Mitochondrial 16S Ribosomal RNA Research Articles

Molecular and morphological examinations show diversity in the genus Amphidromus (Camaenidae: Stylommatophora) in Bangladesh

Establishing a robust taxonomic foundation is crucial for biodiversity conservation. Bangladesh harbours potentially unique biodiversity that remains inadequately explored. Concurrently, its biodiversity faces substantial pressure owing to environmental changes; thus, an urgent assessment of its current status is required. In this study, we focused on the understudied land snails of Bangladesh, the genus Amphidromus. Utilizing 45 samples collected from 13 sites during a 2018–2022 survey, as well as museum specimens and literature sources, we conducted molecular phylogenetic analyses and the haplotype network of the mitochondrial 16S ribosomal RNA, cytochrome c oxidase subunit I and nuclear internal transcribed spacer 1, examined shell shapes using elliptical Fourier features, and evaluated shell size and band patterns. Based on the molecular and morphological examinations, we identified five Amphidromus species in Bangladesh, including two potentially undescribed species, each recorded at a single site in the Chittagong Division. The dominant species, A. globonevilli, is widely distributed in central and eastern Bangladesh, revealing a geographically structured mitochondrial DNA diversity. Furthermore, in the museum specimens, we confirmed the occurrence of A. sinensis vicaria and A. sylheticus, the latter being newly discovered in Bangladesh. These two species may be limited to the eastern part of Bangladesh and represent low-density species. Although the taxonomic relationship of these species with A. globonevilli remains unclear and further studies with molecular data are warranted, our findings provide the basis for further understanding and conservation of biodiversity in Bangladesh.

An innovative molecular approach towards the cost-effective entomological authentication of honey

Honey authentication and traceability are crucial not only for economic purposes but also for ensuring safety. However, the widespread adoption of cutting-edge technologies in practical applications has been hampered by complex, time-consuming sample pre-treatment processes, the need for skilled personnel, and substantial associated expenses. This study aimed to develop a simple and cost-effective molecular technique to verify the entomological source of honey. By utilizing newly designed primers, we successfully amplified the mitochondrial 16S ribosomal RNA gene of honey bees from honey, confirming the high quality of the extracted DNA. Employing RFLP analysis with AseI endonuclease, species-specific restriction patterns were generated for honey derived from six closely related honey bees of the Apis genus. Remarkably, this method was proven equally effective in identifying heat-treated and aged honey by presenting the same RFLP profiles as raw honey. As far as we know, this is the initial research of the simultaneous differentiation of honey from closely related honey bee species using the restriction endonuclease AseI and mitochondrial 16S rRNA gene fragments. As a result, it holds tremendous potential as a standardized guideline for regulatory agencies to ascertain the insect origins of honey and achieve comprehensive traceability.

Generation of an induced pluripotent stem cell line (KEIUi008-A) from a hearing loss patient with an A1555G mutation in mitochondrial DNA

We report the establishment of a human induced pluripotent stem cell (iPSC) line from a 54-year-old male patient with an A1555G mutation in the mitochondrial 12S ribosomal RNA gene (MTRNR1), associated with sensorineural hearing loss. The established iPSC line expressed stemness markers or undifferentiated state markers. We also demonstrated the capacity of the cells to differentiate into the three germ layers, suggesting its pluripotency and utility in the pathological study of sensorineural hearing loss and drug screening for ear disorders.

Genetic Variation in the Pallas’s Cat (Otocolobus manul) in Zoo-Managed and Wild Populations

The Pallas’s cat (Otocolobus manul) is one of the most understudied taxa in the Felidae family. The species is currently assessed as being of “Least Concern” in the IUCN Red List, but this assessment is based on incomplete data. Additional ecological and genetic information is necessary for the long-term in situ and ex situ conservation of this species. We identified 29 microsatellite loci with sufficient diversity to enable studies into the individual identification, population structure, and phylogeography of Pallas’s cats. These microsatellites were genotyped on six wild Pallas’s cats from the Tibet Autonomous Region and Mongolia and ten cats from a United States zoo-managed population that originated in Russia and Mongolia. Additionally, we examined diversity in a 91 bp segment of the mitochondrial 12S ribosomal RNA (MT-RNR1) locus and a hypoxia-related gene, endothelial PAS domain protein 1 (EPAS1). Based on the microsatellite and MT-RNR1 loci, we established that the Pallas’s cat displays moderate genetic diversity. Intriguingly, we found that the Pallas’s cats had one unique nonsynonymous substitution in EPAS1 not present in snow leopards (Panthera uncia) or domestic cats (Felis catus). The analysis of the zoo-managed population indicated reduced genetic diversity compared to wild individuals. The genetic information from this study is a valuable resource for future research into and the conservation of the Pallas’s cat.

Molecular characterisation of Amblyomma integrum circulating in southern India

Amblyomma integrum is a large gooseberry sized longirostrate tick (when fully repleted) found in India and Sri Lanka. In Kerala (India), this tick is commonly found in the forest and its fringe areas frequently infesting deer and hence it is locally known as “maan chellu / maanunny” (deer tick). In the present study, molecular characterisation and phylogenetic analysis of A. integrum collected from the area grazed by the sambar deer (Rusa unicolor) of Kerala, south India was performed using three molecular markers viz., the mitochondrial cytochrome c oxidase subunit 1 (COI), mitochondrial 16S ribosomal RNA, and nuclear 18S ribosomal RNA genes. Cytochrome c oxidase subunit 1 (COI) gene showed better resolving ability for elucidating the evolutionary relationship of A. integrum and identified two distinct clades, viz., A and B. The Tamil Nadu isolates of south India and Marayoor isolate 1 (from Idukki district of Kerala bordering with Tamil Nadu) belonged to clade A. Majority of Wayanad isolates from Kerala, occupied clade B. The intraspecific genetic distance among the A. integrum species ranged from 0.00 to 13.34%. Between clades A and B, the genetic distance observed was 11.49%. The clade B isolates were genetically close to A. geoemydae (GD: 1.22%). Morphological variations between the clades included darker exoskeletal coloration in clade A and distinct differences in the shape of basis capitulum. Further analysis using Assemble Species by Automatic Partitioning (ASAP) and Generalized Mixed Yule Coalescent (GMYC) provided additional insights. Assemble Species by Automatic Partitioning (ASAP) identified 26 Molecular Operational Taxonomic Units (MOTUs) at a threshold distance of 5.38%, supporting the species partition of A. integrum clade B. Generalized Mixed Yule Coalescent (GMYC) analysis retained the same species complex (A. integrum-geoemydae Complex) inferred from the ASAP analyses. It could be inferred from the present study that the A. integrum clades A and B could be two different putative pseudocryptic species.

The systematics of the amphidromous shrimp Macrobrachium tenellum (Smith, 1871) (Decapoda: Caridea: Palaemonidae) from the Mexican Pacific slope

Abstract We had previously revised the systematics of four species of MacrobrachiumSpence Bate, 1868 from the Pacific coast of Mexico, including the Baja California Peninsula (BCP): M. americanumSpence Bate, 1868, M. digueti (Bouvier, 1895), M. hobbsi Nates & Villalobos in Villalobos Hiriart & Nates Rodríguez, 1990, and M. occidentale Holthuis, 1950. Genetic analyses of the mitochondrial markers 16S ribosomal RNA (16S) and cytochrome oxidase I (COI) of individuals of these morphological species from BCP have shown that, even facing the Gulf of California (Sea of Cortés) as an apparent marine ecological barrier, they are conspecific to their respective genetic lineages (species) found from the Pacific slope of mainland Mexico. We discuss the systematics of the fifth species of Macrobrachium from the Mexican Pacific slope, M. tenellum (Smith, 1871) to test the hypothesis that individuals of the putative M. tenellum from BCP belong to the same species from the Pacific slope of mainland Mexico. We revised the morphological characters of taxonomic importance using material obtained in the field and voucher material deposited in Mexican scientific collections, and performed molecular genetic analyses with newly generated fragments of the mitochondrial markers 16S and COI. We conclude, as in our previous studies on Macrobrachium species of the region, that the examined specimens of the putative M. tenellum from BCP belong to the same species found on the Pacific slope of mainland Mexico, and that oceanic dispersal is responsible for the distribution of the species of Macrobrachium in the peninsula rather than by vicariant events.

Development of molecular identification methods for Dryophytes suweonensis and D. japonicus, and their hybrids.

As hybridization can reduce biodiversity or cause extinction, it is important to identify both purebred parental species and their hybrids prior to conserving them. The Suwon tree frog, Dryophytes suweonensis, is an endangered wildlife species in Korea that shares its habitat and often hybridizes with the Japanese tree frog, D. japonicus. In particular, D. suweonensis, D. japonicus, and their hybrids often have abnormal ovaries and gonads, which are known causes that could threaten their existence. We collected 57 individuals from six localities where D. suweonensis is known to be present. High-resolution melting curve (HRM) analysis of the mitochondrial 12S ribosomal RNA gene was performed to determine the maternal species. Thereafter, the DNA sequences of five nuclear genes (SIAH, TYR, POMC, RAG1, and C-MYC) were analyzed to determine their parental species and hybrid status. The HRM analysis showed that the melting temperature of D. suweonensis was in the range of 79.0-79.3°C, and that of D. japonicus was 77.7-78.0°C, which clearly distinguished the two tree frog species. DNA sequencing of the five nuclear genes revealed 37 single-nucleotide polymorphism (SNP) sites, and STRUCTURE analysis showed a two-group structure as the most likely grouping solution. No heterozygous position in the purebred parental sequences with Q values ≥ 0.995 were found, which clearly distinguished the two treefrog species from their hybrids; 11 individuals were found to be D. suweonensis, eight were found to be D. japonicus, and the remaining 38 individuals were found to be hybrids. Thus, it was possible to unambiguously identify the parental species and their hybrids using HRM analysis and DNA sequencing methods. This study provided fundamental information for D. suweonensis conservation and restoration research.

Sympatric occurrence of Taenia saginata and Sarcocystis spp. in cattle from Narok County, Kenya: meat inspection findings with molecular validation.

The epidemiological picture of Taenia saginata infections in Kenya is fragmented with limited available data. Although Sarcocystis species are significant meat-borne parasites, few studies have explored their occurrence in Kenya. This study aimed to estimate the occurrence of bovine cysticercosis and screen for the presence of Sarcocystis spp. A meat inspection-based survey was conducted in ten abattoirs in Narok County, Kenya, and inspection for T. saginata cysticerci was limited to the Triceps brachii muscle. The apparent occurrence of the parasite was 5.4% (95% CI, 3.8, 7.6, n=573). Molecular confirmation of T. saginata was done via nested polymerase chain reaction targeting the mitochondrial 12S ribosomal RNA gene and restricted fragment length polymorphism. Sarcocystis species were identified using a multiplex polymerase chain reaction method targeting the 18S ribosomal RNA gene sequences and the mitochondrial cytochrome c oxidase subunit I gene. Of the 31 cystic lesions tested, 26/31 (83.9%) were confirmed to be T. saginata.Sarcocystis cruzi and S. hominis were detected in 8/31 (25.8%) and 1/31 (3.2%) of the cystic lesions, respectively. Co-infections of S. cruzi and T. saginata were found in 6/31 lesions (19.4%). The confirmation of bovine cysticercosis and S. hominis is suggestive of the presence of risky culinary and sanitation practices that facilitate transmission. This is the first report and molecular confirmation of Sarcocystis spp. in cattle in the country. The presence of both zoonotic S. hominis and pathogenic S. cruzi highlights an underexplored concern of veterinary and human health significance, warranting further epidemiological investigation.

The status of COI and 12S rRNA DNA barcode reference libraries for freshwater fish in South Africa: Implications for future eDNA projects

Environmental DNA metabarcoding (eDNA) is a rapidly emerging field in which high-throughput sequencing is used to catalogue the biodiversity of ecosystems through the amplification of DNA extracted from environmental samples (water, air, faeces and soil). Although eDNA has strong links to DNA barcoding, the molecular marker most often used to detect vertebrates in eDNA studies is a portion of the mitochondrial 12S ribosomal RNA (12S rRNA) and not the standard cytochrome oxidase I (COI) marker used in traditional DNA barcoding. eDNA methods rely on a comprehensive reference library to link sequence data to species, which are often lacking in hyper-diverse countries such as South Africa. In this study, we review the present state of DNA barcode reference databases for both 12S rRNA and COI for freshwater fish (native and introduced) found in South African aquatic systems. Analysis of DNA records available on GenBank and the Barcode of Life Database (BOLD) revealed incomplete records of the examined taxa for both markers. Our findings showed that 34 species, 6 genera and 0 families of native South African freshwater fish lack COI barcode records, while 86 species, 22 genera and 8 families lack 12S rRNA records. Unlike the native freshwater fish, the non-native fish all had barcode records available for both COI and 12S rRNA. Producing comprehensive reference libraries for both markers is an important first step in developing an eDNA protocol for the non-invasive monitoring of native and non-native freshwater fish in South Africa.

Environmental DNA compliments traditional sampling for monitoring fish communities in a Texas estuary

AbstractObjectiveEnvironmental DNA (eDNA) metabarcoding has become an important method for inventorying and monitoring biota in aquatic systems. The Texas Parks and Wildlife Department conducts regular fishery‐independent sampling of biotic communities using traditional sampling gears, such as gill nets and bag seines, in all of the major estuaries of adjacent to the Gulf of Mexico in Texas. Previous studies have shown that eDNA approaches can complement traditional sampling methods.MethodsWe compared fish community structure data in the Cedar Lakes estuary system obtained with traditional sampling gears with data obtained using eDNA sampling using a small sequence of mitochondrial 12S ribosomal RNA gene and a validated taxonomic reference file.ResultFor spring and fall of 2022, eDNA metabarcoding detected a larger number of species than either bag seines or gill nets. Species richness detected via eDNA in two seasons in a single year was comparable with the species richness of agency’s historical record based on traditional gears for Cedar Lakes.ConclusionSeasonal and spatial variation in species richness was similar between traditional and eDNA sampling; however, eDNA metabarcoding allowed detection of several species that would be difficult or impossible to capture with either bag seines or gill nets. We observed two limitations of eDNA metabarcoding. Read depth was not a good index of relative abundance, which limits our ability to infer relative biomass using single samples. Secondly, we observed detection bias in our eDNA results. Specifically, eDNA failed to detect two species of elasmobranchs present when water sampling was performed and eDNA also performed poorly compared to traditional sampling gears for some species of bony fishes. Despite these limitations, eDNA metabarcoding proved to be an efficient and cost‐effective alternative and compliment to traditional fisheries sampling gears for fishery‐independent monitoring of community structure and composition in estuaries of the Gulf of Mexico.

Mare-MAGE curated reference database of fish mitochondrial genes

Biodiversity assessment approaches based on molecular biology techniques such as metabarcoding, RAD-seq, or SnaPshot sequencing are increasingly applied in assessing marine and aquatic ecosystems. Here we present a new reference database for fish meta-barcoding based on mitochondrial genes. The Mare-MAGE database contains quality-checked sequences of the mitochondrial 12S ribosomal RNA and Cytochrome c Oxidase I gene. All sequences were obtained from the National Center for Biotechnology Information- GenBank (NBCI-GenBank), the European Nucleotide Archive (ENA), AquaGene Database and BOLD database, and have undergone intensive processing. They were checked for false annotations and non-target anomalies, according to the Integrated Taxonomic Information System (ITIS) and FishBase. The dataset is compiled in ARB-Home, FASTA and Qiime2 formats, and is publicly available from the Mare-MAGE database website (http://mare-mage.weebly.com/). It includes altogether 231,333 COI and 12S rRNA gene sequences of fish, covering 19,506 species of 4,058 genera and 586 families.

The smallest of its kind: Description of a new cryptic Amnirana species (Amphibia, Anura, Ranidae) from West African rainforests

The distribution of the White-Lipped Frog Amnirana albolabris was long assumed to extend from eastern-central to western African rainforests. However, recent molecular phylogenetic analyses revealed that this taxon likely represents several undescribed species. Because the name-bearing types were collected in Gabon, the distantly related West African populations clearly represent an undescribed species that partly occurs in sympatry with Amnirana fonensis. Based on an integrative taxonomic approach, including molecular, morphological, and acoustic data, we describe the 'albolabris' populations from the Upper Guinean Forest Zone as a new species, and redescribe the morphologically similar A. fonensis on the bases of a larger series of genotyped individuals, including the first known females. We also provide new biological information for A. fonensis, including their advertisement call, habitat, and reproductive data. The new species is sister to A. fonensis and the two species differ by 5.8% in the mitochondrial 16S ribosomal RNA gene. Morphologically, males of the new species are smaller and have relatively smaller humeral glands. Females of the new species seem to have shorter legs than A. fonensis. In comparison to A. fonensis, the advertisement call of the new species has a higher dominant frequency and more pronounced frequency modulation. The two species differ in their distribution and habitat preferences, as revealed by environmental niche modelling. Whereas the new species is restricted to the Upper Guinean forests and thus is a true lowland-rainforest inhabitant, A. fonensis lives predominantly in Guinean montane forests in the forest-savanna mosaic zone.

A new deep-sea palaemonid shrimp assigned to Periclimenes Costa, 1844 (Decapoda: Caridea) from the West Mariana Ridge, northwestern Pacific

A new palaemonid species, Periclimenes variabilis, is described and illustrated on the basis of four specimens from the Ritto Seamount, West Marina Ridge, northwestern Pacific, at depths of 642-666 m. Specimens of the new species were collected with an unidentified hexacoral of Primnoidae (Cnidaria), an antipatharian octocoral Leiopathes sp. (Cnidaria), and an unidentified crinoid (Echinodermata), suggesting association with those invertebrates. Morphological comparison and molecular phylogenetic analyses using the mitochondrial 16S ribosomal RNA and cytochrome C oxidase subunit I genes indicate that the new species is closest to Periclimenes uniunguiculatus Bruce, 1990 although the possession of more than two spiniform setae on either side of the telson in the new species is consistent with the generic diagnosis of Bathymenes Kou, Li & Bruce, 2016. In reference to the close relationship to the species currently assigned to Periclimenes Costa, 1844 and the unsettled taxonomy of the genus, the new species is provisionally assigned to Periclimenes. Differentiating characters between the new species and closely related taxa are discussed.

First detection of Echinococcus multilocularis in Bosnia and Herzegovina.

Echinococcus multilocularis has been spreading through Central Eastern Europe but has not yet been reported in Bosnia and Herzegovina (B&H). Recently, this parasite is confirmed in Croatia suggesting the movement of the parasite's distribution limit further south. Given that there is no surveillance or monitoring system for echinococcosis in B&H, our study was designed as a pilot study of E. multilocularis. A total of 57 red foxes originating from 24 localities all over the country were collected during the routine rabies monitoring, autopsied and examined for the presence of echinococcosis. Based on intestinal scraping technique and microscopy, E. multilocularis adult worms have been detected in one (1/57, 1.75%) red fox. To verify this finding and to differentiate Echinococcus spp., DNA extracted from adult worms was subjected to species-specific PCR targeting part of the mitochondrial 12S ribosomal RNA gene. E. multilocularis PCR-positive samples were further confirmed by NGS sequencing of a 203 bp amplified fragment of 12S rRNA, which has been deposited in GenBank (Accession no.: OP047920). This finding represents the first detection of E. multilocularis in B&H, strongly suggesting its presence in the country. The confirmation of the parasite in the same locality where migrants/refugees temporarily stay on their route to Western Europe highlights the need for a One Health approach in addressing all future questions. Moreover, the first detection of E. multilocularis in B&H warrants the need for the implementation of an appropriate state surveillance program.

Molecular phylogenetic investigation of toads based on sequencing of the mitochondrial 16S ribosomal RNA gene

Amphibians inhabit a wide variety of habitats and environments, such as arboreal, terrestrial and freshwater aquatic ecosystems, and are considered among the vertebrate classes to exhibit one of the most diverse distributions. Their life histories, behaviour and diversity play vital roles in assessing environmental and evolutionary theories. The mitochondrial 16S rRNA gene sequence analysis has been widely used in molecular taxonomy and is frequently used in systematic analyses of families and genera. This study examines taxonomic relationships among 18 specimens of the Arabian toad (Sclerophrys arabica) and the Dhofar toad (Bufo dhufarensis) from Al Madinah Province, Saudi Arabia by estimating phylogenetic relationships using the mitochondrial 16S ribosomal RNA gene (16S rRNA). Results indicate the presence of four new lineages of toads among the samples in this study. The generated phylogenetic tree grouped all samples of Arabian toad in one clade, while the Dhofar toad samples were divided into three subclades. This is the first study to be reported the genetic diversity of Al Madinah Province toads.

Discovery of a new limestone karst-restricted odorous frog from northern Guangdong, China (Anura, Ranidae, Odorrana).

Karstic landscapes play an important role in biodiversity formation and often contain high levels of endemism. However, site-endemic taxa in karstic landscapes are being threatened by exploitation and weak legal protection. In this study, we describe Odorranaconcelata Wang, Zeng, & Lin, sp. nov., a limestone karst-restricted odorous frog from northern Guangdong, China. This new species shows distinctive genetic divergence and morphological differences from its congeners. Phylogenetic results suggest that the new species represents an independent lineage that is grouped with O.lipuensis and O.liboensis based on the mitochondrial 16S and 12S ribosomal RNA genes. We recommend the new species be listed as Vulnerable (VU) in the IUCN categorization as it is only known from the type locality with limited microhabitats and is threatened by habitat degradation.

Similarities and Differences in Fish Community Composition Accessed by Electrofishing, Gill Netting, Seining, Trawling, and Water eDNA Metabarcoding in Temperate Reservoirs

It is difficult to understand the composition and diversity of biological communities in complex and heterogeneous environments using traditional sampling methods. Recently, developments in environmental DNA metabarcoding have emerged as a powerful, non-invasive method for comprehensive community characterization and biodiversity monitoring in different types of aquatic ecosystems. In this study, water eDNA targeting fish (wf-eDNA) and four traditional fish sampling methods (electrofishing, gill netting, seining, trawling) were compared to evaluate the reliability and efficiency of wf-eDNA (vertebrate mitochondrial 12S ribosomal RNA (rRNA) as an alternative approach to assess the diversity and composition of freshwater fish communities. The results of wf-eDNA showed a consistency between the traditional sampling methods regarding species detection. However, some fish species detected using wf-eDNA assay were not detected using traditional sampling methods and vice versa. Comparison of wf-eDNA and traditional sampling methods revealed spatial homogeneity in fish community composition in all reservoirs. Ordination analysis showed that the wf-eDNA approach covers all traditional sampling methods and occupies an intermediate position. In addition, based on the Shannon diversity index, we found that in one reservoir the wf-eDNA method yielded similar fish community diversity to traditional sampling methods. However, in other reservoirs, the calculated Shannon diversity index of the wf-eDNA method was significantly higher than traditional sampling methods. In general, significant positive correlations were found between the wf-eDNA method and almost all traditional sampling methods. We conclude that wf-eDNA seems to be a reliable and complementary approach for biomonitoring and ecosystem management of freshwater ichthyofauna.

Robust environmental DNA assay development and validation: A case study with two vulnerable Australian fish

Abstract Analysis of environmental (e)DNA can facilitate an understanding of the presence and distribution of aquatic species. However, eDNA detection using quantitative PCR requires validated and standardized species‐specific assay designs. This study presents two eDNA assays to detect Murray cod, Maccullochella peelii, and mulloway, Argyrosomus japonicus (two ecologically vulnerable Australian species), based on small fragments of the mitochondrial 12S ribosomal RNA gene. A comprehensive description of species‐specific assay development, from assay design to testing in silico, in vitro and in situ, has been included to guide effective assay design and validation in future studies. The results indicate that the assay was species specific for M. peelii within its natural distribution. However, the assay also amplified genomic DNA from two allopatric and endangered congeners (Maccullochella ikei and Maccullochella mariensis), thus potentially facilitating their eDNA detection elsewhere. In contrast, the A. japonicus assay was highly species specific with no amplification among close relatives. Both target‐species assays are highly sensitive to as few as four and 10 copies per PCR reaction, respectively. This study has demonstrated that the assays assessed are effective tools for detecting the targeted species in situ from environmental DNA samples, which will assist efforts to conserve and manage their stocks.

Species Identification from Suspected Crocodile Leather Products using Mitochondrial 12S Ribosomal RNA and Cytochrome b Genes

법의학 분야에서 종식별은 불법 야생동물 거래의 강력한 규제를 위해 중요한 역할을 한다. 2020 년, 경상남도지방경찰청에서 밀수증거물인 5종의 악어 추정 가죽 제품의 종분석을 의뢰하였다. 이를 위해, 미토콘드리아 12S ribosomal RNA와 cytochrome b 유전자에 대한 PCR 산물의 염기서열 분석을 통해 종식별을 수행하였다. 가죽이나 가죽 안쪽에 위치한 뼈, 발톱, 이빨의 바깥 부분에서는 PCR 산물을얻지 못하였으나, 두개골 안쪽, 턱뼈 안쪽의 혈관 추정 조직, 뼈, 발톱, 이빨의 안쪽 부분에서는 정상적인 PCR 산물을 얻을 수 있었다. 두 유전자의 PCR 산물의 염기서열에 대한BLAST분석 결과, ‘Crocodylus siamensis (샴 악어)’ 종과 100 % 일치하였다. 또한, NCBI에서 얻은 다른 악어 종의 염기서열과 함께샘플 염기서열을 이용한 neighbor-joining phylogenetic tree 분석 결과에서도 ‘Crocodylus siamensis’ 종과 100 % 일치하는 것으로 나타났다. 법의학 분야에서 미토콘드리아 DNA가 종식별에 유용함에도 불구하고, 이번 사례는 가죽 제품에서의 미토콘드리아 DNA가 가죽 공정 과정 동안 손상될 가능성을 보여준다. 따라서, 가죽 공정 과정 동안 DNA가 최대한 손상되지 않은 부위를 찾는 노력이 필요할 것이다

A 16S Next Generation Sequencing Based Molecular and Bioinformatics Pipeline to Identify Processed Meat Products Contamination and Mislabelling.

Simple SummaryMeat adulteration and fraud encompasses the deliberate fraudulent addition or substitution of proteins of animal or plant origin in edible products primarily for economic gain. The mitochondrial 16S ribosomal (rRNA) gene was used to identify species that are present in pure and processed meat samples. The meat samples were sequenced using an Illumina sequencing platform, and bioinformatics analysis was carried out for species identification. The results indicated that pork was the major contaminant in most of the meat samples. The bioinformatics pipeline demonstrated its specificity through identification of species specific and quantification of the contamination levels across all samples. Food business operators and regulatory sectors can validate this method for food fraud checks and manage any form of mislabeling in the animal or plant protein food ecosystem.Processed meat is a target in meat adulteration for economic gain. This study demonstrates a molecular and bioinformatics diagnostic pipeline, utilizing the mitochondrial 16S ribosomal RNA (rRNA) gene, to determine processed meat product mislabeling through Next-Generation Sequencing. Nine pure meat samples were collected and artificially mixed at different ratios to verify the specificity and sensitivity of the pipeline. Processed meat products (n = 155), namely, minced meat, biltong, burger patties, and sausages, were collected across South Africa. Sequencing was performed using the Illumina MiSeq sequencing platform. Each sample had paired-end reads with a length of ±300 bp. Quality control and filtering was performed using BBDuk (version 37.90a). Each sample had an average of 134,000 reads aligned to the mitochondrial genomes using BBMap v37.90. All species in the artificial DNA mixtures were detected. Processed meat samples had reads that mapped to the Bos (90% and above) genus, with traces of reads mapping to Sus and Ovis (2–5%) genus. Sausage samples showed the highest level of contamination with 46% of the samples having mixtures of beef, pork, or mutton in one sample. This method can be used to authenticate meat products, investigate, and manage any form of mislabeling.