DNA Mini-barcode Research Articles

Species authentication and conservation challenges in Chinese fish maw market using Mini-DNA barcoding

Fish maws, derived from the swim bladders of fish, are valued for their high collagen content and nutritional benefits, leading to significant market demand, especially in Asia. This study investigates the species authenticity of fish maw products using mini-DNA barcoding technology to address issues of mislabeling and substitution. Out of 350 samples, 347 were successfully sequenced, revealing a high mislabeling rate of 51.01% among identified species. Species such as Diodon liturosus and Diodon hystrix were found, posing significant health risks due to the presence of tetrodotoxin, a potent neurotoxin. The study also highlights the conservation implications of this market, with endangered species like Totoaba macdonaldi being illicitly traded. These findings underscore the urgent need for accurate species identification, stringent labeling regulations, and enhanced fishery management to protect endangered species and ensure consumer safety. This research advocates for robust international cooperation and reporting to combat illegal fishing and trade, promoting the sustainable development of fish populations and marine ecosystem health.

High-resolution melting analysis to authenticate deer-derived materials in processed products in China using a cytochrome oxidase I mini-barcode.

Deer-derived materials (antler, venison, fetus, penis, bone, tail, and others) are some of the most valuable traditional animal-based medicinal and food materials in China. In production, processing, and trade, the quality of deer products varies. The market is confusing, and counterfeit and shoddy products are common. There is an urgent need to establish an accurate identification method. Two pairs of primers suitable for identifying deer-derived medicinal materials were obtained by screening the cytochrome oxidase I (COI) sequences of 18 species from nine genera of the deer family. The two primers were used to identify the species and adulteration of 22 batches of commercially available deer-derived products with a mini-barcode combining high-resolution melting (HRM) technology and methodical investigation. Deer-derived materials (sika and red deer) were correctly identified by species using varying DNA amounts (1 to 500 ng). The two pairs of primers COI-1FR and COI-2FR yielded melting temperatures (Tm) of 80.55 to 81.00 °C and 82.00 to 82.50 °C for sika deer, and 81.00 to 82.00 °C and 81.40 to 82.00 °C for red deer. Twenty-two batches of commercially available samples were analyzed by HRM analysis and conventional amplification sequencing, and it was found that the species samples had an error rate of species labeling of 31.8%. Four batches of samples were identified as mixed (adulterated) in the HRM analysis. The combination of DNA mini-barcode with HRM analysis facilitated the accurate identification of species of deer-derived materials, especially the identification of samples in an adulterated mixed state. © 2024 Society of Chemical Industry.

Using a DNA mini-barcode within the ITS region to identify toxic Amanita in mushroom poisoning cases

Mushroom poisoning contributes significantly to global foodborne diseases and related fatalities. Amanita mushrooms frequently cause such poisonings; however, identifying these toxic species is challenging due to the unavailability of fresh and intact samples. It is often necessary to analyze residues, vomitus, or stomach extracts to obtain DNA sequences for the identification of species responsible for causing food poisoning. This usually proves challenging to obtain usable DNA sequences that can be analyzed using conventional molecular biology techniques. Therefore, this study aimed to develop a DNA mini-barcoding method for the identification of Amanita species. Following the evaluation and optimization of universal primers for DNA mini-barcoding in Amanita mushrooms, we found that the internal transcribed spacer (ITS) gene sequence primer ITS-a was the most suitable DNA barcode primer for identifying Amanita species. Forty-three Amanita samples were subsequently amplified and sequenced. The sequences obtained were analyzed for intra- and inter-species genetic distances, and a phylogenetic tree was constructed. The findings indicated that the designed primers had strong universality among the Amanita samples and could accurately identify the target gene fragment with a length of 290 bp. Notably, the DNA mini-barcode accurately identified the 43 Amanita samples, demonstrating high consistency with the conventional DNA barcode. Furthermore, it effectively identified DNA from digested samples. In summary, this DNA mini-barcode is a promising tool for detecting accidental ingestion of toxic Amanita mushrooms. It may be used as an optimal barcode for species identification and traceability in events of Amanita-induced mushroom poisoning.Key points• Development of a DNA mini-barcoding method for Amanita species identification without fresh samples.• The ITS-a primer set was optimized for robust universality in Amanita samples.• The mini-barcode is suitable for screening toxic mushroom species in mushroom poisoning cases.Graphical

PCR-Based Amplification of a Cox1 Mini-DNA Barcode Gene from Feces: A Non-Invasive Molecular Technique to Identify Environmental DNA Samples of Maritime Shrew (Sorex maritimensis)

PCR-Based Amplification of a Cox1 Mini-DNA Barcode Gene from Feces: A Non-Invasive Molecular Technique to Identify Environmental DNA Samples of Maritime Shrew (Sorex maritimensis)

Strategy on rapid discrimination of different varieties based on the combination of HS-GC-IMS and DNA Mini-barcode, spore powder of Ganoderma as a case study

The demand for the consumption of Ganoderma (LingZhi) is increasing significantly nowadays. While the wild resources of Ganoderma lucidum and Ganoderma sinense have been reduced severely, and artificially cultivated products are gradually occupying the market. However, it is usually difficult to distinguish the different species of Ganoderma spore powder (GSP). In this research, a rapid and accurate identification method which combined headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) with the DNA mini-barcode was successfully developed to discriminate different GSP. The feasibility of the method was evaluated by both GSP and its Chinese patent medicines. HS-GC-IMS was used to characterize GSP volatile organic compounds (VOCs) and a total of seventy-one VOCs were identified. Principal component analysis could be utilized to show the significantly difference between Korean LingZhi (KL) and American LingZhi (AL) or Taishan LingZhi (TL). Then, we clarified the species information of all samples based on the internal transcribed spacer (ITS) regions. The identification results showed that TL and KL were Ganoderma lucidum, and the AL was Ganoderma resinaceum. Furthermore, according to the mutation sites of the above ITS sequences, the specialized DNA mini-barcode was successfully developed and applied. The artificial mixtures of the three types of GSP and Chinese patent medicines containing GSP were used to authenticate the barcode capability. The mixing experiment found that there was a linear relationship between the sequence numbers and the mixed biomass, indicating that the qualitative and quantitative ability of DNA mini-barcode performed well. The investigation of the ingredients of commercially available GSP products found that the commercial products maybe contain Ganoderma resinaceum, Ganoderma applanatum and Daedaleopsis confragosa, except for Ganoderma lucidum. Therefore, the results suggested that the available GSP products might be adulterated according to Chinese Pharmacopoeia. In this paper, we established a novel approach to distinguish the different GSP samples based on HS-GC-IMS and DNA mini-barcode, and the results indicated that our strategy could not only discriminate different GSP samples, but also identify the origins in their related products.

Integrated DNA barcoding methods to identify species in the processed fish products from Chinese market

DNA-based methods are reliable for a precise identification of species in processed products. In this study, we assessed five typical DNA extraction methods from multiple aspects. Full-length and mini-length DNA barcoding were performed to detect the species substitution and mislabeling of 305 processed fish products from the Chinese market covering six processed fish products. The salt extraction method that exhibited the best overall performance was applied. All samples were successfully extracted; however, only 19.3 % of samples could be amplified using the full-DNA barcode primer set, and 90.2 % of samples could be amplified using the newly designed mini-DNA barcode primer sets (401 and 320 bp). Overall, the molecular identification results revealed that 36.4 % (111/305) of the samples were inconsistent with the labels, with commercial fraud observed in all six types of processed fish products. The survey findings provide technical references for effective fish authentication monitoring, offering insights into the seafood safety in markets.

Classification and identification of mosquitoes in China based on rDNA 28S D5 region

Accurate classification and identification of mosquitoes are essential for the prevention and control of mosquito-borne diseases. In this study, adult mosquitoes were collected from 15 cities across 14 provinces in China. They were identified morphologically with the dominant species determined. Furthermore, representative samples were identified at the molecular level based on rDNA 28S D5. In total, 880 adult mosquitoes were collected belonging to Culex (266), Aedes (473), Armigeres (13), and Anopheles (5). Aedes albopictus and “C. pipiens subgroup” were the dominant species. A total of 140 sequences of 28S D5 region (68 for “C. pipiens subgroup”, 51 for Ae. albopictus, 18 for Ar. subalbatus, and three for An. sinensis) ranging from 148 to 161 bp were obtained, with 100 % success of amplification and sequencing. Molecular identification were consistent with morphological classification. Sequence analysis showed that “C. pipiens subgroup” was identified into three clades: the traditional C. pipiens subgroup (Clade I), the newly discovered C. cf. perexiguus (Clade II), and C. new sp. (Clade III). Clade I contained the most abundant haplotypes (16) widely distributed without geographical differences. Clade II included six haplotypes that were aggregately distributed south of the Yangtze River. Only three sequences in Clade III showed two haplotypes with no geographical differences. Further morphological comparisons demonstrated differences in body color, beaks, and abdomens among the three clades. In conclusion, the rDNA 28S D5 region could effectively distinguish Culex, Aedes, Armigeres, and Anopheles species at the lower category level, demonstrating its potential as a mini-DNA barcode for mosquito identification.

Peran Mini-Barcode Internal Transcribed Spacer 2 (ITS2) untuk Identifikasi Molekuler Spesies Anggrek

AbstractMolecular identification using short orthological DNA sequences (DNA barcoding) has been applied for the classification of orchid species which is a major step in biodiversity management, conservation, breeding, authenticating components of herbal products, and tracking the adulteration of orchid species. One of the most widely used loci for phylogenetic inference at the generic and infrageneric levels in plants was ITS located between 18S rDNA, 5.8S rDNA, and 26S rDNA. The ITS or ITS2 region has been suggested as a plant barcode in some previous studies. However, DNA barcoding using the entire ITS genome is considered less effective and efficient in the process of PCR amplification and sequencing. Besides, complete DNA barcodes are difficult to obtain from herbarium samples and herbal products because some DNA sequences have been degraded. DNA mini-barcodes were developed over the past ten years to overcome issues related to DNA barcoding. DNA mini-barcodes use shorter DNA segments for PCR amplification, so they can identify species effectively and efficiently compared to the regular DNA barcoding. Specific primers that encode the ITS1 and ITS2 regions need to be designed for the PCR amplification. DNA mini-barcoding ITS has proven useful in species identification, classification studies, and authentication of specific orchid species. Therefore, a new rapid identification method based on the ITS mini-barcode is expected to be established, especially for orchid species. Keywords : DNA mini-barcode, Internal Transcribed Spacer, Orchid, Primer Design

Detection of Salmonidae ingredient using mini-DNA barcoding in conjunction with a rapid visual inspection method

Using species outside Salmonidae to prepare salmon products has been widely revealed as a new type of fraud in China and other countries with less stringent labeling stipulations, highlighting the necessity to establish proper methods for rapid screening of Salmonidae ingredient. The present work selected mitochondrial Cytb gene as the target, and developed a novel mini-DNA barcoding assay to identify the salmonids ingredient in processed fish products. Results showed that the novel method is highly specific for salmonids detection and only the seven common salmonids species were successfully amplified. With the inclusion of HRM analysis, we can make an accurate differentiation of Salar salar, Oncorhynchus mykiss, and the other wild-caught Oncorhynchus species, which are deep in the scandal of species mislabeling. Moreover, a closed-tube visual detection can also be achieved by the preaddition of HNB (hydroxyl naphthol blue) and SYTO 9 in the reaction mixture, allowing an easy and rapid way to distinguish positive from negative. Finally, the novel assay was successfully applied on commercial products and the absence of salmonids ingredient indicated mislabeling with 4 commercial products. Overall, mini-DNA barcoding is a robust tool for fish species identification and the novel method is effective in screening Salmonidae ingredient.

A novel biological sources consistency evaluation method reveals high level of biodiversity within wild natural medicine: A case study of Amynthas earthworms as "Guang Dilong".

For wild natural medicine, unanticipated biodiversity as species or varieties with similar morphological characteristics and sympatric distribution may co-exist in a single batch of medical materials, which affects the efficacy and safety of clinical medication. DNA barcoding as an effective species identification tool is limited by its low sample throughput nature. In this study, combining DNA mini-barcode, DNA metabarcoding and species delimitation method, a novel biological sources consistency evaluation strategy was proposed, and high level of interspecific and intraspecific variations were observed and validated among 5376 Amynthas samples from 19 sampling points regarded as "Guang Dilong" and 25 batches of proprietary Chinese medicines. Besides Amynthas aspergillum as the authentic source, 8 other Molecular Operational Taxonomic Units (MOTUs) were elucidated. Significantly, even the subgroups within A. aspergillum revealed here differ significantly on chemical compositions and biological activity. Fortunately, this biodiversity could be controlled when the collection was limited to designated areas, as proved by 2796 "decoction pieces" samples. This batch biological identification method should be introduced as a novel concept regarding natural medicine quality control, and to offer guidelines for in-situ conservation and breeding bases construction of wild natural medicine.

High resolution melting of chloroplast mini-barcode in star anise (Illicium verum) authentication

Star anise (Illicium verum), dubbed the ‘King of Spices’, is one of the most popular spices widely used in cuisine, cosmetics and herbal medicine. However, recent surveys have found that commercial products occasionally replace star anise with some toxic lookalike species. To address this problem, we applied a high resolution melting of chloroplast mini-barcode method to distinguish star anise from poisonous adulterants. We sequenced the complete chloroplast genomes of four reported adulterant species (i.e., Illicium lanceolatum, Illicium simonsii, Illicium henryi and Illicium brevistylum). We carried out a comparative analysis between these species and the published chloroplast genome of star anise. Although the chloroplast genomes are conserved among Illicium species, we found some divergent hotspot regions that can potentially exploit high resolution DNA mini-barcodes for species authentication. Subsequently, we used high resolution melting (HRM) assay to generate standard melting profiles of these mini-barcodes for these species. The melting data showed that the mini-barcodes derived from two divergence regions (i.e., atpF–atpH and ndhF–rpl32) have high resolution for species discrimination and could clearly distinguish star anise from four tested adulterants. We applied these two mini-barcodes to check twelve commercial products alleged or labelled as “star anise”, which were purchased from China traditional markets and internet shops. The HRM curves of eleven samples are clustered with the reference star anise with confidence higher than 95%, indicating that these products contain the correct species as declared. We also found one toxic adulterant, as its melting curve clustered with Illicium simonsii. We conclude that DNA mini-barcode derived from divergent hotspot region of chloroplast genome combined with HRM analysis is an effective and rapid strategy to discriminate star anise from its toxic lookalike adulterants.

DNA Barcode Identification of Fish Products from Guiyang Markets in Southwestern People's Republic of China

Global fish consumption is increasing in tandem with population growth, resulting in the dilemma of overfishing. Overfished high-value fish are often replaced with other fish in markets. Therefore, the accurate identification of fish products in the market is important. In this study, full-DNA and mini-DNA barcoding were used to detect fish product fraud in Guiyang, Guizhou Province, People's Republic of China. The molecular results revealed that 39 (20.42%) of the 191 samples were inconsistent with the labels. The percentages of mislabeling of fresh, frozen, cooked, and canned fish products were 11.70, 20.00, 34.09, and 50.00%, respectively. The average Kimura two-parameter distances of mini-DNA barcoding within species and within genera were 0.56 and 6.42%, respectively, and those of full-DNA barcoding were 0.53 and 7.25%, respectively. Commercial fraud was evident in this study; most high-priced fish were replaced with low-priced fish with similar features. Our findings indicate that DNA barcoding is an effective tool for identifying fish products and could be used to enhance transparency and fair trade in domestic fisheries.

Analysis genetic variation and phylogenetic relationships of local lotus collected in Thua Thien hue province, Vietnam by DNA barcoding

Universal conventional DNA barcodes will become more and more popular in plant species using short sequences of chloroplast DNA identifications. DNA mini-barcode is a solution for such specific purposes. Here we exemplify how to develop the best minibarcodes for specific taxa using the Nelumbo nucifera as an example. Six chloroplast DNA regions (accDpsaI, ndhA, psbE-petL, Rpl32-trnL, trnW – psaJ, trnSGCU-trnGGCC) were tested for their suitability as DNA barcoding regions of thirty three lotus samples (white and pink) which were collected in Thua Thien Hue province, Vietnam. Universal primers were used and sequenced products were analyzed using Minimum Evolution method in the MEGA X program. The sequences accD-psaI; ndhA and psbE-petL did not show any variable sites and none of the regions used in the study allowed the division of white and pink lotus varieties of N. nucifera species according to the adopted classification of the genus Nelumbo. The results confirm that the use of individual six gene regions or combining all six regions together is insufficient for DNA barcoding in white and pink lotus varieties of N. nucifera species. The neutrality tests were negative values for total six gene regions, indicating an excess of rare nucleotide site variants suggesting that either population expansion or background selection has occurred. However following Fu’s Fs test, the hypothesis of netural evolution was significantly rejected for all regions of the studied lotus population. Strobeck’s S, the probability of obtaining equal or fewer haplotypes based on gene frequency and mutation rate, was low in population ranging from 0.183 to 0.549. These results are not consistent with deviation from neutrality due to either selection or population expansion. Our results also indicate the necessity of using a different region.

DNA Barcoding and Mini-DNA Barcoding Reveal Mislabeling of Salmonids in Different Distribution Channels in the Qingdao Area

DNA Barcoding and Mini-DNA Barcoding Reveal Mislabeling of Salmonids in Different Distribution Channels in the Qingdao Area

Authentication of garlic (Allium sativum L.) supplements using a trnLUAA mini-barcode.

Garlic (Allium sativum), a widely distributed plant with great cultural and medicinal significance, is one of the most popular herbal dietary supplements in Europe and North America. Garlic supplements are consumed for a variety of reasons, including for their purported antihypertensive, antibacterial, and anticarcinogenic effects. The steady increase in the global herbal dietary supplement market paired with a global patchwork of regulatory frameworks makes the development of assays for authentication of these products increasingly important. A DNA mini-barcode assay was developed using the P6 loop of the plastid trnLUAA intron to positively identify A. sativum products. Analysis of 43 commercially available garlic herbal dietary supplements produced mini-barcode sequences for 33 supplements, all of which contained detectable amounts of A. sativum. The trnLUAA P6 mini-barcode can be highly useful for specimen identification, particularly for samples that may contain degraded DNA.

DNA Barcode Authentication of Devil's Claw Herbal Dietary Supplements.

Devil’s claw is the vernacular name for a genus of medicinal plants that occur in the Kalahari Desert and Namibia Steppes. The genus comprises two distinct species: Harpagophytum procumbens and H. zeyheri. Although the European pharmacopeia considers the species interchangeable, recent studies have demonstrated that H. procumbens and H. zeyheri are chemically distinct and should not be treated as the same species. Further, the sale of H. zeyheri as an herbal supplement is not legal in the United States. Four markers were tested for their ability to distinguish H. procumbens from H. zeyheri: rbcL, matK, nrITS2, and psbA-trnH. Of these, only psbA-trnH was successful. A novel DNA mini-barcode assay that produces a 178-base amplicon in Harpagophytum (specificity = 1.00 [95% confidence interval = 0.80–1.00]; sensitivity = 1.00 [95% confidence interval = 0.75–1.00]) was used to estimate mislabeling frequency in a sample of 23 devil’s claw supplements purchased in the United States. PCR amplification failed in 13% of cases. Among the 20 fully-analyzable supplements: H. procumbens was not detected in 75%; 25% contained both H. procumbens and H. zeyheri; none contained only H. procumbens. We recommend this novel mini-barcode region as a standard method of quality control in the manufacture of devil’s claw supplements.

The Low Copy Nuclear Gene Region, Granule Bound Starch Synthase (GBSS1), as a Novel Mini-DNA Barcode for the Identification of Different Sage (Salvia) Species.

Morphological similarity within species makes the identification and authentication of Salvia species challenging, especially in dietary supplements that contain processed root or leaf powder of different sage species. In the present study, the species discriminatory power of 2 potential DNA barcode regions from the nuclear genome was evaluated in 7 medicinally important Salvia species from the family Lamiaceae. The nuclear internal transcribed spacer 2 and the exon 9 - 14 region of low copy nuclear gene WAXY coding for granule-bound starch synthase 1 were tested for their species discrimination ability using distance, phylogenetic, and BLAST-based methods. A novel 2-step PCR method with 2 different annealing temperatures was developed to achieve maximum amplification from genomic DNA. The granule-bound starch synthase 1 region showed higher amplification and sequencing success rates, higher interspecific distances, and a perfect barcode gap for the tested species compared to the nuclear internal transcribed spacer 2. Hence, these novel mini-barcodes generated from low copy nuclear gene regions (granule-bound starch synthase) that were proven to be effective barcodes for identifying 7 Salvia species have potential for identification and authentication of other Salvia species.

Authentication of sea cucumber products using NGS-based DNA mini-barcoding

Sea cucumbers are widely consumed in Asia. This study developed a novel DNA mini-barcode mitochondrial cytochrome c oxidase (COI) of 257 bp to discriminate sea cucumbers species in both raw and processed forms. We designed and compared four mini-barcoding primer sets based on PCR amplification rates against 40 authenticated sea cucumber species. The top-performing primer set was successful for most tested sea cucumber varieties. We also tested the DNA mini-barcode COI in 12 highly processed sea cucumber products using both Sanger sequencing and next-generation DNA sequencing (NGS). This is the first report of highly processed sea cucumber products that detected fraudulent replacement of sea cucumber by one or a blend of species using the NGS approach. The established DNA mini-barcoding system revealed that 83.33% (10/12) of highly processed sea cucumber products were mislabeled. This study identified a need for more regulation of the sea cucumber trade and suggested that DNA mini-barcoding combined with NGS could complement existing sea cucumber identification strategies.

DNA barcoding for the identification of shark lips (鱼唇): A nationwide survey for analyzing a never investigated product in the Chinese market

A nationwide survey conducted in China aimed at authenticating species in shark lips products (鱼唇) (n = 252) by full DNACOI barcoding (FDB; 652 bp) is presented. In addition, the efficiency of the FDB and of the mini DNA barcode (MDB; 127 bp) proposed by Fields et al. (2015) (PloS one, 10, e0114844.) in identifying the shark species detected in this study was compared. Despite the manufacturing process, the total DNA of the samples presented a medium low fragmentation degree, and the FDB was obtained from almost all the samples except for two (99.2%) from which the MDB was instead successfully obtained. Samples were allocated to species level in 96.4% of the cases. This confirms the importance to perform a preventive evaluation of the level of DNA degradation before selecting cost and time-consuming procedures. Of the 7 identified species, Prionace glauca was the most recovered (65.5%). The other six detected species were Carcharhinus falciformis (11.5%), Sphyrna lewini (6.7%), S. zygaena (3.6%), Isurus oxyrinchus (3.6%), C. longimanus (3.2%) and C. sorrah (2.4%), 5 of which are threatened and 4 are subject to global commerce regulation. Overall, issues in discriminating among some Carcharhinus spp. were highlighted both using the FDB and the MDB. Outcomes of this study confirms the need to improve the Chinese traceability system. In fact, even though a legislation for seafood labelling supported by an official system for name attribution not always ensure the sector safeguarding from frauds, absent or weak traceability system certainly facilitate illegal practices.

DNA meta-barcoding using rbcL based mini-barcode revealed presence of unspecified plant species in Ayurvedic polyherbal formulations.

Ayurveda takes advantage of the beneficial properties of medicinal plants. High demands in combination with inadequate availability of botanicals and a lack of knowledge with respect to their precise identification lead to adulterations in herbal products. Identification becomes more difficult in complex herbal formulations. Four different polyherbal formulations have been analyzed for the present paper. The targeted plants have different pharmacological properties for various ailments. We aimed to examine the rbcL gene based plant DNA mini-barcode to identify target and non-target plants in polyherbal formulations by using high-throughput next generation sequencing. Degenerate primers of the selected mini-barcode region have been identified from the literature. A blend of 30 authentic medicinal plant species was used to examine the species resolution capacity of the mini-barcode. DNA was isolated from herbal formulations, an amplicon library was prepared, and sequencing was performed on an IonS5 system. Data were analyzed using various bioinformatics tools. Analysis of control pooled samples revealed the optimum resolving power of the DNA mini-barcode. Data analysis of the commercial samples revealed that only one herbal formulation contained all plants and matched with listed contents. In two formulations, only 10 out of 21 and 11 out of 20 plants were detected, respectively. Additionally, several non-listed plants were also detected in these formulations. Two formulations contained >20% reads assigned to non-target plants. Overall, 21.98% of the reads were assigned to non-target plants. The present study clearly demonstrated the successful application and potential of meta-barcoding in the quality control of complex herbal matrices. The results strongly suggest that this approach can be used in pharmacovigilance of processed herbal products.