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Chemical and Biological Technologies in Agriculture
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Dual detection of Bupleurum scorzonerifolium Willd. and Bupleurum chinense DC. using proofman–LMTIA method

Chemical and Biological Technologies in Agriculture volume 11, Article number: 104 (2024) Cite this article

Abstract

A new rapid dual detection method was established to distinguish Bupleurum scorzonerifolium Willd. (BS) and Bupleurum chinense DC. (BC) simultaneously using the proofreading enzyme-mediated probe cleavage coupled with ladder-shape melting temperature isothermal amplification (proofman–LMTIA). The internal transcribed spacer (ITS) sequences of BS and BC were selected as targets for designs of proofman–LMTIA primers and proofman fluorescence probes labeled with FAM or JOE. The reaction temperature optimization, repeatability and reliability assessment, specificity assessment and sensitivity assessment of the proofman–LMTIA performed for dual detection of BS and BC and its application. The results showed that the optimal reaction temperature of the proofman–LMTIA method was at 63℃, which had strongly specificity, repeatability and reliability, as well as sensitivity, and the detection was completed within 20 min with a detection sensitivity of 1 pg/μL. The proofman–LMTIA method realized dual rapid detection of BS and BC, which showed a strong practical value. The 4 kinds were BC, 1 kind was BS, and 2 kinds were counterfeit in the detection of 7 kinds of BS or BC samples from different habitats. Our study successfully established a new approach for dual rapid detection of BS and BC using the proofman–LMTIA, which will provide an effective technique or method for the authenticity detection of authentic Chinese medicinal materials and present a very important practical significance.

Graphical Abstract

Introduction

The Bupleurum L., belonging to Apiaceae family, is constituted by more than 200 confirmed species, which are distributed in the northern temperate zone, especially in China and Europe [1]. As a commonly used herb for over 2000 years in Traditional Chinese Medicines (TCMs), Radix Bupleuri (“Chaihu” in Chinese) contains various bioactive compounds that contribute to its medicinal properties, such as triterpene saponins, rotundiosides, polysaccharide, flavonoids, phenylpropanoids, coumarin, volatile oils, lignanoids, and so on [1,2,3,4,5]. These bioactive compounds work together to provide the potential health benefits associated with Radix Bupleuri. In TCMs, Radix Bupleuri is classified as a “cooling” herb and is believed to have a therapeutic effect on the liver and gallbladder meridians [3]. Radix Bupleuri is commonly used to help regulate the flow of Qi (vital energy) and relieve stagnation in the body [1, 6], cure liver disorders [7, 8], respiratory tract infections [9], as well as improve emotional conditions like irritability, anxiety, and depression [1].

More than 30 Bupleurum species, varieties and variants distribute in China, such as Bupleurum scorzonerifolium Willd., Bupleurum chinense DC., Bupleurum smithii var. parvifolium, Bupleurum marginatum var. stenophyllum, Radices stellariae dichotomae, Bupleuru marginatum var. stenophyllum, and so on [1, 10]. It is worth noting that the pharmacological properties, toxicity and function of Radix Bupleuri may vary depending on factors such as plant species, cultivation conditions and processing methods [5, 11, 12]. Thereinto, Bupleurum scorzonerifolium Willd. (BS) and Bupleurum chinense DC. (BC) gradually evolve into the mainstream of Radix Bupleuri, the dried roots of which are prescribed in The Chinese Pharmacopoeia and are also known as “Nan Chaihu” and “Bei Chaihu” in Chinese, respectively, due to their origin and distribution [13]. Moreover, different varieties with different active ingredients and functions. BC is usually used to treat typhoid fever, while BS is typically used to clear liver heat [14]. Alarmingly, both are confusedly and interchangeably used as Radix Bupleuri [15]. What's worse, there are unscrupulous businesses that adulterate the authentic Chinese herbal medicine market by selling the roots of other plants as Radix Bupleuri [16]. This undermines the integrity of the market and compromises the quality and efficacy of genuine Chinese herbal medicine. To safeguard the authenticity of Chinese herbal medicines and ensure quality control and evaluation, it is crucial to accurately identify and differentiate between southern and northern Bupleurum as well as other plant components.

With the continuous advancement of technology, the screening and identification techniques for authentic Radix Bupleuri are also constantly evolving, leading to a variety of methods, which include traditional identification methods such as physical characteristics and microscopic examination, biological identification such as polymerase chain reaction (PCR) and DNA barcoding, spectral identification such as near-infrared spectroscopy, LC–MS and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry [16,17,18,19,20,21,22]. However, the traditional method is subjective, relying on personal experience, which increases the likelihood of errors, while other methods have longer detection times and higher costs, making them less practical for widespread application. The ladder-shape melting temperature isothermal amplification (LMTIA) technique is a new nucleic acid isothermal amplification method developed by our team in 2021 [23]. This new method enables high specificity and sensitivity nucleic acid amplification in a short period of time without the need for a thermal cycler. It is cost-effective and suitable for translational applications. Currently, LMTIA technology has found wide applications in areas such as food adulteration detection, virus detection and foodborne pathogen detection [24,25,26,27,28], while its application in identification of Radix Bupleuri is still largely unexplored. In addition, probes based on enzymatic cleavage have garnered considerable attention in specific molecular diagnosis [29], and proofreading enzyme-mediated probe cleavage (named proofman) has been proved to enhanced fluorescent signal by cleaving the probe from 3′ end [30]. Therefore, the combined utilization of LMTIA and proofman probes will provide an innovative assay platform for accurate identification of Radix Bupleuri-specific nucleic acid sequences.

In our current study, a new fast, accurate, efficient, and specific method has been developed to distinguish BS and BC simultaneously using proofreading enzyme-mediated probe cleavage coupled with ladder-shape melting temperature isothermal amplification (proofman–LMTIA) through primer design, reaction temperature optimization, repeatability and reliability assessment, specificity assessment and sensitivity assessment of the proofman–LMTIA, which will provide an effective technique or method for the authenticity detection of authentic Chinese medicinal materials and has very important guiding significance.

Materials and methods

Materials

The standard processing of BS and BC were provided by Yuzhou Houshengtang Traditional Chinese Medicine Co., Ltd., the origins and batch numbers of Chinese medicinal materials were shown in Table S1; the plant genomic DNA (gDNA) extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the premix of dNTPs and Bst DNA polymerase were purchased from Shandong Merit Biotech Co., Ltd.; all other reagents used were purchased from Sinopharm Group Chemical Co., Ltd. unless stated otherwise.

Target sequence selection and proofman–LMTIA primer design

As target sequence, the internal transcribed spacer (ITS) sequence of BS and BC, obtained from “The DNA Barcode Database of Plant Species (http://dna.iflora.cn/Home/)”, were used to design proofman–LMTIA primers as described by Wang [23]. ITS sequence alignment between BC and BS was performed to screen difference sites using DNAMAN Version 9 software (Lynnon Biosoft, USA) (Fig. 1A). The ladder-shaped melting temperature curves were selected as the target sequences using Oligo Version 7 software (Molecular Biology Insights, USA) (Fig. 1B, C), and the proofman–LMTIA primers were designed using online software Primer3Plus (http://www.primer3plus.com, accessed on 3 June 2023). In addition, based on the primer LB sequences, the proofman probes were designed, which contained fluorophore and quencher labeled at the end of the 3′ end and 5′ end, respectively [30]. All the primers were synthesized by Anhui Universal Biotech Co., Ltd. and are shown in Table 1.

Fig. 1
figure 1

ITS sequence alignment and melting temperature curves for BC and BS. A ITS sequence alignment between BC and BS; B melting temperature curve of BC target sequence; C melting temperature curve of BC target sequence. BC Bupleurum chinense DC., BS Bupleurum scorzonerifolium Willd

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Table 1 Proofman–LMTIA primers and fluorescence probes
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DNA extraction

The gDNA extraction kit was used to extract DNA from BS, BC, radix angelicae, rhizoma typhonii, soybean, cassava and other plants according to the manufacturer’s instructions. In addition, the DNA was stored at 4 ℃ for further use after the DNA quality assessment using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, USA).

Proofman–LMTIA reaction system

The proofman–LMTIA reaction system was performed using a 10 µL total reaction volume, with the extracted genomic DNA of test samples as the template. The premix of dNTP and Bst DNA polymerase and proofman probe were added for the fluorescence signal collection of proofman–LMTIA reaction. The reaction mixture is presented in Table 2.

Table 2 Proofman–LMTIA reaction system(10 µL)
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Proofman–LMTIA reaction temperature optimization

Independent experiments were performed on BS and BC gDNA using proofman–LMTIA reaction system described in 2.4. The BS or BC gDNA was used as positive control, while ddH2O was used as negative control. The Gentier 96E fully automated medical PCR analysis system (Tianlong, Xian, China) was employed for isothermal amplification reaction of proofman–LMTIA, the temperatures of which were set as 59 ℃, 61 ℃, 63 ℃ and 65 ℃, with fluorescence signal collection every 30 s for a total of 40 cycles, respectively. The isothermal amplification curves of the proofman–LMTIA were then analyzed to determine and screen the optimal reaction temperature for the BS/BC primers.

Specificity assessment of proofman–LMTIA assay

The gDNAs of BS, BC, Angelica Dahurica, Rhizoma Typhonii, Salvia Miltiorrhiza, Angelica Sinensis, Mung bean, soybean, cassava, Zea mays, and olive oil were selected to assess the specificity of proofman–LMTIA assay, and BS or BC was used as positive control, while ddH2O was used as negative control, respectively. The 10 µL reaction system described in Table 2 was used and the results were analyzed using the Gentier 96E fully automated medical PCR analysis system, the reaction temperature of which was set at 63 ℃ with fluorescence signal collection every 30 s for a total of 40 cycles, respectively.

Repeatability and stability assessment of proofman–LMTIA assay

The repeatability and stability assessment of BS and BC detection was performed using proofman–LMTIA reaction system. 22 samples of BS or BC gDNA were used as positive controls, while ten samples of ddH2O were used as negative controls, respectively. Proofman–LMTIA reaction temperature was set at 63℃ with fluorescence signal collection every 30 s for a total of 40 cycles, respectively. The isothermal amplification curves of proofman–LMTIA were then analyzed and evaluated.

Sensitivity assessment of proofman–LMTIA assay

The absolute and relative sensitivity assessments of proofman–LMTIA assay were executed. For absolute sensitivity assessment, diluted gDNA of BS or BC at concentrations of 10 ng/µL, 1 ng/µL, 100 pg/µL, 10 pg/µL, 1 pg/µL and 100 fg/µL were added to proofman–LMTIA assay under the optimal temperature reaction conditions. For relative sensitivity assessment, BS and BC samples were mixed with varying mass fractions of 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99%, respectively. Then, the gDNA of above mixed samples were extracted and used for proofman–LMTIA assay under the optimal temperature reaction conditions.

Dual rapid detection for proofman–LMTIA assay and application

The gDNA of BS, BC and the mixture of them (1:1 mixing) were used for dual rapid detection for proofman–LMTIA assay under the optimal reaction system mentioned above. In addition, seven samples of BS or BC obtained from different provinces of China were subjected to gDNA extraction, and established dual rapid detection for proofman–LMTIA assay was applied for actual sample testing. BS and BC were used as positive control, while ddH2O was used as negative control.

Statistical analysis

All experiments were performed at least in triplicate, and representative data were shown. The Gentier 96E fully automated medical PCR analysis system was employed to collect and analyze isothermal amplification curves of the proofman–LMTIA, and statistical analyses and processing were further performed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, USA).

Results

Target sequence analysis and proofman–LMTIA primer design

The ITS sequences of BS and BC were aligned and the results showed a 97.02% similarity between them (Fig. 1A). Three single nucleotide polymorphism (SNP) sites between BS and BC were screened and selected as “crucial different loci” for proofman–LMTIA primer, occurring at positions 567/569 (A/C), 568/570 (A/T), and 569/571 (T/G), respectively (Fig. 1B, C). In addition, the proofman probes containing fluorophore and quencher were used to enhance the accuracy of the LMTIA reaction, and the proofman–LMTIA primer sequences are shown in Table 1.

Optimization of proofman–LMTIA reaction temperature

The temperatures of 59 ℃, 61 ℃, 63 ℃ and 65 ℃ were chosen for proofman–LMTIA reaction temperature optimization, and the results are shown in Figs. 2 and 3. For BS proofman–LMTIA detection, the amplification plots were presented in all temperatures, and better results occurred at 63 ℃ and 65 ℃, when the amplification curves appeared earlier and had better parallelism (Fig. 2C, D), while the better amplification plot was presented at 63 ℃ for BC proofman–LMTIA detection (Fig. 3C). Overall consideration, 63 ℃ was selected as the best proofman–LMTIA reaction temperature for both BS and BC, which was conducive to the dual detection in a follow-up experiment.

Fig. 2
figure 2

Optimization of proofman–LMTIA reaction temperature for BS. A–D Amplification plots of proofman–LMTIA reaction system for BS at 59 ℃, 61 ℃, 63 ℃ and 65 ℃, respectively. Positive controls, BS gDNAs; negative controls, ddH2O. BS, Bupleurum scorzonerifolium Willd

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Fig. 3
figure 3

Optimization of proofman–LMTIA reaction temperature for BC. A–D Amplification plots of proofman–LMTIA reaction system for BC at 59 ℃, 61 ℃, 63 ℃ and 65 ℃, respectively. Positive controls, BC gDNAs; negative controls, ddH2O. BC Bupleurum chinense DC

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Specificity assessment of proofman–LMTIA assay

The gDNAs extracted from BS, BC, Angelica Dahurica, Rhizoma Typhonii, Salvia Miltiorrhiza, Angelica Sinensis, Mung bean, soybean, cassava, Zea mays, and olive oil were used for specificity of proofman–LMTIA assay, and ddH2O was used as negative control. The results showed that only the gDNA of BS or BC presented the typical “S” type amplification curve at the reaction temperature of 63 ℃, while the gDNAs for other species were not amplified (Fig. 4), indicating that our established proofman–LMTIA assay has a satisfactory specificity.

Fig. 4
figure 4

Specificity assessments of the proofman–LMTIA assay at 63 ℃ for BS and BC. A Amplification plots of proofman–LMTIA reaction system at 63 ℃ for BS specificity assessment. B Amplification plots of proofman–LMTIA reaction system at 63 ℃ for BC specificity assessment. Positive controls, gDNAs of BS or BC; negative controls, gDNAs of BC or BS, Angelica Dahurica, Rhizoma Typhonii, Salvia Miltiorrhiza, Angelica Sinensis, Mung bean, soybean, cassava, Zea mays, and olive oil, as well as ddH2O. BC Bupleurum chinense DC., BS Bupleurum scorzonerifolium Willd

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Repeatability and stability assessment of proofman–LMTIA assay

As shown in Fig. 5, 22 samples of BS or BC gDNA were used as positive controls, while 10 samples of ddH2O were used as negative controls, respectively. As expected, all the positive controls had amplification curve at the reaction temperature of 63 ℃, while negative controls were not amplified, indicating that our established proofman–LMTIA assay has a satisfactory repeatability and stability.

Fig. 5
figure 5

Repeatability and stability assessment of proofman–LMTIA assay at 63 ℃ for BS and BC. A Amplification plots of proofman–LMTIA reaction system at 63 ℃ for BS repeatability and stability assessment. B Amplification plots of proofman–LMTIA reaction system at 63 ℃ for BC repeatability and stability assessment. Positive controls, gDNAs of BS or BC; negative controls, ddH2O. BC Bupleurum chinense DC., BS Bupleurum scorzonerifolium Willd

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Sensitivity assessment of proofman–LMTIA assay

Both absolute and relative sensitivity assessments of proofman–LMTIA assay were executed in our experiment. For absolute sensitivity assessment, diluted gDNA of BS or BC at concentrations of 10 ng/µL, 1 ng/µL, 100 pg/µL, 10 pg/µL, 1 pg/µL and 100 fg/µL were added to proofman–LMTIA assay at the reaction temperature of 63 ℃. The results showed that the detection limit of BS or BC was 1 pg/µL (Fig. 6A, C). For relative sensitivity assessment, BS and BC samples were mixed with varying mass fractions of 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99%, respectively. The results showed that the relative sensitivity of proofman–LMTIA assay in detecting BS could reach at least 5%, while that in detecting BC could reach at least 10% (Fig. 6B, D). All these results present preferable absolute and relative sensitivity of proofman–LMTIA assay.

Fig. 6
figure 6

Sensitivity assessment of proofman–LMTIA assay at 63 ℃ for BS and BC. A Amplification plots of proofman–LMTIA reaction system at 63 ℃ for BS absolute sensitivity assessment. The gDNA of BS diluted at 10 ng/µL, 1 ng/µL, 100 pg/µL, 10 pg/µL, 1 pg/µL and 100 fg/µL. B Amplification plots of proofman–LMTIA reaction system at 63 ℃ for BS relative sensitivity assessment. BS and BC samples were mixed with varying mass fractions of 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99%, respectively. C Amplification plots of proofman–LMTIA reaction system at 63 ℃ for BC absolute sensitivity assessment. The gDNA of BC diluted at 10 ng/µL, 1 ng/µL, 100 pg/µL, 10 pg/µL, 1 pg/µL and 100 fg/µL. D Amplification plots of proofman–LMTIA reaction system at 63 ℃ for BC relative sensitivity assessment. BS and BC samples were mixed with varying mass fractions of 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99%, respectively. Positive controls, gDNAs of BS or BC; negative controls, ddH2O. BC Bupleurum chinense DC., BS Bupleurum scorzonerifolium Willd

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Dual rapid detection of BS and BC for proofman–LMTIA assay

The gDNA of BS, BC and the mixture of them (1:1 mixing) were used for dual-plex proofman–LMTIA detection at the reaction temperature of 63 ℃, and ddH2O was used as negative control. As shown in Fig. 7A, the results showed that both gDNAs of BS and BC presented the exclusive typical “S” type amplification curve for dual-plex proofman–LMTIA assay. However, their mixture showed two typical “S” type amplification curve under different fluoresces, which represented BS and BC, respectively. These results indicated that our established proofman–LMTIA assay achieved BS and BC dual rapid detection.

Fig. 7
figure 7

Dual rapid detection for proofman–LMTIA assay and its application. A Dual rapid detection of BS and BC for proofman–LMTIA assay; B applications for dual rapid detection using proofman–LMTIA assay. Positive controls, gDNAs of BS or/and BC; negative controls, ddH2O or gDNAs of samples. BC Bupleurum chinense DC., BS Bupleurum scorzonerifolium Willd

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Applications for dual rapid detection using proofman–LMTIA assay

In order to verify the practical utility for dual rapid detection using proofman–LMTIA assay, seven samples of BS or BC obtained from different provinces of China were submitted for actual sample testing. The gDNAs of BS or BC were used as positive control, respectively, and ddH2O was used as negative control. The results showed that samples 1–4 were BC, sample 5 was BS, while samples 6 and 7 were neither BS nor BC (Fig. 7B). These results showed that proofman–LMTIA assay realized dual rapid detection for authenticity or adulteration of BS and BC, which revealed preferable practical utility.

Discussion

Originating from the production and clinical practice over thousands of years, genuine Chinese medicinal materials (GCMMs) are synonymous with high-quality TCMs, and functions as a comprehensive standard for the quality evaluation of TCMs with historical and cultural attributes [31]. What’s more, GCMMs have become an important pillar of “Healthy China” and the national medical system in recent years [32]. However, the quality of GCMMs is influenced by many factors, including variety, origin, environment, etc. Variety is one of the most important factors, and different varieties contain different kinds and contents of effective ingredients [33]. Due to the wide range of raw materials, the adulteration of GCMMs has been a concern for decades and continues to be a problem to some extent. As commonly used TCM for over 2000 years, Radix Bupleuri has functions of relieving exterior syndrome, clearing heat, regulating liver-qi, and lifting yang-qi [34]. Due to the wide range of raw materials, however, it is not uncommon to pass off the roots of other plants as Radix Bupleuri or shoddy, which seriously disturbs the market of GCMMs. Among them, the hybird use of BS and BC is becoming more and more severe. Moreover, the complexity and diversity of identification methods have accelerated the adulteration phenomenon due to lack of a unified identification criterion.

There are many methods to identify the authenticity of BS and BC including traditional methods such as physical characteristics and microscopic examination, as well as modern biological methods such as PCR and DNA barcode barcoding [16,17,18]. However, the traditional methods were subjective, relying on personal experience, which increases the likelihood of errors, while other methods have longer detection times, higher costs and expensive equipment, making them less practical for widespread application. And the more sensitive technology needs to be developed on account of their various limitations. Nucleic acid detection technology has become the hotspot in recent years because of its high specificity and sensitivity. In our current study, a new fast, accurate, efficient, and specific dual detection method has been developed to distinguish BS and BC using proofman–LMTIA. The greatest advantage of the proofman–LMTIA method is that it can react at a constant temperature without the need for a thermal cycler. Moreover, the simple primer design, low target sequence length requirements, short reaction times and high visualizations facilitate the range of applications of this technique, which provides an effective technique or method for the authenticity detection of GCMMs.

In our experiment, the primer design and reaction temperature optimization of proofman–LMTIA were firstly carried out, so as to establish the optimal proofman–LMTIA reaction system. The ITS sequences of BS and BC were used for primer design. However, the ITS sequence similarity of BS and BC was as high as 97.02%, which is difficult to design primers for other nucleic acid detection methods and precisely shows the advantage of the LMITIA reaction–low requirement on target sequence length and simple primer design. Of course, these also indirectly show that the proofman–LMTIA method has a strong specificity. In addition, the reactive enzyme system of proofman–LMTIA is simple, it does not require neither thermal denaturation nor enzymes other than Bst DNA polymerase to complete the task. In reaction temperature optimization of proofman–LMTIA, it has a wide range of application, and better reaction temperature (63 ℃) is selected for further experiments. And our experiment also proves it has a satisfactory repeatability and stability. Furthermore, specificity and sensitivity evaluation are key indicators for testing a method. The proofman–LMTIA showed satisfactory specificity, which revealed that each pair of primers was amplified only in a specific species whether in a single or dual detection, but not in other different species including different plants and animals. Both absolute sensitivity detection limit of BS and BC were 1 pg/µL using proofman–LMTIA assay, and the relative sensitivity of proofman–LMTIA assay in detecting BS could reach at least 5%, while that in detecting BC could reach at least 10%. All these results present preferable absolute and relative sensitivity of proofman–LMTIA assay, which cleverly combines LMTIA technology with proofman fluorescent probes to greatly improve the detection sensitivity, facilitating the range of applications of our proofman–LMTIA method.

The ultimate purpose of our established proof–LMTIA method is to be used for the authenticity detection of actual samples. Our current experiment proves that proof–LMTIA can be used for the dual detection of BS and BC, showing strong practical applicability in the authenticity detection of selected commercial samples, and accurately identifying the components of BS or BC. However, the application prospects of the proofman–LMTIA technique go far beyond mentioned above. It can be used for trace detection of GCMMs, origin tracing of seeds and plants, food authenticity detection, rapid typing of different probiotics, rapid detection of viruses, as well as establishment of industry standards and market supervision. Taken together, the proofman–LMTIA method we developed in current study is a very practical detection technology, as well as a key technology that can be rapidly popularized in multiple fields.

Conclusion

In our current study, a new, fast, accurate, and efficient method has been developed to distinguish BS and BC simultaneously using the proofman–LMTIA technology, showing preferable strong specificity, sensitivity, repeatability and practical value, which will provide an effective technique or method for the authenticity detection of GCMMs and has very important guiding significance.

Availability of data and materials

The authors confirm that the data supporting the findings of this study are available within the article. No datasets were generated or analysed during the current study.

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Funding

This work was supported by grants from the National Natural Science Foundation of China (32300991); Central Government Guides the Local Science and Technology Development Special Fund (Z20231811102); the Key R&D and Promotion Special Project of Henan Province (Technology Research, 232102320291, 242102321134 and 242102321128).

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Authors and Affiliations

  1. Food and Pharmacy College, Xuchang University, Xuchang, 461000, China

    Jinxin Liu, Yue Wang, Tingting Li, Kunpeng Huang, Chunmei Song, Pengfei Cui, Juntao Sun, Fugang Xiao & Deguo Wang

  2. Key Laboratory of Biomarker Based Rapid-detection Technology for Food Safety of Henan Province, Xuchang University, Xuchang, 461000, China

    Jinxin Liu, Chunmei Song, Pengfei Cui, Juntao Sun, Fugang Xiao & Deguo Wang

  3. Yuzhou Houshengtang Chinese Medicine Co., LTD, Xuchang, 461675, China

    Bailing Yin

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  1. Jinxin Liu
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Contributions

J.L., C.S. and D.W. conceptualized the study and acquired the funding; J.L., C.S., Y.W., T.L. and K.H. carried out the experiments; P.C., B.Y., J.S. and F.X. contributed new reagents or analytic tools; J.L., C.S., and D.W. analyzed data; J.L. and D.W. wrote the manuscript; C.S., P.C., J.S. and F.X edited the manuscript. Y.W., T.L. and K.H. reviewed the manuscript. All authors read and approved the final manuscript.

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Correspondence to Jinxin Liu or Deguo Wang.

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Liu, J., Wang, Y., Li, T. et al. Dual detection of Bupleurum scorzonerifolium Willd. and Bupleurum chinense DC. using proofman–LMTIA method. Chem. Biol. Technol. Agric. 11, 104 (2024). https://doi.org/10.1186/s40538-024-00637-2

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