The development and application of mini-barcodes from mitochondrial DNA for identifying medicinal leeches from traditional medicines

Abstract

Species-specific efficacy necessitates accurate identification of medicinal leeches, but standard DNA barcoding often fails with degraded DNA from traditional medicines. This deficiency highlights the need for mini-barcoding. This study aimed to develop and validate mini-barcode markers for three Chinese Pharmacopoeia-listed leech species: Whitmania pigra, Whitmania acranulata and Hirudo nipponia. Four novel mini-barcode primer sets (ND1F1/R1, 12SF1/R1, 16SF1/R1 and COX1F1/R1) were developed and validated using seven morphologically identified specimens and subsequently tested on 16 commercial leech products. DNA extractions were performed using both single-tube and column purification kits, with the latter yielding superior DNA quality and meeting the requirements for following PCR amplification. The PCR results confirmed the validation of four candidate mini-barcodes targeting specific genetic regions, which produced results in 13 out of 16 commercial leech products. Mini-barcode sequences from morphologically identified W. pigra specimens exhibit > 95% identity to the complete ND1, 12S rDNA, 16S rDNA, and COX1 sequences (EU304459), whereas sequences from H. nipponia and W. acranulata show < 85% identity, and among leech-derived products only the proprietary Chinese medicine Maxuekang exhibits lower identity. Both the optimal partition of ASAP and phylogenetic tree identified three distinct groups correlating with the morphological species: W. pigra, W. acranulata, and H. nipponia. Mislabeled species have been uncovered in proprietary Chinese medicine, notably the claimed Hirudo nipponia, which was replaced by W. pigra. The results highlight the value of mini-barcodes in enhancing product quality control and offer a reliable method for accurate species identification in traditional and commercial leech-based medicines. This advance supports safer and more effective utilization of medicinal leeches and advocates for their integration into regulatory standards.

Keywords: Hirudo nipponia; Whitmania acranulata; Whitmania pigra; DNA barcoding; DNA extraction; Mahuang; Shuizhi; Species delimitation.

Fig. 1

General information and the quality of extracted DNA from 16 leech derived products. (a) The profiles of each leech derived product contain detailed information about its form (capsules, pills or tablets), pharmaceutical production sites and batch number. (b) A heatmap comparing two different extraction methods in leech derived products based on OD260/OD280 ratios. Each extraction method was repeated three times. The left part of the heatmap represents DNA quality obtained using Ezup Column Animal Genomic DNA Purification Kit, and the right part corresponds to DNA quality using the One-tube Universal Sample DNA Extraction Kit. The gradient bar represents a range of OD260/OD280 ratio values, with 1.0 indicating the lightest shade and 2.0 indicating the darkest shade, where higher values indicated better DNA quality. The abbreviations represent the names of leech products, detailed in Table 1.

Fig. 2

Matrix layout of the PCR profiles of four primer pairs across 23 leech samples. The abbreviations represent the names of leech products, detailed in Table 1. The vertical bar represents the total number of successfully amplified sequences for each leech sample, while the horizontal bar represents the total number of samples that successfully amplified the sequences for each primer pair. The filled or empty dots indicate the success or failure of the corresponding PCR (results of leech derived products were heavily highlighted with gray background), and the number in each dot represents the ratio of the actual sequence obtained to the target amplicon through Sanger single-stranded chain termination sequencing, and the symbol " × " in the dots indicates the presence of PCR products but the absence of sequencing.

Fig. 3

Circular comparison between sequences of Whitmania pigra from GenBank (EU304459) and the mini-barcoding sequences obtained from leech samples visualized by BRIG. The black circle represents the complete ND1, 12S rDNA, 16S rDNA and COX1 sequences obtained from Whitmania pigra (EU304459). Colored arcs of different lengths within the circle represent the mini-barcoding sequences amplified from morphologically identified leech specimens, while arcs outside the circle represent mini-barcoding sequences obtained from leech derived products (including one processed medicinal leech sample, one hang-dried medicinal leech and other commercial traditional medicines). The four solid or hollow dots after each arc located on ND1 consolidate the results and represent the presence or absence of the obtained mini-barcoding sequences of ND1, 12S rDNA, 16S rDNA and COX1. The visualized area shows that the percent identity similar genes between the Whitmania pigra reference sequences and other mini-barcoding sequences in BLASTn was at least 85%. Color saturation in the arcs indicates identity: 100% saturation represents complete identity, 50% represents 95% identity, and grays represent identity below 85%. The abbreviations represent the names of leech products, detailed in Table 1.

Fig. 4

Species delimitation of all leech samples through ASAP based on a concatenated dataset of ND1, 12S rDNA, 16S rDNA and COX1 sequences. The four solid or hollow dots after each sample ID indicate the presence or absence of these sequences for each sample. The best output partition is marked with an asterisk, indicating the existence of three different species. The bar chart displays the results of different species delimitation schemes, with each column representing a unique partitioning result from the ASAP analysis. The numbers above the columns show the ASAP scores and the proposed group number. Colorful segments in each column represent proposed groups, with numbers in each segment indicating the number of leech species assigned to that proposed group. The images in the upper right corner showed the dorsal view of three morphologically identified leech species. GenBank sequences of W. pigra (EU304459), W. acranulata (KC688271 and KM655838) and H. nipponia (NC_023776 and MZ507570) were also included in this matrix and highlighted in boldface. The abbreviations represent the names of leech products, detailed in Table 1.