DNA Purification Research Articles

Bacteriophage-assisted lysis and eluted genomic DNA-based detection of pathogenic bacterial contamination in food

Foodborne diseases represent a significant global health concern, with a substantial number of illnesses and fatalities reported annually owing to the consumption of contaminated food. Current methods for identifying foodborne pathogens cannot effectively distinguish live from dead bacteria or efficiently detect multiple targets. Therefore, we developed a selective detection method for three pathogenic bacteria in food samples that leverages bacteriophage-assisted live-cell lysis and exposed genomic DNA amplification. The antimicrobial activity and host specificity of these three bacteriophages were employed to confirm the target bacteria selectivity. Bacterial DNA purification using bacteriophage-assisted lysis demonstrated the superior activity of the selected phages in extracting DNA from Escherichia coli O157:H7 and Salmonella Typhimurium. The results from reactions involving the three strains, including Bacillus cereus, highlighted that a 1 h incubation period yielded the most efficient DNA extraction and purification. The polymerase chain reaction (PCR) products obtained were comparable in terms of concentration and purity with those generated using bacterial DNA prepared using a commercial kit. In the practical food sample application experiment, bacterial strains were successfully recovered and concentrated using a polyethersulfone filter, resulting in a concentration factor >3 for all samples. Furthermore, the concentration of purified bacterial DNA from contaminated kimchi cabbage exceeded 10 ng/μL. Notably, we could detect E. coli O157:H7, S. Typhimurium, and B. cereus spiked at 103 colony-forming units/mL using multiplex PCR. This innovative bacteriophage-based approach holds substantial promise for the selective and practical detection of live foodborne pathogens, thereby significantly advancing food safety standards.

Variations in Gut Microbiome Profiles Based on Different DNA Extraction Methods: A Comparative Study

Objectives. To compare four DNA extraction methods for recovering bacterial DNA from mammalian faecal samples. Methods and Results. Three commercial kits from QIAamp, TIANamp, and MAGEN, together with the classic cetyltrimethylammonium bromide (CTAB) method, were evaluated for their performance in extracting bacterial DNA from the gut microbiota of five mammals, including humans (Homo sapiens), macaques (Macaca mulatta), dogs (Canis lupus familiaris), golden hamsters (Mesocricetus auratus), and mice (Mus musculus). First, we assessed the efficiency of the four methods based on DNA yield, purity, and integrity. Then, we investigated the impact of these methods on microbial composition and diversity and examined the relative abundance of dominant phyla bacteria based on 16S rRNA gene sequencing and real-time quantitative PCR. Our results showed that the CTAB method yielded relatively larger amounts of DNA, while the MAGEN kit yielded more Firmicutes DNA and reflected the true status of the microbiota more accurately. Conclusions. Of the four methods tested, the traditional CTAB method and commercial MAGEN kit accomplished the best performance in terms of DNA concentration and Firmicutes abundance across most of the tested species. As the CTAB method and MAGEN kit exhibited different advantages, we further tested and compared their DNA extraction performance on a defined microbiota comprising six strains from four dominant phyla to see which one better reflected the true status of the microbiota. In conclusion, the MAGEN kit was found to be superior to the CTAB method, as the testing results were closer to that of the defined microbiota.

Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping.

Next-generation sequencing (NGS) library construction often involves using restriction enzymes to decrease genome complexity, enabling versatile polymorphism detection in plants. However, plant leaves frequently contain impurities, such as polyphenols, necessitating DNA purification before enzymatic reactions. To overcome this problem, we developed a PCR-based method for expeditious NGS library preparation, offering flexibility in number of detected polymorphisms. By substituting a segment of the simple sequence repeat sequence in the MIG-seq primer set (MIG-seq being a PCR method enabling library construction with low-quality DNA) with degenerate oligonucleotides, we introduced variability in detectable polymorphisms across various crops. This innovation, named degenerate oligonucleotide primer MIG-seq (dpMIG-seq), enabled a streamlined protocol for constructing dpMIG-seq libraries from unpurified DNA, which was implemented stably in several crop species, including fruit trees. Furthermore, dpMIG-seq facilitated efficient lineage selection in wheat and enabled linkage map construction and quantitative trait loci analysis in tomato, rice, and soybean without necessitating DNA concentration adjustments. These findings underscore the potential of the dpMIG-seq protocol for advancing genetic analyses across diverse plant species.

Assessment of Common Factors Associated with Droplet Digital PCR (ddPCR) Quantification of Paratrichodorus allius in Soil.

This research investigated the factors associated with the quantitative detection of Paratrichodorus allius in soil using droplet digital PCR (ddPCR). Small-sized nematodes exhibited significantly lower DNA quantities compared to their medium and large counterparts. Soil pre-treatments (room temperature drying and 37 °C oven-drying) demonstrated no substantial impact on ddPCR detection, and soil storage (0-3 months at 4 °C) exhibited negligible alterations in DNA quantities. A commercial DNA purification kit improved the resulting quality of ddPCR, albeit at the cost of a notable reduction in DNA quantity. Upon assessing the impact of inhibitors from soil extracts, a higher inhibitor concentration (5%) influenced ddPCR amplification efficiency. Incorporating bovine serum albumin (BSA) (0.2 μg/μL or 0.4 μg/μL) into the ddPCR setup mitigated the issue. In brief, while ddPCR exhibits minimal sensitivity to soil pre-treatments and storage, higher concentrations of PCR inhibitors and the DNA purification process can influence the results. Despite ddPCR's capability to detect nematodes of all sizes, quantification may not precisely reflect soil population. Incorporating BSA into the ddPCR setup enhances both detection and quantification capacities. This study represents the first comprehensive investigation of its kind for plant-parasitic nematodes, providing crucial insights for application of ddPCR in nematode diagnosis directly from the soil DNA.

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.

A multilevel core-shell structure Fe3O4@SiO2@InGaO@SiO2 magnetic particles for DNA extraction

DNA extraction is greatly significant in biological detection fields, including species identification, pathogen detection, environmental monitoring, biosafety, and food safety. Scientific research and practical application must conduct high-quality trace DNA separation, purification and extraction. Herein, a two-step strategy successfully prepares a multilevel core–shell structure Fe3O4@SiO2@InGaO@SiO2 magnetic particles for trace DNA extraction. The morphologies, structures, elemental compositions, components, and properties of the materials are systematically characterized. Fe3O4@SiO2@InGaO@SiO2 magnetic particles in the chaotropic salt solutions are used to directly target DNA binding due to the existence of Si-O–H bond. The maximum DNA adsorption rate can highly reach 83.1 % after 700 °C annealing in the air for 30 min, far exceeding the adsorption rate of commercial reagent kit, which is attributed to the increased specific surface area. The excellent result is conducive to rapid DNA extraction in practical applications, especially in daily criminal cases, further promoting social security and stability.

Recovering short DNA fragments from minerals and marine sediments: A comparative study evaluating lysis and isolation approaches

AbstractMarine sediments as excellent climate archives, contain among other biomolecules substantial amounts of extracellular DNA. Through mineral binding, some of the DNA remains protected from degradation which aids its preservation. While this pool of DNA represents genomic ecosystem fingerprints spanning over millions of years, the capability of current DNA extraction methods in recovering mineral‐bound DNA remains poorly understood. We evaluated current sedimentary DNA extraction approaches and their ability to recover short DNA fragments from artificially created DNA‐mineral complexes involving pure clay minerals or quartz, as well as from different types of natural marine sediments. We separately investigated lysis (DNA release) and isolation steps (purification of DNA) comparing five different lysis buffers across two commonly used DNA isolation approaches: silica magnetic beads and liquid‐phase organic extraction and purification. The choice of lysis buffer significantly impacted the amount of recovered mineral‐bound DNA and facilitated selective desorption of DNA fragments. High molarity EDTA and phosphate lysis buffers recovered on average an order of magnitude more DNA from clay minerals than other tested buffers, while both isolation approaches recovered comparable amounts of DNA. In marine sediments, however, liquid‐phase organic extraction caused inhibitory effects in subsequent downstream applications (e.g., PCR), across all assessed DNA extracts, while silica magnetic beads induced inhibition only in half of the tested DNA extracts. Thus, the isolation approach, together with the lysis buffer, played a decisive role in successful library preparation with lysis buffer choice ultimately impacting final library fragment distribution. With this study, we underscore the critical importance of lysis buffer selection to maximize the recovery of mineral‐bound DNA and show its profound impact on recovered fragment lengths in sedimentary DNA extractions, a crucial factor alongside existing isolation approaches in facilitating high‐quality DNA extracts for downstream analysis related to ancient environmental DNA research.

Sustainable protein regeneration in encapsulated materials

Zhu etal. introduce MELG (materials engineered by living grafting), combining engineered microbes with non-living scaffolds for functional protein regeneration within. These MELGs can be used for long-term controlled release, enzyme-mediated biocatalysis, and DNA purification. This approach offers enhanced functionality and durability in bioactive materials compared to traditional non-living counterparts.

Assessment of genotoxicity in Clarias gariepinus exposed to lethal and sub-lethal concentrations of oilfield chemicals: DNA concentration, purity, and genetic implications

This study investigates the concentration and purity of DNA extracted from Clarias gariepinus after exposure to lethal concentrations (250 ml/l, 200 ml/l, 150 ml/l, 100 ml/l, 50 ml/l, 25 ml/l and 0.0 ml/l for the control) of Xylene: Diesel (X:D) and Oilfield-Based Emulsifiers (O-BE) over 96 hours using static bioassays and sub-lethal concentrations 0 ml/l (control), 12.8 ml/l, 25.6 ml/l, 38.4 ml/l, 51.2 ml/l and 64.0 ml/l) using renewal bioassay for 28 days. 210 juveniles were used for the experiment. The study used PCR and other methods to analyse DNA concentrations and purity in the fish samples. The results indicated variations in DNA concentration, purity, and the presence of micronuclei, suggesting genetic and cytogenetic responses to chemical exposures. The highest DNA concentration was recorded in the 25 ml/l group for both O-BE and X:D, while the lowest was in the 100 ml/l group. The genetic effects of Xylene: Diesel on the molecular level were also examined, with significant gaps in the alignments of experimental groups indicating notable differences in the sequences compared to the control group without chemical exposure. Through detailed analyses, including gel electrophoresis and sequence alignment, the study explores genetic variations and micronuclei frequencies in blood cells, shedding light on the ecotoxicological impacts and potential evolutionary implications of these oil-based substances on African catfish. Understanding the genetic variations induced by lethal concentrations of O-BE in Clarias gariepinus is crucial for assessing the broader environmental impact, as genetic changes in aquatic organisms can affect population health, adaptation, and ecosystem dynamics.

Biomineralization of Human Genomic DNA into ZIF-8, a Zeolite-Like Metal-Organic Framework.

We studied human genomic DNA isolated from lymphocytes of peripheral blood of healthy volunteers using Proba-NK kit (DNA-Technology LLC, Russia). Genomic DNA purity and concentration was estimated spectrophotometrically at 260/280 nm using Tecan Infinity 200 Pro plate reader (Tecan Instruments, Austria). ZIF-8 was synthesized in the physiological conditions (37°C) by mixing zinc salt and 2-methylimidazole aqueous solutions at different molar ratios. Human genomic DNA was encapsulated into ZIF-8 in similar conditions. The obtained MOF and DNA@ZIF-8 composite were studied using X-ray powder diffraction at the Phaser D2 XRPD device (Bruker, USA), and the specific surface area was estimated using Autosorb iQ porosimetry analyzer (Quantachrome, USA). The encapsulated DNA was quantified by dissolving DNA@ZIF-8 composite in the citrate buffer. DNA integrity was assessed by real-time allele-specific PCR (AS-PCR) using the kits for single nucleotide polymorphisms (Lytech, Russia) at the Quantstudio 6 Pro PCR machine (Thermo Scientific, USA). In case of using the kits with electrophoretic detection, the amplification was performed on the thermal cycler T100 (Thermo Scientific, USA). The polymer ZIF-8 and DNA@ZIF-8 composite were obtained at different molar ratios of zinc ions and 2-methylimidazole. We characterized their structure and specific surface area. Genomic DNA biomineralization efficacy was found to be about 7-8%. PCR indicated the integrity of non-selectively chosen loci within the biomineralized DNA. The study confirmed the possibility of human genomic DNA encapsulation into ZIF-8 metal-organic framework. After the biomineralization, DNA was found to preserve feasibility to be used in studies to investigate genetic constructs.

Подбор и оптимизация методов экстракции ДНК из листьев гледичии трехколючковой (Gleditsia triacanthos L.)

Abstract. Currently, molecular genetic methods using DNA markers are increasingly used in studies of polymorphism of various populations of woody and shrubby plants. The purpose of this work was the evaluation and selection of protocols for the isolation and purification of DNA from the leaves of Gleditsia triacanthos L. for further studies using DNA labeling. Methods. Four protocols were used to isolate DNA from the leaf blade of Gleditsia triacanthos L. Anionic detergent sodium dodecyl sulfate was used in three isolation protocols for cell lysis, potassium acetate was used for purification from polysaccharides and proteins. In the fourth protocol, a cationic surfactant cetyltrimethyl ammonium bromide was used for cell lysis, the extract was purified with a mixture of chloroform-isoamyl alcohol (24 : 1). Precipitation of the isolated DNA was carried out with isopropanol. The quality of the isolated DNA was evaluated by spectrophotometry, horizontal electrophoresis and Real-time PCR with two types of primers. Results. Optimal conditions for DNA extraction from samples of Gleditsia triacanthos L. containing a large number of metabolites affecting the quality of the isolated extract were selected. By electrophoresis, it was found that both the isolation protocol with sodium dodecyl sulfate and the isolation protocol with cetyltrimethyl ammonium bromide make it possible to obtain a sufficient amount of DNA. The most purified DNA was obtained by the third protocol using sodium dodecyl sulfate and dithiotreitol and by the fourth protocol using cetyltrimethylammonium bromide. The results of PCR of the obtained samples with ITS and psbI-psbK primers indicate that a sufficient amount of product has been obtained and the reproducibility of ISSR markers. The scientific novelty of the work consists in choosing the optimal method of DNA extraction from the leaves of Gleditsia triacanthos L., which is a complex object containing a large number of potential PCR inhibitors. The protocol with sodium dodecyl sulfate and dithiotreitol made it possible to obtain DNA in the right amount and of acceptable quality.

Cell-Free Microbial DNA Analysis: Effects of Blood Plasma and Serum Quantity, Biobanking Protocols, and Isolation Kits.

Recent studies highlight the presence of bacterial sequences in the human blood, suggesting potential clinical significance for circulating microbial signatures. These sequences could presumably serve in the diagnosis, prediction, or monitoring of various health conditions. Ensuring the similarity of samples before bacterial analysis is crucial, especially when combining samples from different biobanks prepared under varying conditions (such as different DNA extraction kits, centrifugation conditions, blood collection tubes, etc.). In this study, we aimed to analyze the impact of different sample collection and nucleic acid extraction criteria (blood collection tube, centrifugation, input volume, and DNA extraction kit) on circulating bacterial composition. Blood samples from four healthy individuals were collected into three different sample collection tubes: K2EDTA plasma tube, sodium citrate plasma tube, and gel tube for blood serum. Tubes were centrifugated at standard and double centrifugation conditions. DNA extraction was performed using 100, 200, and 500 μL plasma/serum input volumes. DNA extraction was performed using three different isolation kits: Norgen plasma/serum cell-free circulating DNA purification micro kit, Applied Biosystems MagMAX cell-free DNA isolation kit, and Qiagen QIAamp MinElute cell-free circulating DNA mini kit. All samples were subjected to 16S rRNA V1-V2 library preparation and sequencing. In total, 216 DNA and 18 water control samples were included in the study. According to PERMANOVA, PCoA, Mann-Whitney, and FDR tests the effect of the DNA extraction kit on the microbiota composition was the greatest, whereas the type of blood collection tube, centrifugation type, and sample input volume for the extraction had minor effects. Samples extracted with the Norgen DNA extraction kit were enriched with Gram-negative bacteria, whereas samples extracted with the Qiagen and MagMAX kits were enriched with Gram-positive bacteria. Bacterial profiles of samples prepared with the Qiagen and MagMAX DNA extraction kits were more similar, whereas samples prepared with the Norgen DNA extraction kit were significantly different from other groups.

Development of a high intensity focused ultrasound device for bio-samples preparation

A miniaturized, low-cost high-intensity focused ultrasound device is developed for the problems of cross-contamination and uneven sample fragmentation in conventional ultrasound devices. This device generates ultrasonic waves through a concave spherical self-focusing piezoelectric ceramic piece, and creates a cavitation effect in the focusing area to achieve sample fragmentation. The feasibility of the device is demonstrated by physical simulation, then a driving circuit with adjustable power is designed and manufactured to generate 0 ~ 22.4 W acoustic power, and finally paraffin-embedded sample dewaxing experiments are performed to verify the validation of the device. The experimental results show that the dewaxing efficiency and safety of the high-intensity focused ultrasound device is significantly better than those of traditional chemical methods, and this device is comparable with commercial ultrasonic instruments. In summary, the high-intensity focused ultrasound device is expected to be applied in automated nucleic acid extraction and purification equipment and has a broad application prospect in the field of sample pre-processing.

Technical Note: A simple FTA® based method for the direct STR amplification of human foetal tissues

Short tandem repeats (STRs) or microsatellites are short, tandemly repeated DNA sequences that involve a repetitive unit of 1–6 bp. DNA isolation and purification from a large number and often compromised samples gives problems to forensic labs for STR typing. Many of the conventional methods used in the isolation and purification of DNA from forensic samples are time consuming, expensive, hazardous for health and are often associated with greater risks of cross contamination. FTA® technology is a method designed to simplify the collection, shipment, archiving and purification of nucleic acid from a wide variety of biological samples. We report a new method for the direct STR amplification which can amplify STR loci from human foetal tissues spotted on FTA cards, bye-passing the need of DNA purification. The STR loci amplified by this method was compared with conventional method of STR profiling and was found absolutely matching. Therefore, this new method is demonstrated to be very useful for fast, less expensive and non- hazardous forensic DNA analysis.

Enhancing the authenticity of animal by-products: harmonization of DNA extraction methods from novel ingredients.

Introduction: The increasing global pressure to explore alternative protein sources derived from animal by-products has opened-up opportunities, but it has also created the need to assess their compliance with labelling statements, to ensure consumer's trust in the composition of both feed and food products. Assessing the authenticity of highly processed animal by-products, particularly within the rapidly expanding Halal food market, presents a significant challenge due to the lack of robust and standardized methodologies. However, the success of DNA based authenticity system is highly dependent on the extracted DNA quantity, quality, and purity ratios from heterogeneous matrices. Material and methods: In this work, nine DNA extraction methods were tested on selected processed animal by-products with high-value and interest for the feed industry: meals from poultry meat, blood and feather, and hydrolysates from swine meat and bone, fish, and black soldier fly. The proposed DNA extraction methods are developed to specifically target swine-specific mitochondrial region, as a case study. Results and discussion: Both the conventional CTAB method and the commercial kits, specifically Invisorb® Spin Tissue Mini and NucleoSpin™ Food, demonstrated superior extraction efficiency and quality ratios. Nevertheless, commercial kits enabled faster detection in comparison to the conventional methods. The absence of swine DNA was successfully validated and confirmed in all animal meals and hydrolysates that did not contain swine in their composition beforehand, demonstrating their compliance with the Halal market requirements.

Lateral Flow Biosensor for On-Site Multiplex Detection of Viruses Based on One-Step Reverse Transcription and Strand Displacement Amplification.

Respiratory pathogens pose a huge threat to public health, especially the highly mutant RNA viruses. Therefore, reliable, on-site, rapid diagnosis of such pathogens is an urgent need. Traditional assays such as nucleic acid amplification tests (NAATs) have good sensitivity and specificity, but these assays require complex sample pre-treatment and a long test time. Herein, we present an on-site biosensor for rapid and multiplex detection of RNA pathogens. Samples with viruses are first lysed in a lysis buffer containing carrier RNA to release the target RNAs. Then, the lysate is used for amplification by one-step reverse transcription and single-direction isothermal strand displacement amplification (SDA). The yield single-strand DNAs (ssDNAs) are visually detected by a lateral flow biosensor. With a secondary signal amplification system, as low as 20 copies/μL of virus can be detected in this study. This assay avoids the process of nucleic acid purification, making it equipment-independent and easier to operate, so it is more suitable for on-site molecular diagnostic applications.

Selective hydrophobic interaction chromatography for high purity of supercoiled DNA plasmids.

High purity of plasmid DNA (pDNA), particularly in supercoiled isoform (SC), is used for various biopharmaceutical applications, such as a transfecting agent for production of gene therapy viral vectors, for pDNA vaccines, or as a precursor for linearized form that serves as a template for mRNA synthesis. In clinical manufacturing, pDNA is commonly extracted from Escherichia coli cells with alkaline lysis followed by anion exchange chromatography or tangential flow filtration as a capture step for pDNA. Both methods remove a high degree of host cell contaminants but are unable to generically discriminate between SC and open-circular (OC) pDNA isoforms, as well as other DNA impurities, such as genomic DNA (gDNA). Hydrophobic interaction chromatography (HIC) is commonly used as polishing purification for pDNA. We developed HIC-based polishing purification methodology that is highly selective for enrichment of SC pDNA. It is generic with respect to plasmid size, scalable, and GMP compatible. The technique uses ammonium sulfate, a kosmotropic salt, at a concentration selective for SC pDNA binding to a butyl monolith column, while OC pDNA and gDNA are removed in flow-through. The approach is validated on multiple adeno-associated virus- and mRNA-encoding plasmids ranging from 3 to 12 kbp. We show good scalability to at least 300 mg of >95% SC pDNA, thus paving the way to increase the quality of genomic medicines that utilize pDNA as a key raw material.

Thread-powered cell lysis and isotachophoresis: unlocking microbial DNA for diverse molecular applications.

Central to the domain of molecular biology resides the foundational process of DNA extraction and purification, a cornerstone underpinning a myriad of pivotal applications. In this research, we introduce a DNA extraction and purification technique leveraging polypropylene (PP) threads. The process commences with robust cell lysis achieved through the vigorous agitation of interwoven PP threads. The friction between the threads facilitates cell lysis especially those microbes having tough cell wall. For purification of DNA, thread-based isotachophoresis was employed which makes the whole process swift and cost-effective. Lysed cell-laden threads were submerged in a trailing electrolyte which separated DNA from other cellular contents. The process was performed with a tailored ITP device. An electric field directs DNA, cell debris, trailing electrolyte, and leading electrolyte toward the anode. Distinct ion migration resulted in DNA concentrating on the PP thread's anode-proximal region. The SYBR green dye is used to visualize DNA as a prominent green zone under blue light. The purified DNA exhibits high purity levels of 1.82 ± 0.1 (A260/A280), making it suitable for various applications aiming at nucleic acid detection.

Comparison of Three Methods to Extract Plasmodium falciparum DNA from Whole Blood and Dried Blood Spots.

This study aimed to compare the effectiveness of three DNA extraction methods: the GF-1 Blood DNA Extraction Kit (GF-1 BD Kit), which employs a spin column along with lysing and washing buffers; the tris-ethylenediaminetetraacetic acid and proteinase K (TE-pK) method, which utilizes a combination of TE buffer and proteinase K for cell lysis; and DNAzol® Direct (DN 131), a single reagent combined with heating for the extraction process. Plasmodium falciparum DNA was extracted from both whole blood and dried blook spots (DBSs), with consideration of DNA concentration, purity, cost, time requirement, and limit of parasite detection (LOD) for each method. The target gene in this study was 18S rRNA, resulting in a 395-bp product using specific primers. In the comparative analysis, the DN 131 method yielded significantly higher DNA quantities from whole blood and DBSs than the GF-1 BD Kit and TE-pK methods. In addition, the DNA purity obtained from whole blood and DBSs using the GF-1 BD Kit significantly exceeded that obtained using the TE-pK and DN 131 methods. For LOD, the whole blood extracted using the DN 131, GF-1 BD Kit, and TE-pK methods revealed 0.012, 0.012, and 1.6 parasites/µL, respectively. In the case of DBSs, the LODs for the DN 131, GF-1 BD Kit, and TE-pK methods were 1.6, 8, and 200 parasites/µL, respectively. The results revealed that the TE-pK method was the most cost-effective, whereas the DN 131 method showed the simplest protocol. These findings offer alternative approaches for extracting Plasmodium DNA that are particularly well-suited for large-scale studies conducted in resource-limited settings.

Bioanthropological analysis of human remains from the archaic and classic period discovered in Puyil cave, Mexico.

Determine the geographic place of origin and maternal lineage of prehistoric human skeletal remains discovered in Puyil Cave, Tabasco State, Mexico, located in a region currently populated by Olmec, Zoque and Maya populations. All specimens were radiocarbon (14C) dated (beta analytic), had dental modifications classified, and had an analysis of 13 homologous reference points conducted to evaluate artificial cranial deformation (ACD). Following DNA purification, hypervariable region I (HVR-1) of the mitogenome was amplified and Sanger sequenced. Finally, Next Generation Sequencing (NGS) was performed for total DNA. Mitochondrial DNA (mtDNA) variants and haplogroups were determined using BioEdit 7.2 and IGV software and confirmed with MITOMASTER and WebHome softwares. Radiocarbon dating (14C) demonstrated that the inhabitants of Puyil Cave lived during the Archaic and Classic Periods and displayed tabular oblique and tabular mimetic ACD. These pre-Hispanic remains exhibited five mtDNA lineages: A, A2, C1, C1c and D4. Network analysis revealed a close genetic affinity between pre-Hispanic Puyil Cave inhabitants and contemporary Maya subpopulations from Mexico and Guatemala, as well as individuals from Bolivia, Brazil, the Dominican Republic, and China. Our results elucidate the dispersal of pre-Hispanic Olmec and Maya ancestors and suggest that ACD practices are closely related to Olmec and Maya practices. Additionally, we conclude that ACD has likely been practiced in the region since the Middle-Archaic Period.