Direct wastewater extraction as a simple and effective method for SARS-CoV-2 surveillance and COVID-19 community-level monitoring.

Wastewater surveillance has proven to be an effective tool to monitor the transmission and emergence of infectious agents at a community scale. Workflows for wastewater surveillance generally rely on concentration steps to increase the probability of detection of low-abundance targets, but preconcentration can substantially increase the time and cost of analyses while also introducing additional loss of target during processing. To address some of these issues, we conducted a longitudinal study implementing a simplified workflow for SARS-CoV-2 detection from wastewater, using a direct column-based extraction approach. Composite influent wastewater samples were collected weekly for 1 year between June 2020 and June 2021 in Athens-Clarke County, Georgia, USA. Bypassing any concentration step, low volumes (280 µl) of influent wastewater were extracted using a commercial kit, and immediately analyzed by RT-qPCR for the SARS-CoV-2 N1 and N2 gene targets. SARS-CoV-2 viral RNA was detected in 76% (193/254) of influent samples, and the recovery of the surrogate bovine coronavirus was 42% (IQR: 28%, 59%). N1 and N2 assay positivity, viral concentration, and flow-adjusted daily viral load correlated significantly with per-capita case reports of COVID-19 at the county-level (ρ = 0.69-0.82). To compensate for the method's high limit of detection (approximately 106-107 copies l-1 in wastewater), we extracted multiple small-volume replicates of each wastewater sample. With this approach, we detected as few as five cases of COVID-19 per 100 000 individuals. These results indicate that a direct-extraction-based workflow for SARS-CoV-2 wastewater surveillance can provide informative and actionable results.

Determination of bongkrekic acid in corn flour by direct extraction ultra-high performance liquid chromatography tandem mass spectrometry / 公共卫生与预防医学

Objective To establish a direct extraction ultra-high performance liquid chromatography tandem mass spectrometry method for the determination of bongkrekic acid in corn flour. Methods Bongkrekic acid was directly extracted with 80% methanol from corn flour samples, and the supernatant after vortex and centrifugation was determined after passing through membrane filtration. At the same time, the corn flour samples were extracted by solid phase extraction. The determination results of the two methods were compared. Results The linearity of standard series was good within the range of2-20 μg/L, and the linearity coefficient was＞0.999. The determination result of the positive sample by direct extraction method was 193.40 mg/kg (n=6). Adding the standard to the blank sample at the levels of 2, 6, and 10 μg/L, the calculated recovery rate was 75.82% - 99.33%, and the relative standard deviation was 3.54 % - 8.45%. The detection limit of the method reached 6 μg/kg. After extraction by solid phase extraction, the determination result of the positive sample was 196.84 mg/kg (n=6). The recovery rate was 77.12% -100.83%, with a relative standard deviation of 8.32% - 9.54%. Conclusion Compared with the solid phase extraction, the direct extraction method for the extraction of bongkrekic acid from corn flour has the advantages of rapidity, simplicity, and cost savings.

Quantitation of propionic acid in emmental cheese products by direct solvent extraction.

Propionic acid (PA) is commonly present in Emmental cheese (EM) with relatively high concentration. Even though PA is allowed as GRAS in the USA, it may cause neurological disorders if it is consumed excessively. The maximum level of the preservative PA in cheese is set at 3.0 g/kg by the Korean Food Code. Also, the minimum level of PA in EM (150 mg / 100 g) was set by Codex in 1967. Therefore, PA monitoring in EM, with relatively high level of intrinsic PA, is necessary to recommend the sound use of the PA in the EM ripened. The current official PA analysis method consisting of steam distillation and solvent extraction / evaporation, is too cumbersome to be handled in sample preparation process. In this study, acidic acetone-based direct extraction method was successfully applied to 12 EM samples for the determination of PA level.

Direct Liquid Extraction and Ionization Techniques for Understanding Multimolecular Environments in Biological Systems (Secondary Publication).

A combination of direct liquid extraction using a small volume of solvent and electrospray ionization allows the rapid measurement of complex chemical components in biological samples and visualization of their distribution in tissue sections. This review describes the development of such techniques and their application to biological research since the first reports in the early 2000s. An overview of electrospray ionization, ion suppression in samples, and the acceleration of specific chemical reactions in charged droplets is also presented. Potential future applications for visualizing multimolecular environments in biological systems are discussed.

Sewage, Salt, Silica and SARS-CoV-2 (4S): An economical kit-free method for direct capture of SARS-CoV-2 RNA from wastewater.

Wastewater-based epidemiology is an emerging tool to monitor COVID-19 infection levels by measuring the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. There remains a need to improve wastewater RNA extraction methods' sensitivity, speed, and reduce reliance on often expensive commercial reagents to make wastewater-based epidemiology more accessible. We present a kit-free wastewater RNA extraction method, titled "Sewage, Salt, Silica and SARS-CoV-2" (4S), that employs the abundant and affordable reagents sodium chloride (NaCl), ethanol and silica RNA capture matrices to recover 6-fold more SARS-CoV-2 RNA from wastewater than an existing ultrafiltration-based method. The 4S method concurrently recovered pepper mild mottle virus (PMMoV) and human 18S ribosomal subunit rRNA, both suitable as fecal concentration controls. The SARS-CoV-2 RNA concentrations measured in three sewersheds corresponded to the relative prevalence of COVID-19 infection determined via clinical testing. Lastly, controlled experiments indicate that the 4S method prevented RNA degradation during storage of wastewater samples, was compatible with heat pasteurization, and could be performed in approximately 3 hours. Overall, the 4S method is promising for effective, economical, and accessible wastewater-based epidemiology for SARS-CoV-2, providing another tool to fight the global pandemic.

[Determination of propionic acid and its salts in food by gas chromatography].

A method for the determination of propionic acid and its salts in food by gas chromatography (GC) was developed. Degrease extraction and direct extraction were established for food containing oil and non-oil, respectively. In this study, the effects of different pH values on the solubility and recovery of propionic acid were investigated. The effects of purification conditions, pH values, extraction agent types, and extraction times were studied. After hydrochloric acid solution was added, the pH levels of sample solutions were all found to be less than 2. The sample solutions were degreased with 5 mL n-hexane and extracted twice with 5 mL ethyl acetate. The analytes were detected by GC. The analytes were separated on an HP-INNOWAX chromatographic column and detected by fame ionization detector (FID). The recoveries of degrease extraction were 87.5%-97.6% and relative standard deviation were 3.09%-6.86% (n=6). The recoveries of direct extraction were 90.1%-102.1% and the relative standard deviations were 3.32%-6.33% (n=6). The two methods showed good linearities in the range of 2-1000 mg/L (correlation coefficient is 0.9998). The limit of detection was 0.003 g/kg and the limit of quantification was 0.01 g/kg. The proposed method is accurate, fast, simple, easy, sensitive, and is suitable for the rapid determination of propionic acid and its salts in different food. The proposed method provides a new way for the determination of propionic acid in food.

[Supramolecular solvent-based direct extraction for the determination of polycyclic aromatic hydrocarbons in water].

Supramolecular solvent (SUPRAS) is a nano-structured liquid generated from amphiphiles through a sequential self-assembly process. It is an efficient and excellent solvent for the sample extraction. In this paper, a method to directly extract and rapidly analyze polycyclic aromatic hydrocarbons (PAHs) in water samples by high performance liquid chromatography-fluorescence detection (HPLC-FLD) was developed. The composition and amount of SUPRAS were optimized and practical samples were tested. It indicated that the combination of tetrahydrofuran and 1-octanol was a suitable SUPRAS for the extraction of four PAHs with recoveries between 89.08% and 102.47% and relative standard deviations (RSDs) from 1.38% to 3.92% (n=5). Results showed a good linearity of four PAHs with the correlation coefficients (R2) more than 0.999. The limits of detection (LODs) ranged from 1.26 to 9.23 ng/L. The proposed pretreatment method greatly reduces the analysis time. And the solvent-less approach is in accordance with the development trend of green chemistry and of great application prospects.

Determination of Sulfurous Anhydride in Rhizoma Dioscoreae by Ion Chromatography-Direct Extraction / 中国药师

Objective:To establish a quantitative analysis method to determine the content of sulfurous anhydride in Rhizoma di-oscoreae by ion chromatography-direct extraction. Methods:Sulfurous anhydride was extracted by KOH solution (25 mmol·L-1). An IonPac? AS11-HC column(250 mm × 4 mm, 9. 0 μm) was used. The column temperature was 20℃, the eluent was KOH solution (20 mmol·L-1 ) at flow rate of 1. 00 ml·min-1 and the conductivity temperature was 20℃. Results:There was a good linear rela-tionship between the injection quantity (1.160-29.100 μg)and the peak area of sulfite(r =0.999 9). The average recovery was 98. 9%(RSD=0. 6%, n=9). The quantitation limit was 1. 38 ng·ml-1. Conclusion: The method is simple, accurate and rapid, which is appropriate for the quantitative analysis of sulfite anhydride in Rhizoma dioscoreae.

A simple and rapid modified-new method for DNA extraction of FTA bloodstains / 中国法医学杂志

Objective To establish a simple and rapid new method for DNA extraction of FTA bloodstain samples.Methods Genomic DNA was extracted from FTA bloodstains of 1.2mm diameter by FTA-DNA direct extraction and FTA routine method respectively,and their genotypes were analyzed using ABI IdentifilerTM kit in 10?l and 25?l of reaction volume respectively.Results For 25?l of reaction volume,all DNA extracted by two different methods was successfully genotyped.For 10?l of reaction volume,however,the typing success rate of DNA extracted by FTA routine method was significantly lower than those by FTA-DNA direct extraction procedure.Using FTA routine method,the value of RFU ranged from 100 to 2000,and the peak imbalance result from preferential amplification of the smaller allele was a common phenomenon.Moreover,allelic dropout occurred in approximately nineteen percent of samples,and this was not obviously improved even if performed by automatic DNA workstation.However,using FTA-DNA direct extraction procedure,the typing results were similar to those in 25?l of reaction volume,and better results can be obtained using automatic DNA workstation.Conclusion The FTA-DNA direct extraction method is simple and rapid,and can be used to automatic establishment of DNA database with FTA bloodstains.