Sun Yat-sen University to Transfer DNA-Based Authentication Method for Dilong in Naoxintong Formulations for RMB 200,000

Feb 11, 2026 08:00 CST Updated 08:00

Recently, Sun Yat-sen University released a public notice on the transformation of scientific and technological achievements, proposing to transfer its research outcomes through assignment."A DNA Molecular Identification Method for Pheretima in a Naoxintong Preparation"The relevant patents were assigned to Shaanxi Buchang Pharmaceutical Co., Ltd. for use, with a total transaction amount of RMBRMB 200,000. The inventor of this patented technology isProfessor Su Weiwei and Her Team。

Su Weiwei:Member of the Communist Party of China, Foreign Member of the European Academy of Natural Sciences, Professor and Doctoral Supervisor at the School of Life Sciences, Sun Yat-sen University. Su Weiwei began her career in August 1976. From July 1987 to April 2000, she worked at Guangdong Pharmaceutical University, successively serving as Teaching Assistant, Lecturer, Associate Professor, and Professor. In July 2000, she was appointed Professor at the School of Life Sciences, Sun Yat-sen University. She obtained her Ph.D. from the First Military Medical University of the Chinese People's Liberation Army in July 2005. In 2023, she was elected as a Foreign Member of the European Academy of Natural Sciences. Her primary research interests focus on the research and development of plant-derived new drugs and the analysis of natural medicines.

The present invention is a DNA molecular identification method for the medicinal material Pheretima in the Naoxintong preparation, with the core objective ofAccurately determine whether Naoshintong preparations (such as Naoshintong Capsules) contain authentic Pheretima, and clearly identify the specific species of Pheretima, thereby addressing the challenges in authenticating Pheretima materials and the lack of quality control in traditional Chinese medicine formulations.

Pain Points in the Identification of Earthworm-Based Traditional Chinese Medicine Preparations Are Prominent, with Traditional Methods Facing Dual Bottlenecks of Difficulty in Distinguishing Authenticity and Lack of Quality Control

Naoxintong PreparationsAs a commonly used traditional Chinese medicine for the clinical treatment of cardiovascular and cerebrovascular diseases,EarthwormAs itsCore Deputy Drug, and its authenticity directly affects the efficacy and safety of medication.

According to the 2015 edition of the Chinese Pharmacopoeia, authentic Di Long (Earthworm) is derived from the dried bodies of four specific Pheretima species, including Pheretima aspergillum and Pheretima vulgaris. However, due to the wide variety and morphological similarities of earthworm species in China, adulteration and species confusion are prevalent in the market. Counterfeit products, such as Metaphire pauciseta and Amynthas furcillatus, are frequently encountered, posing significant challenges to quality control of this medicinal material.

Traditional authentication of the medicinal material Dilon (Pheretima) relies on methods such as botanical origin identification, macroscopic characterization, microscopic examination, and thin-layer chromatography. These approaches not only exhibit limited specificity and selectivity but also impose stringent demands on the professional expertise of the examiner, making it difficult for ordinary practitioners to accurately distinguish between authentic and adulterated products.

More critically, after processing such as sun-drying and grinding, the morphological characteristics of Pheretima in Naoxintong preparations are completely lost, rendering traditional methods devoid of a basis for identification and thus unable to meet the practical requirements of pharmaceutical production and regulatory oversight.

Currently, the 2015 edition of the Chinese Pharmacopoeia does not stipulate quality control requirements for Pheretima in Naoxintong preparations, and the industry lacks targeted and effective identification methods, resulting in a lack of scientific support for ensuring the authenticity of Pheretima in these preparations.

In the application of molecular identification techniques, existing studies mostly employ universal primers to amplify genes such as COI via PCR. However, the Pheretima DNA present in Naoxintong preparations is prone to degradation into small fragments during processing and storage, making it difficult to amplify using conventional universal primers.

Meanwhile, the formulation contains other animal-derived medicinal ingredients such as leech (Shuizhi) and scorpion (Quanxie), whose genetic fragments may resemble those of earthworm (Dilong), thereby causing interference in identification and further complicating the authentication of genuine materials. These issues have long left a gap in quality control for the Dilong component in Naoxintong preparations, which not only hinders manufacturing quality assurance by pharmaceutical companies but also poses challenges to drug regulation. There is an urgent need for specialized and efficient identification technologies to address this industry-wide dilemma.

Dual Empowerment via Targeted Amplification and Precise Determination: Earthworm DNA Identification Technology Achieves Dual Breakthroughs in Authentic Product Traceability and Quality Control Upgrade

Addressing industry pain points such as the loss of morphological characteristics, DNA degradation, and the low specificity of traditional identification methods for *Pheretima* (Dilong) in Naoxintong preparations, Sun Yat-sen University has developed a “DNA Molecular Identification Method for *Pheretima* Medicinal Materials in Naoxintong Preparations,” which“Specific Primer Design + Multi-Step Collaborative Validation”as the core,Developed an integrated identification scheme featuring “efficient amplification + precise determination”, achieving comprehensive innovation from technical principles to practical applications and filling the gap in quality control of *Pheretima* in Naoxintong preparations.

The core advantage of this technology lies inTarget-Specific Primers，Overcoming the challenge that traditional molecular identification struggles to align with formulation characteristics. Traditional universal primers can only amplify gene fragments of 350–700 bp, failing to meet the detection requirements for small fragments resulting from DNA degradation of Pheretima in Naoxintong preparations, and are susceptible to interference from other animal-derived medicinal materials such as Hirudo and Scorpio. The present invention specifically designs three pairs of Pheretima-specific primers, respectively targetingGenus Metaphire(Pheretima vulgaris, Pheretima wilsoni) andGenus Amynthas(Pheretima aspergillum and Pheretima pectinifera), enabling precise amplification of small gene fragments (200–350 bp) and completely circumventing the impact of DNA degradation. Among them,COI Metaphire F2/R2 Primer PairThe assay exhibits high specificity, amplifying only the authentic *Metaphire* earthworm genes without cross-reacting with *Amynthas* species or counterfeit products. In contrast, the COI AAF2/R1 and COI AAF3/R1 primer pairs specifically target authentic *Amynthas* species. Sequencing further distinguishes authentic materials from counterfeits, effectively eliminating interference from other animal-derived ingredients in the formulation.

In the authentication process, multi-step collaborative verification enables precise and efficient determination of product authenticity.During the DNA extraction phase, Proteinase K is used to digest animal-derived medicinal components, combined with a broad-spectrum plant genomic DNA rapid extraction kit. This approach preserves DNA information from both Pheretima (earthworm) and plant-based medicines while minimizing component interference. In the PCR amplification step, the reaction system and protocol are optimized: a streamlined 15 μL reaction volume paired with specific annealing temperatures (55°C or 58°C) ensures amplification efficiency and specificity. Dual verification through electrophoresis and sequencing is employed; positive samples are initially screened by the presence of a single, bright band, and precise species溯源 is achieved via BLAST comparison against the GenBank database. Authentic products must meet both criteria—"band match" and "species consistency"—significantly improving identification accuracy.

Furthermore,The technology balances convenience and versatility, meeting quality control needs across diverse scenarios.For Metaphire guillelmi earthworm raw materials commonly used by enterprises such as Shaanxi Buchang Pharmaceutical, direct determination can be made solely based on electrophoresis results following amplification with the COI MetaphireF2/R2 primers, thereby omitting the sequencing step and significantly improving detection efficiency. Three pairs of earthworm-specific primers comprehensively cover the four authentic earthworm species stipulated in the Chinese Pharmacopoeia, making them suitable for Naoxintong Capsules and various traditional Chinese medicine (TCM) preparations containing fine earthworm powder, without being restricted by the processing form of the medicinal materials. Meanwhile, the technical operational process is standardized, with clear and controllable steps from DNA extraction to result interpretation, eliminating reliance on the professional experience of operators. This provides scientific and reliable technical support for production quality control in pharmaceutical enterprises and supervisory testing by drug regulatory authorities, facilitating the upgrading of quality control systems for TCM preparations.

Intensifying Competition in the Earthworm Identification Technology Sector: Multidimensional Innovation Drives Upgrades in Traditional Chinese Medicine Quality Control

With increasingly stringent quality supervision of traditional Chinese medicine (TCM) preparations and the widespread adoption of molecular identification techniques, the demand for precise authentication of Pheretima (Dilong) crude drugs and TCM preparations containing Pheretima is growing. Currently, technological research and development focused on Pheretima identification in the market mainly centers on"Optimization of Traditional Methods"and"Molecular Technology Innovation"In two major directions, domestic and international enterprises and research institutions have addressed industry pain points through primer design, streamlined detection workflows, and the integration of multiple technologies, thereby creating a diversified competitive landscape.

Shenzhen MGI Tech Co., Ltd. Launches the HMBI-G30 All-in-One High-Throughput Gene Sequencing System for DNA Barcoding of Traditional Chinese Medicines, designed for large-scale species identification and traceability of Chinese herbal medicines. The device supports multi-read-length sequencing platforms such as SE50/SE100 and is compatible with degraded DNA samples. It addresses the challenge of DNA fragmentation in traditional Chinese medicine (TCM) preparations through fragmented sequencing and assembly, and performs species comparison using a proprietary database containing millions of sequences. As the world’s first high-throughput identification instrument dedicated to Chinese herbal medicines, it is widely used by drug testing agencies, large pharmaceutical companies, and research institutes. It enables simultaneous detection of multiple species, such as Pheretima (earthworm), but has not yet undergone primer and workflow optimization specifically for compound formulations like Naoxintong.

BioCapital Group Co., Ltd. and the Center for Chinese Materia Medica Resources of the China Academy of Chinese Medical Sciences jointly launched a series of rapid PCR identification kits for Chinese herbal medicines,Focusing on species-specific testing for animal-derived medicinal materials, commercial applications have been successfully implemented for products such as snake-based medicines. The core advantage lies in an integrated workflow combining rapid DNA extraction with fluorescent PCR amplification, enabling completion from sample processing to result interpretation within two hours. This solution is well-suited for scenarios including drug control institutes and quality control (QC) laboratories of manufacturing enterprises. Notably, the rapid PCR test kit for snake species has been incorporated into the Guangdong Provincial Drug Testing Platform, achieving on-site and standardized molecular identification of traditional Chinese medicinal materials, and providing a mature paradigm for the development of kits for other animal-derived medicines such as Pheretima (earthworm). Currently, this series covers various easily confused animal-derived medicinal materials, featuring a comprehensive reagent formulation and quality control system, establishing it as a benchmark domestic product for molecular identification of traditional Chinese medicine.

Hebei Province Traditional Chinese Medicine Quality Inspection and Testing Research Center Co., Ltd. Establishes a DNA Sequence + QR Code Digital Traceability System, converting herbal identification results into portable QR code labels affixed to decoction pieces and pharmaceutical preparations, enabling rapid verification of species information via mobile devices. This technology integrates the entire chain of “identification–coding–distribution–regulation,” facilitating end-to-end traceability of medicinal materials such as Pheretima (Di Long) from raw materials to finished products, thereby reducing the risks of market adulteration and confusion. It has currently been implemented on a large scale in core traditional Chinese medicine distribution hubs such as Anguo, Hebei Province.