FastDNA SPIN Kit For Soil Research Articles

A Comparison of Different Protocols for the Extraction of Microbial DNA Inhabiting Synthetic Mars Simulant Soil.

Compared with typical Earth soil, Martian soil and Mars simulant soils have distinct properties, including pH > 8.0 and high contents of silicates, iron-rich minerals, sulfates, and metal oxides. This unique soil matrix poses a major challenge for extracting microbial DNA. In particular, mineral adsorption and the generation of destructive hydroxyl radicals through cationic redox cycling may interfere with DNA extraction. This study evaluated different protocols for extracting microbial DNA from Mars Global Simulant (MGS-1), a Mars simulant soil. Two commercial kits were tested: the FastDNA SPIN Kit for soil ("MP kit") and the DNeasy PowerSoil Pro Kit ("PowerSoil kit"). MGS-1 was incubated with living soil for five weeks, and DNA was extracted from aliquots using the kits. After extraction, the DNA was quantified with a NanoDrop spectrophotometer and used as the template for 16S rRNA gene amplicon sequencing and qPCR. The MP kit was the most efficient, yielding approximately four times more DNA than the PowerSoil kit. DNA extracted using the MP kit with 0.5 g soil resulted in 28,642-37,805 16S rRNA gene sequence reads and 30,380-42,070 16S rRNA gene copies, whereas the 16S rRNA gene could not be amplified from DNA extracted using the PowerSoil kit. We suggest that the FastDNA SPIN Kit is the best option for studying microbial communities in Mars simulant soils.

Improving the efficiency of DNA extraction from iron incrustations and oilfield-produced water

The quantity and quality of DNA isolated from environmental samples are crucial for getting robust high-throughput sequencing data commonly used for microbial community analysis. The differences in the nature and physicochemical properties of environmental samples impact DNA yields, and therefore, an optimisation of the protocols is always recommended. For instance, samples collected from corroded areas contain high concentrations of metals, salts, and hydrocarbons that can interfere with several steps of the DNA extraction protocols, thereby reducing yield and quality. In this study, we compared the efficiency of commercially available DNA extraction kits and laboratory-adopted methods for microbial community analysis of iron incrustations and oilfield-produced water samples. Modifications to the kits manufacturers’ protocols were included to maximise the yield and quality. For iron incrustations, the modified protocol for FastDNA Spin Kit for Soil yielded higher DNA and resulted in higher diversity, including the recovery of low-abundant and rare taxa in the samples, compared to DNeasy PowerSoil Pro Kit. The DNA extracted with modified phenol–chloroform methods yielded higher DNA but failed to pass quality control PCR for 16S sequencing with and without purification. The protocols mentioned here can be used to maximise DNA recovery from iron incrustations and oilfield-produced water samples.

Optimizing DNA Extraction from Pediatric Stool for Diagnosis of Tuberculosis and Use in Next-Generation Sequencing Applications.

The WHO has endorsed the use of stool samples for diagnosis of tuberculosis (TB) in children, and targeted next-generation sequencing (tNGS) of stool has been shown to support diagnosis and provide information about drug susceptibility (DS). Optimizing extraction of DNA from stool for sequencing is critical to ensure high diagnostic sensitivity and accurate DS information. Human stool samples were spiked with various concentrations of Mycobacterium bovis bacillus Calmette-Guérin (BCG), and DNA was extracted from the samples using four different DNA extraction kits. Each sample was subjected to quantitative PCR for identifying Mycobacterium tuberculosis complex bacteria and underwent further analysis to assess the overall DNA yield, fragment length, and purity. This same process was performed with 10 pediatric participants diagnosed with pulmonary TB, and the samples underwent tNGS. The FastDNA spin kit for soil showed the best results on model samples spiked with known quantities of BCG, compared to the other extraction methods evaluated. For clinical samples, the FastDNA and PowerFecal Pro DNA (PowerFecal) kits both showed an increase in the overall DNA quantity, M. tuberculosis-specific DNA quantity, and successful targeted sequencing when testing was performed on stool samples, compared to the two other kits. Three samples extracted via PowerFecal and three samples extracted via FastDNA (from different patients) provided successful sequencing data, with an average depth of coverage of the rpoB region for FastDNA of 298 (range, 107 to 550) and for PowerFecal of 310 (range, 182 to 474), results that were comparable to one another (P = 0.946). The PowerFecal Pro and FastDNA spin kits were superior for extracting DNA from pediatric stool samples for tNGS. IMPORTANCE This is the first study to compare Mycobacterium tuberculosis DNA extraction techniques from pediatric stool samples for use with sequencing technologies. It provides an important starting point for other researchers to isolate quality DNA for this purpose to further the field and to continue to optimize protocols and approaches.

High throughput method of 16S rRNA gene sequencing library preparation for plant root microbial community profiling

Microbiota are a major component of agroecosystems. Root microbiota, which inhabit the inside and surface of plant roots, play a significant role in plant growth and health. As next-generation sequencing technology allows the capture of microbial profiles without culturing the microbes, profiling of plant microbiota has become a staple tool in plant science and agriculture. Here, we have increased sample handling efficiency in a two-step PCR amplification protocol for 16S rRNA gene sequencing of plant root microbiota, improving DNA extraction using AMPure XP magnetic beads and PCR purification using exonuclease. These modifications reduce sample handling and capture microbial diversity comparable to that obtained by the manual method. We found a buffer with AMPure XP magnetic beads enabled efficient extraction of microbial DNA directly from plant roots. We also demonstrated that purification using exonuclease before the second PCR step enabled the capture of higher degrees of microbial diversity, thus allowing for the detection of minor bacteria compared with the purification using magnetic beads in this step. In addition, our method generated comparable microbiome profile data in plant roots and soils to that of using common commercially available DNA extraction kits, such as DNeasy PowerSoil Pro Kit and FastDNA SPIN Kit for Soil. Our method offers a simple and high-throughput option for maintaining the quality of plant root microbial community profiling.

The Effects of DNA Extraction Kits and Primers on Prokaryotic and Eukaryotic Microbial Community in Freshwater Sediments.

DNA based sequencing technology has revolutionized the field of microbial ecology and environmental studies. However, biases can be introduced at all experimental steps and, thus, affect the interpretation of microbial community. So far, previous studies on the biases introduced from the key steps of DNA extraction and primer sets mainly focused on the bacterial communities in soil or sediment samples, while little is known about the effect on the eukaryotic microbial communities. Here, we studied the effects of three different DNA extraction kits on both prokaryotic and micro-eukaryotic communities by 16S and 18S rRNA gene amplicon sequencing, and further disentangled the influence of primer choice on the micro-eukaryotic communities. Our results showed that the FastDNA SPIN Kit for Soil and DNeasy PowerSoil Kit produced much higher DNA yield with good reproducibility, and observed more eukaryotic OTUs compared to the MinkaGene DNA extraction kit, but all three kits exhibited comparable ability in recovering bacterial alpha diversity. Of the two primer sets, both targeting the V4 region of the 18S rRNA gene, the TAR primer set detected higher number of unique OTUs than the EK primer set, while the EK primer set resulted in longer amplicons and better reproducibility between replicates. Based on our findings, we recommend using the DNeasy PowerSoil Kit with the EK primer set to capture the abundant micro-eukaryotic taxa from freshwater sediment samples. If a more complete picture of the eukaryotic microbial community is desired, the TAR primer set in combination with the FastDNA SPIN Kit is more efficient in this study.

Exploration of vitamin B12-producing bacteria from Indonesia Eutrophic Lake: A new strategy to improve microalgae biomass production

Abstract. Prabaningtyas S, Ardyati T, Suharjono, Retnaningdyah C. 2021. Exploration of vitamin B12-producing bacteria from Indonesia Eutrophic Lake: A new strategy to improve microalgae biomass production. Biodiversitas 22: 4538-4544. Biofuel producing autotrophs and heterotrophs organisms are mostly found in freshwaters such as lakes. Preliminary research showed that Ranu Grati has a high diversity of microalgae and bacteria. Microalgae are known as one source of biofuels due to the high content of lipid. The growth of microalgae is strongly influenced by vitamin B12, which is synthesized by bacteria. The role of vitamin B12 in the metabolism of algae is mainly as a co-enzyme for vitamin B12-dependent methionine synthase. The purpose of this research was to observe the bacterial diversity based on NGS analysis, to explore vitamin B12 produced by bacteria from Ranu Grati lake, and also to identify the highest potency of vitamin B12-producing bacteria based on 16S rRNA gene sequencing. The study was carried out by taking a water sample at a depth of 50 cm at five stations. Isolation of total bacterial DNA was carried out using FastDNA Spin Kit for Soil. The metagenomic method of Next Generation Sequencing (NGS) was used as an initial study of bacterial diversity. Vitamin B12-producing bacteria was isolated using PYBG agar (phytone peptone, trypticase peptone, lab-lemco powder, bacto yeast extract, glucose, agar). Isolated bacteria were screened for production of vitamin B12. The potential isolates were identified base on 16S rRNA gene sequence similarity. The results of the metagenomic study showed that the genus of potential bacteria producing vitamin B12 included Bacillus, Pseudomonas, Propionibacterium, Enterobacter, Escherichia, Acinetobacter, and Flavobacterium. The results of screening with PYBG media obtained 30 isolates of vitamin B12 producing bacteria. Ten efficient vitamin B12-producing isolates were identified as Lysinibacillus fusiformis (isolate G2V1), Bacillus cereus group (isolate G2V25, G2V24, G2V13, G2V9, G2V8), Alcaligenes faecalis (isolate G2V22), Delftia acidovorans (isolate G2V14A) and the genus Delftia (isolate G2V19). The isolate G2V1 (Lysinibacillus fusiformis) was the highest producer of vitamin B12 which was able to produce 33,783 ug/mL of vitamin B12 at 4 days incubation time.

Elucidating biofilm diversity on water lily leaves through 16S rRNA amplicon analysis: Comparison of four DNA extraction kits.

PremiseWithin a broader study on leaf fossilization in freshwater environments, a long‐term study on the development and microbiome composition of biofilms on the foliage of aquatic plants has been initiated to understand how microbes and biofilms contribute to leaf decay and preservation. Here, water lily leaves are employed as a study model to investigate the relationship between bacterial microbiomes, biodegradation, and fossilization. We compare four DNA extraction kits to reduce biases in interpretation and to identify the most suitable kit for the extraction of DNA from bacteria associated with biofilms on decaying water lily leaves for 16S rRNA amplicon analysis.MethodsWe extracted surface‐associated DNA from Nymphaea leaves in early stages of decay at two water depth levels using four commercially available kits to identify the most suitable protocol for bacterial extraction, applying a mock microbial community standard to enable a reliable comparison of the kits.ResultsKit 4, the FastDNA Spin Kit for Soil, resulted in high DNA concentrations with better quality and yielded the most accurate depiction of the mock community. Comparison of the leaves at two water depths showed no significant differences in community composition.DiscussionThe success of Kit 4 may be attributed to its use of bead beating with a homogenizer, which was more efficient in the lysis of Gram‐positive bacteria than the manual vortexing protocols used by the other kits. Our results show that microbial composition on leaves during early decay remains comparable and may change only in later stages of decomposition.

Evaluation of DNA extraction methods for quantifying Helicobacter pylori in water with PCR inhibitors

Abstract DNA extraction methods were evaluated to reduce PCR inhibitors and quantify Helicobacter pylori directly from water samples using real-time PCR. Three nucleic acid extraction methods were evaluated for different types of water samples. While the QIAamp DNA mini kit for tissue was suitable for DNA extraction from treated water, the QIAamp DNA stool mini kit was still efficient in analyzing samples from river water after heavy rain and with high concentration of PCR inhibitors. The FastDNA SPIN Kit for Soil could extract DNA effectively from microbes in river and stream waters without heavy rain. Immunomagnetic separation (IMS) was used prior to DNA extraction and was a useful tool for reducing PCR inhibitors in influent and stream samples. H. pylori in various waters could be quantified directly by real-time PCR while minimizing the effect of PCR inhibitors by an appropriate method through the evaluation of DNA extraction methods considering the characteristics of the matrix water. The findings of the present study suggest that the types or characteristics of water sample by source and precipitation are an important factor in detecting H. pylori and they can be applied when detecting and monitoring of other pathogens in water.

Comparison of DNA Extraction Methods for Metagenomic Sequencing in Anaerobic Digestion

Objectives:This study was performed to compare three commercial kits for the extraction of genomic DNA from anaerobic digestate for subsequent iSeq 100 sequencing and microbial community analysis.Methods:A full-scale mesophilic biogas plant was sampled, and made into aliquots of identical volumes to extract DNA using three commercial kits: FastDNA spin kit for soil (MP Biomedicals, USA), Exgene stool DNA mini (GeneAll, South Korea) and AccuPrep genomic DNA extraction kit (Bioneer, South Korea). To analyze the microbial communities in the purified DNA, 16S rDNA amplicon sequencing (V3-4 region for bacteria and V4-5 region for archaea) was performed on the Illumina iSeq 100 platform. Quality filtered sequence reads were OTU-clustered for taxonomic assignment conducted using the RDP classifier on-line.Results and Discussion:The microbial community structure visualized on the NMDS plot using the weighted UniFrac distance revealed that both bacteria and archaeal communities have phylogenetic differences depending on the DNA extraction kit used. In addition, the abundance of certain microbial populations was significantly different among the DNA extraction methods. For example, Proteobacteria was the least abundant using the soil kit, while this phylum was the most abundant when the stool kit was used. However, in the case of Thermotogae, this tendency was vice versa. The abundance of archaeal genera Methanomethylovorans and Methanosarcina was also affected by DNA extraction methods.Conclusions:The microbial populations analyzed by 16S based sequencing were affected by DNA extraction methods. To compare microbial community changes in the identical set of research, one DNA extraction method should be chosen and used consistently for the whole experiment.

Lack of significant differences between gastrointestinal tract microbial population structure of Helicobacter pylori-infected subjects before and 2years after a single eradication event.

According to recent estimates 80% of Latvian population is infected with Helicobacter pylori thus their susceptibility to numerous gastric tract diseases is increased. The 1st line H. pylori eradication therapy includes treatment with clarithromycin in combination with amoxicillin or metronidazole and a proton pump inhibitor. However, potential adverse events caused by such therapies to microbiome are insufficiently studied. This study aimed to evaluate the long-term effect of H. pylori eradication on human gastrointestinal tract (GIT) microbiome. The assessment of Hpylori eradication impact on GIT microbiome was done by analyzing 120 samples acquired from 60 subjects. Each individual was prescribed the following 10-day eradication regimen: Esomeprazolum 40mg, Clarithromycinum 500mg, and Amoxicillinum 1000mg, BID. Samples from each individual were collected before starting Hpylori eradication therapy, and 2years after the completion of the therapy in OC-Sensor (Eiken Chemical Co.) sample collection containers and stored at -86°C. Prior to DNA extraction, the samples were lyophilized, and total DNA was extracted using FastDNA Spin Kit for Soil. 16S V3 rRNA gene sequencing was done employing Ion Torrent PGM, and the obtained raw sequences were analyzed using vsearch and R (phyloseq, cluster packages). Alpha diversity measurements-observed OTUs, Chao1 and Shannon index did not differ significantly between the pre- and post-eradication states (two-tailed paired t test: P=.95; P=.71, P=.24, respectively). Unweighted and weighted UniFrac distances of beta diversity analysis indicated a non-specific pattern of sample clustering. Enterotype shift was observed for the majority of individuals comparing pre- and post-eradication study groups. Association analysis revealed that certain bacterial genera significantly correlated with age (eg, Dialister, Paraprevotella, Bifidobacterium), individual (eg, Thermotunica, Streptomyces, Faecalibacterium), and history of respiratory and/or allergic diseases (eg, Colinsella, Faecalibacterium). Redundancy analysis confirmed that the individual was a significant determinant of the subject's microbial community composition (ANOVA, 999 perm., P=.001) with the further lower impact of subject-specific medical history (eg, medication used as prescribed: P=.005, history of cardiovascular diseases: P=.005, history of respiratory, and/or allergic diseases: P=.015) and physiological (eg, age: P=.005, gender: P=.02) parameters. In the post-eradication study group, number of influential genera (n=260) was increased compared to the pre-eradication study group (n=209). Modest global differences at the community level exist between individuals before and after the eradication therapy; however, the microbiome structure is more related to the subject-specific parameters rather than by the eradication therapy itself.

Using environmental DNA from sediment samples to detect invasive alligator gar (Atractosteus spatula) in an artificial pond

The early detection and assessment of aquatic species distribution is important in studying conservation and management. However, due to high costs and ineffective conventional methods, this is difficult in developing regions. Molecular genetic studies have led to advanced technology to allow researchers to monitor the presence of trace DNA levels found in the environment. In this study, we show that sediment can be used as an environmental DNA sample to detect the presence of an invasive species, the Alligator gar (Atractosteus spatula). Samples were processed directly using the FastDNA Spin Kit for soil, followed by other downstream applications, including PCR and sequencing. Amplified DNA fragments and sequence analysis revealed successful identification of the Alligator gar. Despite the environmental conditions, which tend to be warmer than typical eDNA samples, the high DNA concentrations in sediment samples allowed the reliable detection of this invasive species.

Molecular Detection of Taeniid Eggs in Beetles Collected in an Area Endemic for Taenia solium.

The aim of this study was to demonstrate the presence of Taenia solium eggs in beetles collected from sources within the natural environment through molecular techniques. Fifty-four pools of beetles were collected in three villages in Piura, Peru. DNA was extracted using the FastDNA spin kit for soil. Molecular identification of Taenia species was then performed through partial amplification of the mitochondrial cytochrome C oxidase subunit I gene. Finally, positive samples were sequenced to determine the tapeworm species. Seven positive samples were obtained through polymerase chain reaction amplification. Sequencing confirmed that two samples were from T. solium and three samples were from Taenia hydatigena. The other two samples could not be specifically identified. Our findings demonstrate that dung beetles ingest T. solium and T. hydatigena eggs under natural conditions and suggest that beetles may play a role in the dynamics of transmission of these cestodes.

Investigation of Detection Limits and the Influence of DNA Extraction and Primer Choice on the Observed Microbial Communities in Drinking Water Samples Using 16S rRNA Gene Amplicon Sequencing.

In recent years, 16S rRNA gene amplicon sequencing has been widely adopted for analyzing the microbial communities in drinking water (DW). However, no comprehensive attempts have been made to illuminate the inherent method biases specifically relating to DW communities. In this study, we investigated the impact of DNA extraction and primer choice on the observed microbial community, and furthermore estimated the detection limit of the 16S rRNA gene amplicon sequencing in these experimental settings. Of the two DNA extraction kits investigated, the PowerWater DNA Isolation Kit resulted in higher yield, better reproducibility and more OTUs identified compared to the FastDNA SPIN Kit for Soil, which is also commonly used within DW microbiome research. The use of three separate primer-sets targeting the V1-3, V3-4, and V4 region of the 16S rRNA gene revealed large differences in OTU abundances, with some of the primers unable to detect entire phyla. Estimations of the detection limit were based on bacteria-free water samples (1 L) spiked with Escherichia coli cells in different concentrations [101–106 cells/ml]. E. coli could be detected in all samples, however, samples with ∼101 cells/ml had several contaminating OTUs constituting approximately 8% of the read abundances. Based on our findings, we recommend using the PowerWater DNA Isolation Kit for DNA extraction in combination with PCR amplification of the V3-4 or V4 region for DW samples if a broad overview of the microbial community is to be obtained.

Effects of sample preservation and DNA extraction on enumeration of antibiotic resistance genes in wastewater.

With the growing application of high-throughput sequencing-based metagenomics for profiling antibiotic resistance genes (ARGs) in wastewater treatment plants (WWTPs), comparison of sample pretreatment and DNA extraction methods are needed to move toward standardized comparisons among laboratories. Three widely employed DNA extraction methods (FastDNA® Spin Kit for Soil, PowerSoil® DNA Isolation Kit and ZR Fecal DNA MiniPrep), with and without preservation in 50% ethanol and freezing, were applied to the influent, activated sludge and effluent of two WWTPs, in Hong Kong and in the USA. Annotated sequences obtained from the DNA extracted using the three kits shared similar taxonomy and ARG profiles. Overall, it was found that the DNA yield and purity, and diversity of ARGs captured were all highest when applying the FastDNA SPIN Kit for Soil for all three WWTP sample types investigated here (influent, activated sludge, effluent). Quantitative polymerase chain reaction of 16S rRNA genes confirmed the same trend as DNA extraction yields and similar recovery of a representative Gram-negative bacterium (Escherichia coli). Moreover, sample fixation in ethanol, deep-freezing and overseas shipment had no discernable effect on ARG profiles, as compared to fresh samples. This approach serves to inform future efforts toward global comparisons of ARG distributions in WWTPs.

Enrichment of functional microbes and genes during pyrene degradation in two different soils

Stimulating microbial degradation is a promising strategy for the remediation of soils contaminated with polycyclic aromatic hydrocarbons (PAHs). To better understand the functional microbial populations and processes involved in pyrene biodegradation in situ, the dynamics of pyrene degradation and functional microbial abundance were monitored during pyrene incubation in soils. We hope our findings will provide new insights into in situ pyrene biodegradation in soils and help to identify functional microbes from soils. Pyrene (60 mg kg−1) was incubated with two different soils, one is lower PAH-containing agricultural soil (LS), and the other is higher PAH-containing industrial soil (HS). During incubation, triplicate samples were collected on days 0, 3, 7, 14, and 35. Pyrene in soil samples was analyzed using an Agilent gas chromatograph (7890A) equipped with a mass-selective detector (model 5897). DNA in soils was extracted with a FastDNA Spin kit for soil (Bio101, USA). The abundance of functional microbes and genes was monitored by a Taqman or SYBR Green based real-time PCR quantification using an iCycler iQ5 themocycler (Bio-Rad, USA). The diversity of PAH-RHDα GP genes was evaluated by constructing clone libraries and sequencing. In both soils, more than 80 % of the added pyrene was degraded within 35 days. After 35-day incubation, there was a significant enrichment of Gram-positive bacteria harboring PAH-ring hydroxylation dioxygenase (PAH-RHDα GP) genes, and the abundance of Mycobacterium increased significantly. In PAH-RHDα GP clone libraries from two soils, Mycobacterium was detected, while most sequences were closely related to uncultured Gram-positive bacteria. In addition, two pyrene catabolic pathways might be involved in pyrene degradation, as pyrene dioxygenase genes, nidA and nidA3, were dramatically enriched during incubation. Moreover, the abundance and diversity of potential degraders in two soils showed significantly difference in responding to pyrene stress. This result indicates that soil condition can significantly affect functional microbial populations and biological process for pyrene biodegradation. These results revealed that Mycobacterium as well as uncultured Gram-positive PAH-RHDα genotypes may be the important group of pyrene degraders in soils, and two pyrene catabolic pathways, targeted by nidA and nidA3, might potentially contribute to in situ biodegradation of pyrene. This study characterized the response pattern of potential pyrene degraders to pyrene stress in two different soils, which would increase our understanding of the indigenous processes of pyrene biodegradation in soil environment.

Back to Basics--The Influence of DNA Extraction and Primer Choice on Phylogenetic Analysis of Activated Sludge Communities.

DNA extraction and primer choice have a large effect on the observed community structure in all microbial amplicon sequencing analyses. Although the biases are well known, no comprehensive analysis has been conducted in activated sludge communities. In this study we systematically explored the impact of a number of parameters on the observed microbial community: bead beating intensity, primer choice, extracellular DNA removal, and various PCR settings. In total, 176 samples were subjected to 16S rRNA amplicon sequencing, and selected samples were investigated through metagenomics and metatranscriptomics. Quantitative fluorescence in situ hybridization was used as a DNA extraction-independent method for qualitative comparison. In general, an effect on the observed community was found on all parameters tested, although bead beating and primer choice had the largest effect. The effect of bead beating intensity correlated with cell-wall strength as seen by a large increase in DNA from Gram-positive bacteria (up to 400%). However, significant differences were present at lower phylogenetic levels within the same phylum, suggesting that additional factors are at play. The best primer set based on in silico analysis was found to underestimate a number of important bacterial groups. For 16S rRNA gene analysis in activated sludge we recommend using the FastDNA SPIN Kit for Soil with four times the normal bead beating and V1-3 primers.

Mo1816 The Impact of NOD2 Variants on Gut Microbiota in Crohn's Disease and Healthy Controls

Introduction Crohn’s disease (CD) is now understood to be caused by the interaction between genetic and environmental factors with dysregulation of gut microbiota playing a pivotal role. NOD2 , the strongest genetic risk factor for CD, encodes a pattern recognition receptor and plays an important role in epithelial defence. Studies of NOD2 -knockout mice have demonstrated shifts in gut microbiota. Human studies to date have been limited by relatively small numbers of individuals homozygous for NOD2 mutations without accurate matching of controls. Methods Individuals with CD of known NOD2 status were identified from the UK IBD genetics consortium. Patients in clinical remission were selected if they carried 2 of the common NOD2 variants (homozygotes or compound heterozygotes). Each NOD2 mutant patient was matched to a NOD2 wild-type patient. For all CD patients a household control was approached. Healthy volunteers stratified by NOD2 genotype were recruited from the Cambridge Bioresource. Faecal samples were frozen within 24h of collection. DNA was extracted using the FASTDNA Spin Kit for Soil. The V1–3 region of the 16S rRNA gene was amplified and amplicons were sequenced with Illumina MiSeq. Sequence data was processed in Mothur. Calprotectin was measured in all samples by ELISA. Results 97/107 individuals were included in the primary analysis (40 CD patients [58% NOD2 mutant], 32 bioresource volunteers [50% NOD2 mutant], 25 household controls). The minimum reads per sample were 3953, mean 21216. There was a significant reduction in diversity (inverse Simpson index), Ruminococcaceae including Faecalibacteria and increase in Enterobacteriaceae in samples from CD patients vs. controls (all p NOD2 status, either within the CD patients or bioresource controls. Conclusion This study confirms previously identified shifts in gut microbiota in CD patients. However, no significant differences in gut microbiota were seen when analysed by NOD2 status. This may be a reflection of sample size or of studying gut bacteria in stool as opposed to the mucosally-associated compartment. We are presently recruiting additional cases and controls to increase study power for additional analysis. Disclosure of Interest None Declared.

First Report of Clubroot on Canola Caused by Plasmodiophora brassicae in North Dakota.

North Dakota leads the United States in canola (Brassica napus L.) production (4). A canola field with a distinct patch of dead plants spreading over an area of approximately 0.4 ha was detected in Cavalier County, North Dakota, in early September 2013. Numerous spots within the patch had plant mortalities >80%. Dead plants pulled from the soil had roots with severe galling and clubbing. Clubbed roots were brittle and disintegrated easily when pressed between fingers. Root and soil samples collected at several locations within and outside the affected patch were pooled in separate groups. All plants collected in the patch were symptomatic but those collected outside were not. In the lab, total genomic DNA from three symptomatic and two healthy root samples was extracted using standard procedures and freehand slices were prepared for observation with a compound microscope. Also, DNA from pooled soil samples was extracted using FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH). Round resting structures ranging from 2.2 to 4.2 μm in diameter were observed by microscopic examination of symptomatic root tissues. These structures resembled those typically produced by Plasmodiophora brassicae Woronin. This initial identification was later confirmed through PCR analysis using the species specific primers TC1F/R and TC2F/R (1). PCR products of 548 bp (TC1F/R) and 519 bp (TC2F/R) were produced in the three symptomatic and two infested soil samples, confirming the presence of P. brassicae. PCR amplicons were not detected in healthy root and soil samples. Pathogenicity tests were conducted in greenhouse to fulfill Koch's postulates. Briefly, five square plastic pots (10 × 10 × 13 cm) were filled with a 10-cm layer of Sunshine Mix #1 potting mix (Fison Horticulture, Vancouver, BC, Canada) and then 1 g of ground root galls (approximately 5 × 105 resting spores) was spread evenly on its surface and covered with 2 cm of soilless mix. A similar number of pots were filled only with soilless mix and used as controls. All pots were planted with two seeds of canola cv. Westar and incubated in greenhouse conditions at 21°C and 16 h light daily. The experiment was conducted twice. Four weeks after planting, all plants in the inoculated pots had developed galls while plants in control pots were symptomless. Presence of P. brassicae resting spores in the newly developed galls was confirmed by microscopic observations and PCR. Based on the symptoms, morphology of resting spores, PCR reactions, and pathogenicity tests, we confirm the presence of P. brassicae on canola. While P. brassicae has been reported as widespread in North America (2), to our knowledge, this is the first report of clubroot on canola in North Dakota and the United States. Clubroot became the most important disease affecting canola production in central Alberta, Canada, within 5 years of its discovery in 2003 (3); since then, the disease has been detected in Saskatchewan and Manitoba (3), Canadian provinces that share borders with North Dakota. Considering the difficulties in management of clubroot, measures should be initiated to limit the spread of the disease before it could pose a threat to United States canola production.

PWE-082 The Impact Of Nod2 Variants On Gut Microbiota In Crohn’s Disease And Healthy Controls

IntroductionCrohn’s disease (CD) is now understood to be caused by the interaction between genetic and environmental factors with dysregulation of gut microbiota playing a pivotal role. NOD2, the strongest genetic...

Recent innovation in microbial source tracking using bacterial real-time PCR markers in shellfish

We assessed the capacity of real-time PCR markers to identify the origin of contamination in shellfish. Oyster, cockles or clams were either contaminated with fecal materials and host-associated markers designed from Bacteroidales or Catellicoccus marimammalium 16S RNA genes were extracted from their intravalvular liquid, digestive tissues or shellfish flesh. Extraction of bacterial DNA from the oyster intravalvular liquid with FastDNA spin kit for soil enabled the selected markers to be quantified in 100% of artificially contaminated samples, and the source of contamination to be identified in 13 out of 38 naturally contaminated batches from European Class B and Class C areas. However, this protocol did not enable the origin of the contamination to be identified in cockle or clam samples. Although results are promising for extracts from intravalvular liquid in oyster, it is unlikely that a single protocol could be the best across all bacterial markers and types of shellfish.