DNA Extraction Procedure Research Articles

Telomere length and chronological age across the human lifespan: A systematic review and meta-analysis of 414 study samples including 743,019 individuals.

Telomere attrition is a proposed hallmark of aging. To evaluate the association of telomere length (TL) with chronological age across the human lifespan, we conducted a systematic review and meta-analysis of 414 study samples comprising 743,019 individuals aged 0-112 years. We examined both cross-sectional and longitudinal data, and evaluated the impact of various biological and methodological factors including sex, health status, tissue types, DNA extraction procedures, and TL measurement methods. The pooled corrected correlation between TL and age from cross-sectional samples was -0.19 (95%CI: -0.22 to -0.15), which weakened with increased chronological age (β=0.003, p<0.001). Z-score change rates of TL across the lifespan showed a gradual decrease in shortening rate until around age 50 and remained at a relatively stable rate towards the elderly period. A greater attrition rate was observed in longitudinal than cross-sectional evaluations. For TL measured in base pairs, the median change rate of TL was -23bp/year in cross-sectional samples and -38bp/year in longitudinal samples. Methodological factors including TL measurement methods and tissue types impacted the TL-age correlation, while sex or disease status did not. This meta-analysis revealed the non-linear shortening trend of TL across the human lifespan and provides a reference value for future studies. Results also highlight the importance of methodological considerations when using TL as an aging biomarker.

Detection of fish sedimentary DNA in aquatic systems: A review of methodological challenges and future opportunities

AbstractEnvironmental DNA studies have proliferated over the last decade, with promising data describing the diversity of organisms inhabiting aquatic and terrestrial ecosystems. The recovery of DNA present in the sediment of aquatic systems (sedDNA) has provided short‐ and long‐term data on a wide range of biological groups (e.g., photosynthetic organisms, zooplankton species) and has advanced our understanding of how environmental changes have affected aquatic communities. However, substantial challenges remain for recovering the genetic material of macro‐organisms (e.g., fish) from sediments, preventing complete reconstructions of past aquatic ecosystems, and limiting our understanding of historic, higher trophic level interactions. In this review, we outline the biotic and abiotic factors affecting the production, persistence, and transport of fish DNA from the water column to the sediments, and address questions regarding the preservation of fish DNA in sediment. We identify sources of uncertainties around the recovery of fish sedDNA arising during the sedDNA workflow. This includes methodological issues related to experimental design, DNA extraction procedures, and the selected molecular method (quantitative PCR, digital PCR, metabarcoding, metagenomics). By evaluating previous efforts (published and unpublished works) to recover fish sedDNA signals, we provide suggestions for future research and propose troubleshooting workflows for the effective detection and quantification of fish sedDNA. With further research, the use of sedDNA has the potential to be a powerful tool for inferring fish presence over time and reconstructing their population and community dynamics.

Optimising faecal sample storage and DNA extraction procedures to help the implementation of forest elephant conservation strategies

AbstractFaecal samples are an important source of genetic information for studies of wild animals. The quality and quantity of faecal DNA can, however, be affected by different factors. Our goal was to compare different dung sample storage and DNA extraction methods to optimise the quality and quantity of DNA extracted from forest elephant dung samples. Dung samples (n = 132) of forest elephants were collected in a forest area in Côte d'Ivoire. Each faecal sample was preserved in 50 mL cryotubes using five different methods: Ethanol 70%, formalin 10%, Sodium acetate, acetic acid, and formalin (SAF), dimethyl sulfoxide, and Sodium chloride (20% DMSO), and silica gel. We used the Cetyl Trimethyl Ammonium Bromide (CTAB) cationic detergent and the phenol chloroform DNA extraction procedures for comparison. DNA was successfully obtained from all 132 faecal samples using both DNA extraction methods. Regarding the total DNA extracted independently from the storage buffers, the phenol chloroform method yields a mean DNA concentration (2163.93 ± 918.62 ng/μL) sevenfold higher than that obtained with the optimised CTAB method (338.15 ± 528.54 ng/μL). Our study indicates optimised faecal sample storage and DNA extraction procedures that can help implement conservation strategies for forest elephants.

Synchrotron-based FTIR microspectroscopy reveals DNA methylation profile in DNA-HALO structure

Fourier transform infrared (FTIR) spectroscopy is a rapid, non-destructive and label-free technique for identifying subtle changes in all bio-macromolecules, and has been used as a method of choice for studying DNA conformation, secondary DNA structure transition and DNA damage. In addition, the specific level of chromatin complexity is introduced via epigenetic modifications forcing the technological upgrade in the analysis of such an intricacy. As the most studied epigenetic mechanism, DNA methylation is a major regulator of transcriptional activity, involved in the suppression of a broad spectrum of genes and its deregulation is involved in all non-communicable diseases. The present study was designed to explore the use of synchrotron-based FTIR analysis to monitor the subtle changes in molecule bases regarding the DNA methylation status of cytosine in the whole genome. In order to reveal the conformation-related best sample for FTIR-based DNA methylation analysis in situ, we used methodology for nuclear HALO preparations and slightly modified it to isolated DNA in HALO formations. Nuclear DNA-HALOs represent samples with preserved higher-order chromatin structure liberated of any protein residues that are closer to native DNA conformation than genomic DNA (gDNA) isolated by the standard batch procedure. Using FTIR spectroscopy we analyzed the DNA methylation profile of isolated gDNA and compared it with the DNA-HALOs. This study demonstrated the potential of FTIR microspectroscopy to detect DNA methylation marks in analyzed DNA-HALO specimens more precisely in comparison with classical DNA extraction procedures that yield unstructured whole genomic DNA. In addition, we used different cell types to assess their global DNA methylation profile, as well as defined specific infrared peaks that can be used for screening DNA methylation.

Shotgun sequencing of sonication fluid for the diagnosis of orthopaedic implant-associated infections with Cutibacterium acnes as suspected causative agent.

Orthopaedic implant-associated infections (OIAIs) due to Cutibacterium acnes can be difficult to diagnose. The aim of this pilot study was to determine if metagenomic next-generation sequencing (mNGS) can provide additional information to improve the diagnosis of C. acnes OIAIs. mNGS was performed on sonication fluid (SF) specimens derived from 24 implants. These were divided into three groups, based on culture results: group I, culture-negative (n = 4); group II, culture-positive for C. acnes (n = 10); and group III, culture-positive for other bacteria (n = 10). In group I, sequence reads from C. acnes were detected in only one SF sample, originating from a suspected case of OIAIs, which was SF and tissue culture-negative. In group II, C. acnes sequences were detected in 7/10 samples. In group III, C. acnes sequence reads were found in 5/10 samples, in addition to sequence reads that matched the bacterial species identified by culture. These samples could represent polymicrobial infections that were missed by culture. Taken together, mNGS was able to detect C. acnes DNA in more samples compared to culture and could be used to identify cases of suspected C. acnes OIAIs, in particular regarding possible polymicrobial infections, where the growth of C. acnes might be compromised due to a fast-growing bacterial species. However, since SF specimens are usually low-biomass samples, mNGS is prone to DNA contamination, possibly introduced during DNA extraction or sequencing procedures. Thus, it is advisable to set a sequence read count threshold, taking into account project- and NGS-specific criteria.

An Improved Method for Efficient DNA Extraction from Grapevine

Grapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops worldwide which has been the subject of extensive genetic studies including gene mapping, genetic transformation, and DNA fingerprinting. Grapevines are rich in polysaccharides, polyphenolic compounds, and various secondary metabolites, many of which have significant importance in food, agrochemical, and pharmaceutical industries. While metabolites are one of the indicators of quality of grapevines, the presence of them makes grapevine one of the most difficult plants to extract DNA from. These metabolites not only affect DNA extraction procedures but also downstream reactions such as restriction digestion and PCR. Development of new genotyping techniques based on sequencing such as genotyping by sequencing (GBS) requires high-quality DNA for digestion and sequencing. To date, several protocols have been developed for DNA extraction from grapevine. In this study, three different protocols with modifications were compared for DNA extraction performance from grapevine leaves from four different cultivars. Efficiencies of these methods were determined by extracted DNA’s quantity and quality. To confirm the suitability for GBS, extracted DNA was digested with restriction enzymes. Although all protocols were based on the traditional CTAB method, they resulted in different DNA yield and restriction digestion efficiency. The modified protocol including PVP-40 and ß-mercaptoethanol was found to be the most efficient method to obtain high quality and quantity grapevine DNA that is amenable to restriction digestion.

Quality analysis of genomic DNA and authentication of fisheries products based on distinct methods of DNA extraction.

Molecular genetic techniques are an effective monitoring tool, but high-quality DNA samples are usually required. In this study, we compared three different protocols of DNA extraction: NaCl (saline); phenol-chloroform and commercial kit (Promega)-from three biological tissues of five individuals of Lutjanus purpureus under two methods of storage. The evaluated items included DNA concentration and purity, processing time and cost, as well as the obtaining of functional sequences. The highest average values of DNA concentration were obtained using the saline procedure and the commercial kit. Pure DNA was only obtained using the saline protocol, evaluated by the ratio of 260/280. The saline and phenol-chloroform protocols were the least expensive methods. The commercial kit costs are counterbalanced by the short time required. The procedure based on phenol-chloroform presented the worst results regarding DNA yield and the time required to perform all steps. The saline and commercial kit protocols showed similar results concerning the amount and quality of extracted DNA. Therefore, the final choice should be based on the available financial resources and the available time for carrying out each procedure of DNA extraction.

Optimisation of Genomic DNA Extraction and PCR Procedure for Sal (Shorea robusta)

Optimisation of Genomic DNA Extraction and PCR Procedure for Sal (Shorea robusta)

Optimizing the pre-analytical phase for accurate HPV detection in skin disorders: insights from a cutaneous warts case study.

In previous years, several cutaneous disorders have been associated with human papillomavirus (HPV); however, the exact role of HPV remains largely unknown. The lack of optimization and standardization of the pre-analytical phase forms a major obstacle. The aim of this study was to develop an accurate/patient-friendly sampling method for skin disorders, with cutaneous warts as a case study. Various sample processing techniques, pre-treatment protocols and DNA extraction methods were evaluated. Several sampling methods were examined, that is, skin scrapings, swabs and a tape-based method. Quantification of DNA yield was achieved by beta-globin real-time polymerase chain reaction (qPCR), and a wart-associated HPV genotyping qPCR was used to determine the HPV prevalence. All samples tested positive for beta-globin. Skin scrapings had significantly higher yield than both swab and tape-based methods (p < 0.01), the latter two did not significantly differ from each other (p > 0.05). No significant difference in DNA yield was found between cotton and flocked swabs (p > 0.05). All swabs were HPV positive, and although there were some discrepancies in HPV prevalence between both swabs, an overall good strength of agreement was found [κ = 0.77, 95% CI (0.71-0.83)]. Although skin scrapings produced the highest DNA yield, patient discomfort was an important limitation of this method. Considering that in combination with our optimized DNA extraction procedure, all samples gave valid results with the less invasive swab methods preferred. Standardization of the pre-analytical phase is the first step in establishing a link between HPV and specific skin disorders and may have significant downstream diagnostic as well as therapeutic implications.

Identification of Leishmania species and frequency distribution of LRV1 and LRV2 viruses on cutaneous leishmaniasis patients in Isfahan Province, Iran

PurposeLeishmaniasis is one of the most serious health problems in developing countries. Iran is one of the endemic regions of cutaneous leishmaniasis. Leishmania RNA virus (LRV) is a dsRNA virus member of the Totiviridae family, which was first detected in the promastigotes of Leishmania braziliensis guyanensis. Our study aimed to investigate possible changes in the predominant and causative strains of CL and screening the LRV1 and LRV2 species genome from Leishmania species isolated from the lesions of patients. Materials and methodsDirect smear samples obtained from 62 patients with leishmaniasis referring to the Skin Diseases and Leishmaniasis Research Center in Isfahan province during 2021–2022 were examined. Total DNA extraction procedures and conservation of site-specific multiplex PCR and nested PCR were performed for detecting Leishmania species. The molecular identification of LRV1 and LRV2 viruses, samples were used for total RNA extraction and real-time (RT)-PCR analysis, followed by conducting a restriction enzyme assay to confirm the PCR products. ResultsOf the total Leishmania isolates, 54 and 8 isolates were identified as L. major and L. tropica, respectively. LRV2 was identified in 18 samples affected by L. major, while LRV1 was only detected in one of the samples with L. tropica. No LRV2 was found in any samples with L. tropica. The results showed that there was a significant relationship between LRV1 and the type of leishmaniasis (Sig. ​= ​0.009, P ​≤ ​0.05), while this relationship was not observed between LRV2 and the type of leishmaniasis. ConclusionsThe presence of a significant number of LRV2 in isolated samples, as well as the recognition of LRV1 in one of the Old World leishmaniasis species, which is a new result, could pave the way for investigating further aspects of this disease and successful treatment strategies in future studies.

DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein

Bovine milk mainly contains two types of β-casein: A1 and A2 variants. In recent years, a new variety of cows’ milk has emerged in dairy sector called “A2 milk”. This novel product is characterised by the absence of A1 β-casein, which has been associated with possible gastrointestinal discomfort due to β-casomorphin-7 (BCM-7) release during gastrointestinal digestion. In this context, methods to verify A1 allele absence in A2 milk are required as a quality control in the A2 milk commercialisation. Therefore, the aim of the present study was to develop a locked nucleic acid (LNA) probe-based duplex real-time PCR (qPCR) assay for A1 allele detection in A2 milk samples. Firstly, four DNA isolation methods from milk somatic cells were optimised and evaluated. Results suggests that the commercial kit NucleoSpin Tissue was the most suitable method in terms of DNA quality and amplificability for downstream applications. Then, optimisation and validation of the qPCR assay were carried out. For both A1 and A2 alleles, the absolute limits of detection of this qPCR assay were 7.3 DNA copies/reaction (2 x 10 −5 ng DNA) and 30.4 DNA copies/reaction (0.1 ng DNA) at a 95% confidence level with synthetic reference DNA samples and heterozygous genotyped DNA sample, respectively. The relative limits of detection were 2% (15 copies) and 5% (152 copies) for A1 allele in A2 samples at a 95% confidence with synthetic reference and genotyped DNA samples, respectively. The qPCR assay was robust, with intra- and inter-assay variability below 4.3%, and specific, differentiating between A1 and A2 alleles with 100% genotyping accuracy. In conclusion, this cost-effective and fast method could be used to discriminate A1 allele in A2 samples and, consequently, to verify A1 allele absence in “A2 milk” by screening commercial product on the market. • The LNA duplex qPCR assay developed could be used to verify A1 allele absence in “A2 milk”. • LNA probes reliably discriminated between A1 and A2 alleles. • The relative limits of detection reach up to 2% (15 copies) for A1 allele in A2 samples at a 95% confidence. • Milk is a challenging matrix for DNA isolation due to the presence of PCR inhibitors that leads to low DNA yield.

A CRISPR/Cas12a-mediated, DNA extraction and amplification-free, highly direct and rapid biosensor for Salmonella Typhimurium

CRISPR/Cas-based biosensors were typically used for nucleic-acid targets detection and complex DNA extraction and amplification procedures were usually inevitable. Here, we report a CRISPR/Cas12a-mediated, DNA extraction and amplification-free, highly direct and rapid biosensor (abbreviated as “CATCHER”) for Salmonella Typhimurium (S. Typhimurium) with a simple (3 steps) and fast (∼2 h) sensing workflow. Magnetic nanoparticle immobilized anti-S. Typhimurium antibody was worked as capture probe to capture the target and provide movable reaction interface. Colloidal gold labeled with anti-S. Typhimurium antibody and DNase I was used as detection probe to bridge the input target and output signal. First, in the presence of S. Typhimurium, an immuno-sandwich structure was formed. Second, DNase I in sandwich structure degraded the valid, complete activator DNA to invalid DNA fragments which can't trigger the trans-cleavage activity of Cas12a. Finally, the integrity of reporter DNA was preserved presenting a low fluorescence signal. Conversely, in the absence of S. Typhimurium, strong fluorescence recovery appeared owing to the cutting of reporter by activated Cas12a. Significantly, the proposed “CATCHER” showed satisfactory detection performance for S. Typhimurium with the limit of detection (LOD) of 7.9 × 101 CFU/mL in 0.01 M PBS and 6.31 × 103 CFU/mL in spiked chicken samples, providing a general platform for non-nucleic acid targets.

Variability of White Spot Syndrome Virus (WSSV) Envelope Protein VP28 from Diseased Shrimp (Litopenaeus vannamei) in Indonesia

White Spot Disease (WSD) is a viral disease affecting crustaceans. Caused by the White Spot Syndrome Virus (WSSV), this disease has caused significant mortality in commercially cultivated marine shrimp species with severe impacts on the shrimp farming industry and may be a threat to wild shrimp stocks. Thorough studies on the molecular biology of this pathogen are urgently needed to improve understanding of the virus at a molecular level, including variation in key viral protein (VP) components of the WSSV virion. This study aimed to isolate and characterize WSSV VP28 gene encoding envelope proteins from Indonesian Pacific white shrimp (Litopenaeus vannamei) isolates. Infected juvenile shrimp were collected from Pangkep, Barru, and Pinrang Districts in South Sulawesi, Indonesia. Genomic DNA was isolated from infected shrimp muscle tissue using a DTAB-CTAB (dodecyle trimethyl ammonium bromide-hexadecyl trimethyl ammonium bromide) DNA extraction procedure. The WSSV VP28 DNA sequences from Pangkep, Barru, and Pinrang isolates were 640-680 bp in length. Homology of Pangkep isolates with isolates from Barru and Pinrang was 97-99%. BLAST-N (Basic Local Alignment Search Tool-Nucleotide) analysis showed isolates from all three sites clustered with WSSV VP28 accessions from China, Indonesia, Japan, South Carolina and Vietnam. These results increase the geographic spread and host taxon coverage of WSSV VP28 sequence data for Indonesia.

ATR-FTIR spectroscopy as a pre-screening technique for the PMI assessment and DNA preservation in human skeletal remains – A review

Skeletal remains from archaeological and forensic contexts are commonly used for various analyses, including DNA.Our research investigates relatively fast and inexpensive ATR-FTIR spectroscopy with further data manipulation as a potential pre-screening technique for assessing the preservation state of skeletal remains. We seek to correlate spectra and chemometric indices with the biological, chemical, and physical composition of the skeletal tissue, and the quality and quantity of DNA preserved in the tissue, also considering the skeletal element, absolute age, and environmental conditions to which the remains have been exposed.Our research focuses on human bones and teeth derived from archaeological, WWII and forensic contexts. The samples are cleaned and pulverized according to established methodological procedures for DNA extraction. DNA is extracted and quantified, while the remaining powder is analysed by ATR-FTIR spectroscopy. The obtained spectra are manipulated to extract the chemometric indices, which are the base for statistical analyses, machine learning, and prediction algorithms.A compilation of results from several studies shows that ATR-FTIR spectra present a solid base for researchers to better understand the preservation state of phosphates, carbonates, and collagen in the skeletal tissues, assessment of post-mortem interval, and the preservation of the DNA. Although many questions still need to be answered, ATR-FTIR spectroscopy with further data manipulation has a high potential for research of taphonomic changes, PMI and DNA preservation in human skeletal remains.

Measurement of yield and quality of DNA in human buffy coat is extraction method dependent

During the last few years, an important element in the improvement of the molecular biology techniques has been the necessity for availability of high quality and functionality DNA. Several DNA extraction procedures with different results in both performance and quality, have been proposed. In this study our objective was to determine the most reliable extraction method that balances DNA quantity, and to assess the sample quantification of the fluorometric DNA quantification methods. For this, blood extracted by venopunction from 20 healthy volunteers was used to obtain DNA from buffy coat, and 4 commercial DNA extraction kits were assessed as well as two fluorometric DNA quantification methods with protocols of different complexity. Results suggest that manual methods achieve higher quality and larger yields of DNA. DNA purity obtained with the 4 extraction kits evaluated through the 260/280 and 260/230 ratio showed that the Qiacube kit fulfilled the criteria established in this work, followed very close by the Flexigene kit. On the other hand, the fluorometric DNA methods used in the samples quantification showed a higher variability when using QuantiFlour method, obtaining better results probably due to the simplicity of this protocol.

Improved assessments of bulk milk microbiota composition via sample preparation and DNA extraction methods.

Although bacterial detection by 16S rRNA gene amplicon DNA sequencing is a widely-applied technique, standardized methods for sample preparation and DNA extraction are needed to ensure accuracy, reproducibility, and scalability for automation. To develop these methods for bovine bulk milk, we assembled and tested a bacterial cell mock community (BCMC) containing bacterial species commonly found in milk. The following protocol variations were examined:: BCMC enumeration (colony enumeration or microscopy), sample volume (200 μl to 30 ml), sample storage condition (frozen in PBS or 25% glycerol or exposure to freeze-thaw cycles), cell lysis method (bead-beating, vortex, enzymatic), and DNA extraction procedure (MagMAX Total, MagMAX CORE, and MagMAX Ultra 2.0, with and without either Proteinase K or RNase A). Cell enumeration by microscopy was more accurate for quantification of the BCMC contents. We found that least 10 mL (≥ 104 cells in high quality milk) is needed for reproducible bacterial detection by 16S rRNA gene amplicon DNA sequencing, whereas variations in storage conditions caused minor differences in the BCMC. For DNA extraction and purification, a mild lysis step (bead-beating for 10 s at 4 m/s or vortexing at 1800 rpm for 10 s) paired with the MagMAX Total kit and Proteinase K digestion provided the most accurate representation of the BCMC. Cell lysis procedures conferred the greatest changes to milk microbiota composition and these effects were confirmed to provide similar results for commercial milk samples. Overall, our systematic approach with the BCMC is broadly applicable to other milk, food, and environmental samples therefore recommended for improving accuracy of culture-independent, DNA sequence-based analyses of microbial composition in different habitats.

Optimised germination protocol for jackfruit seeds and evaluation of methods for extraction of DNA suitable for genetic analysis

Jackfruit (Artocarpus heterophyllus) is an underutilised plant that is promising in curbing food and nutritional security in sub-Saharan Africa. However, high level of secondary metabolites in its tissues significantly hampers its genetic characterisation for breeding purposes. Primarily, the compounds react with DNA during the extraction process, thus reducing its yield and quality. The utilisation of leaves from jackfruit seedlings is a potentially effective approach of addressing the challenge, however, limited information is available on efficient jackfruit seed germination procedures. Elucidating effective methods of jackfruit seed germination, and optimising protocols for DNA extraction is crucial in promoting its genetic characterisation studies for identification of superior varieties for propagation. The objective of this study was to evaluate methods of jackfruit seed germination, and DNA extraction procedures using jackfruit leaves. Pre-treatment of seeds with 3% hydrogen peroxide was effective in enhancing seed germination within a short time, compared to distilled water and 3% hydrochloric acid. We optimised a DNA extraction technique by combining CTAB-SDS based approach with an enhanced solvent extraction method. The technique yielded high quantity and quality of DNA from jackfruit leaves, which was appropriate for downstream polymerase chain reaction analysis. The sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR) amplifications confirmed the effectiveness of the optimised CTAB-SDS based protocol for extraction of high quality DNA.&#x0D;

Truth or Lie: Does the DNA Extraction Procedure Really Affect the Insight in Composition and Diversity of Microbial Communities in Saffron Cultivated Soils?

The aim of this study was to evaluate the performance of two of the most commonly used commercial kits for soil DNA extraction regarding the values of the taxonomic diversity of prokaryotes and community composition of saffron (Crocus sativus) cultivated fields. The impact of the QIAGEN-DNeasy PowerSoil Kit (MO) and Macherey-Nagel™ NucleoSpin™ Soil (MN) kit was tested on the soil of an Italian western alpine experimental site located in Saint Christophe (Aosta Valley, AO). Nine biological replicas of bulk soil were collected and analyzed independently with the two kits. 16S rRNA metabarcoding was applied to characterize soil microbial communities. We first noticed that both DNA extraction kits yielded nearly the same number of OTUs: 1284 and 1268 for MN and MO, respectively. Both kits did not differ in the alpha diversity of the samples, while they had an influence on the beta diversity. The comparative analysis of the microbial community composition displayed differences in microbial community structure depending on which kit was used. These differences were especially highlighted at Phylum and Class levels. On the other hand, the fact that, from a functional point of view, our approach did not highlight any differences allows us to state that the results obtained with the two extraction kits are comparable and interchangeable. Based on these results and those in the literature, we could undoubtedly recommend both commercial kits, especially if the soil target microorganisms are prokaryotes and the study focuses on agricultural sites.

Recovery of DNA from fired and unfired cartridge casings: comparison of two DNA collection methods

For over 10 years, various studies have attempted to increase the recovery of DNA from ammunition by modifying the DNA collection, extraction, purification, and amplification procedures, with varying levels of success. This study focused on the “soaking” method of Montpetit & O’Donnell [1] and the “rinse-and-swab" method of Bille et al. [2]. First, testing for the presence of exogenous DNA, 210 boxed cartridges (brass, steel, and nickel-plated) from nine manufacturers were swabbed and DNA was extracted, concentrated, and quantified. Extracts that quantified > 0 ng/µL (44 of 210) were amplified and genotyped with GlobalFiler™. Of those, only one extract yielded two alleles indicating that the manufacturing and packaging of ammunition was virtually DNA free. Next, to obtain a baseline comparison of two DNA collection methods on a non-metallic substrate and identify a suitable number of cells to spot on cartridges, different DNA input amounts of primary human adult epidermal keratinocytes (HEKa) were tested. Thereafter, 300 brass and 300 nickel-plated, cartridges were spotted with HEKa cells containing ~5 ng of DNA, fired or unfired, and processed with either method. Finally, five methods representing hybrids of the soaking and rinse-and-swab methods were tested to determine if variations of those methods could be used to increase DNA yield and recovery. The results show that the soaking method consistently yielded more DNA than the rinse-and-swab method from a non-metallic substrate. However, the comparison study demonstrated that both methods performed comparably for cartridges. On average, the soaking method recovered 0.25 ng of DNA (5.1% recovery) and the rinse-and-swab method recovered 0.28 ng (5.8% recovery). However, average recoveries were significantly different among three analysts and considerable variation in yields were observed, possibly due to storage time. Furthermore, consistent with prior reports, the DNA recovered from brass casings was only 16% of that recovered from nickel-plated casings and the average yield of DNA from fired casings was reduced to 67% of unfired casings. Moreover, DNA extracts from brass or nickel-plated casings did not appear to contain amplification inhibitors and only 30/596 appeared severely degraded. Finally, both the published rinse-and-swab and soaking methods yielded more DNA than all modifications of the two methods. Overall, both methods yielded equivalent DNA quantities. Additionally, recovery of DNA from any given cartridge casing may be dependent on storage time as well as the skill, proficiency, and experience of the analyst and may reflect stochastic effects, particularly for casings containing low copy and/or degraded DNA.

Optimisation of a bead-beating procedure for simultaneous extraction of bacterial and fungal DNA from pig faeces and liquid feed for 16S and ITS2 rDNA amplicon sequencing

Efficient cell lysis is critical for the extraction of DNA from difficult-to-lyse microorganisms such as Gram-positive bacteria and filamentous fungi. A bead-beating (BB) step is usually included in DNA extraction protocols to improve cell lysis. However, there is no consensus on the duration of BB that is necessary for complete lysis of the microbial communities present in complex microbial ecosystems, but which will still maintain the integrity of DNA released from easy-to-lyse microbes. Another consideration is that most protocols are tailored to one particular target group of microbes, typically either bacteria or fungi, in a given sample matrix. In this study, we investigated the impact of five BB durations (0, 3, 10, 15 and 20 min) during DNA extraction with the QIAamp® Fast DNA Stool Mini Kit, on the bacterial and fungal communities of single pig faecal and liquid feed samples, extracted in triplicate, with the objective of determining a suitable ‘catch-all’ method. Both sample types were subjected to the BB durations in triplicate, followed by 16S (bacterial) and ITS2 (fungal) rDNA amplicon sequencing. The performance of the different BB durations was assessed based on the quantity of total DNA extracted, alpha- and beta-diversity analyses of the resultant microbial communities and differential abundance of bacterial and fungal taxa. Our results suggest that 20 min of BB is most appropriate for maximising the lysis of difficult-to-lyse bacteria and fungi in both pig faeces and liquid feed, while minimising the negative impact on easier-to-lyse microbes. Total DNA yield increased with BB duration for both sample types; however, the yield from faeces decreased after 20 min of BB. Despite this, DESeq2 analysis indicated that changes in the differential abundances of the dominant taxa at this point were limited, which was supported by the Shannon diversity results. Maximising the BB duration appeared to be necessary in order to obtain a representative profile of the Gram-positive bacteria, particularly in liquid feed, and of the filamentous fungi present in both sample types. However, considering the small sample size, along with the reliance on differential as opposed to absolute abundances to validate increases or decreases in taxa, a larger-scale study is necessary to verify the findings of the present study.