Liquid-nitrogen-free CTAB DNA extraction method from silica-dried specimens for next-generation sequencing and assembly.

Next-generation sequencing requires intact and high-quality DNA. However, typical liquid-nitrogen DNA extraction methods are expensive and not practical for field sample collections. Hence, we present a cost-effective method for DNA extraction from silica-dried leaf samples, eliminating the need for liquid nitrogen. Two protocols were evaluated to determine the effectiveness of grinding dried plant samples without liquid nitrogen in comparison to the standard protocol for tissue homogenization and cell lysis. Protocol 1 involved grinding fresh leaf samples with liquid nitrogen, while Protocol 2 entailed incubating dried plant samples at-20 °C for 1 h before grinding in the absence of liquid nitrogen. Both protocols produced comparable DNA yields with an average A260/A280 ratio of 1.78±0.02, suitable for short- and long-read sequencing. Using Protocol 2, we successfully assembled ten plastomes. It also demonstrated versatility as comparable DNA quality was obtained from dried mollusks and actinomycetes, resulting in the successful assembly of two complete mitochondrial genomes. The protocol is advantageous for research workflows involving the collection of samples in the field as a long-term source of genetic material.•Drying: Fresh samples were silica-dried at silica-to-sample ratio of 2:1.•Pre-lysis: Dried samples were frozen at -20 °C for 1 hour before grinding.•Frozen samples were subjected to tissue homogenization followed by the standard CTAB DNA extraction.

Development of Rapid Detection Methods for Fusarium oysporum f. sp. melonis in Melon Seeds.

Melon (Cucumis melo L.) is a global commercial crop that is sensitive to seed-borne wilt infections caused by Fusarium oxysporum f. sp. melonis (Fom). To address the challenge of detecting Fom contamination, we designed a probe-based real-time PCR method, TDCP2, in combination with rapid or column-based DNA extraction protocols to develop reliable molecular detection methods. Utilizing TDCP2, the detection rate reached 100% for both artificially Fom-inoculated (0.25-25%) and pod-inoculated melon seeds in conjunction with DNA samples from either the rapid or column-based extraction protocol. We performed analyses of precision, recall, and F1 scores, achieving a maximum F1 score of 1 with TDCP2, which highlights the robustness of the method. Additionally, intraday and interday assays were performed, which revealed the high reproducibility and stability of column-based DNA extraction protocols combined with TDCP2. These metrics confirm the reliability of our developed protocols, setting a foundation for future enhancements in seed pathology diagnostics and potentially broadening their applicability across various Fom infection levels. In the future, we hope that these methods will reduce food loss by improving the control and management of melon diseases.

Integrated DNA barcoding methods to identify species in the processed fish products from Chinese market.

DNA-based methods are reliable for a precise identification of species in processed products. In this study, we assessed five typical DNA extraction methods from multiple aspects. Full-length and mini-length DNA barcoding were performed to detect the species substitution and mislabeling of 305 processed fish products from the Chinese market covering six processed fish products. The salt extraction method that exhibited the best overall performance was applied. All samples were successfully extracted; however, only 19.3 % of samples could be amplified using the full-DNA barcode primer set, and 90.2 % of samples could be amplified using the newly designed mini-DNA barcode primer sets (401 and 320 bp). Overall, the molecular identification results revealed that 36.4 % (111/305) of the samples were inconsistent with the labels, with commercial fraud observed in all six types of processed fish products. The survey findings provide technical references for effective fish authentication monitoring, offering insights into the seafood safety in markets.

Socioeconomic indices guided linear mixed-effects and meta-regression modelling of the temporal, global and regional prevalence of Helicobacter pylori in environmental waters: A class I carcinogen.

Environmental waters (EW) substantially lend to the transmission of Helicobacter pylori (Hp). But the increase in Hp infections and antimicrobial resistance is often attributed to socioeconomic status. The connection between socioeconomic status and Hp prevalence in EW is however yet to be investigated. This study aimed to assess the impacts of socioeconomic indices (SI: continent, world bank region (WBR), world bank income (WBI), WHO region, Socio-demographic Index (SDI quintile), Sustainable Development Index (SuDI), and Human Development Index (HDI)) on the prevalence of Hp in EW. Hp-EW data were fitted to a generalized linear mixed-effects model and SI-guided meta-regression models with a 1000-resampling test. The worldwide prevalence of Hp in EW was 21.76% [95% confidence interval [CI]: 10.29-40.29], which declined significantly from 59.52% [43.28-74.37] in 1990-99 to 19.36% [3.99-58.09] in 2010-19 and with increasing trend in 2020-22 (33.33%, 22.66-45.43). Hp prevalence in EW was highest in North America (45.12%, 17.07-76.66), then Europe (22.38%, 5.96-56.74), South America (22.09%, 13.76-33.49), Asia (2.98%, 0.02-85.17), and Africa (2.56%, 0.00-99.99). It was negligibly different among sampling settings, WBI, and WHO regions demonstrating highest prevalence in rural location [42.62%, 3.07-94.56], HIEs [32.82%, 13.19-61.10], and AMR [39.43%, 19.92-63.01], respectively. However, HDI, sample size, and microbiological method robustly predict Hp prevalence in EW justifying 26.08%, 21.15%, and 16.44% of the true difference, respectively. In conclusion, Hp is highly prevalence in EW across regional/socioeconomic strata and thus challenged the uses of socioeconomic status as surrogate for hygienic/sanitary practices in estimating Hp infection prevalence.

Faecal metabarcoding provides improved detection and taxonomic resolution for non-invasive monitoring of gastrointestinal nematode parasites in wild moose populations.

BACKGROUND: Although wild ungulate populations are heavily monitored throughout Europe, we understand little of how parasites affect population dynamics, and there is no systematic, long-term monitoring of parasite diversity and parasite loads. Such monitoring is in part hampered by a lack of time- and cost-effective assay methodologies with high sensitivity and good taxonomic resolution. DNA metabarcoding has been successfully used to characterize the parasitic nemabiome with high taxonomic resolution in a variety of wild and domestic hosts. However, in order to implement this technique in large-scale, potentially non-invasive monitoring of gastrointestinal parasitic nematodes (GIN), protocol optimization is required to maximize biodiversity detection, whilst maintaining time- and cost-effectiveness. METHODS: Faecal samples were collected from a wild moose population and GIN communities were characterized and quantified using both parasitological techniques (egg and larva counting) and DNA metabarcoding of the ITS2 region of rDNA. Three different isolation methods were compared that differed in the volume of starting material and cell lysis method. RESULTS: Similar nematode faunas were recovered from all samples using both parasitological and metabarcoding methods, and the approaches were largely congruent. However, metabarcoding assays showed better taxonomic resolution and slightly higher sensitivity than egg and larvae counts. The metabarcoding was not strictly quantitative, but the proportion of target nematode sequences recovered was correlated with the parasitologically determined parasite load. Species detection rates in the metabarcoding assays were maximized using a DNA isolation method that included mechanical cell disruption and maximized the starting material volume. CONCLUSIONS: DNA metabarcoding is a promising technique for the non-invasive, large-scale monitoring of parasitic GINs in wild ungulate populations, owing to its high taxonomic resolution, increased assay sensitivity, and time- and cost-effectiveness. Although metabarcoding is not a strictly quantitative method, it may nonetheless be possible to create a management- and conservation-relevant index for the host parasite load from this data. To optimize the detection rates and time- and cost-effectiveness of metabarcoding assays, we recommend choosing a DNA isolation method that involves mechanical cell disruption and maximizes the starting material volume.

Optimization of bacterial DNA and endotoxin extraction from settled airborne dust.

Collecting and obtaining sufficient amount of airborne particles for multiple microbial component assessments can be challenging. A passive dust sampling device, the electrostatic dust fall collector (EDC) has been established for assessing airborne exposures including endotoxin and glucans. Recently, with advances in next-generation sequencing techniques, EDCs were used to collect microbial cells for DNA sequencing analysis to promote the study of airborne bacterial and fungal communities. However, low DNA yields have been problematic when employing passive sampling with EDC. To address this challenge, we attempted to increase the efficiency of extraction. We compared DNA extraction efficiency of bacterial components from EDCs captured on filters through filtration using five extraction techniques. By measuring the abundance, diversity and structure of bacterial communities using qPCR and amplicon sequencing targeting 16S rRNA genes, we found that two techniques outperformed the rest. Furthermore, we developed protocols to simultaneously extract both DNA and endotoxin from a single EDC cloth. Our technique promotes a high quality to price ratio and may be employed in large epidemiological studies addressing airborne bacterial exposure where a large number of samples is needed.

Global and regional prevalence of Helicobacter pylori in drinking waters: A sustainable, human development and socio-demographic indices based meta-regression-modelling.

Helicobacter pylori (Hp) transmission dynamics via drinking water (DW) has a far much higher direct and indirect public health disease burden than previously thought. This study aimed to assess the global prevalence of Hp in DW, distributions across regions and socioeconomic indices (continent, world bank income, Human Development Index (HDI), Sustainable Development Index (SuDI), Socio-Demographic Index (SDI) quintile, and WHO regions). Hp-DW related data mined from five databases until 10/12/2022 according to PRISMA standard were quality-appraised and fitted to a generalized linear mixed-effects model. Sub-group analysis and meta-regression-modelling coupled with a 1000-permutation test (⁎) were conducted. The global prevalence of Hp in DW was 15.7% (95% confidence interval [CI]: 7.98-27.5), which varied significantly by sampling methods (Moore swabbing (61.0% [0.00-100.0]) vs. grab sampling (13.68%[6.99-25.04])) and detection technique (non-culture (21.35%[9.13-42.31]) vs. cultured-based methods (Psubgroup < 0.01)). The period 1990-99 had the highest prevalence (41.24% [0.02-99.97]). Regarding regional designations, Hp prevalence in DW was significantly different being highest in North America (61.82% [41.03-79.02]) by continents, AMR (42.66% [20.81-67.82]) by WHO group, high HDI (24.64% [10.98-46.43]) by HDI group and North America (61.90% [2.79-98.93]) by world bank region (Psubgroup < 0.01). Generally, sample preparation, SuDI grouping, and detection/confirmation techniques, have significant effects on the detection/prevalence of Hp in DW (Psubgroup < 0.01). Hp prevalence in DW was not significantly different among rural and urban DW (Psubgroup = 0.90), world bank income groups (Psubgroup = 0.15), and SDI quintiles (Psubgroup = 0.07). Among the predictors examined, only sample size (p < 0.1, R∗2(coefficient of determinant) = 15.29%), continent (p∗val = 0.04), HDI (p∗val = 0.02), HDI group (p∗val = 0.05), and microbiological methods (p < 0.1; R∗2=28.09 %) predicted Hp prevalence in DW robustly. In conclusion, Hp prevalence is still endemic in DW regardless of the regional designations/improve DW supplies.

A modified isooctane-based DNA extraction method from crude oil.

Microbes from oil reservoirs shape petroleum composition through processes such as biodegradation or souring. Such processes are considered economically detrimental and might pose health and safety hazards. It is therefore crucial to understand the composition of a reservoir's microbial community and its metabolic capabilities. However, such analyses are hindered by difficulties in extracting DNA from such complex fluids as crude oil. Here, we present a novel DNA extraction method from oils with a wide American Petroleum Institute (API) gravity (density) range. We investigated the ability to extract cells from oils with different solvents and surfactants, the latter both nonionic and ionic. Furthermore, we evaluated three DNA extraction methods. Overall, the best DNA yields and the highest number of 16S rRNA reads were achieved with isooctane as a solvent, followed by an ionic surfactant treatment using sodium dodecyl sulfate and DNA extraction using the PowerSoil Pro Kit (Qiagen). The final method was then applied to various oils from oil reservoirs collected in aseptic conditions. Despite the expected low cell density of 101-103 cells/ml, the new method yielded reliable results, with average 16S rRNA sequencing reads in the order of 41431 (±8860) per sample. Thermophilic, halophilic, and anaerobic taxa, which are most likely to be indigenous to the oil reservoir, were found in all samples. API gravity and DNA yield, despite the sufficient DNA obtained, did not show a correlation.

An efficient procedure for the recovery of DNA from formalin-fixed paraffin-embedded tissue sections.

With the advent of new molecular diagnostic techniques, retrieving DNA from the formalin-fixed paraffin-embedded (FFPE) tissues has become an essential yet challenging step for efficient downstream processes. Owing to low quality and quantity of DNA retrieved from the FFPE sections, the process is often impractical and needs significant improvements. Here, we established an efficient method for the purification of DNA from FFPE specimens by optimizing incubation temperature, incubation time, and the concentration of a formalin scavenger tris(hydroxymethyl)aminomethane (Tris) for reverse-crosslinking. The optimized method, named "Highly concentrated Tris-mediated DNA extraction" (HiTE), yielded three times the DNA yield per tissue slice compared with a representative DNA extraction kit. Moreover, the use of HiTE-extracted DNA increased the yield of the sequencing library three times and accordingly yielded a log higher and more reproducible sequencing library compared with that obtained using the commonly used commercial kit. The sequencing library prepared from HiTE-extracted FFPE-DNA had longer inserts and produced reads that evenly covered the reference genome. Successful application of HiTE-extracted FFPE-DNA for whole-genome and targeted gene panel sequencing indicates its practical usability.

Combination and improvement of conventional DNA extraction methods in Actinobacteria to obtain high-quantity and high-quality DNA.

Background and Objectives: DNA extraction is an important step of any molecular experiment. DNA could not be easily extracted from members of actinomycetes by the usual methods of lysis. Due to the low efficiency of the conventional DNA extraction methods, development of an effective technique for DNA extraction of actinobacteria in emergency cases seems to be necessary. Since most of the DNA extraction techniques and commercial kits are not efficient enough to extract DNA from actinobacteria, this study was conducted to improve an efficient method obtained from conventional one to extract DNA from this group of bacteria. Materials and Methods: DNA extraction was performed using five methods (an improved method, Invisorb Spin Plant Mini Kit, EZ-10 Spin Column, Sarrbrucken method (HZI, Germany) and Kirby Bauer's method). To evaluate the quantity and quality of extracted genomic DNA, UV absorbance of all samples and efficiency of polymerase chain reaction (PCR) were evaluated. Results: Overall, the results revealed that the highest quantity (up to 4000 ng/µl) and good quality of DNA was obtained using introduced DNA extraction method. Conclusion: Results indicated that recently introduced improved method was more efficient for extraction of DNA from actinobacteria for DDH (DNA-DNA hybridization) test and for those require the high concentration of DNA.

Development and validation of a flexible DNA extraction (PAN) method for liquid biopsy of multiple sample types.

BACKGROUND: Liquid biopsy is gaining increasing popularity in cancer screening and diagnosis. However, there is no relatively mature DNA isolation method or commercial kit available that is compatible with different LB sample types. This study developed a PAN-sample DNA isolation method (PAN method) for liquid biopsy samples. METHODS: The PAN method has two key steps, including biosample-specific pretreatments for various LB sample types and high concentration guanidine thiocyanate buffer for lysis and denaturation procedure. Subsequently, the performance of PAN method was validated by a series of molecular analyses. RESULTS: The PAN method was used to isolate DNA from multiple sample types related to LB, including plasma, serum, saliva, nasopharyngeal swab, and stool. All purified DNA products showed good quality and high quantity. Comparison of KRAS mutation analysis using DNA purified using PAN method versus QIAamp methods showed similar efficiency. Epstein-Barr virus DNA was detected via Q-PCR using DNA purified from serum, plasma, nasopharyngeal swab, and saliva samples collected from nasopharyngeal carcinoma patients. Similarly, methylation sequencing of swab and saliva samples revealed good coverage of target region and high methylation of HLA-DPB1 gene. Finally, 16S rDNA gene sequencing of saliva, swab, and stool samples successfully defines the relative abundance of microbial communities. CONCLUSIONS: This study developed and validated a PAN-sample DNA isolation method that can be used for different LB samples, which can be applied to molecular epidemiological research and other areas.

Comparative analysis of DNA extraction protocols for ancient soft tissue museum samples.

DNA studies of endangered or extinct species often rely on ancient or degraded remains. The majority of ancient DNA (aDNA) extraction protocols focus on skeletal elements, with skin and hair samples rarely explored. Similar to that found in bones and teeth, DNA extracted from historical or ancient skin and fur samples is also extremely fragmented with low endogenous content due to natural degradation processes. Thus, the development of effective DNA extraction methods is required for these materials. Here, we compared the performance of two DNA extraction protocols (commercial and custom laboratory aDNA methods) on hair and skin samples from decades-old museum specimens to Iron Age archaeological material. We found that apart from the impact sample-specific taphonomic and handling history has on the quantity and quality of DNA preservation, skin yielded more endogenous DNA than hair of the samples and protocols tested. While both methods recovered DNA from ancient soft tissue, the laboratory method performed better overall in terms of DNA yield and quality, which was primarily due to the poorer performance of the commercial binding buffer in recovering aDNA.

Modern, archaeological, and paleontological DNA analysis of a human-harvested marine gastropod (Strombus pugilis) from Caribbean Panama.

Although protocols exist for the recovery of ancient DNA from land snail and marine bivalve shells, marine conch shells have yet to be studied from a paleogenomic perspective. We first present reference assemblies for both a 623.7 Mbp nuclear genome and a 15.4 kbp mitochondrial genome for Strombus pugilis, the West Indian fighting conch. We next detail a method to extract and sequence DNA from conch shells and apply it to conch from Bocas del Toro, Panama across three time periods: recently-eaten and discarded (n = 3), Late Holocene (984-1258 before present [BP]) archaeological midden (n = 5), and mid-Holocene (5711-7187 BP) paleontological fossil coral reef (n = 5). These results are compared to control DNA extracted from live-caught tissue and fresh shells (n = 5). Using high-throughput sequencing, we were able to obtain S. pugilis nuclear sequence reads from shells across all age periods: up to 92.5 thousand filtered reads per sample in live-caught shell material, 4.57 thousand for modern discarded shells, 12.1 thousand reads for archaeological shells, and 114 reads in paleontological shells. We confirmed authenticity of the ancient DNA recovered from the archaeological and paleontological shells based on 5.7× higher average frequency of deamination-driven misincorporations and 15% shorter average read lengths compared to the modern shells. Reads also mapped to the S. pugilis mitochondrial genome for all but the paleontological shells, with consistent ratios of mitochondrial to nuclear mapped reads across sample types. Our methods can be applied to diverse archaeological sites to facilitate reconstructions of the long-term impacts of human behaviour on mollusc evolutionary biology.

Isolation and identification of Streptomyces sp. Act4Zk, a good producer of Staurosporine and some derivatives.

In this study, strain Streptomyces sp. Act4Zk was isolated based on a method developed for the isolation of myxobacteria. Due to the low efficiency of the majority of conventional DNA extraction techniques, for molecular identification of the strain Streptomyces sp. Act4Zk, a new technique for DNA extraction of Actinobacteria was developed. In order to explore potential bioactivities of the strain, extracts of the fermented broth culture were prepared by an organic solvent (i.e. ethyl acetate) extraction method using. These ethyl acetate extracts were subjected to HPLC fractionation against standard micro-organisms, followed by LC/MS analysis. Based on morphological, physiological, biochemical and 16S rRNA gene sequence data, strain Streptomyces sp. Act4Zk is likely to be a new species of Streptomyces, close to Streptomyces genecies and Streptomyces roseolilacinus. Antimicrobial assay indicated high antifungal activity as well as antibacterial activity against Mycobacterium smegmatis and Gram-positive bacteria for the new strain. HPLC and LC/MS analyses of the extracts led to the identification of three different compounds and confirmed our hypothesis that the interesting species of the genus Streptomyces being a good producer of staurosporine and some derivatives.

Optimization of high molecular weight DNA extraction methods in shrimp for a long-read sequencing platform.

Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.

Impact of DNA Extraction Method on Variation in Human and Built Environment Microbial Community and Functional Profiles Assessed by Shotgun Metagenomics Sequencing.

Both the host microbiome and the microbiome of the built environment can have profound impacts on human health. While prior studies have suggested that the variability introduced by DNA extraction method is less than typical biologic variation, most studies have focused on 16S rRNA amplicon sequencing or on high biomass fecal samples. Shotgun metagenomic sequencing provides advantages over amplicon sequencing for surveying the microbiome, but is a challenge to perform in lower microbial biomass samples with high human DNA content such as sputum or vacuumed dust. Here we systematically evaluate the impact of four different extraction methods (phenol:choloroform, and three high-throughput kit-based approaches, the Promega Maxwell gDNA, Qiagen MagAttract PowerSoil DNA, and ZymoBIOMICS 96 MagBead). We report the variation in microbial community structure and predicted microbial function assessed by shotgun metagenomics sequencing in human stool, sputum, and vacuumed dust obtained from ongoing cohort studies or clinical trials. The same beadbeating protocol was used for all samples to focus our evaluation on the impact of kit chemistries on sequencing results. DNA yield was overall highest in the phenol:choloroform and Promega approaches. Only the phenol:choloroform approach showed evidence of contamination in negative controls. Bias was evaluated using mock community controls, and was noted across all extraction methods, although Promega exhibited the least amount of bias. The extraction method did not impact the proportion of human reads, although stool had the lowest proportion of human reads (0.1%) as compared to dust (44.1%) and sputum (80%). We calculated Bray-Curtis dissimilarity and Aitchison distances to evaluate the impact of extraction method on microbial community structure by sample type. Extraction method had the lowest impact in stool (extraction method responsible for 3.0-3.9% of the variability), the most impact in vacuumed dust (12-16% of the variability) and intermediate values for sputum (9.2-12% variability). Similar differences were noted when evaluating microbial community function. Our results will inform investigators planning microbiome studies using diverse sample types in large clinical studies. A consistent DNA extraction approach across all sample types is recommended, particularly with lower microbial biomass samples that are more heavily influenced by extraction method.

Nails as a primary sample type for molecular genetic identification of highly decomposed human remains.

For identification of badly preserved cadavers, only a few tissues can be used as a source of DNA, mostly bones and teeth, from which sampling and DNA extraction are difficult and time-consuming. In most highly decomposed remains, the nails are preserved. The aim of this study was to evaluate nails as an alternative source of DNA instead of bones and teeth in demanding routine identification cases. An automated extraction method was optimized on nails obtained from 33 cadavers with a post-mortem interval (PMI) up to 5 years. The commercially available EZ1 Investigator Kit (Qiagen) was used for extraction, and the G2 buffer included in the kit was replaced with TNCa buffer, and DTT was added for digestion of 5 mg of nail. The DNA was purified in a Biorobot EZ1 device (Qiagen), quantified using the PowerQuant System (Promega), and STR typing was performed with the NGM kit (TFS). From 0.3 to 270 µg DNA/g of nail was obtained from the samples analyzed, with an average yield of 36 µg DNA/g of nail. Full STR profiles were obtained from all nails except one. The optimized extraction method proved to be fast and highly efficient in the removal of PCR inhibitors, and it yields high amounts of DNA for successful STR typing. Nails were implemented as the primary sample type for obtaining DNA from highly decomposed and partially skeletonized cadavers in routine forensic identification cases in our laboratory.

Impact of sampling and DNA extraction methods on skin microbiota assessment.

The skin microbiota is characterized by high intra- and inter-variability among individuals, due to a multitude of intrinsic and extrinsic parameters such as genetics, lifestyles or pollution. This variability may be heightened due to sampling method as the skin is a multilayer organ and its outermost layer consists of dead cells. In order to investigate this biological variability in a reproducible way, we studied how sampling procedure and DNA extraction methods influence the qualitative and quantitative gathering of bacterial communities. Here, we tested a new sampling procedure that consists in exerting a slight abrasion (scrubbing) on the skin prior to swabbing and extracting DNA in order to remove squames and access deeper ecological niches. Scrubbed and non-scrubbed samples were collected from a panel of six volunteers, and four DNA extraction methods were performed on the samples. The abundance, diversity and structure of the bacterial communities were measured using qPCR technics and 16S rDNA gene-metabarcoding. Bacterial community abundance was significantly impacted by the DNA extraction method (in favor of a method designed for tissues) but not by sampling procedure, as scrubbing does not increase bacterial biomass gathered. Bacterial α- and ß-diversities were both affected by DNA extraction methods and sampling procedure. Scrubbing reveals different microbial composition by gathering bacteria living in deeper skin layer, resulting in a lower intra-personal variability. The taxonomic analysis showed that more bacteria belonging to anaerobes groups were present in scrubbed samples. We conclude that DNA extraction methods designed for tissue are not necessarily associated with high qualitative efficiency and slight scrubbing prior DNA extraction reduces intrapersonal variability and give access to a new microbial diversity.

Rapid and in-situ detection of fecal indicator bacteria in water using simple DNA extraction and portable loop-mediated isothermal amplification (LAMP) PCR methods.

Contamination of water by fecal matter and potential human enteric pathogens is a serious health concern. Microbiological water quality has been assessed by conventional culture-based methods of fecal indicator bacteria (FIB). Recently, molecular techniques for FIB have been introduced as alternative tools for rapid detection. However, such molecular techniques require a modern laboratory setting, expensive equipment, and skilled personnel. In this study, we developed a simple and rapid DNA extraction method based on a syringe filter without any specialized equipment. Furthermore, loop-mediated isothermal amplification (LAMP) PCR for fecal indicator bacteria (FIB) (i.e. E. coli and E. faecalis) was carried out using the DNA extracts from the syringe-filter based DNA extraction method. The efficiency of the extracted DNA from the syringe-filter based method was comparable to the results of the commercial kit method. We also tested fresh and marine-water collected directly from different locations in Singapore that were spiked with E. coli or E. faecalis. The LAMP assays combined with our DNA extraction method showed higher sensitivity and more tolerance to PCR inhibitors than that of conventional PCR methods. We further developed a portable LAMP device to conduct isothermal PCR reactions for rapid on-site measurement of FIB. As the color changes in the end point of the LAMP reaction can be observed with the naked eye, the portable LAMP device was easily operated and quick, obtaining results in 30 min. The simple, portable and user-friendly platform can be used as an initial screening for the rapid detection of the presence FIB in lower-resource settings. In conclusion, the portable LAMP device coupled with a syringe-filter based DNA extraction method enables us to detect the presence of FIB for assessing microbial water quality within 1 h without any sophisticated laboratory equipment or highly trained personnel.

Optimization of DNA extraction from dental remains.

Efficient DNA extraction procedures is a critical step involved in the process of successful DNA analysis of such samples. Various protocols have been devised for the genomic DNA extraction from human tissues and forensic stains, such as dental tissue that is the skeletal part that better preserves DNA over time. However DNA recovery is low and protocols require labor-intensive and time-consuming step prior to isolating genetic material. Herein, we describe an extremely fast procedure of DNA extraction from teeth compared to classical method. Sixteen teeth of 100-year-old human remains were divided into two groups of 8 teeth and we compared DNA yield, in term of quantity and quality, starting from two different sample preparation steps. Specifically, teeth of group 1 were treated with a classic technique based on several steps of pulverization and decalcification, while teeth of group 2 were processed following a new procedure to withdraw dental pulp. In the next phase, the samples of both group underwent the same procedure of extraction, quantification and DNA profile analysis. Our findings provide an alternative protocol to obtain a higher amount of good quality DNA in a fast time procedure, helpful for forensic and anthropological studies.