Acid Extraction Method Research Articles

Diagnosis of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) by RT-PCR using different extraction protocols

RT-PCR method was performed using four different nucleic acid extraction methods to identify viroids; peach latent mosaic viroid (PLMVd) and hop stunt viroid (HSVd) which causes serious damage to stone fruits. Leaf samples were collected from fruit orchards showing viroid-like symptoms in İzmir province. Silica capture, citric buffer, lithium chloride, and ames buffer methods were used to extract total nucleic acids. The four extraction methods were compared using samples collected during the vegetation period from naturally infected trees (plum, apricot, and peach). They were evaluated with RT-PCR tests. In 64 stone fruit tree samples, only the silica capture method gave reliable and accurate results in RT-PCR molecular tests for the detection of PLMVd and HSVd. The other wielded nucleic acid extraction methods were found to be ineffective for the isolation of the viroid RNAs.

A NitroPure Nitrocellulose Membrane-Based Grapevine Virus Sampling Kit: Development and Deployment to Survey Japanese Vineyards and Nurseries.

We developed a NitroPure Nitrocellulose (NPN) membrane-based method for sampling and storing grapevine sap for grapevine virus detection. We devised an efficient nucleic acid extraction method for the NPN membrane, resulting in 100% amplification success for grapevine leafroll-associated virus 2 (GLRaV2) and 3 (GLRaV3), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine virus A, grapevine virus B, and grapevine red blotch virus (GRBV). This method also allowed the storage of recoverable nucleic acid for 18 months at room temperature. We created a sampling kit to survey GLRaV2, GLRaV3, and GRBV in Japanese vineyards. We tested the kits in the field in 2018 and then conducted mail-in surveys in 2020-2021. The results showed a substantial prevalence of GLRaV3, with 48.5% of 132 sampled vines being positive. On the other hand, only 3% of samples tested positive for GLRaV2 and none for GRBV.

Effect of enzyme‐assisted acid extraction on citrus high‐methoxyl pectin riched in RG‐I: preparation, structure characterisation and physico‐chemical properties

SummaryThe properties of pectin extracted by enzyme‐assisted nitric acid (EAE‐N) and pectin extracted by enzyme‐assisted oxalic acid (EAE‐O) were analysed and compared with pectin extracted by nitric acid (AE‐N) and pectin extracted by oxalic acid (AE‐O) systematically. The yield of pectin obtained by the enzyme‐assisted acid method (23.73% ~ 25.60%) was significantly higher than that by acid extraction method (19.75% ~ 20.60%). Monosaccharide analyses demonstrated that the enzyme‐assisted acid method was able to attain more branched rhamnogalacturonan I (RG‐I) regions (36.59% ~ 42.06%) than using acid solely (31.01% ~ 34.58%). Fourier transform infrared spectroscopy (FT–IR) and nuclear magnetic resonance (1H NMR) analyses indicated that there was no obvious difference in the characteristic structure of pectin. The atomic force microscopy (AFM) images revealed that EAE‐N and EAE‐O had more short side chains and cross‐link compared with AE‐N and AE‐O. Thermogravimetric analysis (TGA) shows that EAE‐N and EAE‐O have good thermal stability. All pectin samples showed a shear‐thinning fluid flow behaviour and an elastic gel‐like behaviour. The results suggested that enzyme‐assisted acid provided a viable alternative method for the extraction of pectin.

B-392 Standardization of Nucleic acid Extraction Method From Thyroid Fine Needle Aspiration (FNA) Cells on Stained and Fixed Slides for Next-Generation Sequencing Protocols

Abstract Background Thyroid Fine Needle Aspiration (FNA) cytopathology is the best method for histological classification of thyroid nodules. However, between 20%–30% of thyroid nodules show indeterminate classification and are often referred for surgery due to the malignancy risk. Post-surgical histological reports indicate that 84% of these cases are benign, highlighting a high rate of unnecessary surgeries. In this sense, molecular approaches using next-generation sequencing (NGS) have been useful to confirm benignity or malignancy. As important as the choice of a powerful methodology to evaluate different genes simultaneously such as NGS, is the standardization of nucleic acid extraction methods to allow sufficient quality recovery of these molecules. Pre-analytical steps involved in FNA slide preparation include fixation and stain methods that contribute to the low quality and poor recovery of nucleic acid, representing a great challenge to NGS analysis. Thus, this work aims to describe a standardization of nucleic acid extraction method from FNA slides for NGS protocols. Methodology Nucleic acid isolation from 30 FNA samples collected between 2016–2022 were performed by Trizol or RecoverAll kits, according to the manufacturer’s instructions (ThermoScientific). Initially, cells’ slides were counted in an optical microscope, considering cell clusters >10 (equivalent to 1 counter unit). Before extractions with Trizol, the slides were submerged in xylene for 15 min. For RecoverAll extractions, the varnished slides were previously exposed to dry ice for 5 min, while the slides containing coverslip were also exposed to dry ice and submerged in xylene for 15 min. In all cases, the slides were hydrated before scraping and then extracted. Subsequently, the extraction of whole slides and split slides was tested. To split slides, one-half of the slide was separated for RNA extraction, while the remaining half was for DNA extraction. Then, the DNA/RNA were quantified using the Qubit™ 4 Fluorometer equipment (ThermoScientific) and concentrated using Amicon® Ultra filters (Millipore). Results The DNA and RNA quantifications range from 0.0720–1.77 ng/µL and 0.508–7.43 ng/µL, respectively. Among samples, 12% (DNA) and 62.5% (RNA) presented concentration below the quantification limit (too low). Four samples extracted with Trizol did not obtain recovery of nucleic acid and this methodology was discontinued. The use of Amicon® filter improved all samples’ yield. The number of thyroid cell clusters was not proportional to the amount of DNA/RNA recovered. The split slides showed worst RNA/DNA recovery, being ideal to use at least two slides for each patient since co-extraction using the RecoverAll kit was not possible. Conclusion The isolations of nucleic acid from FNA slides are essential to performing subsequent molecular tests, such as NGS. However, specific protocols for nucleic acid extraction from FNA slides are hardly found in the literature. This work showed the combination of methodologies that allowed the scraping of material from FNA slides by exposure to dry ice in samples with varnish or coverslip. The nucleic acid recovery capacity described for this type of sample indicates a way of possibilities to improve the FNA extraction methodology.

Agrofood Waste and By-Product Valorization, Extraction, and Characterization of Pectin from the Waste Biomass Fruit Peel of Aframomum angustifolium Using Response Surface Methodology as Alternative Sources of a Functional Pectin

Applications of pectin in the food industry are strongly influenced by their source, structures, and extraction methods, which affect their functionalities. This research aims to extract and assess pectin’s physicochemical and functional properties from waste biomass peels of Aframomum angustifolium as an alternate source using acid (AAE) and microwave extraction (MAE) methods. Pectin extracted from A. angustifolium was compared based on yield, color, moisture, equivalent weight, methoxyl content (MC), and degree of esterification (DegE). Response surface experimental design was used to study the effect of the extraction process pectin such as the yield and the DegE. MAE had a significantly higher % yield of 4.74 ± 0.1% and a lower equivalent weight of 852.49 ± 16.59 mg/ml than AAE with 3.09 ± 0.03% and 882.1 ± 9.04 mg/ml, respectively, with light brown color. The lower moisture contents of 6.5%, MC of 33.06%, and DegE of 67.96% were obtained by MAE compared to 6.9%, 31.85%, and 66.61%, respectively, for AAE. The time and temperature had a positive significant effect ( p < 0.05) on % pectin yield for MAE and AAE, while time and pH squared had a negative. Temperature squared had a negative significance on % DE, and pH had a positive significance using AAE and MAE. Optimal conditions for MAE obtained were the power of 555.18W, pH of 2.79, and time of 40.69 min with optimum desirability of 0.829, while for acid extraction, the temperature of 72.95°C, pH of 2.31, and time of 142.55 min with the desirability of 0.88. A highly functionalized pectin can be extracted from the peels of A. angustifolium as an alternate source.

Extraction, characterization, and vascular response of proteins from catfish (Clarias batrachus L.) mucus

The mucus of fish is its first line of defense to the surrounding environment and has been found to possess a range of bioactivities. This study was conducted to extract, characterize, and evaluate the vascular response of mucus from catfish (Clarias batrachus L.) against an irritant. The fish mucus was extracted through acidic and aqueous extraction methods yielding 0.585±0.00675 mg/mL protein in the acidic extract while the aqueous extract contained 1.18±0.0106 mg/mL. The extracts also analyzed through FT-IR spectroscopy showed characteristic bands of Amide A at 3225 to 3280 cm−1, Amide I at around 1600 and 1700 cm−1, and Amide II from 1510 to 1580 cm−1. The crude protein extracts also showed vascular response using CAM assay, with the aqueous extracts having higher activity, 82.05%±8.882 irritating inhibition than the acidic extract, 48.72%±8.882 irritating inhibition. This study suggests that the mucus of catfish could potentially be a novel anti-irritant agent and could be administered in drug application.

Quantitative analysis of respiratory viruses based on lab-on-a-chip platform.

The quantitative analysis of respiratory viruses is of great importance for rapid diagnosis, precision medicine, and prognosis. Several current quantitative analysis systems have been proposed and commercialized. Although they have been proven in trials, quantitative analyzes based on real samples are still complex, time-consuming, and expensive. Therefore, they are not able to directly quantify real samples. In this work, we presented a lab-on-a-chip platform combined with an automated control system to achieve quantitative analysis from samples to results. We developed a multilayer integrated chip to rapidly extract and quantify RNA of coronavirus disease 2019 (COVID-19) pseudovirus from large-volume nasal swab samples. The dependence of the magnetic bead size and the interfacial effect was studied for the first time, and the conditions of immiscible filtration assisted by surface tension (IFAST) method for nucleic acid extraction were optimized to increase the nucleic acid recovery rate up to 85%. Inside the chip, a pneumatic valve was developed for automatic opening and closing of the liquid channel. The integrated chip platform and automatic control system presented here are advantageous for use in resource-limited settings (RLS). In addition, our method can be extended to other respiratory viruses and other sample types.

An improved digestion and analysis procedure for silicon in plant tissue.

Silicon (Si) in plant tissues reduces abiotic and biotic stress, but it is incorporated as silica (SiO2), which is difficult to solubilize for analysis. We modified an oven-induced tissue-digestion and analysis method to improve Si solubilization and validated its accuracy by quantifying the mass-balance recovery of Si from the hydroponic solution and plant tissues of cucumber (Cucumis sativus). Leaf, stem, and root tissues were dried, finely-ground, and digested in 12.5 molar sodium hydroxide at 95°C for 4 hours. Solutions were then acidified with 6 molar hydrochloric acid to achieve a pH below 2 for measurement of Si using the molybdate blue colorimetric method. Interference of phosphorus in the analysis was minimized by increasing the addition of oxalic acid from 0.6 to 1.1 molar. We recovered 101% ± 13% of the expected Si, calculated using mass-balance recovery, in leaf, stem, and root tissues across 15 digestions. This Si recovery was fourteen-fold higher than the standard acid-extraction method and similar to a USDA-ARS alkaline-extraction method. Our procedure offers a low-cost, accurate method for extraction and analysis of Si in plant tissues.

Comparisons of pulsed ultrasound-assisted and hot-acid extraction methods for pectin extraction under dual acid mixtures from onion (Allium cepa L.) waste.

The aim of this study is to compare the physicochemical properties and yields of pectins extracted from onion waste under hot acid (HAE) and pulsed ultrasound-assisted extraction (PUAE) methods using different organic-inorganic acids, their mixtures, and pure water. The extraction temperature for experiments carried out under HAE was kept at 90°C for 90 min, whereas PUAE experiments were accomplished at RT in 15 min. In general, HAE gave better pectin yields compared with PUAE due to the significance of the increasing extraction temperature for the release of pectin from the plant matrix. While the maximum pectin yield from onion waste was 16.22% for HAE, the highest yield for PUAE was 9.83%. PUAE provides less time- and energy-consuming extraction of pectin within 15 min and thus seems to be more economic compared with the HAE. According to the physicochemical properties (equivalent weight (EW), degree of esterification (DE), methoxyl (MeO), and galacturonic acid (Gal-A) contents) of obtained pectins, extracted pectins were mostly high methoxy pectin. While the DE and MeO values of pectins extracted in organic acid conditions under HAE were higher, these values were found to be higher for pectins extracted in inorganic acids under PUAE. For acid mixtures, the DE and MeO values of pectins under HAE were mostly found to be lower than those under PUAE. Sequential PUAE and HAE methods for the extraction of pectin from onion waste were also found to be useful in terms of obtaining higher yields and better physicochemical properties. The highest pectin yield was 20.32% for the sequential PUAE and HAE methods. FT-IR analyses of the extracted pectins by both HAE and PUAE methods showed similar vibration bands compared with those of commercial citrus pectin.

Development of RNA/DNA automated extraction and purification device for infectious disease diagnosis

Genetic tests using RNA/DNA are the most accurate for diagnosing infectious diseases and assessing disease susceptibility, including COVID-19. However, manual specimen handling and the risk of secondary infections by medical staff highlight the need for automated equipment. Automation methods, such as bead purification, have limitations with high-viscosity specimens, while column purification requires complex equipment. This study aimed to develop an automated device using the column purification method for safe and reliable infectious disease diagnosis. We compared the yield and purification of three nucleic acid extraction methods (centrifugation, pressurization, and depressurization) and examined the adaptation of the extraction methods to automated device. Furthermore, we examined the feasibility of extracting SARS-CoV-2 RNA from COVID-19 patients and using qPCR analysis to determine whether the extraction method could be used as a clinical analyzer. Results varied with different columns and reagents, but pressurization method was selected for the automated device's RNA/DNA extraction. Using an automated device equipped with a pressurization method, RNA extracted from pharyngeal fluids from COVID-19 patients who had already been diagnosed with SARS-CoV-2 by qRT-PCR again tested positive. These findings demonstrate the device's effectiveness for nucleic acid extraction and virus-targeted diagnostics. Moreover, it holds potential for genetic testing in fields like food and environmental measurements. The automated device addresses specimen handling challenges and provides a reliable tool for infectious disease diagnosis.

A simple and purification-free nucleic acid extraction method for rapid diagnosis of malaria

A simple and purification-free nucleic acid extraction method for rapid diagnosis of malaria

Dry Swab-Based Nucleic Acid Extraction vs. Spin Column-Based Nucleic Acid Extraction for COVID-19 RT-PCR Testing: A Comparative Study.

Conventional nucleic acid extraction involves usage of spin columns to isolate the RNA, but this is labor intensive. This study compares the spin column method with a dry swab-based method of extraction using a proteinase K buffer and subsequent heat inactivation. A total of 56 subjects were tested for COVID-19 by RT-PCR with probes targeting the E and RdRp genes by collecting two nasopharyngeal and two oropharyngeal swabs and subjecting one set to nucleic acid extraction by spin column and the other set to dry swab-based methods. Out of the 56 samples tested, 27 were positive for VTM-based extraction and 29 were negative. Dry swab-based extraction produced 22 positive results (sensitivity = 81.48%) and 34 negative results. The E gene was detectable in 25 samples by the dry swab method out of 27 samples that tested positive by the VTM-based method (sensitivity = 92.5%). The RdRp gene was detectable in 22 samples by the dry swab method out of 27 samples that tested positive by the VTM-based method (sensitivity = 81.48%). Concordance was 91% with discordance at 9% and a Kappa value of 0.82, indicating almost perfect agreement between the two methods. Our findings indicate that the dry swab method of nucleic acid extraction is a useful alternative to conventional spin column-based extraction with comparable sensitivity and specificity. The trial was registered with the Clinical Trials Registry of India (CTRI) with a CTRI registration number of CTRI/2021/12/038792.

Evaluation of the Autof ms1000 mass spectrometry for rapid clinical identification of filamentous fungi

BackgroundMatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized microbial identification. However, there is a lack of data on its performance in identifying filamentous fungi. The objective of our study was to evaluate the accuracy of the Autof ms1000 mass spectrometry for identifying filamentous fungi in the clinical microbiology laboratory.ResultsAmong 106 samples tested using the Autof ms1000 system, 101 (95.28%) were identified at the genus or species level, and 81 (76.41%) were accurately identified at the species level. Additionally, we developed a new rapid formic acid extraction method with simple pretreatment for filamentous fungi that saved time and provided accurate results.ConclusionsThe Autof ms1000 mass spectrometer proved to be a valuable tool for identifying filamentous fungi. However, upgrading the database is recommended for correctly identifying rare strains.

Hyaluronic Acid-Extraction Methods, Sources and Applications.

In this review, a compilation of articles in databases on the extraction methods and applications of hyaluronic acid (HA) was carried out. HA is a highly hydrated component of different tissues, including connective, epithelial, and neural. It is an anionic, linear glycosaminoglycan (GAG) primarily found in the native extracellular matrix (ECM) of soft connective tissues. Included in the review were studies on the extraction methods (chemical, enzymatical, combined) of HA, describing advantages and disadvantages as well as news methods of extraction. The applications of HA in food are addressed, including oral supplementation, biomaterials, medical research, and pharmaceutical and cosmetic industry applications. Subsequently, we included a section related to the structure and penetration routes of the skin, with emphasis on the benefits of systems for transdermal drug delivery nanocarriers as promoters of percutaneous absorption. Finally, the future trends on the applications of HA were included. This final section contains the effects before, during, and after the application of HA-based products.

A high-throughput plate method for nucleic acid extraction from beet leafhopper (Hemiptera: Cicadellidae) and potato psyllid (Hemiptera: Triozidae) for pathogen detection.

Plant pathogens that are transmitted by insect vectors cause considerable damage to crops when pests or pathogens are not detected early in the season and populations are not controlled. Knowledge of pathogen prevalence in insect pest populations can aid growers in their insect pest management decisions but requires the timely dissemination of results. This process requires that specimen capture, identification, nucleic acid extraction, and molecular detection of a pathogen(s) occur alongside a platform for sharing results. The potato psyllid (Bactericera cockerelli, Sulc; Hemiptera: Triozidae) and beet leafhopper (Circulifer tenellus, Baker; Hemiptera: Cicadellidae) transmit pathogens to potato and other vegetable or seed crops each season in the northwestern United States. While the potato psyllid has been tested for pathogen occurrence for the past decade, testing of the beet leafhopper is a new endeavor and substantially increases the specimen number that must be tested by our laboratories each season. To aid in the rapid processing of individual insect specimens, we optimized and validated a new high-throughput 96-well plate nucleic acid extraction method for use in place of a standard 1.5-ml single-tube extraction method. Processing efficiency, in terms of total specimens processed over a 2-day period, improved 2.5-fold, and the cost associated with processing a single sample was nearly cut in half with this newly developed plate nucleic acid extraction method. Overall, this method has proven to be an excellent tool for the rapid testing of large numbers of small, individual insect vectors to enable timely dissemination of data on pathogen prevalence to growers.

Optimization of nucleic acid extraction methods for rapid detection in pandemic situations or diseases with high prevalence

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Miniaturization and Automation Protocol of a Urinary Organic Acid Liquid-Liquid Extraction Method on GC-MS.

The aim of this study was to improve the extraction method for urinary organic acids by miniaturizing and automating the process. Currently, manual extraction methods are commonly used, which can be time-consuming and lead to variations in test results. To address these issues, we reassessed and miniaturized the in-house extraction method, reducing the number of steps and the sample-to-solvent volumes required. The evaluated miniaturized method was translated into an automated extraction procedure on a MicroLab (ML) Star (Hamilton Technologies) liquid handler. This was then validated using samples obtained from the ERNDIM External Quality Assurance program. The organic acid extraction method was successfully miniaturized and automated using the Autosampler robot. The linear range for most of the thirteen standard analytes fell between 0 to 300 mg/L in spiked synthetic urine, with low (50 mg/L), medium (100 mg/L), and high (500 mg/L) levels. The correlation coefficient (r) for most analytes was >0.99, indicating a strong relationship between the measured values. Furthermore, the automated extraction method demonstrated acceptable precision, as most organic acids had coefficients of variation (CVs) below 20%. In conclusion, the automated extraction method provided comparable or even superior results compared to the current in-house method. It has the potential to reduce solvent volumes used during extraction, increase sample throughput, and minimize variability and random errors in routine diagnostic settings.

Virus Inactivation by Formaldehyde and Common Lysis Buffers.

Numerous mammalian viruses are routinely analyzed in clinical diagnostic laboratories around the globe or serve as indispensable model systems in viral research. Potentially infectious viral entities are handled as blood, biopsies, or cell and tissue culture samples. Countless protocols describe methods for virus fixation and inactivation, yet for many, a formal proof of safety and completeness of inactivation remains to be shown. While modern nucleic acid extraction methods work quite effectively, data are largely lacking on possible residual viral infectivity, e.g., when assessed after extended culture times, which maximizes the sensitivity for low levels of residual infectiousness. Therefore, we examined the potency and completeness of inactivation procedures on virus-containing specimens when applying commonly used fixatives like formaldehyde or nucleic acid extraction/lysis buffers. Typical representatives of different virus classes, including RNA and DNA viruses, enveloped and non-enveloped, such as adenovirus, enterovirus, lentivirus, and coronavirus, were used, and the reduction in the in vitro infectiousness was assessed for standard protocols. Overall, a 30-minute incubation with formaldehyde at room temperature effectively inactivated all tested enveloped and non-enveloped viruses. Full inactivation of HIV-1 and ECHO-11 was also achieved with all buffers in the test, whereas for SARS-CoV-2 and AdV-5, only five of the seven lysis buffers were fully effective under the tested conditions.

Evaluation of A Simple DNA Extraction Method and Its Combination with Loop-Mediated Isothermal Amplification Assays for Rapid Plasmodium knowlesi Diagnosis.

The initial and vital stage in the diagnosis of malaria involves extracting DNA. The efficiency of malaria testing is restricted by the multiple steps involved in commercial DNA extraction kits. We attempted to improve an existing loop-mediated isothermal amplification (LAMP) for the detection of Plasmodium knowlesi by using a simple DNA extraction approach, making it a feasible option for mass screening. We utilized a simple nucleic acid extraction method directly from whole blood for the detection of P. knowlesi, taking only 5 min to complete. The extracted DNA was evaluated by two fluorescent-based LAMP and one colorimetric-based LAMP assay. The detection limit for both SYTO-LAMP and SYBR green-LAMP was 0.00001% and 0.0001% parasitemia, respectively. Meanwhile, neutral red-LAMP had a detection limit of 0.01% parasitemia. Combining this simple and inexpensive DNA extraction method, SYTO-LAMP could serve as an alternative molecular diagnosis for the detection of P. knowlesi and other human Plasmodium spp.

Antinutrients and metabolomic compounds of Bambara groundnut (Vigna subterranean) as affected by traditional processing by smallholder farmers.

Bambara groundnut (BG) (Vigna subterranean) is an underutilized, indigenous crop in South Africa that has nutritional and associated health benefits. Decreasing the antinutrients in food sources can potentially increase the digestibility of proteins and mineral absorption. To determine the effect of traditional processing (cooking) on the antinutrient content and metabolome of this crop, BG was sampled from 12 rural farms in three districts of the Mpumalanga province, South Africa. The four main colors that were identified (cream, orange, brown, and purple) were pooled together according to the district they were obtained from. One-half of each color sample obtained from each of the three districts was dehulled, color sorted, milled, and subjected to subsequent antinutrient and metabolome analyses, while the other half was cooked, air-dried, and milled prior to analyses. Samples were analyzed for phytate and tannins (antinutrients) by hydrochloric acid extraction methods as well as metabolome constituents by ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Phytate, tannins, as well as other metabolomic constituents, namely, catechin, epicatechin, procyanidin, as well as citric acid, were identified in all raw and cooked BG samples. The cooking process resulted in a significant decrease (p<0.05) in the phytate and tannin content as well as an increase in the health-associated phenolic compounds.