SEQ ID NO: 10.

[0058] It should be noted that the above-mentioned 5 groups of primer pairs were obtained through design and screening in the present invention. After PCR amplification of honeysuckle and Chinese honeysuckle by the above-mentioned 5 groups of primer pairs, it was found by analyzing the high-resolution melting curve and gel electrophoresis bands of the PCR amplification products that the typing effect was good, and honeysuckle and Chinese honeysuckle samples of different mass ratios could be distinguished, and the identification of honeysuckle and Chinese honeysuckle could be realized; especially primer pair 1, the amplification fluorescence value was the highest, indicating that the detection effect was better.

[0059] In a second aspect, an embodiment of the present invention provides a detection reagent for identifying honeysuckle and Chinese honeysuckle, which includes the above-mentioned primer pair and a fluorescent dye.

[0060] It should be noted that the present invention needs to achieve the purpose of identifying honeysuckle and Chinese honeysuckle based on the melting curve of the primer pair amplification, so during amplification, it is necessary to use fluorescent materials for labeling. Fluorescent materials are well known in the art, such as fluorescent dyes.

[0061] In some specific examples, the fluorescent dye is selected from EvaGreen and / or LC Green.

[0062] It should be noted that fluorescent dyes have the advantages of low cost and no need for specific design. In addition, fluorescent dyes are well known in the art, such as one or more combinations of EvaGreen or LC Green, of which EvaGreen is more preferred and more suitable for HRM analysis.

[0063] In a third aspect, an embodiment of the present invention provides a method for identifying honeysuckle and Chinese honeysuckle, the method comprising:

[0064] Step 1: Obtain a high-resolution melting curve of the sample to be tested;

[0065] Step 2: Compare the high-resolution melting curve of the sample to be tested with the standard high-resolution melting curves in the standard library to determine the standard high-resolution melting curve with the highest similarity;

[0066] Step 3: Determine the mass ratio of honeysuckle and Chinese honeysuckle in the sample to be tested based on the mass ratio of honeysuckle and Chinese honeysuckle corresponding to the standard dissolution curve with the highest similarity;

[0067] Among them, the high-resolution melting curve in the sample to be tested is obtained by amplifying the DNA genome of the test sample with the above-mentioned detection reagent; the standard high-resolution melting curve in the standard library is obtained by amplifying the DNA genome of the standard sample with the above-mentioned detection reagent; the standard sample types include honeysuckle standard samples and / or honeysuckle standard samples.

[0068] In some specific examples, the high-resolution melting curve of the sample to be tested and the standard high-resolution melting curve in the standard library are both normalized curves.

[0069] It should be noted that normalizing the high-resolution melting curve can eliminate experimental variation, improve the comparability of high-resolution melting curves between different samples, and thus improve identification accuracy.

[0070] In some specific examples, the above-mentioned standard sample types include honeysuckle standard samples, Chinese honeysuckle standard samples and mixed standard samples of honeysuckle standards and Chinese honeysuckle standards; in the mixed standard samples, the mass proportion of honeysuckle standards is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% and 10% respectively.

[0071] It should be noted that the more and wider the standard high-resolution melting curves of standards with different mass ratios in the standard library of the present invention, the higher the accuracy of identification. In order to reduce the size of the database while ensuring a certain degree of accuracy, it is preferred to set the standard high-resolution melting curves according to the mass ratios of the honeysuckle standards listed above, which can ensure identification accuracy within a certain range. In addition, it should be understood that the standard library can be expanded at any time.

[0072] It should also be noted that in addition to manual judgment, similarity can also be determined by methods known in the art, such as similarity based on vector space models (such as cosine similarity, Euclidean distance or Manhattan distance), similarity of sets (such as Jaccard similarity coefficient or Dice coefficient), or similarity based on probability and statistics (Pearson correlation coefficient or KL divergence).

[0073] In a fourth aspect, an embodiment of the present invention provides a device for identifying honeysuckle and Chinese honeysuckle, the device comprising:

[0074] A high-resolution melting curve acquisition module is configured to acquire a high-resolution melting curve of a sample to be detected;

[0075] a high-resolution melting curve comparison module configured to traverse the comparison and identify the standard high-resolution melting curve with the highest similarity between the high-resolution melting curve of the sample to be tested and the standard high-resolution melting curve in the standard library;

[0076] The identification module is configured to output the mass ratio result of honeysuckle or Chinese honeysuckle in the sample to be tested based on the mass ratio of honeysuckle or Chinese honeysuckle in the standard sample corresponding to the standard high-resolution melting curve with the highest similarity.

[0077] Among them, the high-resolution melting curve in the sample to be tested is obtained by amplifying the DNA genome of the test sample with the above-mentioned detection reagent; the standard high-resolution melting curve in the standard library is obtained by amplifying the DNA genome of the standard sample with the above-mentioned detection reagent; the standard sample types include honeysuckle standard samples and / or honeysuckle standard samples.

[0078] It should be noted that the curves in the present invention can be compared by human naked eyes or by a device, which can improve the efficiency of identification.

[0079] In a fifth aspect, an embodiment of the present invention provides an electronic device, comprising: a processor, and a memory communicatively connected to the processor;

[0080] Memory stores computer-executable instructions;

[0081] The processor executes the computer-executable instructions stored in the memory to implement the above-mentioned method for identifying honeysuckle and Chinese honeysuckle.

[0082] In a sixth aspect, an embodiment of the present invention provides a computer-readable storage medium, in which computer execution instructions are stored. When the computer execution instructions are executed by a processor, they are used to implement the above-mentioned method for identifying honeysuckle and Chinese honeysuckle.

[0083] In a sixth aspect, an embodiment of the present invention provides an application of the above-mentioned primer pair and the above-mentioned detection reagent in identifying honeysuckle and Chinese honeysuckle.

[0084] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0085] In the following example, nine medicinal materials were collected (Table 1). The control materials, honeysuckle (DZ121060) and honeysuckle (DZ121595), were purchased from the China Food and Drug Administration. The remaining three samples were collected from the China (Bozhou) Traditional Chinese Medicine Trading Center. Another four samples were collected from pharmacies in Dezhou, Shandong Province. The control material powder was stored in a medicinal bottle. The commercially available materials were ground with liquid nitrogen and stored in 15 mL centrifuge tubes.

[0086] Table 1. Collection and identification results of Chinese medicinal materials samples

[0087]

[0088]

[0089] In the following examples, the instruments used include: compound rotor centrifuge (D1008, Dalong Xingchuang Laboratory Instrument Co., Ltd.), high pressure sterilizer (LS-75HD, Jiangyin Binjiang Medical Equipment Co., Ltd.), clean bench (SW-CJ-2D, Suzhou Purification Equipment Co., Ltd.), electric constant temperature water bath (HWS-12, Shanghai Yiheng Scientific Instrument Co., Ltd.), refrigerated high-speed centrifuge (5401, Germany Eppendorf AG) and fluorescence quantitative PCR instrument (Light Cycler 96, Roche), etc.

[0090] In the following examples, DNA was extracted using a plant genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP305) (the extraction method was referred to the kit instructions). After extraction, the DNA stock solution was stored at -20°C.

[0091] Example 1

[0092] The embodiment of the present invention designs and screens primer pairs suitable for identifying honeysuckle and Chinese honeysuckle.

[0093] (1) Confirmation of variant sites

[0094] (1) Using the plant genomic DNA extraction kit, extract the honeysuckle standard and the Chinese honeysuckle standard (both powders, no need to grind) to obtain a mixed standard DNA;

[0095] (2) PCR amplification of the standard medicinal material DNA ITS2 sequence fragment (the primers in the present application are derived from the ITS2 sequence of honeysuckle flower and flos lonicerae) to obtain PCR amplification products; the PCR amplification system and PCR amplification procedure are shown in Table 2; in addition, the sequence of the forward primer ITS2F is 5'-ATGCGATACTTGGTGTGAAT-3', and the sequence of the reverse primer ITS3R is 5'-GACGCTTCTCCAGACTACAAT-3';

[0096] Table 2 PCR amplification system and procedure of ITS2 sequence

[0097]

[0098] (3) After using agarose gel electrophoresis to detect the PCR amplification products to obtain amplification bands, the PCR amplification products are sent to the Jinan sequencing network of Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing;

[0099] (4) The Codoncode Aligner software (version 11.0.2) is used to process the spliced sequencing peak map, and the MEGA software (version 11.0.10) is used to analyze and compare the ITS2 sequence obtained in the experiment to determine the variation sites between honeysuckle flower and flos lonicerae.

[0100] (II) Primer design

[0101] According to the variation sites between honeysuckle flower and flos lonicerae, the upstream and downstream of the interspecific variation sequence fragment are determined, the conserved sequence between honeysuckle flower and flos lonicerae is found, and the forward and reverse primers (primer positions are shown in Figure 1 , and primer information is shown in Table 3) for PCR-HRM analysis of honeysuckle flower and flos lonicerae are designed.

[0102] Table 3 Primer information table for PCR-HRM analysis of honeysuckle flower and flos lonicerae

[0103]

[0104]

[0105] (III) Primer pair combination and screening

[0106] The 10 primers in Table 3 are combined to form 25 primer pair combinations, which are as follows:

[0107] JYH1F / JYH1R, JYH1F / JYH2R, JYH1F / JYH3R, JYH1F / JYH4R, JYH1F / JYH5R,

[0108] JYH2F / JYH1R, JYH2F / JYH2R, JYH2F / JYH3R, JYH2F / JYH4R, JYH2F / JYH5R,

[0109] JYH3F / JYH1R, JYH3F / JYH2R, JYH3F / JYH3R, JYH3F / JYH4R, JYH3F / JYH5R,

[0110] JYH4F / JYH1R, JYH4F / JYH2R, JYH4F / JYH3R, JYH4F / JYH4R, JYH4F / JYH5R,

[0111] JYH5F / JYH1R, JYH5F / JYH2R, JYH5F / JYH3R, JYH5F / JYH4R, JYH5F / JYH5R;

[0112] The above primer pairs were used to perform PCR-HRM analysis on the standard DNA of honeysuckle and the standard DNA of Chinese honeysuckle. The PCR amplification system and PCR-HRM amplification analysis procedure are shown in Table 4 below. After the reaction was completed, the high-resolution melting curves of the PCR amplification products of honeysuckle and Chinese honeysuckle at 66℃ to 96℃ were statistically analyzed. The results are as follows: Figures 2 to 6 shown.

[0113] Table 425 primer pair combination PCR-HRM amplification analysis system and procedure

[0114]

[0115]

[0116] The results showed that all 25 primer pairs amplified the sequence bands of honeysuckle and Chinese honeysuckle, as follows:

[0117] Among them, the typing effects of primer pairs such as JYH1F / JYH1R, JYH1F / JYH2R, JYH2F / JYH1R, JYH2F / JYH2R, JYH3F / JYH1R, JYH3F / JYH2R, and JYH3F / JYH4R were poor, and they could not clearly distinguish Lonicera japonica from Lonicera japonica.

[0118] The typing effect of primer pairs such as JYH1F / JYH5R, JYH2F / JYH3R, JYH2F / JYH4R, JYH2F / JYH5R, JYH3F / JYH5R, JYH4F / JYH1R, JYH4F / JYH2R, JYH4F / JYH4R, JYH5F / JYH1R, JYH5F / JYH2R, JYH5F / JYH3R, JYH5F / JYH4R, and JYH5F / JYH5R was average, and they could distinguish the two, but the discrimination was not enough for subsequent typing of samples with different ratios;

[0119] The primer pairs with better typing effects include JYH1F / JYH3R, JYH1F / JYH4R, JYH3F / JYH3R, JYH4F / JYH3R, and JYH4F / JYH5R, which can distinguish between Lonicera japonica and Lonicera japonica, and the discrimination is sufficient for typing samples with different proportions.

[0120] In addition, the amplification curves of the five primer pairs with good typing were studied ( Figure 7 ), it was found that the amplification fluorescence value of the JYH1F / JYH3R primer pair was the highest, indicating that the JYH1F / JYH3R primer pair had better adaptability.

[0121] Example 2

[0122] The embodiment of the present invention establishes a high-resolution melting curve standard database of a mixed sample of honeysuckle and Chinese honeysuckle standard DNA.

[0123] The honeysuckle standard DNA was mixed with the honeysuckle standard DNA according to 11 content gradients of 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% and 0% to obtain honeysuckle DNA stock solution, honeysuckle DNA stock solution and different DNA mixtures, which were used as DNA templates respectively. PCR amplification was performed according to the PCR amplification system and procedure in Table 4 above (the amplification primer pair was JYH1F / JYH3R) to obtain different PCR amplification products; then the different PCR amplification products were subjected to sequencing analysis and HRM analysis, respectively.

[0124] (1) Sequencing analysis

[0125] The different PCR amplification products obtained from the above PCR amplification were analyzed by agarose gel electrophoresis and then sent to the Testing Center of the Chinese Academy of Agricultural Sciences for sequencing. Based on the returned results, a 138-bp sequence amplified from JYH1F / JYH3R was intercepted and analyzed. Representative SNP bases from the sequencing peaks were selected and analyzed using CodoncodeAligner (version 11.0.2) to analyze the base peaks of the SNP sites in the different DNA mixture sequencing peaks of honeysuckle and honeysuckle, and to examine the correlation between the base peak ratio and the content gradient.

[0126] The results are as follows Figure 8 As shown, honeysuckle is a C base (blue) at this position, and Chinese honeysuckle is a T base (red); when the honeysuckle content ratio is 0%, the sequencing base only shows a T peak graph at this site, and when the honeysuckle content ratio gradually increases, the base peak at this site changes, such as when the honeysuckle content ratio is 30%, the T peak graph is high and the C peak graph is low; when the honeysuckle content ratio is 50%, the T and C base peak graphs are highly consistent; and when the honeysuckle content ratio is 70%, the C base peak begins to be higher than the T base peak, until it reaches 100% honeysuckle, and only the C base peak appears.

[0127] In addition, since the principle of HRM analysis is based on the differences in the types and numbers of bases contained in the sequence, the types and numbers of bases in the 138 bp sequence were statistically analyzed, and the results are shown in Table 5.

[0128] Table 5 DNA base content of samples

[0129] Honeysuckle content GC content AT content 0％ 63％ 37％ 30％ 63％ 37％ 50％ 64％ 36％ 70％ 65％ 35％ 100％ 66％ 34％

[0130] It can be seen from Table 5 above that as the honeysuckle content in the mixed sample increases, the GC content continues to increase, and the C base height in the sequencing peak graph continues to increase and gradually exceeds the T base height. When the honeysuckle content is 100%, there is only a C base peak.

[0131] The above data demonstrate that the mixtures of different percentages in the present invention are uniformly mixed, and that both honeysuckle and Chinese honeysuckle are present in the mixtures, with their content varying in a gradient. Subsequent HRM analysis of the standard mixtures will be performed based on these experimental results to determine whether the resulting HRM curves also exhibit a linear relationship with the sample DNA content, thus ensuring the accuracy of the database.

[0132] (2) HRM analysis

[0133] After PCR amplification, the PCR amplification products were analyzed using Light Cycler 96 (version 1.1.0.1320). The melting peak curves, HRM normalization curves, and HRM differentiation curves of the honeysuckle DNA stock solution, the Chinese honeysuckle DNA stock solution, and the DNA mixture were obtained, and a high-resolution melting curve database of honeysuckle and Chinese honeysuckle was established.

[0134] The results are as follows Figure 9 As shown in the figure, the peak values ​​(Tm values) and peak shapes of the melting peak curves of honeysuckle and Chinese honeysuckle are quite different, and the two can be effectively distinguished; the melting peak curves of the mixtures with different percentages are all double peaks, indicating that honeysuckle and Chinese honeysuckle are present in the mixture at the same time; and the melting peak curves of the mixtures with different percentages show a regular gradient change between honeysuckle and Chinese honeysuckle ( Figure 9 A); From the normalized curve, it can be seen that between 84℃ and 92℃, the curves of the mixtures with different percentages have good differentiation and can be effectively distinguished. As the proportion of one party in the mixture increases, its normalized curve gradually approaches the curve corresponding to 100% of the sample ( Figure 9 B); From the differentiation curve, the curves of different percentage mixtures also show regular changes between honeysuckle and Chinese honeysuckle, which can be effectively distinguished ( Figure 9 C).

[0135] Example 3

[0136] In the embodiment of the present invention, an identification test was performed on a mixture of a honeysuckle standard powder and a Chinese honeysuckle standard powder in different proportions.

[0137] (1) Base peak map comparison

[0138] After mixing the powder of the standard honeysuckle and the powder of the silver flower at a 50% mass ratio (three parallel experiments were designed, mixed powder 1, mixed powder 2, and mixed powder 3), DNA was extracted to obtain mixed powder DNA. The mixed powder DNA was then used as a DNA template for PCR amplification according to the PCR amplification system and procedure in Table 4 above (the primer pair was JYH1F / JYH3R) to obtain a PCR amplification product. After the PCR amplification product was detected by agarose gel electrophoresis, it was sent to the Testing Center of the Chinese Academy of Agricultural Sciences for sequencing. Based on the returned results, the base peaks of the sample SNP sites in the mixed powder DNA sequencing peaks were analyzed using CodoncodeAligner (version 11.0.2).

[0139] The sequencing results are as follows Figure 10As shown, representative SNP site bases were selected for analysis. Honeysuckle is C base (blue) at this position, and Chinese honeysuckle is T base (red). The peaks of T and C bases at this site of 50% honeysuckle powder are highly consistent, and the base peaks of 50% DNA mixed solution at this site in Example 2 are Figure 1 This indicates that the content of the two medicinal powders can still be detected after they are mixed and processed into Chinese patent medicine. Therefore, the present invention uses HRM-PCR technology to detect the mixed powder and compare it with the standard database to realize the content detection of the mixed sample.

[0140] (2) HRM curve comparison

[0141] The honeysuckle standard powder and the silver flower standard powder were mixed at a mass ratio of 50%, and DNA was extracted to obtain mixed powder DNA; the mixed powder DNA was then used as a DNA template to perform PCR amplification according to the PCR amplification system and procedure in Table 4 above (the amplification primer pair was JYH1F / JYH3R) to obtain a PCR amplification product (50% honeysuckle self-mixed powder);

[0142] Similarly, the honeysuckle standard powder and the silver flower standard powder were mixed at a ratio of 75% by mass of honeysuckle, and DNA was extracted to obtain mixed powder DNA; the mixed powder DNA was then used as a DNA template, and PCR amplification was performed according to the PCR amplification system and procedure in Table 4 above (the amplification primer pair was JYH1F / JYH3R) to obtain a PCR amplification product (70% honeysuckle self-mixed powder);

[0143] Light Cycler 96 (version 11.0.2) was used to obtain PCR amplification products (50% honeysuckle self-mixed powder) and PCR amplification products (70% honeysuckle self-mixed powder), respectively; they were compared with the HRM normalized high-resolution melting curves corresponding to the high-resolution melting curve database obtained in Example 2.

[0144] The results are as follows Figure 11 As shown in the figure, the normalized curve of 50% mixed powder and the curve of 50% honeysuckle have a good degree of overlap, and the normalized curve of 75% mixed powder is between the curve of 70% honeysuckle sample and the curve of 80% honeysuckle sample. The above results show that the method constructed by the present invention can more accurately detect the content ratio of honeysuckle and Chinese honeysuckle in mixed samples.

[0145] Example 4

[0146] The embodiment of the present invention performs identification tests on honeysuckle and Chinese honeysuckle medicinal materials purchased from the market.

[0147] Commercially available honeysuckle and honeysuckle medicinal materials (see Table 1) were crushed and DNA was extracted. PCR amplification was performed according to the PCR amplification system and program in Table 4 above (the amplification primer pair was JYH1F / JYH3R). Light Cycler 96 (version 11.0.2) was used to obtain the medicinal material melting peak curve and HRM normalization curve, which were compared with the high-resolution melting curve database curve established in Example 2 to identify the species composition of the commercially available medicinal materials.

[0148] The results are as follows Figure 12 As shown:

[0149] Among them, the normalized high-resolution melting curves of commercially available honeysuckle samples YC06MT14 and YC06MT15 are similar to those of the honeysuckle standard and can roughly overlap;

[0150] The normalized high-resolution melting curve of honeysuckle sample YC06MT02 is similar in shape to that of the honeysuckle standard and roughly overlaps with it, indicating that the three commercially available medicinal materials are indeed the commercially available honeysuckle or honeysuckle. This result is also consistent with the DNA barcode detection results.

[0151] However, the normalized high-resolution melting curves of commercially available honeysuckle samples YC06MT01, YC06MT05, YC06MT06 and YC06MT07 were similar to those of the honeysuckle standard, indicating that the above four commercially available honeysuckle samples were inconsistent with the nominal ones and were actually detected as honeysuckle. This result was also consistent with the DNA barcode test results.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A primer pair for identifying honeysuckle and Chinese honeysuckle, characterized in that: Select one of the following primer pairs: Primer pair 1: the forward primer sequence is shown in SEQ ID NO: 1, and the reverse primer sequence is shown in SEQ ID NO: 8; Primer pair 2: the forward primer sequence is shown in SEQ ID NO: 1, and the reverse primer sequence is shown in SEQ ID NO: 9; Primer pair 3: the forward primer sequence is shown in SEQ ID NO: 3, and the reverse primer sequence is shown in SEQ ID NO: 8; Primer pair 4: the forward primer sequence is shown in SEQ ID NO: 4, and the reverse primer sequence is shown in SEQ ID NO: 8; Primer pair 5: The sequence of the forward primer is shown in SEQ ID NO: 4, and the sequence of the reverse primer is shown in SEQ ID NO:

10.

2. A detection reagent for identifying honeysuckle and Chinese honeysuckle, characterized in that: The method comprises the primer pair according to claim 1 and a fluorescent dye.

3. The detection reagent according to claim 2, characterized in that The fluorescent dye is selected from EvaGreen and / or LCGreen.

4. A method for identifying honeysuckle and Chinese honeysuckle, characterized in that: Methods include: Step 1: Obtain the melting curve of the sample to be tested; Step 2: Compare the melting curve of the sample to be tested with the standard melting curves in the standard library to determine the standard high-resolution melting curve with the highest similarity; Step 3: Determine the mass ratio of honeysuckle and Chinese honeysuckle in the sample to be tested based on the mass ratio of honeysuckle and Chinese honeysuckle corresponding to the standard dissolution curve with the highest similarity; Wherein, the high-resolution melting curve in the sample to be detected is obtained by amplifying the DNA genome of the detection sample by the detection reagent according to claim 2 or 3; The standard high-resolution melting curve in the standard library is prepared by amplifying the DNA genome of the standard sample by the detection reagent described in claim 2 or 3; the standard sample types include honeysuckle standard samples and / or Chinese honeysuckle standard samples.

5. The method according to claim 4, characterized in that The high-resolution melting curve of the sample to be tested and the standard high-resolution melting curve in the standard library are both normalized curves.

6. The method according to claim 4 or 5, characterized in that The standard sample types include honeysuckle standard samples, Chinese honeysuckle standard samples and mixed standard samples of honeysuckle standard and Chinese honeysuckle standard; in the mixed standard samples, the mass proportion of honeysuckle standard is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% and 10% respectively.

7. A device for identifying honeysuckle and Chinese honeysuckle, characterized in that: The device includes: A high-resolution melting curve acquisition module is configured to acquire a high-resolution melting curve of a sample to be detected; a high-resolution melting curve comparison module configured to traverse the comparison and identify the standard high-resolution melting curve with the highest similarity between the high-resolution melting curve of the sample to be tested and the standard high-resolution melting curve in the standard library; The identification module is configured to output a mass ratio result of the honeysuckle or Chinese honeysuckle in the sample to be tested based on the mass ratio of the honeysuckle or Chinese honeysuckle in the standard sample corresponding to the standard high-resolution melting curve with the highest similarity; Wherein, the high-resolution melting curve in the sample to be detected is obtained by amplifying the DNA genome of the detection sample by the detection reagent according to claim 2 or 3; The standard high-resolution melting curve in the standard library is prepared by amplifying the DNA genome of the standard sample by the detection reagent described in claim 2 or 3; the standard sample types include honeysuckle standard samples and / or Chinese honeysuckle standard samples.

8. An electronic device, characterized in that: include: a processor, and a memory communicatively connected to the processor; Memory stores computer-executable instructions; The processor executes the computer-executable instructions stored in the memory to implement the method for identifying honeysuckle and Chinese honeysuckle according to any one of claims 4 to 6.

9. A computer-readable storage medium, characterized in that The computer-readable storage medium stores computer-executable instructions, which, when executed by a processor, are used to implement the method for identifying honeysuckle and Chinese honeysuckle according to any one of claims 4 to 6.

10. Use of the primer pair according to claim 1 or the detection reagent according to claim 2 or 3 in identifying honeysuckle and Chinese honeysuckle.

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