Qiagen DNeasy Blood Research Articles

Using Environmental DNA to Detect and Identify Sweetpotato Whitefly Bemisia argentifolii and Twospotted Spider Mite Tetranychus urticae in Greenhouse‐Grown Tomato Plants

ABSTRACTEnvironmental DNA (eDNA) consists of genetic material shed by living organisms, including those that are deceased, offering a unique opportunity to detect and identify terrestrial insect pests without requiring visual identification. The sweetpotato whitefly, Bemisia argentifolii, and the twospotted spider mite, Tetranychus urticae, are notorious for causing crop losses through virus transmission and direct feeding. Our study aimed to: (1) assess the effectiveness of B. argentifolii literature‐based PCR primers compared to newly developed primers for eDNA amplification, (2) evaluate the sensitivity of conventional PCR (cPCR) and real‐time quantitative PCR (qPCR) for detecting eDNA of B. argentifolii and T. urticae, (3) establish a rapid eDNA processing methodology using the LGC Biosearch Technologies QuickExtract DNA extraction kit and the Qiagen DNeasy Blood and Tissue kit, and (4) test the specificity of the developed primers against non‐target species. B. argentifolii and T. urticae were confined to tomato leaves (Solanum lycopersicum) using clip cages for 24 h, after which eDNA was collected from leaf surfaces using a water spray method, filtered, and processed for DNA amplification. While literature‐based primers showed sufficient sensitivity, their specificity for eDNA applications was inadequate, prompting the design of novel PCR primers for both pest species. Positive eDNA detection was achieved with both amplification methods, with qPCR proving more reliable than cPCR due to the latter's inconsistent performance with positive control samples. We also introduced a rapid eDNA processing approach using the QuickExtract DNA extraction kit, contrasting it with the more conventional Qiagen DNeasy Blood and Tissue kit. We believe that our findings are the first step toward the practical use of eDNA as a highly sensitive, early detection technique.

Detection of rat lungworm (Angiostrongylus cantonensis) infection by real-time PCR from the peripheral blood of animals: a preliminary study

Rat lungworm disease or neuroangiostrongyliasis is a cerebral parasitic infection that affects humans and animals alike. Its clinical signs and symptoms can range from mild self-resolving to serious life-threatening conditions. Studies suggest therapeutic interventions during the early stages of infection to be more effective than in later stages. However, early diagnosis of infection is usually problematic without the knowledge of exposure and/or detection of the parasite’s DNA or antibody against the parasite in the cerebrospinal fluid. This requires a lumbar puncture, which is an invasive procedure that generally requires hospitalization. This study evaluates an affordable and less invasive alternative to detect parasitic DNA by PCR from the peripheral blood of potentially infected animals. Blood samples from 58 animals (55 dogs and 3 cats) with clinical suspicion of infection were submitted to our lab between February 2019 and August 2022 by local, licensed veterinarians. DNA was extracted from whole blood, plasma, serum, and/or packed cells using the Qiagen DNeasy Blood & Tissue Kit as per the manufacturer’s protocol. All 58 animals were tested by real-time PCR using the AcanITS1 assay and 32 of these animals (31dogs; 1 cat) were also tested using the AcanR3990 assay. The PCR results for both assays were classified into strongly positive > positive > weakly positive > negative, and equivocal for ambiguous results, based on the strength of the signal. The percent infection detected using the AcanITS1 and AcanR3990 assays was 12.72% (7/55) and 20.68% (6/29), respectively. The overall percent infection detected was 34.37% (11/32), with only two animals testing positive by both assays. The three cats involved in this study tested negative by both assays. These results are promising and warrant further investigations to increase sensitivity including variables that might affect detection in the blood, such as parasite load, and laboratory methodologies.

Comparison of DNA Extraction Methods for the Detection of Canned Tuna Species with DNA Mini-Barcoding

Tuna is susceptible to species mislabeling due to its high demand, quick rate of production, and wide range of price points. DNA barcoding, a sequencing-based technique, allows for the detection of species mislabeling by targeting a standardized region of DNA. A mitochondrial control region (CR) DNA barcode has been found to be capable of species discrimination for tuna, but it is challenging to recover the entire DNA fragment from canned tuna. While a short fragment of CR, referred to as a “mini-barcode,” has shown some success with canned tuna species identification, more research is needed to improve identification rates. The objective of this study was to determine the optimal DNA extraction method for species identification of canned tuna using CR mini-barcoding. Four commercial DNA extraction kits were compared using a sample set of 24 different cans of tuna labeled as albacore, light tuna, skipjack, or yellowfin. All samples were tested in duplicate. The greatest success was found using the Qiagen DNeasy Blood and Tissue Kit and the Qiagen DNeasy Mericon Food Kit, which resulted in species identification for 42% of the samples. In comparison, the MP Biomedicals FastPrep-24 + Machery-Nagel NucleoSpin Tissue Kit resulted in species identification for 30% of the samples and the Qiagen DNeasy Blood and Tissue Kit + PowerClean Pro Cleanup Kit resulted in species identification for 21% of the samples. Overall, the top-performing DNA extraction methods for use with CR mini-barcoding of canned tuna products were determined to be the DNeasy Blood and Tissue Kit and the DNeasy Mericon Food Kit.

Evaluation of DNA extraction methods for the detection of Shiga toxin producing Escherichia coli in food by polymerase chain reaction

Reported food-borne outbreaks of Shiga toxin producing Escherichia coli (STEC) have involved a very diverse range of foods. Contemporary analytical methods for the detection of STEC in foods typically include PCR screening of enrichment media. However, PCR inhibitors present in food enrichments can produce false negative results when screening for DNA sequences associated with the pathogen. To avoid false negative results in enrichment screening, it is advantageous to have DNA extraction methods that are effective at removing PCR inhibitors from a wide range of foods. The standard Canadian STEC method MFLP-52 uses Bio-Rad Instagene Matrix for DNA extraction. In this study, three DNA extraction protocols using commercial kits (Instagene Matrix with Beckman Coulter Ampure XP Beads; Qiagen Gentra Puregene Yeast/Bact. Kit; Qiagen DNeasy Blood & Tissue) were assessed as alternative DNA extraction methods for the detection of the Shiga toxin gene by PCR in enrichments from sixteen different foods inoculated with STEC O157. The inoculated foods were bean sprouts, blackberries, blue cheese, cilantro, cocoa powder, coleslaw, cream of mushroom dried soup mix, cream of vegetable dried soup mix, flaxseed, guacamole, peanut butter, soft cheese, soy butter, spinach, walnut, and wheat flour. Two of the protocols, Instagene Matrix with Ampure XP Beads, and Gentra Puregene Yeast/Bact, produced no false-negative or false positive results in the analysis of triplicate enrichment samples from sixteen inoculated foods.

(108) The Vaginal and Vulvar Microbiomes of Post-menopausal Women with Lichen Sclerosus

Abstract Introduction Lichen sclerosus is a chronic, inflammatory disorder of the genital and extragenital skin that can lead to sexual dysfunction. The possible causes of this disorder include genetic predisposition, chronic irritation, infection and autoimmunity. Post-menopausal women are at higher risk for Lichen sclerosus. The vaginal microbiome is important for reproductive health and is known to shift after menopause to a Lactobacillus-depleted community due to changes in estrogen levels. However, little is known about the role of the vaginal and vulvar microbiome in Lichen sclerosus and its pathogenesis. Objective Here, we describe and compare microbial taxa between matching vaginal and vulvar samples collected from post-menopausal women with Lichen sclerosus. Methods Matching vaginal and vulvar swabs were collected from 27 women with Lichen sclerosus. Genomic DNA was extracted from swab eluates using the QIAGEN DNeasy Blood and Tissue kit and PCR amplification was performed on the V3-V4 regions of the 16s rRNA gene using 745R and 341F customized primers. 16S rRNA sequencing was performed using the Illumina MiSeq platform. Data was analyzed using the Mothur pipeline with alignment using SILVA, and ribosomal database project (RDP) for taxonomic classification. Sequencing analysis and plots were performed with R programming software packages: reshape2, ggplot, dplyr, compositions, and vegan. Statistical tests were performed on proportional and CLR-transformed data. Results Of the 27 vaginal samples, 51.9% (n=14) were polymicrobial, 22.2% (n=6) were Gardnerella co-dominant, 14.8% (n=4) were Lactobacillus dominant, 3.7% (n=1) was Atopobium co-dominant, 3.7% (n=1) was Corynebacterium dominant, and 3.7% (n=1) was Staphylococcus dominant. Of the 27 matching vulvar samples, 55.6% (n=15) were polymicrobial, 14.8% (n=4) Staphylococcus co-dominant, 7.4% (n=2) were Gardnerella co-dominant, 7.4% (n=2) Atopobium co-dominant, 7.4% (n=2) Lactobacillus dominant, and 7.4% (n=2) Lactobacillus/Bifidobacterium co-dominant. Principal coordinate analysis demonstrated that samples grouped based on the participant they were collected from suggesting similar beta diversity between vaginal and vulvar microbiomes within the same woman (Bray-Curtis distances, p =0.001, r2=0.833, perMANOVA). However, there were a number of bacteria (n=14) that significantly differed between the vaginal and vulvar compartments including Actinomyes, Corynebacterium, Campylobacter, Peptoniphilus, Staphylococcus, Ezakiella, Finegoldia, Porphyromonas, and Actinotignum which were increased in vulvar microbiome whereas Enhydrobacter, Lactobacillus, Micrococcus, Granulicatella and Bacillus were increased in the vaginal microbiome (p<0.05, paired t-test). Vulvar samples were also significantly more diverse than matching vaginal samples (Shannon’s H index, paired t-test, p=0.042). Conclusions Women with Lichen sclerosus have diverse, non-Lactobacillus dominant vaginal and vulvar microbiomes, which are considered less optimal for reproductive health. Understanding the role of these bacteria in Lichen sclerosus pathogenesis will be an important future investigation. Disclosure No

Evaluation of five commercial DNA extraction kits using Salmonella as a model for implementation of rapid Nanopore sequencing in routine diagnostic laboratories.

Oxford Nanopore long-read sequencing offers advantages over Illumina short reads for the identification and characterization of bacterial pathogens for outbreak detection and surveillance activities within a diagnostic public health laboratory context. Compared to Illumina, Nanopore is more cost-effective for small batches, has a lower capital cost and has a faster turnaround time, in addition to the ability to assemble complete bacterial genomes. The quantity and quality of DNA required for Nanopore sequencing are greater than for Illumina, and the DNA extraction methods recommended for obtaining high-molecular-weight DNA are different from those typically used in diagnostic laboratories. Using a Salmonella isolate with a previously closed PacBio genome as a model Enterobacteriaceae organism, we evaluated the quantity, quality and fragmentation of five commercial DNA extraction kits. Nanopore sequencing performance was evaluated for the top three methods: Qiagen EZ1 DNA Tissue, Qiagen DNeasy Blood and Tissue, and a modified, in-house version of the MasterPure Complete DNA and RNA purification. To evaluate the effect of post-extraction DNA purification methods, we subjected extracted DNA from the three selected extraction methods to purification by AMPure beads or ethanol precipitation and compared these outputs with untreated DNA as a control. All methods are suitable for routine whole-genome sequencing (WGS), since all 60 replicates had very high genome recovery rates, with ≥98 % of the reference genome covered by mapped Nanopore reads. For 85 % of the replicates, assembly was able to produce a complete, circular chromosome using either Flye or Canu. In most cases, it is recommended to move directly from extraction to sequencing, as untreated DNA had the highest rates of genome closure regardless of extraction method. Using our evaluation criteria, the Qiagen DNeasy Blood and Tissue kit was found to be the best overall method due to its low cost, ability to scale from single tubes to 96-well plates, and high consistency in yield and sequencing performance.

DNA analysis and validation for species identification of seized helmeted hornbill (Rhinoplax vigil) casques

Helmeted hornbills (Rhinoplax vigil, J.R. Forster, 1781) are ‘Critically Endangered’ due to illegal hunting for their casques which are carved and traded for ornamental purposes. DNA species identification techniques can aid enforcement efforts, and validated wildlife forensic techniques for the species identification of R. vigil are needed. Here we tested multiple methods for sampling and extracting DNA from R. vigil casques and a validated a previously published assay using cytochrome B (cytB) primers to identity species and origin of traded casques. Phenol-chloroform: isoamyl alcohol extractions resulted in samples with higher quantity and quality of DNA than those extracted using the commercial Qiagen DNeasy Blood and Tissue kit. Samples collected from the caudal side of the casque yielded higher DNA quantity and quality than rostral and lateral sides, regardless of sampling method. We then assessed the repeatability, reproducibility, robustness, sensitivity, specificity, and phylogenetic resolution of a previously published species identification assay. We confirm the ability of this method to phylogenetically distinguish between R. vigil and closely related hornbills with high bootstrap support (99%). We also report the first genetic evidence of illegally traded R. vigil in Hong Kong using confiscated casques and provide more reference samples of R. vigil for future work. Overall, we provide multiple protocols for sampling and extracting DNA, and a validated species identification assay for amplifying DNA from R. vigil casques with potential to aid law enforcement in illegal wildlife crimes.

S5.5a Standing up FungiNet: Genomic epidemiology and surveillance of fungal diseases

S5.5 Genomic Epidemiology of Fungal Infections, September 22, 2022, 3:00 PM - 4:30 PM ObjectivesWith the advent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing, the public health landscape for genomic epidemiology and surveillance has transformed for a variety of pathogens. For fungal diseases, the U.S. Centers for Disease Control and Prevention (CDC) is working with global partners to stand up FungiNet, a network that aims to equip scientists with laboratory, bioinformatics, and informatics resources to harness genomic data. FungiNet partners will use genomic and epidemiologic data to detect outbreaks, identify introductions, and characterize transmission of fungal infections. In 2022, FungiNet aims to onboard nine state and local health departments in the United States and two global partners, the Instituto Nacional de Salud in Colombia and the National Institute for Communicable Diseases in South Africa, with a focus on Candida auris.MethodsTo streamline the onboarding process, CDC generated standardized operating procedures (SOPs) specific to C. auris. For DNA extraction, SOPs were created for workflows using the Zymo Research Quick-DNA™ (ZR) Fungal/Bacterial Miniprep, Qiagen Dneasy Blood and Tissue, and Epicentre (Illumina) MasterPure Yeast DNA Purification kits. For library preparation and Illumina sequencing, PulseNet methods used for foodborne pathogens were validated for C. auris. For NCBI data submissions, required data elements were defined. For SNP and phylogenetic analyses, the bioinformatics workflow MycoSNP was adapted to use Nextflow software and the Terra platform. For visualization with epidemiologic data, guidance documents and tutorials for Microreact were created. Finally, for data reporting, processes are being designed in REDCap and in laboratory information management systems to rapidly share genomic-related data.ResultsTo date, 11 partners have committed to building capacity for C. auris genomic sequencing and analysis as a FungiNet partner. Of these, seven have validated methods for DNA extraction, and nine have generated high-quality sequencing data. Only one partner has installed and locally run MycoSNP, and none have submitted raw sequence data to NCBI.ConclusionsCurrently, 11 FungiNet partners are working to onboard C. auris genomic sequencing and bioinformatics analysis in 2022. This process is complex, requiring several laboratories, bioinformatics, and informatics workflows. For many partners, bioinformatics analysis and NCBI submission are the most challenging activities with the installation of MycoSNP and the ability to batch upload data to NCBI as the main barriers. Next steps will focus on the validation of informatics methods to link genomic and epidemiologic data.

Moving environmental DNA (eDNA) technologies from benchtop to the field using passive sampling and PDQeX extraction

AbstractEnvironmental DNA (eDNA) metabarcoding has shown great promise as an effective, non‐invasive monitoring method for marine biomes. However, long filtration times and the need for state‐of‐the‐art laboratories are restricting sample replication and in situ species detections. Methodological innovations, such as passive filtration and self‐contained DNA extraction protocols, have the potential to alleviate these issues. We explored the implementation of passive sampling and a self‐contained DNA extraction protocol by comparing fish diversity obtained from active filtration (1 L; 0.45 μm cellulose nitrate [CN] filters) to five passive substrates, including 0.45 μm CN filters, 5 μm nylon filters, 0.45 μm positively charged nylon filters, artificial sponges, and fishing net. Fish diversity was then compared between the PDQeX Nucleic Acid Extractor and the conventional Qiagen DNeasy Blood & Tissue protocol. Experiments were conducted in both a controlled mesocosm and in situ at the Portobello Marine Laboratory, New Zealand. No significant differences in fish diversity were observed among active filtration and more porous passive materials (artificial sponges and fishing net) for both the mesocosm and harbor waters. For the in situ comparison, all passive filter membranes detected a significantly lower number of fish species, resulting from partial sample drop‐out. While no significant differences in fish eDNA signal diversity were observed between either DNA extraction methods in the mesocosm, the PDQeX system was less effective at detecting fish for the in situ comparison. Our results demonstrate that a passive sampling approach using porous substrates can be effectively implemented to capture eDNA from seawater, eliminating vacuum filtration processing. The large variation in efficiency observed among the five substrate types, however, warrants further optimization of the passive sampling approach for routine eDNA applications. The PDQeX system can extract high‐abundance DNA in a mesocosm with further optimization to detect low‐abundance eDNA from the marine environment.

Comparison of DNA Extraction and Amplification Techniques for Use with Engorged Hard-Bodied Ticks.

Tick-borne infections are a serious threat to humans, livestock, and companion animals in many parts of the world, often leading to high morbidity and mortality rates, along with decreased production values and/or costly treatments. The prevalence of the microbes responsible for these infections is typically assessed by the molecular identification of pathogens within the tick vectors. Ticks sampled from animals are often engorged with animal blood, presenting difficulties in the amplification of nucleic acids due to the inhibitory effects of mammalian blood on the enzymes used in polymerase chain reactions (PCRs). This study tested two tick preparation methods, three methods of DNA extraction, and four commercially available DNA polymerases to determine the most reliable method of extracting and amplifying DNA from engorged ticks. Our study found that the phenol–chloroform extraction method yielded the highest concentration of DNA, yet DNA extracted by this method was amplified the least successfully. Thermo Scientific’s Phusion Plus PCR Master Mix was the best at amplifying the tick 16s rRNA gene, regardless of extraction method. Finally, our study identified that using the Qiagen DNeasy Blood & Tissues kit for DNA extraction coupled with either Phusion Plus PCR Master Mix or GoTaq DNA polymerase Master Mix is the best combination for the optimized amplification of DNA extracted from engorged ticks.

DNA Methylation in Degenerating Human Intervertebral Discs

Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP) and is associated with widespread changes in gene expression, extracellular matrix (ECM) degradation, inflammation, and innervation. Epigenetic modifications, including DNA methylation, can drive changes in gene expression without modifying DNA sequences. To date, little is known about epigenetic dysregulation in degenerated IVDs. The objective of this study was to investigate changes in DNA methylation in degenerating human IVDs from chronic LBP patients. Human lumbar IVD segments at L4/5 level including control (n=4) and degenerating (n=6) were homogenized in liquid nitrogen with a mortar and pestle and genomic DNA was extracted using the Qiagen DNeasy Blood and Tissue kit. Epigenome-wide 5mC mapping was performed following bisulfite sequencing using the Infinium MethylationEPIC BeadChip 850k Arrays. All analysis was performed in the R package RnBeads 2.0. Principal component analysis and hierarchical clustering analysis revealed group differences between healthy and degenerating IVDs. Pathway analysis revealed enrichment in genes related to ECM organization, signaling by receptor tyrosine kinases and signaling by interleukins. Further analysis at the single gene promoter level showed hypermethylation in ECM-related genes linked to IVD degeneration including sparc and col2a1 and hypomethylation of pro-inflammatory cytokines associated with IVD degeneration including IL-1b and IL-8. These changes in methylation would be expected to result in loss of ECM and increased inflammation. Persistent reprogramming of gene expression by DNA methylation could drive IVD degeneration and inflammation. Understanding the role of DNA methylation in IVD pathology may provide a scientific framework for the development of new therapeutic approaches.

Novel polymorphisms in GPX5 gene and their association with reproductive and productive traits in Lumsniang (Hampshire x Niang Megha) pigs under subtropical hilly region of Meghalaya

Aim: To detect genetic variants of GPX5 gene and their associations with reproductive and productive traits in the Lumsniang (Hampshire x Niang Megha) pigs. Methodology: In this study, a total of 87 Lumsniang pigs were selected for collecting blood samples (3-5 ml) from Nucleus Pig Breeding Farm of ICAR Research Complex for North Eastern Hilly region, Umiam, Meghalaya. Genomic DNA was extracted from the blood leukocytes using the Qiagen DNeasy Blood and tissue kits and diluted to working concentration (50 ng μl-1) and stored at -20°C, which were used as templates for association of GPX5 variants with polymerase chain reaction. The data for reproductive and productive traits were recorded from all the animals taken in this study. The reproductive and productive traits were analyzed using a general linear model (GLM) procedure of SPSS Version 16.0. Results: Polymorphisms at Intron 1 of the GPX5 gene was determined by PCR-RFLP with HinfI restriction enzyme and DNA sequencing analyses. Two alleles and three genotypes were identified by HinfI digestion of the GPX5 gene. In the SNP-HinfI (g.1896A>G) locus, significant associations were found between GPX5 genotypes and litter size at birth. The genotype AA demonstrated significantly (P<0.05) superior litter size at birth as compared to AG and GG genotypes at the SNP-HinfI locus in Lumsniang pig. Interpretation: The study reported probable associations of GPX5 variants with reproduction and production traits in Lumsniang pig. The genotype AA at locus SNP-HinfI improved the litter size at birth in the studied population. The GPX5 gene may be considered as a DNA marker for marker-assisted selection after validation in a large pig population.

A Review of Environmental DNA Field and Laboratory Protocols Applied in Fish Ecology and Environmental Health

Environmental DNA (eDNA) has been used in research relevant to fish ecology such as species diversity and conservation studies, threatened and invasive species monitoring, and analyses of population structure and distribution. How to choose the optimal laboratory protocols on the basis of the research targets is the first question to be considered when conducting an eDNA study. In this review, we searched 554 published articles using the topic subject ((eDNA or environmental DNA) and (fish)) within the time span 2011–2021 via Thompson Reuters Web of Science (WoS) and China National Knowledge Infrastructure (CNKI) literature databases, and screened 371 articles related to eDNA research on fish ecology. These articles were categorized into “article (334)”, “review (36)”, and “letter (1)” based on the type, and “article” was divided into “article (method research)” and “article (eDNA application)” in line with the study objectives. The experimental methods adopted in each study were reviewed, and advantages and disadvantages of the main protocols were analyzed for each step. We recommend a set of optimal protocols for regular eDNA-based fish diversity detection and present the following suggestions for water sample collection and subsequent sample processing and experiments. Sample size is suggested to be 2 L regardless of the type of water bodies; three water replicates are recommended per sampling site, and water collection sites should be designed to cover various water layers and micro-habitats within research areas. Filtration is the best method for collecting eDNA from the larger water samples; 0.45 μm glass fiber/glass microfiber (GF) filters and mixed cellulose acetate and nitrate (MCE) filters are recommended for use, and MCE filters are suitable for use in turbid waters; pre-filtration (>10 μm filtering membranes) can be used to prevent clogging. Freezing temperature storage can slow eDNA degradation, and this is the optimal way to store DNA no matter what filtering method is applied. The Qiagen DNeasy Blood and Tissue DNA extraction kit was the most economical and efficient DNA extraction method compared to other commercial kits. The 12S rRNA gene is the first choice for detecting interspecies variation in fishes, and five 12s primer sets, Ac12S, MDB07, Mi-Fish, Vert-12SV5, and Teleo, are recommended. The TruSeq DNA PCR-free LT Sample Prep kit and NEBNext DNA Library Prep Master Mix Set for the 454 kit can be chosen. The Illumina HiSeq platform can obtain sufficient data depth for fish species detection. QIIME and OBITools are independent software packages used for eDNA sequences analysis of fishes, and bioinformatic analyses include several indispensable steps such as filtering raw reads, clustering filtered reads into molecular operational taxonomic units (MOTUs) or amplicon sequence variants (ASVs), and completing taxon annotation. Contamination, inhibition, lack of reference DNA data, and bioinformatic analysis are key challenges in future eDNA research, and we should develop effective experimental techniques and analysis software regarding these aspects. This review intends to help eDNA beginners to quickly understand laboratory protocols applied in fish ecological research; the information will be useful for the improvement and development of eDNA techniques in the future.

Comparing DNA yield from fish scales following different extraction protocols

Studies on genetic diversity, adaptive potential and fitness of species have become a major tool in conservation biology. These studies require biological material containing a reliable source of DNA which can be extracted and analysed. Recently, non-invasive sampling has become the preferred sampling method of such biological material; particularly when studying endangered species. Elasmoid scales from teleost fish are an example of non-invasive samples from which DNA can successfully be extracted. This study compared different extraction protocols to find an optimal method for extracting DNA from teleost fish scales. This was done with the intent to use the protocol that yielded the highest quantity of DNA on dried, archived scales. The protocols tested in this study included (1) phenol/chloroform with a TNES-urea digestion buffer, (2) phenol/chloroform with an amniocyte digestion buffer and (3) Qiagen DNeasy Blood and Tissue Kit with variations in incubation times and temperatures of each protocol. While the phenol/chloroform with TNES-urea digestion buffer yielded significantly higher concentrations of DNA compared to the other protocols, all protocols followed in this study yielded sufficient quantities of DNA for further downstream applications. Therefore, while there are multiple viable options when selecting a DNA extraction protocol, each research project’s individual needs, requirements and resources need to be carefully considered in order to choose the most effective protocol.

Salivary microbial dysbiosis may predict lung adenocarcinoma: A pilot study.

Adenocarcinoma is a more common type of Non-small cell lung cancer (NSCLC). Lung cancer showed a statistically significant increment in the Kamrup Urban district of Assam, Tripura, Sikkim, and Manipur of India. The goal of our pilot study is to identify non-invasive microbial biomarkers to detect lung adenocarcinoma (LAC). DNA extraction from saliva samples of five LAC patients and five healthy controls was performed by Qiagen DNeasy blood and tissue kit using Lysozyme (3mg/ml) treatment. 16S rRNA genes of distinct regions (V3-V4) were amplified from saliva DNA by PCR. Paired-end sequencing targeting the V3-V4 region of the 16S rRNA gene has been performed on the Illumina MiSeq platform. Raw sequences were analyzed using the QIIME(Quantitative Insights Into Microbial Ecology) software package. Our preliminary results showed that Rothia mucilaginosa, Veillonella dispar, Prevotella melaninogenica, Prevotella pallens, Prevotella copri, Haemophilus parainfluenzae, Neisseria bacilliformis and Aggregatibacter segnis were significantly elevated in saliva of LAC which may serve as potential non-invasive biomarkers for LAC detection. Functional prediction analysis showed that bacterial genes involved in glycosyltransferase, peptidases, amino sugar, and nucleotide sugar metabolism, starch and sucrose metabolism were significantly enriched in LAC. These salivary bacteria may contribute to the development of LAC by increasing expression of glycosyltransferase and peptidases. However to understand their role in pathobiology, studies are required to perform in large cohort.

Whole genome sequencing of marine organisms by Oxford Nanopore Technologies: Assessment and optimization of HMW-DNA extraction protocols.

Marine habitats are Earth's largest aquatic ecosystems, yet little is known about marine organism's genomes. Molecular studies can unravel their genetics print, thus shedding light on specie's adaptation and speciation with precise authentication. However, extracting high molecular weight DNA from marine organisms and subsequent DNA library preparation for whole genome sequencing is challenging. The challenges can be explained by excessive metabolites secretion that co‐precipitates with DNA and barricades their sequencing. In this work, we sought to resolve this issue by describing an optimized isolation method and comparing its performance with the most commonly reported protocols or commercial kits: SDS/phenol–chloroform method, Qiagen Genomic Tips kit, Qiagen DNeasy Plant mini kit, a modified protocol of Qiagen DNeasy Plant kit, Qiagen DNeasy Blood and Tissue kit, and Qiagen Qiamp DNA Stool mini kit. Our method proved to work significantly better for different marine species regardless of their shape, consistency, and sample preservation, improving Oxford Nanopore Technologies sequencing yield by 39 folds for Spirobranchus sp. and enabling generation of almost 10 GB data per flow cell/run for Chrysaora sp. and Palaemon sp. samples.

New geographic records for Echinococcus canadensis in coyotes and moose from Nova Scotia, Canada.

Echinococcus spp. tapeworms can cause serious diseases in mammals, including humans. Within the E. granulosus species complex, metacestodes produce unilocular cysts that are responsible for cystic echinococcosis in animal intermediate hosts. Canids are definitive hosts, harbouring adult cestodes in their intestines. Adult E. canadensis were recovered from the small intestine of 1 of 262 coyotes (Canis latrans) from Nova Scotia, Canada. Subsequently, we found unilocular cysts in lungs and livers of 4 of 8 sympatric moose (Alces alces) from Cape Breton Island. DNA was extracted from three cysts using the Qiagen DNeasy Blood and Tissue kit and assayed by polymerase chain reaction (PCR) with primers (cest4 and cest5) for a 117-bp region of the small subunit of ribosomal RNA of E. granulosus sensu lato, and further validated as E. canadensis G8 using primers targeting nicotinamide adenosine dinucleotide dehydrogenase subunit 1 (ND1) and cytochrome c oxidase subunit 1 (CO1) mitochondrial genes. These are the first records of E. canadensis in any of the three Maritime provinces, which include Nova Scotia, New Brunswick, and Prince Edward Island. The parasite was thought to be absent in this region due to extirpation of wolves (Canis spp.) in the 1800s. These findings suggest that further wildlife surveillance and risk assessment is warranted.

Development of Keratinocyte Cell Lines Containing Extrachromosomal Human Papillomavirus Genomes.

Human papillomaviruses (HPVs) cause persistent infections in stratified cutaneous and mucosal epithelia. In these infections, the viral DNA replicates as low-copy-number, extrachromosomal, double-stranded-DNA circular plasmids in the nucleus of the dividing basal cells. When the infected cells begin the process of differentiation, the viral DNA amplifies to a high copy number and virions are assembled in the superficial cells. To study HPV DNA replication, our laboratory generates primary keratinocyte cell lines that contain replicating extrachromosomal HPV genomes. Here, we describe protocols to culture human keratinocytes, to transfect viral DNA into cells using electroporation, to determine the efficiency of genome establishment in cells with a colony-forming assay, and to measure the copy number and extrachromosomal status of viral genomes using Southern blotting. These methods can be used to study DNA replication of different oncogenic Alphapapillomavirus HPV types. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Electroporation to transfect keratinocytes with recircularized HPV genomes Alternate Protocol: Use of HPV replicon containing selection marker in keratinocyte transfection Support Protocol 1: Rheinwald-Green method of co-culture of irradiated J2 3T3 feeders and human keratinocytes Support Protocol 2: Recircularization of HPV genomes Basic Protocol 2: Quantitative colony formation assay to measure the efficiency of HPV genome establishment Basic Protocol 3: Southern blot analysis of extrachromosomal viral DNA Support Protocol 3: Hirt extraction of low-molecular-weight DNA Support Protocol 4: Qiagen DNeasy Blood & Tissue DNA extraction Support Protocol 5: Generation of a 32 P-labeled HPV DNA probe.

Morphological Identification of Bolbosoma turbinella (Acanthocephala) in Sei Whale (Balaenoptera borealis) from Straits of Malacca, Malaysia

HighlightIdentification of Morphological characteristic of the Bolbosoma sp.Genetic confirmation of the specific species of Bolbosoma turbinella.The first report in Straits of Malacca.Health status of Sei Whale (Balaenoptera borealis).AbstractThe study of ectoparasite and endoparasites of marine mammals are not habitually done because some species are endangered and protected by law. A stranded Sei Whale, Balaenoptera borealis (Anderson, 1878) from the East Coast of Peninsular Malaysia was examined for endoparasites. The objective of this study is to identify the species of acanthocephalan in the intestine of the Sei Whale found in the straits of Malacca. A total of ten parasite specimens were collected from the fresh intestine, and were fixed in 10% buffered formalin for further histological procedures. The morphological features of this parasite viewed under Scanning Electron Microscope (SEM) are referred to as the proboscis armature and variations in the spination of the area between the anterior and posterior cephalic bulb. Genomic DNA extracted by using QIAGEN DNeasy Blood & Tissue Kit DNA and sequenced with First Base Sequencer showed that this species belonged to Bolbosoma turbinella. This was the first record of a sei whale carrying the endoparasites Bolbosoma turbinella, in Malaysian waters.

Benchmarking DNA isolation kits used in analyses of the urinary microbiome

The urinary microbiome has been increasingly characterized using next-generation sequencing. However, many of the technical methods have not yet been specifically optimized for urine. We sought to compare the performance of several DNA isolation kits used in urinary microbiome studies. A total of 11 voided urine samples and one buffer control were divided into 5 equal aliquots and processed in parallel using five commercial DNA isolation kits. DNA was quantified and the V4 segment of the 16S rRNA gene was sequenced. Data were processed to identify the microbial composition and to assess alpha and beta diversity of the samples. Tested DNA isolation kits result in significantly different DNA yields from urine samples. DNA extracted with the Qiagen Biostic Bacteremia and DNeasy Blood & Tissue kits showed the fewest technical issues in downstream analyses, with the DNeasy Blood & Tissue kit also demonstrating the highest DNA yield. Nevertheless, all five kits provided good quality DNA for high throughput sequencing with non-significant differences in the number of reads recovered, alpha, or beta diversity.