HRM primer and application thereof in identifying plant-derived components of tara and gleditsia

Abstract

The invention discloses an HRM primer and application of the HRM primer to identification of plant-derived components of tara and gleditsia. Aiming at the defect of lack of a product and a method for identifying high-value gleditsia sinensis lam pretended by low-cost tara seed endosperm at present, nuclear gene DNA of tara, gleditsia sinensis lam for producing gleditsia sinensis lam and common congeneric related species thereof are analyzed; an HRM primer capable of distinguishing plant-derived components of tara and gleditsia is obtained based on ITS sequence design. On the basis of the HRM primer, the invention also provides a kit and a method for identifying the tara and the gleditsia plant-derived components, and the tara can be rapidly and specifically detected, so that the identification of the gleditsia sinensis seeds adulterated with the tara seed endosperm is realized. The method is beneficial to inspection of adulterated gleditsia sinensis fruits, and economic benefits of consumers and gleditsia sinensis fruit commercial tenants and enterprises are protected.

Description

Technical Field

[0001] The invention belongs to the technical field of plant identification, and more specifically, relates to an HRM primer and an application thereof in identifying components derived from Tara and Gleditsia sinensis plants. Background Art

[0002] Gleditsia sinensis, commonly known as snow lotus seeds, gleditsia kernels, and gleditsia essence, is a product made from the dried mature fruit of the leguminous plant Gleditsia sinensis, which is made through processes such as de-podding, seed extraction, soaking, steaming, endosperm extraction, and drying. The main component of gleditsia sinensis is galactomannan, which is a high-energy, low-fat food. It is also rich in plant dietary fiber and a variety of mineral elements. It has the effects of nourishing the heart and meridians, clearing the liver and improving eyesight, and refreshing the mind and replenishing qi. It is usually used as a healthy meal replacement food and is deeply loved by consumers.

[0003] At present, part of the soapberry rice sold on the market comes from the endosperm of soapberry seeds, and part comes from the endosperm of seeds of soapberry related species in the genus Gleditsia, such as Gleditsia japonica, Gleditsia microphylla, and Gleditsia delavayi, all of which are sold as soapberry rice. Since the soapberry used to produce soapberry rice grows slowly, the output of soapberry rice is very limited, and its production and processing mainly rely on manual labor, which is inefficient, so the market price is relatively high. In order to obtain higher profits, illegal vendors will use other plant seed endosperms similar to soapberry rice, such as the seed endosperm of Tara, to impersonate it.

[0004] Tara, also known as Caesalpinia spinosa, has a seed endosperm that is very similar in shape and color to real sapodilla rice, and is also rich in plant polysaccharides. After being processed into a finished product of "sapodilla rice", it is extremely difficult for consumers to accurately distinguish it based on appearance alone. According to relevant information, Tara has a short growth cycle, and generally blooms and bears fruit in 3 years, with an acreage yield of 300 to 400 kilograms of pods, and a wholesale price of 5 to 10 yuan per kilogram. The sapodilla used to produce sapodilla rice requires about 6 to 8 years of vegetative growth to bloom and bear fruit, and the pod yield during the peak fruiting period is about 169 kilograms per mu, and the seed price is 50 to 140 yuan per kilogram. The huge price difference induces unscrupulous vendors to use cheap Tara seed endosperm to impersonate the more expensive sapodilla rice for sale. Although Tara seed endosperm can be used as a raw material for the production of food additive Tara gum, can it be directly eaten as food? How safe is it? There is no relevant research conclusion at home and abroad. In addition to food safety risks, the use of Tara seed endosperm to impersonate sapodilla rice for adulteration and sale will also damage the economic interests of consumers and law-abiding sapodilla rice merchants and enterprises, forming the unhealthy phenomenon of "bad money driving out good money", which seriously hinders the healthy development of the sapodilla rice industry. However, there is a lack of products or methods that can be used to detect the use of Tara seed endosperm to impersonate sapodilla rice for adulteration. Therefore, it is urgent to establish products and methods that can accurately distinguish between Tara and sapodilla plant-derived ingredients for rapid identification of adulterated sapodilla rice.

[0005] High-resolution melting (HRM) analysis mainly relies on physical properties such as the size of nucleic acid molecule fragments and GC content to achieve differentiation. It can identify extremely small base differences in product fragments, including single nucleotide polymorphisms, etc. It has the advantages of fast, accurate, high throughput, no need for fluorescent labeled probes, and low cost. It is suitable for the identification of mixed samples. This technology provides the possibility for rapid species identification of plant-derived ingredients. Although there are reports on the use of HRM analysis for the detection of plant-derived ingredients, such as a method developed based on the ITS2 fragment of Araliaceae plants to identify whether there are Araliaceae plant components in botanical medicinal samples and whether they are adulterated using HRM technology. However, the HRM primers required for the identification of different plants are different, which cannot inspire technicians on how to design HRM primers that can accurately distinguish between Tara and Gleditsia plants. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides an HRM primer and application thereof in identifying components derived from Tara and Gleditsia sinensis plants.

[0007] The first object of the present invention is to provide an HRM primer.

[0008] The second object of the present invention is to provide the use of the HRM primers in identifying components derived from Tara and Gleditsia sinensis plants.

[0009] The third object of the present invention is to provide the application of the HRM primer in the preparation of a product for identifying tara and Gleditsia plant-derived components.

[0010] The fourth object of the present invention is to provide a kit for identifying tara and Gleditsia plant-derived components.

[0011] The fifth object of the present invention is to provide a method for identifying tara and Gleditsia plant-derived components.

[0012] The above objects of the present invention are achieved by the following technical solutions:

[0013] To achieve the identification of tara seed endosperm adulterated with Gleditsia sinensis, based on HRM analysis, with the ITS sequence of the nuclear gene of tara DNA as the target, through homology alignment analysis of the nuclear gene ITS sequences of 7 species including tara, Gleditsia sinensis, and its closely related species Gleditsia japonica, Gleditsia microphylla, Gleditsia delavayi, Gleditsia triacanthos, and Gleditsia fera in the genus Gleditsia, a HRM primer capable of distinguishing tara and Gleditsia plant-derived components was designed and screened. Using the HRM primer, the tara seed endosperm adulterated with Gleditsia sinensis can be identified. The HRM primer has good universality and high detection sensitivity (10 ng / reaction), can identify and amplify trace DNA extracted from high-sugar and multi-gum Gleditsia sinensis products, and does not require complex enrichment and purification steps for sample DNA, realizing the rapid identification of tara components in Gleditsia sinensis products. Therefore, the present invention requests protection for the HRM primer and its related applications.

[0014] The present invention provides a HRM primer, and the nucleotide sequences of the primer are shown as SEQ ID NO.1 and SEQ ID NO.2, or as SEQ ID NO.3 and SEQ ID NO.4.

[0015] Using the HRM primer of the present invention and combining HRM analysis can achieve the identification of tara and Gleditsia plant-derived components. Therefore, the present invention requests protection for the application of the HRM primer in identifying tara and Gleditsia plant-derived components.

[0016] The present invention also requests protection for the application of the HRM primer in the preparation of a product for identifying tara and Gleditsia plant-derived components.

[0017] The present invention also provides a kit for identifying tara and Gleditsia plant-derived components, and the kit contains the HRM primer of the present invention.

[0018] Specifically, the kit also contains reagents required for fluorescence PCR reaction.

[0019] Optionally, the fluorescent dye used in the fluorescent PCR reaction is SYBR Green I.

[0020] In a specific embodiment of the present invention, the reagents required for the fluorescent PCR reaction are 2×qPCR PreMix reagents premixed with SYBR Green dye.

[0021] The present invention also provides a method for identifying the plant-derived components of Caesalpinia spinosa and Gleditsia, comprising the following steps:

[0022] S1. Extract the DNA of the sample to be tested;

[0023] S2. Using the DNA obtained in S1, positive control Caesalpinia spinosa, negative control Gleditsia DNA, and blank control as templates respectively, perform fluorescent PCR reaction using the HRM primers or the kit of the present invention, perform HRM analysis on the PCR amplification products, and compare the Tm values of the products;

[0024] S3. Result interpretation; if there is no amplification curve and melting curve in the blank control, both the positive control Caesalpinia spinosa and the tested sample have typical amplification curves and there is no significant difference in the Tm values of the melting curves of the amplification products, and the negative control Gleditsia has a typical amplification curve and the Tm value of the melting curve of the amplification product is greater than the Tm value of the melting curve of the positive control Caesalpinia spinosa, then the tested sample contains plant-derived components of Caesalpinia spinosa; wherein, the Tm values of the melting curves of the amplification products of the positive control Caesalpinia spinosa and the tested sample are evaluated for significant difference by t-test, and no significant difference means P>0.05.

[0025] Optionally, when extracting the DNA of the sample to be tested, the kit method or the CTAB method is used.

[0026] In a specific embodiment of the present invention, the kit used for extracting sample DNA is a plant genomic DNA extraction kit (DP360, Tiangen Biochemical Technology Co., Ltd.). Other recognized DNA extraction kits with the same efficacy can also be used.

[0027] Specifically, in the reaction system of the fluorescent PCR reaction in S2, the final concentration of the HRM primers is 0.4 - 0.6 μM.

[0028] More specifically, the final concentration of the HRM primers is 0.5 μM.

[0029] In a specific embodiment of the present invention, the fluorescent PCR reaction system used is: 10 μL 2×Fast Real qPCRPreMix (SYBR Green), the final concentration of HRM primers (274-F / 493-R) is 0.5 μM, 1 - 50 ng of DNA template, and the total system is 20 μL.

[0030] Specifically, the fluorescence PCR reaction described in S2 includes real-time fluorescence nucleic acid amplification reaction and HRM melting curve analysis of the amplification product; among them, the reaction conditions of the amplification reaction are: pre-denaturation at 95°C for 2-3 min; denaturation at 95°C for 4-6 s, annealing at 55°C for 10-12 s, 38-40 cycles; extension at 72°C for 14-16 s; the conditions of HRM melting curve analysis are: reaction at 95°C for 50-60 s; reaction at 60°C for 14-16 s; heating to 98°C for reaction for 4-6 s, and collecting fluorescence signals every 0.5°C increase in temperature.

[0031] More specifically, the reaction conditions of the amplification reaction are: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 5 s, annealing at 55°C for 10 s, 39 cycles; extension at 72°C for 15 s; the conditions of HRM melting curve analysis are: reaction at 95°C for 1 min; reaction at 60°C for 15 s; heating to 98°C for reaction for 5 s, and collecting fluorescence signals every 0.5°C increase in temperature.

[0032] In a specific embodiment of the present invention, when identifying tara and Gleditsia plant-derived components, the HRM primers used are the primers shown in SEQ ID NO.1 and SEQ ID NO.2.

[0033] When identifying tara and Gleditsia plant-derived components, if there is no amplification curve in the positive control or an amplification curve appears in the blank control, it indicates that there is an operation error or the reagent is contaminated, and re-detection is required.

[0034] Specifically, the Gleditsia plants described in the present invention are Gleditsia sinensis, Gleditsia japonica, Gleditsia microphylla, Gleditsia delavayi, Gleditsia triacanthos, and Gleditsia fera.

[0035] In a specific embodiment, the Gleditsia plants are Gleditsia sinensis, Gleditsia japonica, Gleditsia microphylla, and Gleditsia delavayi.

[0036] The present invention has the following beneficial effects:

[0037] 1. The HRM primers of the present invention have good amplification universality, and can universally identify and amplify the nuclear gene target fragments of tara and common species of Gleditsia seeds, such as Gleditsia sinensis, Gleditsia microphylla, Gleditsia japonica, and Gleditsia delavayi. The detection method for identifying tara adulteration in Gleditsia seeds constructed based on the HRM primers can specifically distinguish tara from common Gleditsia seed source species Gleditsia sinensis, Gleditsia japonica, Gleditsia microphylla, and Gleditsia delavayi, and is applicable to the detection and identification of tara components in Gleditsia seed products, with accurate results.

[0038] 2. Using different amounts of Tara genomic DNA as templates, the recognition absolute sensitivity of detection using the HRM primer pairs of the present invention was tested. The results showed that when the amounts of Tara genomic DNA were 25, 20, 15, and 10 ng respectively, amplification was carried out using the HRM primer pairs, and the melting curve Tm values of the finally formed amplification products ranged from 90°C to 92°C, which were significantly different from the melting curve Tm values (92°C to 95°C) of the amplification products of the negative control Gleditsia sinensis; when the amount of Tara genomic DNA was 1 ng, the HRM primer could not recognize and amplify it, and the amplification and analysis curve results were the same as those of the no-template control group (blank control, NTC). That is, when using the HRM primer pairs of the present invention to identify Tara plant-derived components, the detection sensitivity is as low as 10 ng, which can fully meet the detection requirements for adulterated Gleditsia sinensis with Tara.

[0039] 3. When using the HRM primer pairs of the present invention to identify Tara plant-derived components, by combining with a small portable fluorescence PCR instrument (such as the Gentier mini fluorescence quantitative PCR instrument (Tianlong Technology)), rapid identification and detection of Tara components in Gleditsia sinensis can be achieved under conditions without a laboratory environment. The operation is convenient, the results are easy to judge, it is suitable for on-site rapid detection of adulterated Gleditsia sinensis with Tara, and is especially suitable for rapid analysis by front-line detection personnel, with strong practicability. At the same time, due to the high detection sensitivity, it is also possible to recognize and amplify and analyze trace DNA of the extracted Gleditsia sinensis products, and the entire analysis process can be shortened to 1 h, greatly improving the detection efficiency and reducing the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Test results for identifying Tara and plants of the genus Gleditsia using 16 pairs of designed HRM primer pairs; the bold HRM analysis curves in the figure are Tara samples, and the remaining curves are Gleditsia sinensis, Gleditsia microphylla, and Gleditsia japonica respectively.

[0041] Figure 2 Amplification and HRM analysis methods established using the designed HRM primer set (6) (274-F / 493-R) for the recognition amplification universality of Tara and Gleditsia sinensis, Gleditsia microphylla, Gleditsia japonica, and Gleditsia delavayi targets and the specific detection results for identifying Tara; A in the figure is the detection result of recognition amplification universality, where the red amplification curve is the Tara sample, the blue horizontal line is the blank control (ddH 2 O), and the remaining 4 amplification curves are Gleditsia sinensis, Gleditsia microphylla, Gleditsia japonica, and Gleditsia delavayi samples respectively; B in the figure is the specific detection result for identifying Tara, where the red melting curve is the Tara sample, the blue horizontal line is the blank control, and the remaining 4 melting curves are Gleditsia sinensis, Gleditsia microphylla, Gleditsia japonica, and Gleditsia delavayi samples respectively.

[0042] Figure 3Detection limit test results of the amplification and HRM analysis method established using the designed HRM primer set (6) (274-F / 493-R); A in the figure is the sample amplification result, and B in the figure is the HRM melting curve analysis result; the amounts of Tara genomic DNA detected are 0 (NTC), 1, 10, 15, 20, 25 ng in sequence; Gleditsia sinensis genomic DNA (10 ng) is used as a control; the blank control (NTC) is ddH 2 O.

[0043] Figure 4 Target recognition amplification results of the amplification and HRM analysis method established using the designed HRM primer set (6) (274-F / 493-R) for 30 commercially available Gleditsia sinensis blind samples (Samples 1 - 30); the horizontal curve is the blank control (ddH 2 O); Tara control group (Tara DNA); G. sinensis control group (Gleditsia sinensis DNA).

[0044] Figure 5 Melting curve analysis results of the target amplification products of the amplification and HRM analysis method established using the designed HRM primer set (6) (274-F / 493-R) for 30 commercially available Gleditsia sinensis blind samples (Samples 1 - 30); the horizontal curve is the blank control (ddH 2 O); Tara control group (Tara DNA); G. sinensis control group (Gleditsia sinensis DNA).

[0045] Figure 6 For Figure 5 Statistical analysis results of the melting curve peaks corresponding to the Tm values of the melting curve analysis results of the shown target amplification products; the vertical axis is the Tm value, and the horizontal axis is the names of the Tara and G. sinensis control samples and the numbers (1 - 30) of the Gleditsia sinensis blind samples. Detailed implementation mode

[0046] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0047] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0048] Example 1 Design and screening of HRM primers

[0049] The present invention aims to identify adulterated Gleditsia sinensis with the endosperm of Caesalpinia spinosa seeds. Based on HRM analysis, the ITS sequence of the nuclear gene of Caesalpinia spinosa DNA is used as a target. Through homology alignment analysis of the nuclear gene ITS sequences of a total of 7 species, namely Caesalpinia spinosa, Gleditsia sinensis, and its closely related species in the genus Gleditsia, including Gleditsia japonica, Gleditsia microphylla, Gleditsia delavayi, Gleditsia triacanthos, and Gleditsia fera (the sequence information is from the GenBank database, and the species names, their Latin names, and the accession numbers of the sequences are shown in Table 1), a pair of HRM primers capable of distinguishing the plant-derived components of Caesalpinia spinosa from those of the genus Gleditsia are designed and screened out.

[0050] Table 1 Species and sequence information involved in the design of HRM primers

[0051]

[0052]

[0053] 1. Primer design

[0054] After homology alignment analysis of the sequences of the above 7 species, 16 pairs of HRM primers are preliminarily designed. The nucleotide sequences of the primers are shown in Table 2; some HRM primers share the same forward primer (Forward) or reverse primer (Reverse).

[0055] Table 2 Sequence information of the initially screened HRM primers

[0056]

[0057]

[0058] 2. Primer screening

[0059] The genomic DNA of Caesalpinia spinosa, Gleditsia sinensis, Gleditsia microphylla, and Gleditsia japonica is extracted respectively using a commercial plant genomic DNA extraction kit (DP360, Tiangen Biochemical Technology Co., Ltd.). Using this as a template, nucleic acid real-time fluorescence amplification reactions and HRM melting curve analyses of the amplification products are carried out respectively using the 16 pairs of HRM primers shown in Table 2 to screen the primers. Among them, the fluorescence PCR reaction system used for nucleic acid real-time fluorescence amplification is: 10 μL of 2×Fast Real qPCR PreMix (SYBR Green), the final concentration of the HRM primer is 0.5 μM, 1 - 50 ng of DNA template, and the total system is 20 μL; the reaction conditions for nucleic acid real-time fluorescence amplification are: 95°C, 2 min; 95°C, 5 s, 55°C, 10 s, cycle 39 times; 72°C, 15 s; the conditions for HRM melting curve analysis of the amplification products are: 95°C, 1 min; 60°C, 15 s, heating up to 98°C, collecting fluorescence signals once every 0.5°C increase in temperature; 98°C, 5 s; finally 35°C, 30 s.

[0060] The test results of using 16 pairs of designed HRM primers to identify Taraxacum mongolicum and Gleditsia plants are as follows Figure 1 shown; the bold HRM analysis curves in the figure are Taraxacum mongolicum samples, and the remaining curves are Gleditsia sinensis, Gleditsia microphylla, and Gleditsia japonica, respectively; the specific numerical values of the horizontal and vertical coordinates in the figure do not affect the screening of HRM primers. From Figure 1 the results shown, it can be seen that the 16 pairs of HRM primers designed in the present invention can all identify and successfully amplify the genomic DNA of Taraxacum mongolicum, Gleditsia sinensis, Gleditsia microphylla, and Gleditsia japonica, indicating that the designed primers have good universality for the target amplification of the genomic DNA of common species of Taraxacum mongolicum and Gleditsia plants.

[0061] HRM analysis of the amplification products of 16 pairs of HRM primers found that except for the primer sets (8) and (11) which may have a small amount of non-specific amplification, most of the remaining curves showed single peaks, indicating that the amplification products were single and could be used for the next analysis. Among them, there were obvious differences in the Tm values of the amplification products of the primer sets (6) and (7) for Taraxacum mongolicum and other Gleditsia plants. Therefore, the primer sets (6) (primer nucleotide sequences are shown in SEQ ID NO.1 - 2) and (7) (primer nucleotide sequences are shown in SEQ ID NO.3 - 4) can be used as alternative primer sets for distinguishing Taraxacum mongolicum and Gleditsia plants. In view of the fact that the amplification fragment length of the primer set (6) in the figure is longer than that of the primer set (7), with a size of 219bp, it may be convenient for direct Sanger sequencing of the products in the subsequent analysis process. Therefore, the primer set (6) was finally selected as the universal amplification primer for the establishment of a rapid HRM identification method for the adulteration of Taraxacum mongolicum in Gleditsia sinensis.

[0062] That is, in the present invention, the detection effect of the HRM primer set (6) was further analyzed and tested, and the primer sequences are as follows

[0063] 274 - F (SEQ ID NO.1): 5’-AACGACTCTCGGCAACGGA-3’;

[0064] 493 - R (SEQ ID NO.2): 5’-AGTCGCCCACGGGAGGCCAT-3’.

[0065] Example 2 Detection of Target Recognition Amplification Universality and Discrimination Specificity

[0066] 1. Detection of Target Recognition Amplification Universality

[0067] Based on the HRM primer set (6) (274-F / 493-R) designed and screened in Example 1 and the method for amplifying and performing HRM analysis on tara and plants of the genus Gleditsia using the same, in this example, genomic DNA of tara, Gleditsia sinensis, Gleditsia japonica, Gleditsia microphylla, and Gleditsia delavayi was used as a template, and the recognition and amplification universality of the HRM primers for the targets of plants of the genus Gleditsia was tested using the said method. The results are as Figure 2 shown in A of

[0068] As can be seen from Figure 2 A of

[0069] , the HRM primers of the present invention can quickly recognize and amplify tara, Gleditsia sinensis, Gleditsia japonica, Gleditsia microphylla, and Gleditsia delavayi, generating typical amplification curves, indicating that the use of the said HRM primers for target recognition and amplification of tara and common species of gleditsia seeds (Gleditsia sinensis, Gleditsia japonica, Gleditsia microphylla, Gleditsia delavayi) has high universality.

[0069] 2. Specificity of tara identification in gleditsia seeds

[0070] Based on the HRM primer set (6) (274-F / 493-R) designed and screened in Example 1 and the method for amplifying and performing HRM analysis on tara and plants of the genus Gleditsia using the same, in this example, genomic DNA of tara, Gleditsia sinensis, Gleditsia japonica, Gleditsia microphylla, and Gleditsia delavayi was used as a template, and the specificity of tara identification in gleditsia seeds was tested using the said method (judged by the HRM analysis results, i.e., the Tm value of the amplification product). The results are as Figure 2 shown in B of

[0071] As can be seen from Figure 2 B of

[0072] and Table 3, the HRM analysis results show that the Tm value of the amplification product of tara is 91.24 ± 0.26 °C, between 90 °C and 92 °C, while the Tm value of the genomic DNA amplification product of Gleditsia sinensis is 92.80 ± 0.05 °C, and the Tm values of the amplification products of Gleditsia microphylla, Gleditsia japonica, and Gleditsia delavayi are all between 92 °C and 95 °C. Since only the Tm value of tara among the genomic amplification products of the above five species is lower than 92 °C, while the Tm values of Gleditsia sinensis, Gleditsia microphylla, Gleditsia japonica, and Gleditsia delavayi are all higher than 92 °C. Therefore, a Tm value of 92 °C can be used as the dividing line between tara and the amplification products of all gleditsia products.

[0072] The above results show that the use of the HRM primers of the present invention and the HRM analysis method established using the same have high recognition specificity for tara in Gleditsia sinensis and its related species, and the low-value adulterant tara in high-value gleditsia seeds can be accurately distinguished and identified using the difference in the Tm values of their amplification products.

[0073] Table 3 Tm values of amplification products of tara and Gleditsia sinensis

[0074]

[0075] Example 3 Detection Limit Test

[0076] Based on the HRM primer set (6) (274-F / 493-R) screened and designed in Example 1 and the method for amplifying and performing HRM analysis on tara and Gleditsia plants using the same, in this example, using the extracted tara genomic DNA as a template, systems with different DNA contents were configured for detection to test the detection limit of the HRM primers of the present invention. In the configured reaction system, the amounts of tara genomic DNA were 0 (NTC), 1, 10, 15, 20, and 25 ng in sequence.

[0077] The detection limit test results are as Figure 3 shown. As Figure 3 can be seen, when the amounts of tara genomic DNA are 25, 20, 15, and 10 ng respectively, the amplification and HRM analysis methods described in Example 1 of the present invention can all identify and amplify them ( Figure 3 A in), and the Tm values of the melting curves of the finally formed amplification products are all between 90°C and 92°C, significantly different from the Tm values of the melting curves of the Gleditsia control sample products located between 92°C and 95°C ( Figure 3 B in); when the amount of tara genomic DNA is 1 ng, the amplification and HRM methods cannot identify and amplify it, and the amplification and melting curve analysis results are the same as those of the no-template control group (NTC). Therefore, the detection limit of the method for amplifying and performing HRM analysis on tara and Gleditsia plants established by the designed HRM primer set (6) (274-F / 493-R) of the present invention can be as low as 10 ng.

[0078] Example 4 Actual Detection

[0079] Based on the HRM primer set (6) (274-F / 493-R) screened and designed in Example 1 and the method for amplifying and performing HRM analysis on tara and Gleditsia plants using the same, the present invention tested its actual detection effect.

[0080] On the network platform, the present invention purchased 30 commercially available horned soapwort products (30 horned soapwort blind samples) from different regions and stores, extracted the sample DNA, and performed amplification and HRM analysis on it using the sample DNA as a template; the sample DNA extraction, HRM detection system, and method were all the same as those in Example 1; among them, it was difficult to obtain high-quality DNA for samples 26 and 29. The identification and amplification results of the 30 horned soapwort blind samples are as Figure 4 shown, and the melting curve analysis results of their target amplification products are as Figure 5 shown, and in the melting curve analysis results of the target amplification products, the statistical analysis results of the Tm values corresponding to the melting curve peaks are as Figure 6 shown.

[0081] It can be seen from Figures 4 - 6 that the target amplification products of the 30 samples of commercially available raw gleditsia sinensis blind samples purchased in this invention were successfully obtained ( Figure 4 ), and HRM analysis was carried out. For samples 26 and 29 from which it was difficult to obtain high-quality DNA, the method described in this invention can also successfully amplify and analyze them, indicating that the method described in this invention has a highly sensitive target recognition and amplification ability, and can better identify and amplify the raw gleditsia sinensis samples with low-quality or trace genomic DNA, facilitating the analysis and identification of actual samples. HRM analysis was carried out on 30 commercially available raw gleditsia sinensis blind samples, and the results showed ( Figure 5 and 6 ) that the HRM analysis curves of the genomic DNA amplification products of sample 3 and sample 14 were both single peaks, and the Tm values were 91.66 °C and 91.31 °C respectively, which were not statistically significantly different from the Tm values of 91.35 °C and 91.08 °C of the respective positive control tara amplification products (P>0.05, 0.29787 and 0.16850 respectively) (Table 4), and their peak positions were also similar, both between 90 °C and 92 °C. Therefore, it can be determined that this sample is counterfeit raw gleditsia sinensis sourced from tara or adulterated with tara. The HRM analysis peak positions of the amplification products of the remaining 28 samples were all between 92 °C and 95 °C, and the peak patterns and Tm values were significantly different from those of the tara samples ( Figure 6 ). From the above results, it can be seen that the HRM primers and method described in this invention have good practicability for identifying the adulteration of low-value tara in high-value commercially available raw gleditsia sinensis products.

[0082] Table 4 Statistical analysis of Tm values for the identification of samples 3 and 14 by HRM analysis

[0083]

[0084] In addition, to prove the identification accuracy of the method for amplifying and performing HRM analysis on tara and plants of the genus Gleditsia using the HRM primer set (6) (274-F / 493-R) designed and screened, this invention adopted DNA barcoding technology in the early stage of work, and sequenced and analyzed the ITS sequences of the 30 commercially available raw gleditsia sinensis products by Sanger sequencing (Zhao Gang, et al. Foods, 2024), and determined their species sources. The detection results of the 30 commercially available raw gleditsia sinensis products obtained by Sanger sequencing using DNA barcoding technology are shown in Table 5. It can be seen from Table 5 that the detection results using the DNA barcoding technology detection method are completely consistent with the detection results of this invention (Table 5), thus proving that the identification method described in this invention has high accuracy and can be fully applied to the rapid identification of tara adulteration in market raw gleditsia sinensis products.

[0085] Table 5 Comparison of the detection results between the HRM analysis method and the DNA barcoding technology of the present invention

[0086]

[0087] The above embodiments are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A HRM primer, characterized in that: The nucleotide sequences of the primers are shown as SEQ ID NO.1 and SEQ ID NO.2, or as SEQ ID NO.3 and SEQ ID NO.

4.

2. Use of the HRM primers according to claim 1 in identifying components derived from Tara and Gleditsia sinensis plants.

3. Use of the HRM primers according to claim 1 in the preparation of a product for identifying Tara and Gleditsia plant-derived ingredients.

4. A kit for identifying Tara and Gleditsia sinensis plant-derived ingredients, characterized in that: The kit contains the HRM primers according to claim 1.

5. The kit according to claim 4, characterized in that: The kit also contains reagents required for fluorescent PCR reaction.

6. The kit according to claim 5, characterized in that The fluorescent dye used in the fluorescent PCR reaction is SYBRGreen I.

7. A method for identifying Tara and Gleditsia sinensis plant-derived ingredients, characterized in that: The following steps are involved: S1. Extract DNA from the sample to be tested; S2. Using the DNA obtained in S1, the positive control Tara, the negative control Gleditsia sinensis DNA and the blank control as templates, respectively, using the HRM primers described in claim 1 or the kit described in any one of claims 4 to 6 to perform fluorescent PCR reaction, perform HRM analysis on the PCR amplification products, and compare the product Tm values; S3. Interpretation of results; if the blank control has no amplification curve and melting curve, the positive control Tara and the tested sample both have amplification curves and there is no significant difference in the Tm value of the melting curve of the amplified product, and the negative control Gleditsia sinensis has an amplification curve and the Tm value of the melting curve of the amplified product is greater than the Tm value of the melting curve of the positive control Tara, then the tested sample contains Tara plant-derived ingredients; the Tm value of the melting curve of the amplified product of the positive control Tara and the tested sample is evaluated by t-test to see whether there is a significant difference.

8. The method according to claim 7, characterized in that: In the reaction system of the fluorescent PCR reaction in S2, the final concentration of the HRM primer is 0.4-0.6 μM.

9. The method according to claim 7, characterized in that: The fluorescent PCR reaction in S2 includes a nucleic acid real-time fluorescent amplification reaction and an amplification product HRM melting curve analysis; The reaction conditions of the amplification reaction are as follows: pre-denaturation at 95°C for 2 to 3 min; denaturation at 95°C for 4 to 6 s, annealing at 55°C for 10 to 12 s, 38 to 40 cycles; extension at 72°C for 14 to 16 s; The conditions for HRM melting curve analysis were as follows: reaction at 95°C for 50-60 s; reaction at 60°C for 14-16 s; heating to 98°C for 4-6 s, and collecting fluorescence signals every 0.5°C increase in temperature.

10. The method according to claim 9, characterized in that: The reaction conditions of the amplification reaction were as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 5 s, annealing at 55°C for 10 s, 39 cycles; extension at 72°C for 15 s; The conditions for HRM melting curve analysis were as follows: 95°C for 1 min; 60°C for 15 s; heating to 98°C for 5 s, with fluorescence signals collected every 0.5°C increase in temperature.

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