Clinical metagenomics.

Clinical metagenomic next-generation sequencing (mNGS), the comprehensive analysis of microbial and host genetic material (DNA and RNA) in samples from patients, is rapidly moving from research to clinical laboratories. This emerging approach is changing how physicians diagnose and treat infectious disease, with applications spanning a wide range of areas, including antimicrobial resistance, the microbiome, human host gene expression (transcriptomics) and oncology. Here, we focus on the challenges of implementing mNGS in the clinical laboratory and address potential solutions for maximizing its impact on patient care and public health.

An Improved Method and Device for Nucleic Acid Isolation Using a High-Salt Gel Electroelution Trap.

The success of DNA analytical methods, including long-read sequencing, depends on the availability of high-quality, purified DNA. Previously, we developed a method and device for isolating high-molecular-weight (HMW) DNA for long-read sequencing using a high-salt gel electroelution trap. Here, we present an improved version of this method for purifying nucleic acids with high yield and purity from even the most challenging biological samples. The proposed method is a significant improvement over the previously published procedure, offering a simple, fast, and efficient solution for isolating HMW DNA and smaller DNA and RNA molecules. The method utilizes vertical gel electrophoresis in two nested, partially overlapping electrophoretic columns. The upper, smaller-diameter column has a thin layer of agarose gel at the bottom, which separates nucleic acids from impurities, and an electrophoresis buffer on top. After the target nucleic acid has been gel-purified on the upper column, a larger-diameter column with a layer of high-salt gel overlaid with electrophoresis buffer is inserted from below. The purified nucleic acid is then electroeluted into the buffer-filled gap between the separating gel and the high-salt gel, where excess counterions from the high-salt gel slow its migration and cause it to accumulate. The proposed vertical purification system outperforms the previously described horizontal system in terms of ease of use, speed, scalability, and compatibility with high-throughput workflows. Furthermore, the vertical system allows for the sequential purification of several nucleic acid species from the same sample using interchangeable salt-gel columns.

Programmable Lanthanide Metal-Organic Framework for Ultra-Efficient Nucleic Acids Extraction and Interaction Analysis.

Efficient, mild, and reversible adsorption of nucleic acids onto nanomaterials represents a promising analytical approach for medical diagnosis. However, there is a scarcity of efficient and reversible nucleic acid adsorption nanomaterials. Additionally, the lack of comprehension of the molecular mechanisms governing their interactions poses significant challenges. These issues hinder the rational design and analytical applications of the nanomaterials. Herein, we propose an ultra-efficient nucleic acid affinity nanomaterial based on programmable lanthanide metal-organic frameworks (Ln-MOFs). Through experiments and density functional theory calculations, a rational design guideline for nucleic acid affinity of Ln-MOF was proposed, and a modular and flexible preparation scheme was provided. Then, Er-TPA (terephthalic acid) MOF emerged as the optimal candidate due to its pore size-independent adsorption and desorption capabilities for nucleic acids, enabling ultra-efficient adsorption (about 150% mass ratio) within 1 min. Furthermore, we elucidate the molecular-level mechanisms underlying the Ln-MOF adsorption of single- and double-stranded DNA and G4 structures. The affinity nanomaterial based on Ln-MOF exhibits robust nucleic acid extraction capability (4-fold higher than commercial reagent kits) and enables mild and reversible CRISPR/Cas9 functional regulation. This method holds significant promise for broad application in DNA/RNA liquid biopsy and gene editing, facilitating breakthroughs in analytical chemistry, pharmacy, and medical research.

Sensitive and rapid analysis of plasmid DNA topology isoforms by capillary gel electrophoresis with laser induced fluorescence in uncoated capillary.

The work describes the use of SYBR Gold to improve the detection sensitivity of plasmid DNA topoisomers by capillary gel electrophoresis with laser induced fluorescence in an uncoated capillary. The impact of different dyes, including ethidium bromide, SYBR Green and SYBR Gold, was compared based on detection and separation of DNA plasmid topoisomers. Use of SYBR Gold enabled improvement of detection sensitivity by 15-fold while maintaining good separation resolution of the different topoisomers. The baseline dropped with the use SYBR Gold but was overcome by the employment of a capillary with longer ineffective length (40 vs. 20 cm). Separation resolution and reproducibility were impacted by the concentration of SYBR Gold and hydroxypropyl methylcellulose. With the use of a short capillary (10 cm effective length and 50 cm total length), fast separations of supercoiled, linear, open circular, and other isoforms were accomplished within 8 min. Appropriate capillary cleaning with 0.1 M sodium hydroxide/0.1 M hydrochloric acid and capillary storage with 0.1 M hydrochloric acid ensured good separation reproducibility of 217 runs during an extended period of use.

Assessment of the efficiency of DNA isolation and profiling applying a temperature-driven method in human remains.

The identification of human remains is of utmost importance in a variety of scenarios. One of the primary identification methods is DNA. DNA extraction from human remains could be difficult, particularly in situations where the remains have been exposed to environmental conditions and other insults. Several studies tried to improve extraction by applying different approaches. ForensicGEM Universal (MicroGem) is a single-tube approach to DNA extraction and a temperature-driven method that could have some advantages with respect to previous techniques, among them, reducing the risk of contamination, not requiring specialized equipment, or several steps to perform. The aim of this study was to assess, for the first time, the efficiency of DNA extraction and quality of STR profiles applying the MicroGem protocol and modifications of this protocol from tooth samples in comparison with automatic extraction (AE). Our results indicated that AE and MicroGem performed similar, though with variability depending on the MicroGem modifications, increasing the DNA yield and STR profile quality when DNA is concentrated with Microcon. These findings demonstrated the efficiency of this methodology for DNA extraction from human remains while also providing a simple and quick technique suitable to apply in a variety of forensic scenarios.

Rapid molecular identification of Rana dybowskii by species-specific primers.

Oviductus Ranae is the dried oviduct from Rana dybowskii, a forest frog species with medicinal, tonic, and cosmetic properties. Due to the high price and resource shortage, counterfeit varieties of Oviductus Ranae often appear in the market. However, traditional identification methods cannot accurately differentiate between Oviductus Ranae and its adulterants. In this study, a rapid molecular identification method has been established. The method involves extracting genomic DNA in just 30 s using filter paper purification, species-specific rapid polymerase chain reaction (PCR) amplification, and finally, fluorescence detection of the products. It can accurately identify Oviductus Ranae and its three common adulterants in about 30 min, making the process simple, fast, and highly specific.

Extraction of Genomic DNA from Soil Samples by Polyethylene Glycol-Modified Magnetic Particles via Isopropanol Promotion and Ca2+ Mediation.

Obtaining reliable and informative DNA data from soil samples is challenging due to the presence of interfering substances and typically low DNA yields. In this work, we prepared poly(ethylene glycol)-modified magnetic particles (PEG@Fe3O4) for DNA purification. The particles leverage the facilitative effect of calcium ions (Ca2+), which act as bridges between DNA and PEG@Fe3O4 by coordinating with the phosphate groups of DNA and the hydroxyl groups on the particles. The addition of 2-propanol further enhances this Ca2+-mediated DNA adsorption by inducing a conformational change from the B-form to the more compact A-form of DNA. PEG@Fe3O4 demonstrates a DNA adsorption capacity of 144.6 mg g-1. When applied to the extraction of genomic DNA from soil samples, PEG@Fe3O4 outperforms commercial kits and traditional phenol-chloroform extraction methods in terms of DNA yield and purity. Furthermore, we developed a 16-channel automated DNA extraction device to streamline the process and reduce the extraction time. The successful amplification of target bacterial and fungal amplicons underscores the potential of this automated, device-assisted method for studying soil microbial diversity.

Cleaning protocols in forensic genetic laboratories.

It is pivotal to avoid cross-sample contamination in forensic genetic laboratories and optimal cleaning protocols for the removal of DNA are essential. A survey was performed, and ten forensic genetic laboratories shared their cleaning protocols in pre-PCR and post-PCR laboratories. The cleaning frequencies on different surface areas were somewhat similar, whereas none of the laboratories used the same cleaning reagents. Therefore, the efficiencies of the cleaning protocol utilised were tested and compared. The results showed that freshly made household bleach and Virkon® removed all amplifiable DNA from the surfaces, whereas DNA AWAY™ and the disinfection reagents ethanol, isopropanol, and ChemGene HLD4L did not.

Evaluation of metal ions and DNA recovery from the surface of fired and unfired brass ammunition to improve STR profiling.

Interest in recovering DNA from the surface of ammunition evidence for genotyping has increased over the past few years. Numerous studies have examined a variety of methods to maximize DNA recovery from these types of challenging samples, but successful DNA profiling has been inconsistent. Low amounts of DNA and PCR inhibition due to metal ions have been suggested as the leading causes of poor results; however, no study quantitatively examined the presence of metal ions at various stages of the DNA analysis workflow from DNA collection through to amplification. In this study, the effectiveness of six different DNA collection and purification methods commonly used by forensic laboratories to process brass ammunition for DNA evidence was investigated. The amount of copper, zinc, and other metals co-recovered from fired and unfired brass casings during DNA collection (using numerous soaking, swabbing, and direct PCR protocols) was quantified via Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES). This same panel of metals was subsequently quantified after DNA lysis and purification steps. Results demonstrated that low amounts of DNA, DNA damage, and degradation are more detrimental to STR typing results than PCR inhibition, as metal ions were successfully removed by all DNA purification methods tested. Furthermore, the use of metal ion chelators increased the amount of DNA recovered and number of reportable STR alleles. This research informs the forensic community on the most effective way to collect and process trace amounts of biological material from brass ammunition and similar evidence.

Evaluation of DNA extracted from residual blood clots after serological testing.

DNA quality is of paramount importance for molecular biology research. This study aimed to assess the DNA extracted from residual blood clots after serological testing, focusing on the impact of blood clot segments, extraction kits, temporary storage durations (TSDs), and thawing methods on DNA quality. We divided the residual blood clot column (BCC) from healthy donors into three segments and utilized two different extraction kits. The BCCs were subjected to four TSDs at 4°C (7 days, 10 days, 1 month, and 2 months) and three thawing methods (4°C, room temperature, and 37°C). We found that the TIANamp Blood Clot DNA Kit yielded consistently high-quality DNA from each segment with stable A260/280 and A260/230 ratios. The DNA yield showed a strong positive correlation with leukocyte concentration, and a satisfactory median DNA yield of 28.79 µg/g BCC was obtained across all segments. DNA integrity, as measured by the DNA integrity number and DNA fragment peak size, decreased with increasing TSD at 4°C, with a notable decrease after 10 days of storage. Thawing at 37°C resulted in the lowest DNA fragment peak size. In conclusion, BCC could be an ideal DNA source with satisfactory yield and purity. A prolonged TSD at 4°C leads to an obvious decrease in DNA integrity, and thawing the frozen BCC at 37°C decreases DNA fragment sizes. To maintain DNA integrity, BCCs should be cryopreserved as soon as possible after short TSDs at 4°C and thawed at 4°C.

Application of biomolecular techniques on tsetse fly puparia for species identification at larvipostion sites.

Puparia are commonly found in tsetse fly larviposition sites during studies on larval ecology. This chitinous shell is representative of past or ongoing exploitation of these sites by tsetse flies. The morphological characteristics of the puparium are not sufficiently distinctive to allow identification of the species. This study explores the applicability of biomolecular techniques on empty puparia for tsetse fly species identification. Five techniques were compared for DNA extraction from tsetse fly puparia, 1/Chelex® 100 Resin, 2/CTAB, 3/Livak's protocol, 4/DEB + proteinase K and 5/QIAamp® DNA Mini kit, using two homogenisation methods (manual and automated). Using a combination of two primer pairs, Chelex, CTAB, and DEB + K proved the most efficient on fresh puparia with 90, 85, and 70% samples identified, respectively. Shifting from fresh to one- to nine-month-old puparia, the Chelex method gave the best result allowing species identification on puparia up to seven months old. The subsequent testing of the Chelex extraction protocol identified 152 (60%) of 252 field-collected puparia samples at species level. The results show that reliable genetic identification of tsetse flies species can be performed from empty puparia, what can prove of great interest for future ecological studies on larviposition sites. The Chelex technique was the most efficient for DNA extraction, though the age-limit of the samples stood at seven months, beyond which DNA degradation probably compromises the genetic analysis.

Molecular identification of haemoparasites in animals using blood lysate PCR: a quick and inexpensive alternative to purified whole genomic DNA.

Haemoparasitic diseases constitute a significant constraint to economic livestock farming. Diagnostic techniques that are inexpensive, rapid, reliable, and precise are crucial for the management of diseases. In this context, PCR assays are very valuable yet expensive since the samples must be processed before being included in the PCR reaction. Accordingly, the goal of the current study was to lower the PCR costs without jeopardizing the assay's sensitivity and specificity. For that purpose, the alkaline solution was optimized for low cost and quick DNA extraction (blood lysate), and PCR reagents were modified for optimum reaction. In comparison to purified whole blood genomic DNA, the currently developed and optimized blood lysate method was found to be 95.5% less expensive, as well as being equally sensitive and specific for the molecular detection (PCR) of haemoparasites like Babesia, Theileria, Trypanosoma and rickettsiales in cattle, buffaloes, horses, and dogs. The assay was also demonstrated to be quick, less likely to cross-contaminate, and appropriate for use in laboratories with limited resources. Therefore, the currently developed and optimized blood lysate method could serve as a viable alternative to purified whole blood genomic DNA for molecular detection (PCR) of haemoparasites in animals particularly in resource-limited settings.

Preparation of DNA nanoflower-modified capillary silica monoliths for chiral separation.

Innovative chiral capillary silica monoliths (CSMs) were developed based on DNA nanoflowers (DNFs). Baseline separation of enantiomers such as atenolol, tyrosine, histidine, and nefopam was achieved by using DNF-modified CSMs, and the obtained resolution value was higher than 1.78. To further explore the effect of DNFs on enantioseparation, different types of chiral columns including DNA strand containing the complementary sequence of the template (DCT)-modified CSMs, DNF2-modified CSMs, and DNF3-modified CSMs were prepared as well. It was observed that DNF-modified CSMs displayed better chiral separation ability compared with DCT-based columns. The intra-day and inter-day repeatability of model analytes' retention time and resolution kept desirable relative standard deviation values of less than 8.28%. DNF2/DNF3-modified CSMs were able to achieve baseline separation of atenolol, propranolol, 2'-deoxyadenosine, and nefopam enantiomers. Molecular docking simulations were performed to investigate enantioselectivity mechanisms of DNA sequences for enantiomers. To indicate the successful construction of DNFs and DNF-modified CSMs, various charaterization approaches including scanning electron microscopy, agarose gel electrophoresis, dynamic light scattering analysis, electroosmotic flow, and Fourier-transform infrared spectroscopy were utilized. Moreover, the enantioseparation performance of DNF-modified CSMs was characterized in terms of sample volume, applied voltage, and buffer concentration. This work paves the way to applying DNF-based capillary electrochromatography microsystems for chiral separation.

From Jane Doe to Sofia: DNA Extraction Protocol from Bones and Teeth without Liquid Nitrogen for Identifying Skeletal Remains.

DNA analysis plays a crucial role in forensic investigations, helping in criminal cases, missing persons inquiries, and archaeological research. This study focuses on the DNA concentration in different skeletal elements to improve human identification efforts. Ten cases of unidentified skeletal remains brought to the Institute of Forensic Medicine in Timisoara, Romania, underwent DNA analysis between 2019 and 2023. The results showed that teeth are the best source for DNA extraction as they contain the highest concentration of genetic material, at 3.68 ng/µL, compared to the petrous temporal bone (0.936 ng/µL) and femur bone (0.633 ng/µL). These findings highlight the significance of teeth in forensic contexts due to their abundant genetic material. Combining anthropological examination with DNA analysis enhances the understanding and precision of identifying human skeletal remains, thus advancing forensic science. Selecting specific skeletal elements, such as the cochlea or teeth, emerges as crucial for reliable genetic analyses, emphasizing the importance of careful consideration in forensic identification procedures. Our study concludes that automated DNA extraction protocols without liquid nitrogen represent a significant advancement in DNA extraction technology, providing a faster, more efficient, and less labor-intensive method for extracting high-quality DNA from damaged bone and tooth samples.

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.

Evolved DNA Duplex Readers for Strand-Asymmetrically Modified 5-Hydroxymethylcytosine/5-Methylcytosine CpG Dyads.

5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), the two main epigenetic modifications of mammalian DNA, exist in symmetric and asymmetric combinations in the two strands of CpG dyads. However, revealing such combinations in single DNA duplexes is a significant challenge. Here, we evolve methyl-CpG-binding domains (MBDs) derived from MeCP2 by bacterial cell surface display, resulting in the first affinity probes for hmC/mC CpGs. One mutant has low nanomolar affinity for a single hmC/mC CpG, discriminates against all 14 other modified CpG dyads, and rivals the selectivity of wild-type MeCP2. Structural studies indicate that this protein has a conserved scaffold and recognizes hmC and mC with two dedicated sets of residues. The mutant allows us to selectively address and enrich hmC/mC-containing DNA fragments from genomic DNA backgrounds. We anticipate that this novel probe will be a versatile tool to unravel the function of hmC/mC marks in diverse aspects of chromatin biology.

Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation.

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.

Detection of deletion/insertion polymorphism profiles from single human hair shafts.

BACKGROUND: Hair is a frequently encountered biological evidence in personal identification. The amount of nuclear DNA that can be extracted from a single strand of rootless hair is most limited, making the detection of short tandem repeat (STR) polymorphisms difficult. To overcome these limitations, deletion/insertion polymorphisms (DIP) as a new type of genetic marker have shown their benefits in detecting low-copy-number DNA. The Investigator DIPplex kit contains 30 biallelic autosomal DIP and amelogenin. The analysis of DIPs combines the advantages of both STR and single nucleotide polymorphism analyses. Thus, this study aimed to detect the DIP distribution of individual hair shafts from individuals. METHODS AND RESULTS: DNA was extracted from the shaft of fresh, aged, and shed hair. After DNA was evaluated, the DIP profiles were detected by capillary electrophoresis. The results indicated that the amount of DNA extracted from hair roots was much higher than that from the hair shafts in the same individual for all samples. The degradation index values of DNA from the aged hair shafts were highest. It is classified to be "mildly degraded." Compared with their hair roots, the full DIP profiles were detected for fresh hair, 70% for aged hair, and 92% for shed hair. Contrarily, except for fresh hair shafts, only three STR loci of the aged and shed strands of hair could be genotyped using AmpFlSTR MiniFiler PCR Amplification Kit. CONCLUSIONS: These results indicate that the detection of DIP profile is an effective tool for personal identification from hair shafts, including aged hair.