Direct PCR Approach Research Articles

Development of a Selective Medium for Surveillance of Fusarium Head Blight Disease.

Fusarium head blight (FHB), predominantly caused by Fusarium graminearum and F. asiaticum, is a significant fungal disease impacting small-grain cereals. The absence of highly resistant cultivars underscores the need for vigilant FHB surveillance to mitigate its detrimental effects. In 2023, a notable FHB outbreak occurred in the southern region of Korea. We assessed FHB disease severity by quantifying infected spikelets and grains. Isolating fungal pathogens from infected samples often encounters interference from various microorganisms. We developed a cost-effective, selective medium, named BGT (Burkholderia glumae Toxoflavin) medium, utilizing B. glumae, which is primarily known for causing bacterial panicle blight in rice. This medium exhibited selective growth properties, predominantly supporting Fusarium spp., while substantially inhibiting the growth of other fungi. Using the BGT medium, we isolated F. graminearum and F. asiaticum from infected wheat and barley samples across Korea. To further streamline the process, we used a direct PCR approach to amplify the translation elongation factor 1-α (TEF-1α) region without a separate genomic DNA extraction step. Phylogenetic analysis of the TEF-1α region revealed that the majority of the isolates were identified as F. asiaticum. Our results demonstrate that BGT medium is an effective tool for FHB diagnosis and Fusarium strain isolation.

Drop it all: extraction-free detection of targeted marine species through optimized direct droplet digital PCR.

Molecular biomonitoring programs increasingly use environmental DNA (eDNA) for detecting targeted species such as marine non-indigenous species (NIS) or endangered species. However, the current molecular detection workflow is cumbersome and time-demanding, and thereby can hinder management efforts and restrict the "opportunity window" for rapid management responses. Here, we describe a direct droplet digital PCR (direct-ddPCR) approach to detect species-specific free-floating extra-cellular eDNA (free-eDNA) signals, i.e., detection of species-specific eDNA without the need for filtration or DNA extraction, with seawater samples. This first proof-of-concept aquarium study was conducted with three distinct marine species: the Mediterranean fanworm Sabella spallanzanii, the ascidian clubbed tunicate Styela clava, and the brown bryozoan Bugula neritina to evaluate the detectability of free-eDNA in seawater. The detectability of targeted free-eDNA was assessed by directly analysing aquarium marine water samples using an optimized species-specific ddPCR assay. The results demonstrated the consistent detection of S. spallanzanii and B. neritina free-eDNA when these organisms were present in high abundance. Once organisms were removed, the free-eDNA signal exponentially declined, noting that free-eDNA persisted between 24-72 h. Results indicate that organism biomass, specimen characteristics (e.g., stress and viability), and species-specific biological differences may influence free-eDNA detectability. This study represents the first step in assessing the feasibility of direct-ddPCR technology for the detection of marine species. Our results provide information that could aid in the development of new technology, such as a field development of ddPCR systems, which could allow for automated continuous monitoring of targeted marine species, enabling point-of-need detection and rapid management responses.

A direct PCR approach with low-biomass insert opens new horizons for molecular sciences on cryptogam communities.

Molecular sequence data have transformed research on cryptogams (e.g., lichens, microalgae, fungi, and symbionts thereof) but methods are still strongly hampered by the small size and intermingled growth of the target organisms, poor cultivability and detrimental effects of their secondary metabolites. Here, we aim to showcase examples on which a modified direct PCR approach for diverse aspects of molecular work on environmental samples concerning biocrusts, biofilms, and cryptogams gives new options for the research community. Unlike traditional approaches, this methodology only requires biomass equivalent to colonies and fragments of 0.2 mm in diameter, which can be picked directly from the environmental sample, and includes a quick DNA lysis followed by a standardized PCR cycle that allows co-cycling of various organisms/target regions in the same run. We demonstrate that this modified method can (i) amplify the most widely used taxonomic gene regions and those used for applied and environmental sciences from single colonies and filaments of free-living cyanobacteria, bryophytes, fungi, and lichens, including their mycobionts, chlorobionts, and cyanobionts from both isolates and in situ material during co-cycling; (ii) act as a tool to confirm that the dominant lichen photobiont was isolated from the original sample; and (iii) optionally remove inhibitory secondary lichen substances. Our results represent examples which highlight the method's potential for future applications covering mycology, phycology, biocrusts, and lichenology, in particular.IMPORTANCECyanobacteria, green algae, lichens, and other cryptogams play crucial roles in complex microbial systems such as biological soil crusts of arid biomes or biofilms in caves. Molecular investigations on environmental samples or isolates of these microorganisms are often hampered by their dense aggregation, small size, or metabolism products which complicate DNA extraction and subsequent PCRs. Our work presents various examples of how a direct DNA extraction and PCR method relying on low biomass inserts can overcome these common problems and discusses additional applications of the workflow including adaptations.

Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities.

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively-both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1-B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.

Application of direct PCR for phylogenetic analysis of Fusarium fujikuroi species complex isolated from rice seeds.

Plant pathogenic fungi cause severe yield losses and mycotoxin contamination in crops. The precise and rapid detection of fungal pathogens is essential for effective disease management. Sequencing universal DNA barcodes has become the standard method for the diagnosis of fungal diseases, as well as for identification and phylogenetic analysis. A major bottleneck in obtaining DNA sequence data from many samples was the laborious and time-consuming process of sample preparation for genomic DNA. Here, we describe a direct PCR approach that bypasses the DNA extraction steps to streamline the molecular identification of fungal species. Using a direct PCR approach, we successfully sequenced the nuclear ribosomal internal transcribed spacer (ITS) region for the representatives of major fungal lineages. To demonstrate the usefulness of this approach, we performed a phylogenetic analysis of the Fusarium fujikuroi species complex, which causes bakanae ("foolish seedling") disease of rice and mycotoxin contamination. A total of 28 candidate strains were isolated from rice seeds in the Republic of Korea, and the identity of the isolates was determined using the DNA sequence of both ITS and translation elongation factor 1-α regions. In addition, 17F. fujikuroi isolates were examined for fumonisin (FB) production in rice medium using an enzyme-linked immunosorbent assay. Phylogenetic and toxigenic analyses showed that the F. fujikuroi strains could be distinguished into two groups: FB producers (B14-type) and non-producers (B20-type). These results will accelerate the molecular identification of fungal pathogens and facilitate the effective management of fungal diseases.

Further spread of perch rhabdovirus on European percid farms.

Variants of perch rhabdovirus (PRV) circulate across European percid farms via the fish trade. To trace their circulation, they are usually isolated by cell culture and subsequently identified genetically by sequencing partial or complete genes. Here, a newly developed nested PCR-based method was used to amplify and sequence the complete N and P genes directly from clinical samples obtained during an outbreak on a farm as well as from four batches of fish sampled from two other farms in another country. In an attempt to trace the origin of the five detected viruses, their N and P sequences were concatenated and compared with related viruses. One virus found in pike-perch was highly related to a virus isolated in 2016 in Belgium. Two other viruses detected on a single farm were distinct from one another, with one being almost identical to another virus isolated in 2016 in Belgium and the other being more closely related to a subgroup with different origins, France and Belgium. Two other viruses found in perch from a third farm were identical and were more related to a subgroup of viruses isolated in France. Identifying variants by a direct PCR approach will help to prevent further dissemination in farms.

A Simple, Cost-Effective, and Automation-Friendly Direct PCR Approach for Bacterial Community Analysis.

ABSTRACTBacterial communities in water, soil, and humans play an essential role in environmental ecology and human health. PCR-based amplicon analysis, such as 16S rRNA sequencing, is a fundamental tool for quantifying and studying microbial composition, dynamics, and interactions. However, given the complexity of microbial communities, a substantial number of samples becomes necessary for analyses that parse the factors that determine microbial composition. A common bottleneck in performing these kinds of experiments is genomic DNA (gDNA) extraction, which is time-consuming, expensive, and often biased based on the types of species present. Direct PCR method is a potentially simpler and more accurate alternative to gDNA extraction methods that do not require the intervening purification step. In this study, we evaluated three variations of direct PCR methods using diverse heterogeneous bacterial cultures, including both Gram-positive and Gram-negative species, ZymoBIOMICS microbial community standards, and groundwater. By comparing direct PCR methods with DNeasy Blood and Tissue Kits for microbial isolates and DNeasy PowerSoil Kits for microbial communities, we found that a specific variant of the direct PCR method exhibits an overall efficiency comparable to that of the conventional DNeasy PowerSoil protocol in the circumstances we tested. We also found that the method showed higher efficiency for extracting gDNA from the Gram-negative strains compared to DNeasy Blood and Tissue protocol. This direct PCR method is 1,600 times less expensive ($0.34 for 96 samples) and 10 times simpler (15 min hands-on time for 96 samples) than the DNeasy PowerSoil protocol. The direct PCR method can also be fully automated and is compatible with small-volume samples, thereby permitting scaling of samples and replicates needed to support high-throughput large-scale bacterial community analysis.IMPORTANCE Understanding bacterial interactions and assembly in complex microbial communities using 16S rRNA sequencing normally requires a large experimental load. However, the current DNA extraction methods, including cell disruption and genomic DNA purification, are normally biased, costly, time-consuming, labor-intensive, and not amenable to miniaturization by droplets or 1,536-well plates due to the significant DNA loss during the purification step for tiny-volume and low-cell-density samples. A direct PCR method could potentially solve these problems. In this study, we developed a direct PCR method which exhibits similar efficiency as the widely used method, the DNeasy PowerSoil protocol, while being 1,600 times less expensive and 10 times faster to execute. This simple, cost-effective, and automation-friendly direct-PCR-based 16S rRNA sequencing method allows us to study the dynamics, microbial interaction, and assembly of various microbial communities in a high-throughput fashion.

Timely gene detection assay and reliable screening of genetically engineered plants using an improved direct PCR-based technology.

Engineered plants have been widely produced for fundamental and practical use. Several methods have been developed for genetically modified crop detection and quantification; however; they still laborious and expensive. Efforts are needed to set-up diagnosis-oriented techniques as alternatives to overcome DNA extraction which remains a tedious and time-consuming procedure. Here, we established a standard direct PCR workflow using a regular Taq polymerase without prior DNA purification over a wide range of plant species. Only a small amount of fresh tissue allowed direct amplification of target gene sequences. Evaluation of accuracy, sensitivity, and reproducibility of direct PCR assay was investigated for proof-of-concept, and subsequently applied to gene detection assays and rapid transgenic revealing. The newly established method achieved full success and has amplified constitutive housekeeping genes from several plant specimens in a reproducible manner with high-quality sequencing profiles. In our case, the screening of transgenic plants confirmed that both the gfp-ER reporter gene and the npt II selectable marker were integrated into the plant genome. This direct PCR approach provides a powerful tool for large-scale PCR-based gene detection making DNA purification irrelevant. It could be easily implemented for downstream applications in the field of genetic fingerprinting, plant biotechnology, and functional genomics.

Optimization and Application of direct PCR in community metabarcoding

Direct PCR allows the amplification of DNA from animal or plant tissue samples without the need for DNA extraction and purification steps. For this procedure, dry tissue is homogenized, dissolved in water and subsequently amplified, thus, its successful application largely depends on the absence of PCR inhibitors. Although this method has been successfully applied in barcoding approaches of invertebrates, it has not yet been attempted in metabarcoding approaches. We used nonbiting midges (Diptera: Chironomidae) to test if amplicons produced by direct PCR could be used for next-generation sequencing. To access whether direct PCR is applicable for a variety of chironomid species, we tested 236 adult specimens randomly selected from emergence traps of an artificial pond mesocosm. We used ground tissue, corresponding to 0.1% of the specimens’ biomass, and a direct PCR protocol following Wong et al. (2014) for amplification. In total, 98 % of the samples were successfully amplified and we found a diverse community comprised of 20 different genera. In order to compare direct PCR and ´traditional´ DNA isolation-PCR, we created mock communities (14 species) and used both approaches for the amplification of a 421 bp COI fragment. After a second PCR for indexing and adapter ligation, samples were sequenced on an Illumina sequencer. We found only slightly lower recovery rates for mock communities with the direct PCR approach compared to traditional protocols. These recovery rates were further improved for both methods when an equal biomass (ca. 0.006 mg) of chironomid specimens was used. With our approach, it was possible to detect species which constituted only 1% of the entire biomass of a sample. Generally, direct PCR did not have a large effect on sequence read abundance. However, read abundance varied strongly between species. We are currently investigating whether this was caused by primer bias or an artifact of differently degraded tissue. This study is a proof of principle that the amplicons produced by direct PCR can be used for next-generation sequencing, with possible applications for future biomonitoring projects and portable laboratory technologies. We are currently using this technique to monitor a large-scale chironomid community experiment (artificial pond mesocosm facility) covering weekly samples taken over two summer half-years.

Direct PCR of fired shotgun casings: a South Australian evaluation

ABSTRACT Recovery of touch DNA from fired ammunition casings may provide vital forensic evidence for investigation and/or prosecution of firearm offences. Previous studies employing a direct PCR approach, where the traditional DNA extraction process is bypassed, have demonstrated an improved profiling success rate from some types of ammunition. To assess the potential value of these techniques to recover touch DNA from fired shotgun ammunition, direct PCR was evaluated for its ability to recover DNA profiles from fired 12 gauge Buckshot OOSG shotgun cartridges as compared to routine swabbing with DNA extraction. In this study, swabs subjected to direct PCR gave a significantly lower recovery of alleles than those undergoing DNA extraction. The presence of PCR inhibitors that were produced or deposited during the firing process may be the cause of these poorer results. The success of a direct PCR approach may therefore be directly related to the type of ammunition or firearm used and should therefore be specifically tested for individual case scenarios prior to employing it in forensic casework.

Evaluation of direct PCR for routine DNA profiling of non-decomposed deceased individuals

Forensic DNA profiling is a standard method used in the attempt to identify deceased individuals. In routine investigations, and if available, the preferred sample type is usually blood. However, this requires the invasive re-opening of the body, days or weeks after the autopsy, which is undesirable in resource-constrained mortuary settings. Motivated by the ease of sampling as well as reduced health and safety risks, this study aimed to establish the success rate of generating a full DNA profile on first attempt from buccal swab lysates using a direct PCR approach. Buccal swab samples were collected from 100 unidentified deceased males, and were subjected to direct DNA profiling with use of the Promega PowerPlex® Y23 Kit. At the time of sample collection, these individuals had been stored for between 1 and 887 days. This study shows that full DNA profiles were initially obtained from 73% of samples, which constitutes the first empirical data pertaining to first time success rates of direct PCR from post-mortem buccal lysates. Further investigation of partial and failed DNA profiles using real-time PCR showed that samples did not contain PCR inhibitors, DNA was not degraded, but DNA concentration was particularly low. Repeating DNA profiling with increased lysate input and extra PCR cycles yielded an additional six full DNA profiles, resulting in an overall success rate of 79%. Overall, DNA profile success rate was not associated with the duration of storage (p = 0.387). Lastly, massively parallel sequencing with the ForenSeq™ Signature DNA Prep kit provided more informative profiles for three additional samples. These results indicate that blood should therefore remain the sample of choice in a post-mortem setting, yet buccal lysates hold potential to be optimised further, which may ease the human identification workflow.

An assessment of tape-lifts

Tape-lifting is a non-destructive alternative to swabbing for collection of biological materials deposited on surfaces, especially on porous substrates. While there have been a number of studies looking at the efficiency of tapes in terms of recovery and their effect on downstream processes, none has been able to visually monitor cellular material collection.We report on a comparative study of a range of tapes regarding their collection efficiency of cellular material visualised using fluorescence microscopy, their background fluorescence after staining with DD diluted with three different solvent types and inhibition of subsequent PCR using direct PCR. Three of the fourteen tapes were selected for further testing. These were brown packing tape (Packmate™), clear tape (Sellotape®), and DNA-free tape (Lovell Surgical Solutions). These did not inhibit direct STR amplification; the other eleven tapes exhibited either high background fluorescence and/or inhibition of subsequent PCR. The effectiveness of the three tapes for the collection of cellular material was tested after tape-lifting a fingermark once, twice and ten times. The amount of cellular material within fingermarks stained with Diamond™ dye (DD) was recorded using fluorescence microscopy before and after tape-liftings. The DNA-free tape (Lovell) used in many forensic laboratories gave poor recovery compared to the other two tapes. After a tape-lift, an average of 30 % of cellular material was recovered by using DNA-free tape (Lovell), contrasting with an average recovery of 59.5 % for the clear tape (Sellotape®) and 88.8 % for the brown packing tape (Packmate™). The results presented show that standard crime scene sampling tape does collect DNA but is less effective than shop-bought tapes. Full DNA profiles can be generated from all of touched fabric samples that were collected DNA using the three tapes, triaged by DD staining and amplified by direct PCR approach.

Simultaneous Detection and Genotyping of Hydatid Cysts in Slaughtered Livestock via a Direct PCR Approach

The article's abstract is not available.

Anthrax Laboratory Diagnostic Methods at the Laboratory of the Ministry of Agriculture (LMA)

ObjectiveLaboratory of the Ministry of Agriculture (LMA) conducts Anthrax diagnostics using Bacteriology and Molecular Biology Methods: Isolated cultures through the classical bacteriology methods are always confirmed by Molecular Biology assay (PCR).In the study the samples were screened for the presence of B. anthracis via two concurrent approaches to compare classical methods and a novel PCR method. Before the TAP-7 project, PCR was only used to confirm the identity of cultures isolated by the Bacteriology. New SOPs and algorythm was created for better laboratory diagnostic.IntroductionBacillus anthracis, the etiologic agent of anthrax, is a member of a highly diverse group of endospore-forming bacteria. Bacillus anthracis spores are typically found in soil, from which they may spread via contaminated dust, water, and materials of plant and animal origin. Although anthrax is primarily a disease of herbivores, humans may contract anthrax directly or indirectly from animals.Laboratory of the Ministry of Agriculture (LMA) conducts Anthrax diagnostics using Bacteriology and Molecular Biology Methods: Isolated cultures through the classical bacteriology methods are always confirmed by Molecular Biology assay (PCR).In 2014, within Tap7 project ,Identification and Mapping of Anthrax foci in Georgia’’ Anthrax suspected soil samples were tested using two lab diagnostic methods and they were compared to each other.MethodsAnthrax suspected samples were tested by two methods - classical method and new method.Classical method included isolation of bacterium from soil samples using standard bacteriology tests and then PCR confirmed its identity. New method was initial PCR testing of soil samples.302 soil samples were tested by classical method.At the same time, approximately 10% (32 samples) of the already mentioned 302 soil samples were also tested by initial PCR.Results24 cultures isolated through bacteriology tests (Gram staining; lysis by gamma phage; motility testing; detection of polyDglutamic acid capsule by direct fluorescent antibody (DFA) were confirmed by PCR.Out of the above mentioned 32-suspected samples, 11 were confirmed positive using the classical methods, versus 9 confirmed positive using the direct PCR approach. Two bacteriologically positive samples appeared negative by the direct PCR method, i.e. only two samples did not match.ConclusionsThe samples were screened for the presence of B. anthracis via two concurrent approaches to compare classical methods and a novel PCR method. Before the TAP-7 project, PCR was only used to confirm the identity of cultures isolated by the Bacteriology.The purpose of the investigation of the new method was to identify if a less labor-intensive process with fewer points of operator manipulation was as efficacious as the classical method of bacteriology followed by PCR analysis of suspected samples.Despise the limited sampling and the little difference in the efficacy of the two methods, classical method stays prior to new one.

A single ectomycorrhizal plant root system includes a diverse and spatially structured fungal community.

Although only a relatively small proportion of plant species form ectomycorrhizae with fungi, it is crucial for growth and survival for a number of widespread woody plant species. Few studies have attempted to investigate the fine scale spatial structure of entire root systems of adult ectomycorrhizal (EcM) plants. Here, we use the herbaceous perennial Bistorta vivipara to map the entire root system of an adult EcM plant and investigate the spatial structure of its root-associated fungi. All EcM root tips were sampled, mapped and identified using a direct PCR approach and Sanger sequencing of the internal transcribed spacer region. A total of 32.1% of all sampled root tips (739 of 2302) were successfully sequenced and clustered into 41 operational taxonomic units (OTUs). We observed a clear spatial structuring of the root-associated fungi within the root system. Clusters of individual OTUs were observed in the younger parts of the root system, consistent with observations of priority effects in previous studies, but were absent from the older parts of the root system. This may suggest a succession and fragmentation of the root-associated fungi even at a very fine scale, where competition likely comes into play at different successional stages within the root system.

Rapid identification of genome-edited mesenchymal stem cell colonies via Cas9.

Mesenchymal stem cells (MSCs) have been intensively investigated and widely applied in regenerative medicine and immune modulation. However, their efficacy declines during the aging or disease process. Thus, genome-edited MSCs with over-expression or inhibition ofspecific genes hold a great deal ofpromise in terms of theirtherapeutic application. Here we optimized the direct PCR approach for rapid identification of genome-edited MSCs with only ten cells required, which reduces the time and labor to expand the MSC colonies. Combined with our previouslyoptimized guide RNA structure and plasmid construction strategy for Cas9, we successfully identified MSCcolonies over-expressing IL-10 in the AAVS1 locus.

DNA profiles generated from a range of touched sample types

Direct PCR from touch DNA has a range of potential applications in the field of forensic investigation for exhibit examination that, under standard extraction methods, rarely produce informative DNA profiles. Previous studies from ‘touch DNA’ have focussed on fingermarks created under laboratory conditions. Here we report on successful STR DNA profiling from a range of touched items. Direct PCR, with no increase in cycle number, was performed after eight different sample types, typical of those submitted for forensic investigation, were handled by volunteers for a maximum of 15 s to deposit trace amounts of their DNA. Amplifications were performed using either GlobalFiler® or Identifiler® Plus following manufacturer’s instructions. These two kits were chosen deliberately as many laboratories worldwide have adopted and validated them in their workflow, thus allowing for direct PCR to be incorporated within their practises easily. It was found that informative STR profiles were obtained from all eight substrates using both STR kits. Identifiler® Plus out-performed GlobalFiler® in terms of the percentage of alleles amplified using the direct PCR approach. Both generated informative profiles from all items and all individuals, at different rates, with Identifiler® Plus being informative in a larger percentage of samples. GlobalFiler® produced profiles with an average of 60% ± 24% (36 ± 15 alleles) alleles present while Identifiler® Plus produced profiles with an average of 96% ± 4% (31 ± 1 alleles) alleles present. A comparison was made between the direct PCR approach and subjecting touched samples to a standard DNA extraction process, both using Identifiler®. An average of 4% of profiles were informative for samples that underwent extraction with 100% being informative from the same subset of samples amplified by direct PCR. Our findings further demonstrate the success of direct PCR to enhance the STR DNA profiles from touch DNA. Further, Identifiler® Plus was found to generate informative profiles more often than GlobalFiler®. Direct PCR is fast, simple, and non-destructive of evidence with the ability to generate informative genetic data where standard methods are likely to fail.

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease. Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample. Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease. Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample. Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

Priama PCR bez pou�itia izol�cie DNA ako platforma genotypovania Hedera helix, L.

Hedera helix, L. is a specie that is used for its ornamental and medicinal properties widely. In spite of its very good biochemical characterization, the knowledge about the DNA variability is very limited and no DNA markers were used to analyse the genomic variability of the ivy populations, up to date. Here, a direct PCR approach was used to evaluate a cytochrome P450 sequences based polymorphism by PBA technique. A set of 4 samples from Slovakia, 2 samples from Czech Republic, 2 samples originated in Poland, 2 samples from Croatia, 2 samples from Scotland and samples from Germany, Austria and Spain were used in analysis. Using a set of three PBA primers and their combinations, the number of amplified fragment levels obtained by individual primer combinations was for both of them 11 and the obtained polymorphism was 91% or 100% respectivelly. The PCoA analysis was performed and the genotypes belonging to a different geographic localities were grouped together.