Search Results (403)

Thread-based analytical devices are low-cost, portable, and easy to use, making them ideal for detecting various biomolecules like glucose and DNA with minimal sample requirements, while also offering environmental benefits through their biodegradability. This study explores the potential of zwitterionic poly(sulfobetaine methacrylate) brushes modified cotton thread (PSBMA@threads) as an innovative substitute for DNA solid-phase extraction. The PSBMA polymer brushes were synthesized on cotton threads via surface-initiated atom transfer radical polymerization (SI-ATRP). The usability of the PSBMA@threads for DNA extraction from cell lysates containing cell debris, proteins, and detergents was evaluated. Characterization using SEM, FTIR, and EDS confirmed the successful functionalization with PSBMA polymer brushes. The antifouling properties of PSBMA@threads, including resistance to non-specific protein adsorption and underwater oil repellency, were assessed. The results demonstrated selective DNA capture from protein and lipid-rich lysates. Optimized extraction parameters improved DNA yield, enabling efficient extraction from tumor cells, which successfully underwent PCR amplification. Comparative experiments with commercial silica membrane-based columns revealed that PSBMA@threads exhibited comparable DNA extraction capability. The PSBMA@threads maintained extraction capability after six months of ambient storage, highlighting its stability and cost-effectiveness for nucleic acid isolation in analytical applications. Full article

The root-knot nematode Meloidogyne enterolobii has emerged as a devastating pathogen in global agricultural systems. Its geographic distribution is progressively expanding from tropical to temperate zones, leading to difficulties in discerning the symptoms it causes from those of congeners such as M. incognita. Currently, some molecular diagnostic technologies (e.g., qPCR) have been established for detecting M. enterolobii, but these methods fail to meet field-based detection demands due to their reliance on laboratory-grade thermocyclers. We thus developed a method for detecting M. enterolobii based on enzyme-mediated duplex exponential amplification (EmDEA) technologies to address this issue. The EmDEA detection method demonstrated strict specificity for the target species, showing no amplification in 13 non-target nematodes or host tissue samples. Sensitivity analyses revealed detection limits of 3.6 × 10⁻⁴ ng/μL (purified DNA), 1/1000 of an individual nematode (single-organism detection), 8.97 nematodes/g sweet potato, and 4.08 nematodes/100 g soil, achieving equivalent performance to qPCR. Field validation confirmed successful on-site detection, with significantly higher nematode loads in root tissues (50.41–97.62 nematodes/g) than in rhizospheric soil (1.07–1.28 nematodes/g). The established detection method employs a 42 °C isothermal amplification technology paired with a palm-sized thermal module, enabling field-deployable detection. Its unique duplex exponential amplification mechanism achieves threshold determination 10 cycles (~10 min) faster than conventional qPCR. When integrated with rapid DNA extraction protocols, the entire workflow is completed within 40 min, improving detection efficiency. This study provides a molecular tool for the precise monitoring of M. enterolobii, offering critical support for formulating targeted control strategies. Full article

Background: Helicobacter pylori (HP) and Epstein–Barr virus (EBV) coinfection lead to chronic inflammation and contribute to the development of gastric cancer. However, studies examining the association between HP virulence factors and EBV infection in gastric cancer are limited. This study investigated the polymorphisms of HP virulence factors associated with EBV infection and their effects on clinical outcomes in EBV-associated gastric cancer (EBVaGC). Methods: A total of 96 HP isolates from 54 patients with gastric cancer were divided and analyzed based on EBV coinfection status. Polymerase chain reaction amplifications of virulence factors were conducted using DNA extracts from HP isolates cultured from gastric mucosal specimens. Results: EBV infection was significantly associated with gastric carcinoma with lymphoid stroma morphology and a proximal location in the stomach. Most HP strains from patients with gastric cancer were positive for cagA (100.0%), vacA (100.0%), and iceA1 (87.5%). Among HP isolates with EBV coinfection, the prevalence of iceA2 (21.7% vs. 0.0%, p < 0.001) and ureA (21.7% vs. 4.0%, p = 0.009) was significantly more frequent, and that of iceA1 (78.3% vs. 96.0%, p = 0.009) and vacA s1a (4.3% vs. 22.0%, p = 0.012) was less frequent than those of EBV– colonies. Multivariate analysis indicated that ureA (odds ratio, 6.148; 95% confidence interval [CI], 1.221 to 30.958; p = 0.028) was associated with EBVaGC. No significant difference in clinical outcomes was observed based on the presence of ureA expression in EBVaGC. Conclusions: In gastric cancer, regardless of EBV infection, most HP strains were highly virulent, testing positive for cagA, vacA, and iceA1. Although ureA was significantly associated with EBV infection, it did not influence the clinical outcomes of EBVaGC. Full article

Background: The identification of body fluids collected from crime scenes is crucial for determining the type and nature of assaults and for advancing the resolution of crimes. Objectives: The primary aim of this study was to investigate tissue-specific DNA methylation markers that can effectively distinguish buccal samples from blood, semen, and vaginal epithelial tissue. Methods: We screened various markers and selected four genomic locations for further analysis. Genomic DNA was extracted from tissue samples, followed by bisulfite conversion, locus-specific polymerase chain reaction (PCR) amplification, and pyrosequencing. Results: Four loci—cg-9652652, cg-11536474, cg-3867465, and cg-10122865—along with several adjacent CpG sites, were found to be hypermethylated in buccal samples compared to other tissue types. The difference in DNA methylation of buccal samples was statistically significant (p < 0.0001) compared to other tissues, indicating the potential usefulness of these loci for forensic tissue identification. Two additional studies were conducted: (a) a species specificity study and (b) a mixture study involving two different tissue types. The species specificity study showed that the primers used in the assay were specific to primates and humans. They did not amplify five non-primate samples, while the two primate samples—chimpanzee and rhesus—provided usable methylation data. The mixture study involved DNA from two different tissues—buccal samples and semen—combined in varying proportions. The results showed a decrease in the overall percentage of DNA methylation at the locus cg-9652652 as well as five adjacent CpG sites when the amount of buccal cell DNA in the mixture was reduced. Conclusion: The specificity of the primers and the significant differences in percent DNA methylation between buccal cells and other tissues make these markers excellent candidates for forensic tissue identification. Full article

(1) Background: A safe and effective nucleic acid sample transportation method was developed that is suitable for underdeveloped areas which lack advanced sequencing capabilities, specifically for virus genomic sequencing and infectious disease monitoring. (2) Methods: This study evaluated the use of Flinders Technology Associates (FTA) cards for transporting amplified whole-genome DNA from 120 SARS-CoV-2-positive nasopharyngeal swab samples in Sierra Leone. Nucleic acid extraction and whole-genome amplification were conducted at a local laboratory. Amplified products were applied to FTA Elute cards for room temperature shipment to China CDC for elution and sequencing. (3) Results: The FTA card method achieved a 9.6% recovery rate for amplicons, sufficient for viral genome sequencing. In total, 86 (71.7%) high-quality SRAS-CoV-2 genomic sequences were obtained, with the majority reaching depths exceeding 100X. Sequence analysis revealed co-circulation of Delta, Omicron, and B.1 lineages. Higher Ct values in the original sample significantly reduced coverage and depth, with Ct ≤ 27; 73.6% of samples yielded effective sequences. (4) Conclusions: Transportation of amplified nucleic acid samples using FTA cards enables virus genomic sequencing in resource-limited areas. This approach can potentially improve local virus surveillance and outbreak response capabilities. Further optimizations could improve sequence recovery rate. Implementing this method could significantly enhance sequencing accessibility in underdeveloped regions. Full article

High-throughput 16S rRNA metagenomic sequencing has advanced our understanding of the gut microbiome, but its reliability depends on upstream processes such as DNA extraction and bacterial library preparation. In this study, we evaluated the impact of three different DNA extraction methods (a manual method with an ad hoc-designed pre-extraction phase (PE-QIA), and two automated magnetic bead-based methods (T180H and TAT132H)) and two bacterial library preparation protocols (home brew and VeriFi) on the 16S rRNA-based metagenomic profiling of faecal samples. T180H and TAT132H produced significantly higher DNA concentrations than PE-QIA, whereas TAT132H yielded DNA of lower purity compared to the others. In the taxonomic analysis, PE-QIA provided a balanced recovery of Gram-positive and Gram-negative bacteria, TAT132H was enriched in Gram-positive taxa, and T180H was enriched in Gram-negative taxa. An analysis of Microbial Community Standard (MOCK) samples showed that PE-QIA and T180H were more accurate than TAT132H. Finally, the VeriFi method yielded higher amplicon concentrations and sequence counts than the home brew protocol, despite the high level of chimeras. In conclusion, a robust performance in terms of DNA yield, purity, and taxonomic representation was obtained by PE-QIA and T180H. Furthermore, it was found that the impact of PCR-based steps on gut microbiota profiling can be minimized by an accurate bioinformatic pipeline. Full article

Flagellates of the genus Giardia are intestinal parasites with a broad host range. Several Giardia duodenalis variants (assemblages) recently elevated to species rank—G. duodenalis (assemblage A1), G. intestinalis (A2) and Giardia enterica (B) are human pathogens. Giardia enterica has been reported in some hystricomorph rodents such as wild crested porcupines (Hystrix cristata), but no data were previously available from Brazilian porcupines (Coendou prehensilis) and naked mole rats (Heterocephalus glaber). The aim of this study is to genetically identify the Giardia isolates from these three rodent species, all housed in a zoological institution. Fecal samples were processed using the Bailenger concentration method, and DNA was extracted from the sediments using commercial kits. Partial PCR amplification and sequencing of the glutamate dehydrogenase, beta-giardin, and triose-phosphate isomerase genes revealed that all isolates belonged to G. enterica, showing 99–100% identity with sequences available in GenBank. Prevalences could not be reliably estimated due to small group sizes and the resulting proportions may be biased. To our knowledge, this is the first report identifying Giardia (G. enterica) in C. prehensilis and H. glaber, thus expanding the known host range of this parasite species and reinforcing the importance of surveillance in captive wild hosts. Full article

Evidence suggests that a cross-talk between the gut microbiota and joint health exists in a paradigm known as the gut–joint axis. Recent studies have also reported the presence of microorganisms potentially involved in the pathogenesis and progression of arthritis in synovial joints, previously believed to be sterile. This systematic review describes in detail the methodologies employed to characterize the microbiota in human synovial fluid (SF). A literature search was conducted in PubMed, Embase, and Web of Science up to 5 February 2025. Nine studies aimed to characterize the SF microbiome using next-generation sequencing or polymerase chain reaction. Eight studies detected bacterial DNA in SF. However, significant heterogeneity and incomplete reporting in methodologies, including sample collection and preparation, contamination management, DNA extraction and amplification, sequencing technology, targeted 16S rRNA or ITS regions, and bioinformatics processing, limit the comparability and significance of findings. Given the potential implications for understanding arthritis mechanisms and developing targeted treatments, a standardized methodological and reporting approach in SF microbiota characterization is needed to enhance the reproducibility and the relevance of results. Full article

This study aimed to investigate the potential role of the vasoactive intestinal peptide receptor-1 (VIPR-1) gene polymorphisms and haplotypes in influencing egg production performance and egg quality parameters in laying-type quail. Genomic DNA was extracted from 150 quail across three strains: Chinese yellow (CY, n = 50), Beijing white (BW, n = 50), and Korean (KO, n = 50). We designed two pairs of primers and initiated PCR amplification, after which the amplified products were sent to a testing company for purification. Sanger sequencing was employed to identify single nucleotide polymorphisms (SNPs) within the VIPR-1 gene. Two SNP sites were selected for genotyping; g.1603402T>G was analyzed using PCR-RFLP with the BsrD I enzyme, while g.1614884A>G was genotyped using the HpyCH4 IV enzyme. The association results revealed that the g.1603402T>G site showed significant association with egg shell thickness (EST) in the BW strain (p < 0.05). There were no significant associations between these two loci and the remaining egg quality traits in the BW and KO strains (p > 0.05). Differences in egg quality and laying performance among haplotype combinations were not significant (p > 0.05). In conclusion, the VIPR-1 gene, with its identified polymorphisms and haplotypes, has potential as a molecular marker that could improve egg shell thickness in BW quail. Full article

Efficient extraction of high-quality DNA from plants is a critical challenge in molecular research, especially in species such as Gossypium barbadense L., native to Peru, due to the presence of inhibitors such as polysaccharides and phenolic compounds. This study presents a modified CTAB-based protocol with silica columns that is designed to overcome these limitations without the need for liquid nitrogen or expensive reagents. Native cotton samples were collected in Lambayeque, Peru, and processed using a simplified procedure that optimizes the purity and concentration of the extracted DNA. Eight cultivars of G. barbadense L. with colored fibers (cream, fifo, light brown, dark brown, orange-brown, reddish, fine reddish, and white) were evaluated, yielding DNA with A260/A280 ratios between 2.14 and 2.19 and A260/A230 ratios between 1.8 and 3.14; these values are higher than those obtained with the classical CTAB method. DNA quality was validated by PCR amplification using ISSR and RAPD molecular markers, which yielded clear and well-defined banding patterns. Furthermore, the extracted DNA was suitable for advanced applications, such as Sanger sequencing, by which high-quality electropherograms were obtained. The results demonstrate that the proposed protocol is an efficient, economical, and adaptable alternative for laboratories with limited resources, allowing the extraction of high-quality DNA from Gossypium barbadense L. and other plant species. This simplified approach facilitates the development of genetic and biotechnological research, contributing to the knowledge and valorization of the genetic resources of Peruvian native cotton. Full article

Background/Objectives: Bacterial vaccines were first developed and used in the late 1800s to prevent chicken cholera and anthrax. Bacterial pneumonia vaccines were widely used during the 1918 influenza pandemic, despite the influenza A/H1N1 virus not yet being identified. Studies showed that bacterial pathogens, including Haemophilus influenzae, Streptococcus pneumoniae, and Streptococcus pyogenes, contributed significantly to fatal secondary bacterial pneumonias during the pandemic. In this study, we aimed to characterize the microbial composition of two ampules of a mixed bacterial influenza vaccine produced in 1916, which were labeled as containing killed Bacillus influenzae, Pneumococci, and Streptococcus pyogenes. Methods: DNA was extracted from two 1916-era vaccine ampules, and due to low DNA yields, whole genome amplification (WGA) was performed prior to construction of Illumina sequencing libraries. Deep sequencing was conducted, followed by bioinformatic analysis to identify bacterial DNA content. Consensus genomes were assembled for predominant species, and further analyzed for serotype, phylogeny, and antibiotic resistance genes. Results: The amount of recoverable DNA from these century-old vaccine ampules was limited. The sequencing results revealed minimal detectable S. pneumoniae DNA. The first ampule contained predominantly H. influenzae DNA, while the second vial primarily contained Enterococcus faecium DNA, in addition to S. pyogenes DNA. Consensus genomes for H. influenzae, S. pyogenes, and E. faecium were assembled and analyzed for serotype, phylogeny, and antibiotic resistance genes. Conclusions: This study presents the first genomic analysis of century-old bacterial pneumonia vaccine ampules from the 1918 influenza pandemic era. The findings provide a unique historical perspective on early vaccine formulations and highlight the limitations of early vaccine production. Full article

The search for life beyond Earth currently hinges on the detection of biosignatures that are indicative of current or past life, with terrestrial life being the sole known example. Deoxyribonucleic acid (DNA), which acts as the long-term storage of genetic information in all known organisms, is considered a biosignature of life. Techniques like the Polymerase Chain Reaction (PCR) are particularly useful as they allow for the amplification of DNA fragments, allowing the detection of even trace amounts of genetic material. This study aimed to detect DNA extracted from colonies of an Antarctic black fungus both when (i) alone and (ii) mixed with a Sulfatic Mars Regolith Simulant (S-MRS), after exposure to increasing doses of Fe ions (up to 1 kGy). PCR-based amplification methods were used for detection. The findings of this study revealed no DNA amplification in samples mixed with Sulfatic Mars Regolith Simulant, providing important insights into the potential application of these techniques for in situ DNA detection during future space exploration missions or for their application on the Mars sample return program; it also gives input in the planetary protection discussions. Full article

Seafood is a valuable commodity with increasing demand, traded for billions of USD each year. The volatility in supply chains and fluctuating prices contribute to the susceptibility of the seafood market to food fraud. Analytical methods are required to identify seafood in processed foods to ensure food authenticity and compliance with European laws. To address this need, we developed and validated a DNA metabarcoding method for the authentication of crustaceans and cephalopods in processed food samples, as both are prone to food fraud, especially in mixed products. A ~200 bp barcode of the mitochondrial 16S rDNA was selected as the marker for identification and sequenced on Illumina platforms. The DNA metabarcoding method utilizes two primer systems, one for the amplification of crustacean DNA and another for cephalopods. The crustacean primer system comprises two forward and two reverse primers, while the cephalopod primer system includes three forward and one reverse primer. DNA extracts from reference materials, model foods, processed foodstuffs, and DNA extract mixtures were investigated. Even species with a close phylogenetic relationship were successfully identified and differentiated in commercial samples, while single species were detected at amounts as low as 0.003% in model foods. However, false-negative results were obtained for certain species in DNA extract mixtures, which are most likely due to degraded or low-quality DNA and can best be prevented by optimized DNA extraction procedures. Our DNA metabarcoding method demonstrates strong potential as a qualitative screening tool in combination with other in-house DNA metabarcoding methods for food authentication in routine analysis. Full article

This study introduces a one-pot colorimetric nucleic acid test (NAT) platform that integrates silver nanoparticle (AgNP)-based DNA isolation and colorimetric detection of bacterial genes. The NAT platform is comprised with purification and reaction units that enable cell lysis, DNA purification, loop-mediated isothermal amplification (LAMP), and colorimetric detection. In the purification unit, polyethyleneimine (PEI)-capped AgNPs were used as cell lysis agents because of their cell-disrupting and antimicrobial properties and were immobilized on a glass fiber membrane for DNA capture and isolation. The reaction unit enabled colorimetric detection of DNA amplicons, achieved by the synthesis of AgNPs on chromatography paper formed via the reduction of silver ions present on the paper, mediated by the use of sodium ascorbate, a reducing agent, present in the LAMP reagent, after the reaction. AgNPs were formed only in the presence of the target amplicons in the positive samples after reaction at 65 °C for 5 min. Bacterial DNA was efficiently extracted using this method, and Enterococcus faecium was detected with a detection limit of 10² CFU/mL. This platform is a promising alternative for rapid and cost-effective nucleic acid testing in resource-limited settings. Full article

Although lactic acid bacteria (LABs) possess unique metabolic and physiological characteristics that have crucial effects on the transport of substances both into and out of the cell, there is still a lack of systematic research on membrane transporters in LABs and their roles in material transport. In this study, genomic data for the species Lactobacillus delbrueckii, Streptococcus thermophilus, Leuconostoc lactis, Pediococcus lactis, Lactococcus garvieae, and Bifidobacterium lactis were analyzed to identify the associated transport systems, including what kind of substances are transported. As part of a comparative genomics approach, we used the G-BLAST and AveHAS programs in the TCDB database to screen for transport proteins and clarify the distribution of these proteins in different Lactobacillus strains, allowing for further prediction of their transport substrates. Studies have shown that the distributions of these transporters differ among the selected LAB strains. Through screening and tabulation, we found that the content of transporters in the six LAB proteomes was greater than 20%, with the dominance of the large transporter group indicating complex metabolic and probiotic effects. Furthermore, it was found that the LAB strains contain a variety of homologs of drug-efflux proteins, which may make them resistant to antibiotics, as well as a large number of toxin-related transporters. This study allowed for reasonable predictions of the roles of toxin-related proteins in LABs, and further research on these proteins may be valuable for understanding the probiotic effects of LABs that arise through competition. The study of LAB transporters and the prediction of their functions might support a better understanding of the metabolic and physiological activities of these bacteria. In the future, we aim to extract DNA from laboratory strains and perform PCR amplification using suitable primers designed by us. Through comparison of the obtained gene sequences with those reported in this study, we can explore the differences among them. Full article