Reference DNA Research Articles

Development and Evaluation of a Semi-Nested PCR Method Based on the 18S ribosomal RNA Gene for the Detection of Babesia aktasi Infections in Goats.

We developed and evaluated a semi-nested PCR assay for the detection of Babesia aktasi infection in goats based on the sequence of the B. aktasi 18S ribosomal RNA gene. Following in silico screening, the specificity of the primers was assessed using reference DNA samples, including B. ovis, B. motasi, B. crassa, B. venatorum, B. divergens, B. capreoli, Theileria ovis, and T. annulata. To determine the sensitivity of the method, blood infected with 2% parasitemia of B. aktasi was diluted to 10-fold serial dilutions. The method specifically amplified a 438 bp fragment of B. aktasi DNA, but did not demonstrate cross-amplification with the other hemoparasites tested. The sensitivity assay indicated that this PCR method was able to detect infection at a dilution of 10-8 of 2% parasitemia (0.074 parasites/200 µL). Ninety-seven blood samples collected from goats were used to analyze for B. aktasi, and the infection was detected in 18.5% of the goats. Additionally, the method was also applied to 44 field DNA samples that were detected to be positive for B. aktasi by reverse line blotting (RLB), and showed 84.1% agreement. The findings revealed that newly developed semi-nested PCR can detect B. aktasi infections in goats with high sensitivity and specificity.

168 American mink Genome Sequencing Project

Abstract The long-term goal of American Mink (Neogale vison) Genome Sequencing Project is to develop novel genomics tools to study the genome biology and evolution of mink. This research agenda is organized around three major goals: 1) development of first chromosome-level genome assembly with high quality for American mink to help understanding the biology and evolution of the order Carnivora, 2) designing the first mink single nucleotide polymorphism (SNP) genotyping array for genomic discovery in mink, and 3) using genomics to understand the genetic architecture of economically important traits in mink. An initial draft of the genome assembly is generated using 2,884,047 PacBio long reads. Integration of Hi-C data into the initial draft led to an assembly with 183 scaffolds and scaffold N50 of 220 Mb. This gap-free genome assembly of American mink (ASM_NN_V1) has a length of 2.68 Gb in which about 98.6% of the whole genome is covered by 15 chromosomes. Significant improvements are observed in genome contiguity, the number of scaffolds, and annotation compared with the first draft of mink genome assembly. A custom genotyping array is developed using the developed mink reference genome and DNA panel composed of 100 whole-genome sequences data of American mink from five color-types. This Affymetrix Mink 70K SNP array created 70,000 SNPs, which is sufficient for conducting genomic studies in the mink research program and beyond. This project would facilitate the discovery of genomic regions, markers and genetic networks underlying economically important traits in American mink, which will contribute novel insights into the genetic architecture of key traits and is a critical step to implement genomic selection in mink breeding programs. The results of this research have the potential to impact the mink industry, especially in North America, and improve the overall performance of the mink industry.

EDNAqua-Plan—Standardisation Overview for eDNA Sequencing of Aquatic Organisms and the Downstream Data Ecosystem

The eDNAqua-Plan project stands as a beacon of innovation in the biomonitoring of marine and freshwater ecosystems, propelled by the urgent need to integrate DNA-based approaches in aquatic bioassessment and monitoring frameworks. The broad utilisation of cutting-edge environmental DNA (eDNA) and DNA barcoding methodologies is dependent on complete, reliable, and accessible reference DNA sequence data (Rimet et al. 2021). Complete and interoperable metadata is crucial to allow a broad reuse of (e)DNA data and analysis outputs, and for a broader uptake of results by end users. The eDNAqua-Plan project aims to address key limitations to the routine implementation of eDNA-based monitoring methods in Europe by developing plans for federated DNA barcode reference libraries and eDNA data repositories to support DNA-based environmental monitoring. This will ensure a sustainable and reliable infrastructure to underpin its broad use, thereby paving the way for more effective conservation and management strategies. The project is working towards creating a comprehensive overview of standardisation efforts and data workflows, through collaborations with other projects, initiatives and infrastructures for aquatic monitoring across the European Union (EU) and associated countries. We are analysing existing archives (e.g., International Nucleotide Sequence Database Collaboration (INSDC), Barcode of Life Data System (BOLD), Global Biodiversity Information Facility (GBIF), Ocean Biodiversity Information System (OBIS)), portals, and papers to determine current and best practices through the use of questionnaires, manual evaluation of repositories, and machine learning methods (LLMs). This includes an overview of the usage of existing metadata and data standards (e.g., Minimum Information about any (X) Sequence Specifications from the Genomics Standards Consortium (GSC), Darwin Core standard). The results are being integrated by a team of experts in marine and freshwater biomonitoring. With a diverse consortium comprising 18 partner institutions from 11 countries and one international institute, eDNAqua-Plan brings together experts in marine and freshwater monitoring, eDNA analysis, and data science. The collective effort by this consortium will lay the groundwork for the creation of a digital ecosystem of eDNA repositories and an integrated reference library of marine and freshwater species, adhering to FAIR (Findable, Accessible, Interoperable, and Reusable) principles.

Quantitative analysis of dose dependent DNA fragmentation in dry pBR322 plasmid using long read sequencing and Monte Carlo simulations

Exposure to ionizing radiation can induce genetic aberrations via unrepaired DNA strand breaks. To investigate quantitatively the dose–effect relationship at the molecular level, we irradiated dry pBR322 plasmid DNA with 3 MeV protons and assessed fragmentation yields at different radiation doses using long-read sequencing from Oxford Nanopore Technologies. This technology applied to a reference DNA model revealed dose-dependent fragmentation, as evidenced by read length distributions, showing no discernible radiation sensitivity in specific genetic sequences. In addition, we propose a method for directly measuring the single-strand break (SSB) yield. Furthermore, through a comparative study with a collection of previous works on dry DNA irradiation, we show that the irradiation protocol leads to biases in the definition of ionizing sources. We support this scenario by discussing the size distributions of nanopore sequencing reads in the light of Geant4 and Geant4-DNA simulation toolkit predictions. We show that integrating long-read sequencing technologies with advanced Monte Carlo simulations paves a promising path toward advancing our comprehension and prediction of radiation-induced DNA fragmentation.

Use of the EmsB microsatellite-based next generation sequencing for genotyping of Echinococcus granulosus sensu lato in hydatid cyst tissue samples from animals and humans.

Echinococcus granulosus (Batsch, 1786), a cestode of the Teniidae family, causes human cystic echinococcosis (CE) also known as hydatid disease. Echinococcus granulosus sensu lato includes the G1, G3, G4, G5, G6/7 and G8/10 genotypes which are known to cause human CE. This study aimed to differentiate genotypes of E. granulosus s.l. complex by employing EmsB, a tandemly repeated multilocus microsatellite, using next-generation sequencing (MIC-NGS). Human and animal histopathology-confirmed hydatid cyst tissue samples and reference DNA samples of E. granulosus G1, G3, G4, G5, G6/7 and G10 underwent MIC-NGS assay with custom primers amplifying a 151 bp EmsB DNA fragment. NGS data were analysed using online Galaxy analysis pipeline, a phylogenetic tree was constructed by MEGA software, and haplotype networking was performed with PopArt 1.7. All sixty samples (49 from animals and 11 from humans) included were successfully identified and genotyped with a 100 % success rate. The study showed improved discrimination power to distinguish all study samples including closely related E. granulosus s.s. genotypes G1-G3. The maximum likelihood tree reaffirmed the monophyly of E. granulosus s.l. The median-joining haplotype networking revealed 12 distinct haplotypes. In conclusion, MIC-NGS assay was shown to be sensitive, specific and simple to apply to clinical samples offering a powerful discriminatory tool for the genotyping of E. granulosus s.l.

Discordance of Penta D x.4 microvariant alleles results between three capillary electrophoresis and one massively parallel sequencing short tandem repeat kits

Forensic Biology is contingent upon matching DNA profiles between a crime sample and a reference sample. There are several capillary electrophoresis kits available to generate a short tandem repeat (STR) profile from DNA samples, while newer methods using massively parallel sequencing are slowly being implemented in forensic laboratories worldwide. During evaluation of a newer capillary electrophoresis kit, Applied Biosystems™ VeriFiler™ Plus, a discordance was observed in the Penta D locus. The previous kit, Promega PowerPlex 21® System produced a 13.4,14 genotype, whilst VeriFiler™ Plus produced a 14,14 genotype. An expanded investigation into Penta D microvariant alleles revealed that multiple discordances were observed for DNA profiles containing larger x.4 variants. There was full concordance between PowerPlex® 21 and QIAGEN Investigator® 26plex, however discordances were observed between VeriFiler™ Plus and the other three kits tested, including the massively parallel sequencing kit, Verogen ForenSeq® MainstAY. Notably, four of these discordances resulted in null alleles with the VeriFiler™ Plus kit. A review of the Penta D DNA sequences in MainstAY revealed fully concordant microvariant alleles involved deletions within the repeat region, whilst variability in the discordances observed were dependent on the location of the variation outside the repeat region and the analysis method used. Variations observed within the 5’ flanking region produced the same allele designation across all capillary electrophoresis kits. However, deletions within the 3’ region either produced a null allele for VeriFiler™ Plus where the deletion is thought to overlap the primer binding site, or microvariant alleles for the PowerPlex® 21 and Investigator 26plex kits, which produced longer Penta D amplicons. The discovery of these variations in the Penta D flanking sequences is informative as it increases the awareness of Penta D discordances between different kit chemistries in nominated reference DNA profile comparisons and DNA database searching and matching alike, and provides support for this phenomenon when providing evidence as to the admissibility of such results in trial proceedings.

Enhancing metabarcoding efficiency and ecological insights through integrated taxonomy and DNA reference barcoding: Acase study on beach meiofauna.

Molecular techniques like metabarcoding, while promising for exploring diversity of communities, are often impeded by the lack of reference DNA sequences available for taxonomic annotation. Our study explores the benefits of combining targeted DNA barcoding and morphological taxonomy to improve metabarcoding efficiency, using beach meiofauna as a case study. Beaches are globally important ecosystems and are inhabited by meiofauna, microscopic animals living in the interstitial space between the sand grains, which play a key role in coastal biodiversity and ecosystem dynamics. However, research on meiofauna faces challenges due to limited taxonomic expertise and sparse sampling. We generated 775 new cytochrome c oxidase I DNA barcodes from meiofauna specimens collected along the Netherlands' west coast and combined them with the NCBI GenBank database. We analysed alpha and beta diversity in 561 metabarcoding samples from 24 North Sea beaches, a region extensively studied for meiofauna, using both the enriched reference database and the NCBI database without the additional reference barcodes. Our results show a 2.5-fold increase in sequence annotation and a doubling of species-level Operational Taxonomic Units (OTUs) identification when annotating the metabarcoding data with the enhanced database. Additionally, our analyses revealed a bell-shaped curve of OTU richness across the intertidal zone, aligning more closely with morphological analysis patterns, and more defined community dissimilarity patterns between supralittoral and intertidal sites. Our research highlights the importance of expanding molecular reference databases and combining morphological taxonomy with molecular techniques for biodiversity assessments, ultimately improving our understanding of coastal ecosystems.

N-Acetyl Cysteine-Cadmium Telluride NAC-CdTe QDs for Detecting the Damaged DNA in Cancerous Diseases

In this study water-soluble N-Acetyl Cysteine Capped-Cadmium Telluride QDs (NAC/CdTe nanocrystals) using N-acetyl cysteine as a stabilizer were prepared to investigate the utility of quantum dots (QDs) in distinguishing damaged DNA, (extracted from blood samples of leukaemia patients), from intact DNA (extracted from blood samples of healthy individuals) to be used for biosensing application. Based on the optical characterization of the prepared QDs, the XRD results revealed the formation of the NAC-CdTe-QDs with a grain size of 7.1nm. Whereas, the SEM test showed that the spherical size of the NAC-CdTe-QDs lies within 11~33nm. NAC-CdTe-QDs have superior PL emission properties at of 550nm and UV-Vis absorption peak at 300nm. The energy gap measurement through PL and UV–Vis was found to be 2.2eV and 2.3 eV, respectively. The interaction between the synthesized QDs and the extracted genomic DNA (both cancer damaged DNA and healthy undamaged DNA) was analysed optically, and compared to the normal reference DNA. The results showed a shift in the maximum fluorescence emission intensities (observed at 540nm nm for a damaged sample and 535 for a reference cell). Based on the obtained fluorescence results, the present study reached the conclusion that the prepared core/shell QDs could be employed as probes for diagnosing genetically disrupted DNA that is associated with malignant diseases from healthy DNA.

Oxford nanopore sequencing-based assay for BTD gene screening: Design, clinical validation, and variant frequency assessment in the Turkish population

Biotinidase deficiency (BTD) is an autosomal recessive disorder characterized by impaired recycling of the water-soluble vitamin biotin which leads to a spectrum of clinical manifestations ranging from mild to severe, including mainly neurological and cutaneous symptoms. Biotin supplementation is a cornerstone of treatment, but diagnosis often relies on measuring serum enzyme activity, which needs to be confirmed by genetic analysis. Thus, molecular methods become necessary in the differential diagnosis of BTD. Accordingly, countries with a high-incidence have implemented next-generation sequencing (NGS) techniques to newborn screening programs for BT. Nevertheless, NGS platforms, while well-established, present challenges in cost, labor, accessibility, and duration for newborn screening programs targeting BTD, therefore these limitations necessitate the exploration of alternative systems to ensure efficient and widespread screening. Here, third-generation sequencing platforms, notably Oxford Nanopore Technology (ONT), present promising solutions to the associated challenges. Hence, in the present study, we aimed to develop an ONT-based assay for the screening of BTD gene. After designing and optimizing primers for long-PCR using reference DNA, we assessed the performance of the ONT assay in BTD patients previously diagnosed by enzyme assay and confirmed using Illumina-based sequencing. The results demonstrate a strong correlation between the two methods, indicating the reliability of the ONT-based assay. Moreover, this first in-house single gene testing specifically tailored for BTD successfully detected previously known genetic variants with high sequencing depths, affirming the effectiveness of ONT-based sequencing in human genetics.

A DNA barcode reference library of Croatian mosquitoes (Diptera: Culicidae): implications for identification and delimitation of species, with notes on the distribution of potential vector species

BackgroundMosquitoes pose a risk to human health worldwide, and correct species identification and detection of cryptic species are the most important keys for surveillance and control of mosquito vectors. In addition to traditional identification based on morphology, DNA barcoding has recently been widely used as a complementary tool for reliable identification of mosquito species. The main objective of this study was to create a reference DNA barcode library for the Croatian mosquito fauna, which should contribute to more accurate and faster identification of species, including cryptic species, and recognition of relevant vector species.MethodsSampling was carried out in three biogeographical regions of Croatia over six years (2017–2022). The mosquitoes were morphologically identified; molecular identification was based on the standard barcoding region of the mitochondrial COI gene and the nuclear ITS2 region, the latter to identify species within the Anopheles maculipennis complex. The BIN-RESL algorithm assigned the COI sequences to the corresponding BINs (Barcode Index Number clusters) in BOLD, i.e. to putative MOTUs (Molecular Operational Taxonomic Units). The bPTP and ASAP species delimitation methods were applied to the genus datasets in order to verify/confirm the assignment of specimens to specific MOTUs.ResultsA total of 405 mosquito specimens belonging to six genera and 30 morphospecies were collected and processed. Species delimitation methods assigned the samples to 31 (BIN-RESL), 30 (bPTP) and 28 (ASAP) MOTUs, with most delimited MOTUs matching the morphological identification. Some species of the genera Culex, Aedes and Anopheles were assigned to the same MOTUs, especially species that are difficult to distinguish morphologically and/or represent species complexes. In total, COI barcode sequences for 34 mosquito species and ITS2 sequences for three species of the genus Anopheles were added to the mosquito sequence database for Croatia, including one individual from the Intrudens Group, which represents a new record for the Croatian mosquito fauna.ConclusionWe present the results of the first comprehensive study combining morphological and molecular identification of most mosquito species present in Croatia, including several invasive and vector species. With the exception of some closely related species, this study confirmed that DNA barcoding based on COI provides a reliable basis for the identification of mosquito species in Croatia.Graphical

Molecular study of KRAS mutations in Iraqi patients with gastrointestinal tract cancer

Gastrointestinal cancers, including stomach, liver, oesophageal, pancreatic, and colorectal cancers, represent more than a quarter of all cancers. Many abnormal gene expressions and dysregulated signalling pathways have been found in human cancer. Cancer often has activating mutations of the KRAS (Kirsten rat sarcoma virus) oncogene. Fifty blood samples from gastrointestinal cancer patients were gathered from the Merjan Teaching Hospital in Babylon, Iraq, and were used for a case-control study in the Oncology Center. According to the results, the most common cancers were found in the colon (29%), followed by the liver (27%), pancreas (19%), stomach (13%), and other (12%). In this work, we evaluated the distribution of KRAS mutations across the gastrointestinal tract. Sequencing data re¬vealed a significant regional difference in the frequency of KRAS mutations, while the alignment results revealed the presence of six variations in the analysed samples when compared with the referring reference DNA sequences. Six highly interesting nucleic acid polymorphisms were detected in the investigated samples. When combined with addi¬tional carcinogenic markers such as the patient sex, age, consistent molecular subtypes, and tumour stage, KRAS mutation is not the deterministic carcinogenic factor for gastrointestinal malignancies.

Island mosquitoes of Thailand: an update on species diversity and DNA barcoding.

Mosquitoes (Diptera: Culicidae) are among the most medically significant insects, with several species acting as vectors for human pathogens. Although there are frequent reports of mosquito-borne diseases in the border island areas of Thailand, comprehensive data on the diversity and DNA barcoding of these mosquito species remain limited. This study investigated mosquito diversity in two main archipelagos in Thailand-the Trat archipelago (comprising Chang Island and Kood Island) and the Ranong archipelago (comprising Chang Island and Phayam Island)-and generated DNA barcode data from the mosquitoes found there. The survey across these islands discovered a total of 41 species, highlighting the presence of several species known to be vectors for human diseases. Thirty-seven mosquito species from the island areas were documented to provide reference DNA barcode sequences for mosquitoes in Thailand's island regions. Two species, Aedes fumidus and Finlaya flavipennis, have been added as new COI sequence records in the database. DNA barcoding was highly effective in classifying almost all species by identifying barcoding gaps, except for Anopheles baimaii and Anopheles dirus, which could not be distinguished. Additionally, the study noted that geographical variations might influence certain mosquito species, such as Anopheles barbirostris A3 and Mansonia dives, causing them to be split into two distinct subgroups. The findings of this study are crucial, as they aid in classifying mosquito species using molecular techniques and expand our knowledge of disease vectors in these biodiverse regions.

Dual-centre evaluation of the FluoroType MTBDR version 2 assay for detection of Mycobacterium tuberculosis complex and resistance-conferring mutations in pulmonary and extrapulmonary samples from Denmark, Germany and Sierra Leone

ObjectivesWe evaluated the ability of FluoroType MTBDR version 2 (FTv2; Hain Lifescience), a second-step real-time PCR assay, to simultaneously detect Mycobacterium tuberculosis complex (MTBC) DNA and mutations conferring resistance to rifampicin (RIF) and isoniazid (INH), in pulmonary and extrapulmonary samples from patients and compared them with corresponding cultures. MethodsFTv2 MTBC was evaluated on 1815 and 432 samples from Denmark (DK) and Germany (DE), respectively. RIF and INH resistance mutations were assessed in the German samples and 110 samples from Sierra Leone and subsequently compared to phenotypic antimicrobial susceptibility testing and a composite reference DNA (CRD) based on the GenoType MTBDR line-probe assay and Sanger sequencing or whole-genome sequencing. ResultsOf the 584 (557 smear-negative) Danish and 277 (85 smear-negative) German sputum samples, 42 (16) and 246 (54) were culture positive, and 44 (18) and 222 (35) were FTv2 positive, providing an FTv2 sensitivity and specificity of 0.86 (0.63) and 0.98 (DK), 0.90 (0.65) and 1.00 (DE), respectively. The count, sensitivities, and specificities for all pulmonary samples were 1434, 0.79, and 0.99 (DK) and 347, 0.86, and 1.00 (DE), respectively; for extrapulmonary samples, 381, 0.33, 0.99 (DK) and 83, 0.50, and 1.00 (DE). The valid count, sensitivity, and specificity compared with CRD for detecting resistance mutations were RIF 355, 0.99, 0.96, and INH 340, 1.00, and 0.98, respectively. DiscussionFTv2 reliably detects MTBC DNA in pulmonary and extrapulmonary samples and detects resistance mutations for INH and RIF resistance in inhA promoter, katG, and rpoB genes.

Physiological and morphological plasticity in response to nitrogen availability of a yeast widely distributed in the open ocean.

Yeasts are prevalent in the open ocean, yet we have limited understanding of their ecophysiological adaptations, including their response to nitrogen availability, which can have a major role in determining the ecological potential of other planktonic microbes. In this study, we characterized the nitrogen uptake capabilities and growth responses of marine-occurring yeasts. Yeast isolates from the North Atlantic Ocean were screened for growth on diverse nitrogen substrates, and across a concentration gradient of three environmentally relevant nitrogen substrates: nitrate, ammonium, and urea. Three strains grew with enriched nitrate while two did not, demonstrating that nitrate utilization is present but not universal in marine yeasts, consistent with existing knowledge of nonmarine yeast strains. Naganishia diffluens MBA_F0213 modified the key functional trait of cell size in response to nitrogen concentration, suggesting yeast cell morphology changes along chemical gradients in the marine environment. Meta-analysis of the reference DNA barcode in public databases revealed that the genus Naganishia has a global ocean distribution, strengthening the environmental applicability of the culture-based observations. This study provides novel quantitative understanding of the ecophysiological and morphological responses of marine-derived yeasts to variable nitrogen availability in vitro, providing insight into the functional ecology of yeasts within pelagic open ocean environments.

A new deep-sea species of Halirages Boeck, 1871 (Crustacea: Amphipoda: Calliopiidae) inhabiting sponges

In the vast abyssal plains northwest of Iceland, white glass sponges of the genus Caulophacus Schulze, 1886 were inhabited by reddish Bythocaris G.O. Sars, 1870 shrimps and pinkish amphipods. After in situ observations at 3700 m depth, in -1°C waters by a remotely operated vehicle, members of this assemblage were collected and preserved for molecular studies. Based on integrative taxonomic analyses, the amphipods were identified as a new species of the genus Halirages Boeck, 1871 – Halirages spongiae sp. nov. Lörz, Nack & Tandberg –, as described in detail below. Part of our integrative approach was to establish reference DNA barcodes for known species of Halirages. However, our investigation of material of Calliopiidae G.O. Sars, 1895 collected around Iceland and Norway revealed slight morphological discrepancies in all the described species of Halirages. Except for Halirages fulvocinctus (M. Sars, 1858), none of the encountered specimens of Calliopiidae fully matched a current species description. We illuminate the morphological characteristics of nine operational taxonomic units, which also represented clades in COI and 28S. We set the Icelandic samples in the context of Halirages from Canada and Norway. A key to the world species of Halirages is provided.

DNA Barcoding for Species Identification of Moss-Dwelling Invertebrates: Performance of Nanopore Sequencing and Coverage in Reference Database

In view of the current biodiversity crisis and our need to preserve and improve ecosystem functioning, efficient means for characterizing and monitoring biodiversity are required. DNA barcoding, especially when coupled with new sequencing technologies, is a promising method that can, in principle, also be employed by taxonomic lay people. In this study we compare the performance of DNA barcoding by means of a third-generation sequencing technology, nanopore sequencing with classical Sanger sequencing, based on a sample of invertebrates collected from moss pads in a bog in Austria. We find that our nanopore sequencing pipeline generates DNA barcodes that are at least as good as barcodes generated with Sanger sequencing, with the MinION producing better results than the Flongle flowcell. We further find that while many arthropod taxa are well covered in the international reference DNA barcode database BOLD, this clearly is not the case for important taxa like mites and springtails, which hampers large-scale biodiversity assessments. Based on examples from our study we further highlight which factors might be responsible for ambiguous species identification based on BOLD and how this can, at least partly, be solved.

Abstract 6423: KRAS mutation detection by Sanger sequencing using the SeqStudio Flex capillary electrophoresis instrument

Abstract The detection of mutations in the KRAS gene highly associated with cancer is one of the most widely used Sanger sequencing capillary electrophoresis (CE) applications in molecular diagnostics. In addition to their traditional role in cancer diagnostic, KRAS mutations have recently gained additional relevance as pharmacogenomics (PGx) markers for a significant number of new oncology therapeutics. KRAS mutation detection assays are used as companion diagnostics (CDx) for these cancer treatments. Analysis by Sanger Sequencing of KRAS mutations is cost effective and lends itself for use in resource limited healthcare settings. While there are currently no commercial kits for targeted sequencing of the KRAS mutation hotspots available, the online Applied Biosystems Primer Designer™ Tool (thermofisher.com) allows standardization of the PCR primer design between labs and repeat orders of the KRAS assays by using these commercially available assay ID numbers. Here we demonstrate the use of Primer Designer KRAS mutation detections assays for mutation in codon 12, 13 and 61 totaling to 8 allele variants. The data was generated on the SeqStudio Flex CE instrument using reference standard DNA as well as DNA extracted from FFPE solid tumor samples. We show accuracy of variant call (OPA 100% (64/64)) and concordance (OPA 98.3% (59/60)) in a side-by-side comparison of the SeqStudio Flex with the legacy Model 3500 Genetic Analyzer instrument using a set of commercially available (Horizon Discovery) KRAS reference DNA samples. Primary solid tumor samples are processed to Fresh Frozen Paraffin Embedded samples (FFPE) for further disease marker evaluation. These samples are stable for long term at ambient temperatures and provide the opportunity to evaluate prospective biomarkers at large scale to understand their potential utility. Discovery Life Sciences has generated a large-scale biorepository of human biospecimen, including FFPE, plasma/sera, buffy coat, and PBMCs. Archival FFPE samples from oncology indications associated with KRAS mutations, such as non-small cell lung cancer and colorectal cancer, were analyzed and identified samples with KRAS mutations, demonstrating the potential diagnostic utility of these assays. Finally, cell-free (cf) DNA from double spun plasma from relevant indications was also successfully evaluated for KRAS mutations, highlighting the potential for these assays in liquid biopsy studies. The study demonstrates the robustness of the Primer Designer KRAS mutation detection sequencing assays using reference standard DNA as well as DNA derived from FFPE DNA and cfDNA from patient derived tumor samples. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Achim Karger, Ami Patel, Kaylee Tseng, Camila Ornelas, Mary Napier, Rod Beck, Kerri Collwell, Shawn Fahl, Carole Bornarth. KRAS mutation detection by Sanger sequencing using the SeqStudio Flex capillary electrophoresis instrument [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6423.

Expanding the swimmer’s itch pool of the Benelux: a first record of the neurotropic Trichobilharzia regenti and potential link to human infection

BackgroundSwimmer's itch, an allergic contact dermatitis caused by avian and mammalian blood flukes, is a parasitic infection affecting people worldwide. In particular, avian blood flukes of the genus Trichobilharzia are infamous for their role in swimmer’s itch cases. These parasites infect waterfowl as a final host, but incidental infections by cercariae in humans are frequently reported. Upon accidental infections of humans, parasite larvae will be recognized by the immune system and destroyed, leading to painful itchy skin lesions. However, one species, Trichobilharzia regenti, can escape this response in experimental animals and reach the spinal cord, causing neuroinflammation. In the last few decades, there has been an increase in case reports across Europe, making it an emerging zoonosis.MethodsFollowing a reported case of swimmer’s itch in Kampenhout in 2022 (Belgium), the transmission site consisting of a private pond and an adjacent creek was investigated through a malacological and parasitological survey.ResultsSix snail species were collected, including the widespread Ampullaceana balthica, a well-known intermediate host for Trichobilharzia parasites. Shedding experiments followed by DNA barcoding revealed a single snail specimen to be infected with T. regenti, a new species record for Belgium and by extension the Benelux. Moreover, it is the most compelling case to date of the link between this neurotropic parasite and cercarial dermatitis. Additionally, an Echinostomatidae sp. and Notocotylus sp. were isolated from two other specimens of A. balthica. However, the lack of reference DNA sequences for these groups in the online repositories prevented genus- and species-level identification, respectively.ConclusionsThe presence of T. regenti in Belgium might have severe clinical implications and its finding highlights the need for increased vigilance and diagnostic awareness among medical professionals. The lack of species-level identification of the other two parasite species showcases the barcoding void for trematodes. Overall, these findings demonstrate the need for a Belgian framework to rapidly detect and monitor zoonotic outbreaks of trematode parasites within the One Health context.Graphical

Three novel multiplex PCR assays for rapid detection of virulence, antimicrobial resistance, and toxin genes in Acinetobacter calcoaceticus-baumannii complex species.

The Acinetobacter calcoaceticus-baumannii (ACB) complex is an often-overlooked group of nosocomial pathogens with a significant environmental presence. Rapid molecular screening methods for virulence, antimicrobial resistance, and toxin (VAT) genes are required to investigate the potential pathogenicity of environmental isolates. This study aimed to develop and apply novel ACB complex-specific multiplex PCR (mPCR) primers and protocols for the rapid detection of eight VAT genes. We optimized three single-tube mPCR assays using reference DNA from ACB complex and other Acinetobacter species. These assays were then applied to detect VAT genes in cultured ACB complex isolates recovered from clinical and environmental sources. Widespread detection of VAT genes in environmental isolates confirmed the validity, functionality, and applicability of these novel assays. Overall, the three newly developed ACB complex species-specific mPCR assays are rapid and simple tools that can be adopted in diagnostic and clinical lab settings. The detection of VAT genes in environmental isolates suggests that environmental niches could serve as a reservoir for potentially pathogenic ACB complex and warrants further investigation. The newly developed mPCR assays are specific, sensitive, and efficient, making them well-suited for high-throughput screening in epidemiological studies and evaluating the potential pathogenicity of ACB complex recovered from various sources.

Environmental DNA reveals temporal variation in mesophotic reefs of the Humboldt upwelling ecosystems of central Chile: Toward a baseline for biodiversity monitoring of unexplored marine habitats.

Temperate mesophotic reef ecosystems (TMREs) are among the least known marine habitats. Information on their diversity and ecology is geographically and temporally scarce, especially in highly productive large upwelling ecosystems. Lack of information remains an obstacle to understanding the importance of TMREs as habitats, biodiversity reservoirs and their connections with better-studied shallow reefs. Here, we use environmental DNA (eDNA) from water samples to characterize the community composition of TMREs on the central Chilean coast, generating the first baseline for monitoring the biodiversity of these habitats. We analyzed samples from two depths (30 and 60 m) over four seasons (spring, summer, autumn, and winter) and at two locations approximately 16 km apart. We used a panel of three metabarcodes, two that target all eukaryotes (18S rRNA and mitochondrial COI) and one specifically targeting fishes (16S rRNA). All panels combined encompassed eDNA assigned to 42 phyla, 90 classes, 237 orders, and 402 families. The highest family richness was found for the phyla Arthropoda, Bacillariophyta, and Chordata. Overall, family richness was similar between depths but decreased during summer, a pattern consistent at both locations. Our results indicate that the structure (composition) of the mesophotic communities varied predominantly with seasons. We analyzed further the better-resolved fish assemblage and compared eDNA with other visual methods at the same locations and depths. We recovered eDNA from 19 genera of fish, six of these have also been observed on towed underwater videos, while 13 were unique to eDNA. We discuss the potential drivers of seasonal differences in community composition and richness. Our results suggest that eDNA can provide valuable insights for monitoring TMRE communities but highlight the necessity of completing reference DNA databases available for this region.