Development of an aptasensor for dibutyl phthalate detection and the elucidation of assay inhibition factors.

We developed a fluorescence aptasensor (hereafter 'SG-aptasensor') using SYBR Green I, a newly truncated 20-mer aptamer, and probe DNA to detect dibutyl phthalate (DBP). The detection range of DBP was 0.1-100 ng L-1 with 0.08 ng L-1 as the limit of detection. To adapt the assay to environmental samples in the near future, possible inhibition factors (experimental and environmental) have been tested and reported. The experimental inhibitors included the incubation time, temperature, pH, and ionic strength. Consequently, temperature (2-25 °C) and pH (7.0-9.0) ranges did not significantly inhibit the assay. The incubation time required for sufficient reaction was at least 4 h, and a relative humidity <20% may have induced fluorescence quenching. Tris-HCl-based incubation buffer with excess ionic strength (more than 0.2 M NaCl) demonstrated an abnormal increase in fluorescence. Environmental inhibitors including cations (Mg2+, Ca2+, and Cu2+) and humic acids were tested. The fluorescence signal was significantly reduced (∼99%) by 100 mM Cu2+ compared to that by 0 mM Cu2+. In contrast, the reduction in fluorescence signal was marginal (<15%) when Mg2+ or Ca2+ ions were present. Inhibition of the assay was observed (∼28%) in the presence of 100 mg L-1 humic acids.

Tackling Challenges of Long-Term Electrode Stability in Electrochemical Treatment of 1,4-Dioxane in Groundwater.

Electrochemical advanced oxidation is an appealing point-of-use groundwater treatment option for removing pollutants such as 1,4-dioxane, which is difficult to remove by using conventional separation-based techniques. This study addresses a critical challenge in employing electrochemical cells in practical groundwater treatment─electrode stability over long-term operation. This study aims to simulate realistic environmental scenarios by significantly extending the experimental time scale, testing a flow-through cell in addition to a batch reactor, and employing an electrolyte with a conductivity equivalent to that of groundwater. We first constructed a robust titanium suboxide nanotube mesh electrode that is utilized as both anode and cathode. We then implemented a pulsed electrolysis strategy in which reactive oxygen species are generated during the anodic cycle, and the electrode is regenerated during the cathodic cycle. Under optimized conditions, single-pass treatment through the cell (effective area: 2 cm2) achieved a remarkable 65-70% removal efficiency for 1,4-dioxane in the synthetic groundwater for over 100 h continuous operation at a low current density of 5 mA cm-2 and a water flux of 6 L m-2 h-1. The electrochemical cell and pulse treatment scheme developed in this study presents a critical advancement toward practical groundwater treatment technology.

Multi-target aptamer assay for endocrine-disrupting phthalic acid ester panel screening in plastic leachates.

A multi-target aptamer assay was developed as a phthalic acid ester (PAE) panel to screen selected PAEs in plastic leachate samples. The panel comprises 13 PAEs (PAE-13), namely dimethyl phthalate, diethyl phthalate, di-n-butyl phthalate, di-n-hexyl phthalate, diisobutyl phthalate, diisononyl phthalate, diisodecyl phthalate, mono-2-ethylhexyl phthalate, di-2-ethylhexyl phthalate, diphenyl phthalate, butyl benzyl phthalate, dicyclohexyl phthalate, and phthalic acid. Herein, we proposed an aptamer assay using a newly truncated aptamer (20-mer) and the 7-aminoactinomycin D fluorophore, which selectively binds to guanine in single-stranded DNA, resulting in increased fluorescence intensity. The assay is highly selective for PAE-13 clusters. The selectivity of the assay was evaluated using 13 different PAEs and mixtures depending on the side chain structure. The quantitative detection of PAEs was demonstrated by adopting mixed PAE-13 simulants and achieved a limit of detection of ∼1.4 pg/mL. The repeatability and reproducibility of the assay were also evaluated by presenting acceptable coefficients of variation (%CV less than 10% and 15%, respectively). The performance of the assay was demonstrated by analyzing the plastic leachate samples, and the positive correlation (correlation coefficient, r = 0.985) was confirmed by comparing them with the total sum of individual PAE peak areas obtained by gas chromatography mass spectrometry analysis.

Peroxymonosulfate-Based Electrochemical Advanced Oxidation: Complication by Oxygen Reduction Reaction.

Peroxymonosulfate (PMS)-based electrochemical advanced oxidation processes (EAOPs) have received widespread attention in recent years, but the precise nature of PMS activation and its impact on the overall process performance remain poorly understood. This study presents the first demonstration of the critical role played by the oxygen reduction reaction in the effective utilization of PMS and the subsequent enhancement of overall pollutant remediation. We observed the concurrent generation of H2O2 via oxygen reduction during the cathodic PMS activation by a model nitrogen-doped carbon nanotube catalyst. A complex interplay between H2O2 generation and PMS activation, as well as a locally increased pH near the electrode due to the oxygen reduction reaction, resulted in a SO4•-/•OH-mixed oxidation environment that facilitated pollutant degradation. The findings of this study highlight a unique dependency between PMS-driven and H2O2-driven EAOPs and a new perspective on a previously unexplored route for further enhancing PMS-based treatment processes.

Palladium Single-Atom (In)Stability Under Aqueous Reductive Conditions.

Here, we investigate the stability and performance of single-atom Pd on TiO2 for the selective dechlorination of 4-chlorophenol. A challenge inherent to single atoms is their high surface free energy, which results in a tendency for the surface migration and aggregation of metal atoms. This work evaluates various factors affecting the stability of Pd single-atoms, including atomic dispersion, coordination environment, and substrate properties, under reductive aqueous conditions. The transition from single atoms to clusters vastly enhanced dechlorination kinetics without diminishing carbon-chlorine bond selectivity. X-ray absorption spectroscopy analysis using both in situ and ex situ conditions followed the dynamic transformation of single atoms into amorphous clusters, which consist of a unique unsaturated coordination environment and few nanometer diameter. The intricate relationship between stability and performance underscores the vital role of detailed characterization to properly determine the true active species for dehalogenation reactions.

Pulsed Electrolysis of Boron-Doped Carbon Dramatically Improves Impurity Tolerance and Longevity of H2O2 Production.

Electrocatalytic water treatment has emerged in the limelight of scientific interest, yet its long-term viability remains largely in the dark. Herein, we present for the first time a comprehensive framework on how to optimize pulsed electrolysis to bolster catalyst impurity tolerance and overall longevity. By examining real wastewater constituents and assessing different catalyst designs, we deconvolute the complexities associated with key pulsing parameters to formulate optimal sequences that maximize operational lifetime. We showcase our approach for cathodic H2O2 electrosynthesis, selected for its widespread importance to wastewater treatment. Our results unveil superior performance for a boron-doped carbon catalyst over state-of-the-art oxidized carbon, with high selectivity (>75%) and near complete recoveries in overpotentials even in the presence of highly detrimental Ni2+ and Zn2+ impurities. We then adapt these fine-tuned settings, obtained under a three-electrode arrangement, for practical two-electrode operation using a novel strategy that conserves the desired electrochemical potentials at the catalytic interface. Even under various impurity concentrations, our pulses substantially improve long-term H2O2 production to 287 h and 35 times that attainable via conventional electrolysis. Our findings underscore the versatility of pulsed electrolysis necessary for developing more practical water treatment technologies.

A multi-functional reagent suitable for 1-step rapid DNA intercalation fluorescence-based screening of total bacteria in drinking water.

The prerequisites for rapid screening of total bacteria in drinking water are low detection limit and convenience. Inspired by commercial adenosine 5'-triphosphate (ATP) based total bacterial detection kits, we pursued likewise convenience but with much lower detection limit. Existing intercalation fluorescence-based techniques employ multiple reagents to permeate the cell membrane and intercalate dye into the DNA in discrete sequential steps. A simple multi-functional reagent is proposed to do the same within one step. Surfactants (TritonX and SDS), and intercalating dyes (SYBR green, SYBR gold) were examined for their mutual compatibility and augmented with EDTA. Evaluation was performed with Gram negative Escherichia coli K12 (E. coli K12) and Gram positive Bacillus subtilis (B. subtilis) at serial dilution ratios from 10-6 to 10-2. Comparison was made with absorbance (600 nm) measurements and a commercial ATP kit. Using charge integrated photodetection, the proposed 1-step reagent achieved an LOD (1.00 × 10-6, B. subtilis) that is two orders of magnitude lower than that of ATP kit (LOD = 1.06× 10-4). This means it could detect minute quantity of total bacteria that is otherwise undetected by the ATP kit.

Impact of COVID-19 Pandemic on Biomedical Publications and Their Citation Frequency.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has resulted in enormous related publications. However, the citation frequency of these documents and their influence on the journal impact factor (JIF) are not well examined. We aimed to evaluate the impact of COVID-19 on biomedical research publications and their citation frequency. METHODS: We searched publications on biomedical research in the Web of Science using the search terms "COVID-19," "SARS-Cov-2," "2019 corona*," "corona virus disease 2019," "coronavirus disease 2019," "novel coronavirus infection" and "2019-ncov." The top 200 journals were defined as those with a higher number of COVID-19 publications than other journals in 2020. The COVID-19 impact ratio was calculated as the ratio of the average number of citations per item in 2021 to the JIF for 2020. RESULTS: The average number of citations for the top 200 journals in 2021, per item published in 2020, was 25.7 (range, 0-270). The average COVID-19 impact ratio was 3.84 (range, 0.26-16.58) for 197 journals that recorded the JIF for 2020. The average JIF ratio for the top 197 journals including the JIFs for 2020 and 2021 was 1.77 (range, 0.68-8.89). The COVID-19 impact ratio significantly correlated with the JIF ratio (r = 0.403, P = 0.010). Twenty-five Korean journals with a COVID-19 impact ratio > 1.5 demonstrated a higher JIF ratio (1.31 ± 0.39 vs. 1.01 ± 0.18, P < 0.001) than 33 Korean journals with a lower COVID-19 impact ratio. CONCLUSION: COVID-19 pandemic infection has significantly impacted the trends in biomedical research and the citation of related publications.

Switchable inhibitory behavior of divalent magnesium ion in DNA hybridization-based gene quantification.

Contrary to the understanding that divalent cations only result in under-estimation of gene quantification via DNA hybridization-based assays, we have discovered that Mg2+ could cause either under or over-estimation at different concentrations. Its switchable inhibitory behavior is likely due to its rigid first solvation (hydrated) shell and hence it is inclined to form non-direct binding with DNA. At low concentrations, it caused under-estimation by occupying the hybridization sites. At high concentrations, it caused probe, signaling and target DNA to aggregate non-specifically via Coulomb forces. By quantifying target DNAs at a range of Mg2+ concentrations using a gene quantification assay (NanoGene assay), a Mg2+ inflection concentration of ∼10-3 M was observed for both target ssDNA and dsDNA. Field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDS), and Fourier transform infrared spectroscopy (FT-IR) were employed to observe Mg2+-induced non-specific binding in the complexes that mimicked the presence of target DNA. Together with two other divalent cations Ca2+ and Cu2+, they were further examined via zeta potential measurements as well as NanoGene assay. This study revealed the importance of Mg2+ in achieving accurate gene quantification. Through a better mechanistic understanding of this phenomenon, it will be possible to develop strategies to mitigate the impact of Mg2+ on DNA hybridization-based gene quantification.

Toxic Effects on Oxidative Stress, Neurotoxicity, Stress, and Immune Responses in Juvenile Olive Flounder, Paralichthys olivaceus, Exposed to Waterborne Hexavalent Chromium.

Juvenile Paralichthys olivaceus were exposed to waterborne hexavalent chromium at various concentrations (0, 0.5, 1.0, and 2.0 mg/L) for 10 days. After chromium exposure, the activities of superoxide dismutase and glutathione S-transferase, which are oxidative stress indicators, were significantly increased; however, the glutathione level was significantly reduced. Acetylcholinesterase activity as a neurotoxicity marker was significantly inhibited upon chromium exposure. Other stress indicators, including plasma cortisol and heat shock protein 70, were significantly increased. The immune response markers (lysozyme and immunoglobulin M) were significantly decreased after chromium exposure. These results suggest that exposure to environmental toxicity in the form of waterborne chromium at concentrations higher than 1.0 mg/L causes significant alterations in antioxidant responses, neurotransmitters, stress, and immune responses in juvenile olive flounders. This study will provide a basis for an accurate assessment of the toxic effects of hexavalent chromium on aquatic organisms.

Clustered Detection of Eleven Phthalic Acid Esters by Fluorescence of Graphene Quantum Dots Displaced from Gold Nanoparticles.

A gold nanoparticle-quenched graphene quantum dot-based aptasensor was developed to perform clustered detection of 11 phthalic acid esters (PAEs). The binding of the target PAEs to the aptasensor frees the graphene quantum dots that are otherwise quenched by the carrier gold nanoparticle. The resultant fluorescence upon excitation is proportional to the number of freed graphene quantum dots and hence the target PAE concentration. The synthesis of the proposed aptasensor was first verified step-by-step via FT-IR measurement, scanning electron microscopy, and fluorescence measurement. Selectivity was evaluated for individual and combined target PAEs and compared against seven non-PAE endocrine disrupting compounds. The proposed aptasensor successfully quantified 11 PAEs in test samples with varying concentrations of 0.001-50 ng PAEs/mL and demonstrated a limit of detection of ∼4 pg./mL. Finally, the AuNP-gQD aptasensor was employed to detect multiple combinations of commonly regulated PAEs (DBP, DIBP, DEHP, and BBP). The recovery (%) for all four PAEs combination in environmentally relevant concentrations of 0.5, 1, 5, and 10 ng/mL were ∼100%.

Differential virulence of infectious hematopoietic necrosis virus (IHNV) isolated from salmonid fish in Gangwon Province, Korea.

The present study investigated the virulence and expression of innate immunity genes in isolates of infectious hematopoietic necrosis virus (IHNV) in Gangwon province, South Korea, by challenging rainbow trout, Atlantic salmon, and coho salmon. Eight IHNV isolates were used to infect RTG-2 cells for viral replication using plaque assays. Three isolates with the highest replication rates, the RtPc0314g and RtPc0314c isolates of the JRt-Shizuoka type and the RtPc0816g isolate of the JRt-Nagano type, were experimentally infected into the fish. In rainbow trout, both RtPc0314c and RtPc0314g isolates showed 100% cumulative mortality while the RtPc0816g isolate showed 60% cumulative mortality for 14 days. In contrast, all three isolates showed <60% cumulative mortality in Atlantic salmon and coho salmon. The expression of G genes in the kidney was higher than that in the spleen-infected fish, with the highest expression observed in the kidneys of rainbow trout. The relative expression levels of innate immunity genes were higher in rainbow trout than in Atlantic salmon and coho salmon. The expression level of immunoglobulin M increased until day 7, and the expression of type I interferon was higher in the spleen than in other tissues. The expression of Mx-1 was higher in the kidney and liver than other tissues. These results indicate that IHNV isolates from Gangwon province show host-specific virulence in rainbow trout and that their virulence and replication were higher in JRt-Shizuoka type than in JRt-Nagano type isolates.

Chia seed-assisted separation and detection of polyvinyl chloride microplastics in water via gas chromatography mass spectrometry.

Chia seeds were used to significantly improve the separation efficiency of polyvinyl chloride (PVC) microplastics from water samples via centrifugation. Upon hydration, the mucilage of chia seeds were able to capture PVC microplastics with sizes ranging from tens to hundreds of micrometers. Since PVC microplastics contained di-2-etylhexyl phthalate (DEHP) as a plasticizer (verified via Fourier transform infrared spectrometry), DEHP was used as an indicator in the subsequent quantification via gas chromatography - mass spectrometry (GC-MS) analysis. Specifically after verifying the DEHP peak in the GC spectrum using DEHP reference standard as a positive control, the GC spectral area of that peak was used to quantify the amount of DEHP in the sample. Using nominal operation settings at 10 min and 1000 rpm with 100 mg of chia seeds, the separation efficiency could be improved by 5 times (500%) as compared to the absence of chia seeds. Furthermore, chia seeds were also compatible with simulated synthetic wastewater samples. Most importantly, the use of chia seeds did not interfere with GC-MS quantification protocol and accuracy. The result suggested the proposed method can be used as a simple screening tool of microplastics entering wastewater treatment plant, even though a series of follow-up studies are needed in future.

Single-stranded DNA probe paired aptasensor with extra dye binding sites to enhance its fluorescence response in the presence of a target compound.

The purpose of this study is to investigate the possibility of improving the performance of a DNA binding dye water quenching based aptasensor without changing or truncating the aptamer. To demonstrate the possibility of increasing the change in fluorescence of the aptasensor by pairing it with a suitable ssDNA probe, three ssDNA probes (probe 1, 2, and 3) were employed and the fluorescence from the bound dyes was measured. This showed that ssDNA probe 2 created the most additional binding sites. By varying the target compound concentration (0, 0.05, 0.5, 5, 50, and 500 mg L-1 4-n-nonylphenol), the corresponding change in the fluorescence signal of the unpaired and ssDNA probe paired aptasensors were measured and compared over a range of emission wavelengths. The response of all three ssDNA probe paired aptasensors showed good fit (R 2 = 0.88-0.92) to a logarithmic response. The sensitivity of the aptasensor paired with ssDNA probe 2 was improved by ∼60%, whereas that of the aptasensor paired with ssDNA probe 3 was only improved by a marginal ∼3%. This study is a demonstration of using an appropriate ssDNA probe to increase the number of binding sites and hence the performance of a DNA binding dye and water quenched aptasensor. It is a possibility that can be extended to similar aptasensors without having to change or truncate the aptamer.

Development of non-equilibrium rapid replacement aptamer assay for ultra-fast detection of phthalic acid esters.

In this paper, we developed a non-equilibrium rapid replacement aptamer (NERRA) assay that performed ultra-fast (in 30 s) quantitative detection of phthalic acid esters (PAEs) without waiting for the reaction to reach equilibrium. NERRA assay employed fluorescence PoPo3 dye intercalated in an ssDNA aptamer to selectively detect and quantify the PAEs in water. As the intercalated dye was replaced by the PAEs and quenched in the water, the rate of fluorescence change became proportional to PAEs concentration. The sensitivity of NERRA assay was first evaluated with a commercial spectrofluorometer. The selectivity for PAE mixture, individual PAEs, and non-phthalate compounds were also investigated. NERRA assay was also able to quantitatively detect the PAEs in a common plastic product (picnic mat), and the results were compared with those of gas chromatography mass spectrometry. Finally, a custom analyzer (8.5 cm × 8.5 cm × 16.5 cm) was built to demonstrate the portability of the NERRA assay. Using a commercial spectrofluorometer, NERRA assay was able to quantitatively detect a PAE mixture in 30 min with an LOQ of 0.1 µg/L. Using the portable custom analyzer, the detection time was shortened to 30 s with a tradeoff in the LOQ (1 µg/L). In both cases, the LOQs remain within the environmentally relevant PAE concentrations of 0.1-1472 µg/L.

Waterborne manganese modulates immunity, biochemical, and antioxidant parameters in the blood of red seabream and black rockfish.

Immunotoxic effects of manganese (Mn) were investigated in the blood of the economically important marine fish, red seabream (Pagrus major) and black rockfish (Sebastes schlegelii) when exposed to different concentrations of Mn (0, 0.5, 1, and 2 mg L-1) for 14 days. During exposure, the levels of alternative complement activity in both fish were significantly lowered at 2 mg L-1 of Mn of exposure. Lysozyme activity was significantly decreased in black rockfish in all concentrations of Mn after 14 days, while in red seabream, the decrease was significant with concentrations of 1 and 2 mg L-1 of Mn after 7 and 14 days of exposure. A significantly low level was observed only in the 2 mg L-1-exposed red seabream on day 14 of exposure. The concentrations of hemoglobin, red blood cells, white blood cells, and total serum proteins were significantly decreased in both fish under exposure to 1 and 2 mg L-1 of Mn, while cortisol, alanine transferase, aspartate transaminase, and alkaline phosphatase levels were significantly increased compared to the levels of control groups. No significant change was found in serum glucose and albumin except in red seabream exposed to 2 mg L-1 of Mn for 14 days. The responses of the antioxidant defense system were significantly induced in both fish after exposure to 1 and 2 mg L-1 of Mn on day 7 and 14 of exposure. Taken together, alterations of these parameters suggest the immunotoxicity of waterborne Mn produced by the modulation of hematological components and the induction of oxidative stress in the blood of these marine fish.

Constant exposure to environmental concentrations of the antifouling biocide Sea-Nine retards growth and reduces acetylcholinesterase activity in a marine mysid.

Sea-Nine (4,5-dichloro-2-n-octyl-4-isothiazoline3-one; DCOIT) antifoulant has been widely used owing to its broad spectrum of biocide activity against major fouling organisms. In this study, several physiological parameters of a marine mysid were analyzed upon exposure to sublethal environmental concentrations (1 and 100 ng L-1) of Sea-Nine in two exposure conditions, intermittent (weekly; once per week) and constant (daily; once per 24 h) exposure, for 4 weeks. In both experimental conditions, growth retardation, acetylcholinesterase (AChE) activity, glutathione S-transferase (GST) activity, and number of newborn juveniles as second generation, together with their survival were measured. Morphometric parameters of total body, antennal scale, exopod, endopod, and telson were significantly retarded by 22%, 14%, 13%, and 24%, respectively, by daily exposure to 100 ng L-1 Sea-Nine for 4 weeks. Significant inhibition of AChE activity was observed at week 4 in the 100 ng L-1 daily Sea-Nine-exposed groups, whereas no significant GST activity was measured at the same experimental conditions. Inhibition of AChE activity would be associated with impairment of cholinergic system and may adversely modulate growth parameters of the mysid. The number of newly hatched juveniles from females that were exposed daily to 100 ng L-1 Sea-Nine was significantly lower than that of the control. Although no significant differences were observed between survival percentages of newborn juveniles for 30 days, mortality (NOEC and LC50) increased in the surviving offspring from the 100 ng L-1-exposed 1st generation of mysids. These findings suggested that constant exposure to Sea-Nine has detrimental effects on the growth parameters of marine mysids with inhibition of AChE activity.

Development of quantum dot aptasensor and its portable analyzer for the detection of di-2-ethylhexyl phthalate.

We have developed a quantum dot aptasensor (QD-aptasensor) and its accompanying portable analyzer for the detection of di-2-ethylhexyl phthalate (DEHP). This sensor is based on a newly screened aptamer (60-mer) via SELEX and shows a binding affinity of 213 nmol/L with DEHP. The 60-mer aptamer together with its three shorter truncated aptamers (45, 28, and 22-mer) as well as three different DNA probes (12, 9, and 13-mer) were further investigated to form the best combination for the QD-aptasensor. Using a 22-mer-truncated aptamer and a 12-mer DNA probe combination, the QD-aptasensor demonstrated excellent DEHP sensitivity with an LOQ = 0.5 pg/mL as well as good selectivity in the presence of other phthalate analogs. The binding between the truncated aptamers and DEHP was also characterized. Finally, a QD-aptasensor-based portable analyzer was also developed, and its equivalence to the laboratory protocol was established with a correlation coefficient r = 0.86 for DEHP concentrations ranging from 0.0005 to 100 ng/mL.

Quantitative screening for endocrine-disrupting bisphenol A in consumer and household products using NanoAptamer assay.

The NanoAptamer assay is a bisphenol A (BPA) quantification method that uses magnetic beads, quantum dot nanoparticles, and a BPA-specific aptamer. In this study, screening of various consumer and household products for BPA was demonstrated utilizing the NanoAptamer assay. First, the experimental conditions suitable for BPA detection using the NanoAptamer assay were examined in terms of incubation time, temperature, and buffer composition. The range of BPA quantification via the NanoAptamer assay was determined to be 0.005-1000 ng/mL of BPA. The selectivity was confirmed by detecting BPA in an analog mixture containing bisphenol S and bisphenol F. Finally, a leaching experiment using 20 consumer and household products classified into 4 categories was performed to demonstrate the capability of the NanoAptamer assay for BPA detection. The experiment was validated by high-performance liquid chromatography analysis (correlation coefficient, r = 0.99).

Necessity of purification during bacterial DNA extraction with environmental soils.

Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for polymerase chain reaction (PCR) assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification) method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification). The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium [Mg]) showed that sand samples containing less than 10 µg/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of Mg ion was different from other inhibitors due to the complexation interaction of Mg ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 µg/g of humic acids, less than 70% clay content and less than 0.01% Mg ion content.