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A suitable enzymatic method for starch quantification in different organic matrices.

ABSTRACT: Starch can represent 70-80% of the cereals grains (on a dry matter basis) used for livestock feeding. Several methods have been developed to estimate the feed starch contents of energy feed sources. However, the efficiency of these methods to evaluate the starch content in other feed sources, as well as other types of samples used to evaluate starch availability in the gastrointestinal tract, such as digesta and faeces, remains unclear. Furthermore, most of the currently used starch analysis methods have not been effectively evaluated, being only applied to samples of sporadic experiments, without a wide-ranging validation of the procedures and results. Here, we propose a modification of a method for analysing the starch content in different organic matrices normally evaluated in ruminant nutrition studies. The evaluated organic matrices were: soybean meal, soybean hull, Tifton 85 Bermuda grass hay, abomasal digesta, and faeces. •The modified method is more feasible than the original procedures.•The modified method estimates the starch contents in different organic matrices with accuracy and precision.

SUBMITTER: Silva BC

PROVIDER: S-EPMC6812349 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A suitable enzymatic method for starch quantification in different organic matrices.

Silva Breno de Castro BC Godoi Letícia Artuzo LA Valadares Filho Sebastião de Campos SC Zanetti Diego D Benedeti Pedro Del Bianco PDB Detmann Edenio E

MethodsX 20191004

Starch can represent 70-80% of the cereals grains (on a dry matter basis) used for livestock feeding. Several methods have been developed to estimate the feed starch contents of energy feed sources. However, the efficiency of these methods to evaluate the starch content in other feed sources, as well as other types of samples used to evaluate starch availability in the gastrointestinal tract, such as digesta and faeces, remains unclear. Furthermore, most of the currently used starch analysis met ...[more]

PMID: 31667131

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Project description:Hepatitis B virus (HBV) is a para-retrovirus that reverse transcribes its pregenomic RNA into relaxed circular DNA inside viral nucleocapsids. The number of HBV genomes produced in vitro is typically quantified using commercial silica-membrane-based nucleic acid purification kits to isolate total DNA followed by HBV-specific quantitative PCR (qPCR). However, despite the convenience of commercial kits, this procedure is costly and time-consuming due to multiple centrifugation steps, which produce unnecessary waste. Here, we report a rapid, cost-effective, and environmentally friendly total DNA preparation method. The assay is based on the simple incubation of detergent and proteinase K with cells or cell-free supernatants to permeabilize cells and disrupt viral particles. After heat inactivation and subsequent centrifugation to clear the lysates, DNA samples are directly subjected to qPCR to quantify HBV genomes. As a proof of concept, the assay was developed in 12-well plates to assess intra- and extracellular HBV genome equivalents (GEqs) of stably viral-replicating cell lines (e.g., HepAD38) and HBV-infected HepG2-NTCP cells, both treated with lamivudine (LMV), an HBV replication inhibitor. Viral DNA was also prepared from the serum of patients chronically infected with HBV. To validate the assay, a representative commercial DNA isolation kit was used side-by-side to isolate intra- and extracellular HBV DNA. Both methods yielded comparable amounts of HBV GEqs with comparable LMV 50% efficient concentration (EC50) values. The assay was subsequently adapted to 96- and 384-well microtiter plates using HepAD38 cells. The EC50 values were comparable to those obtained in 12-well plates. In addition, the calculated coefficient of variation, Z' values, and assay window demonstrated high reproducibility and quality. We devised a novel, robust, reproducible, high-throughput microtiter plate DNA preparation method suitable for quantifying HBV GEqs by qPCR analysis. This strategy enables rapid and convenient quantitative analysis of multiple viral DNA samples in parallel to investigate intracellular HBV replication and the secretion of DNA-containing viral particles.

Project description:BackgroundChildren are vulnerable to environmental exposure of contaminants due to their small size, lack of judgement skills, as well as their proximity to dust, soil, and other environmental sources. A better understanding about the types of contaminants that children are exposed to or how their bodies retain or process these compounds is needed.ObjectiveIn this study, we have implemented and optimized a methodology based on non-targeted analysis (NTA) to characterize chemicals in dust, soil, urine, and in the diet (food and drinking water) of infant populations.MethodsTo evaluate potential toxicological concerns associated with chemical exposure, families with children between 6 months and 6 years of age from underrepresented groups were recruited in the greater Miami area. Samples of soil, indoor dust, food, water, and urine were provided by the caregivers, prepared by different techniques (involving online SPE, ASE, USE, QuEChERs), and analyzed by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Data post-processing was performed using the small molecule structure identification software, Compound Discoverer (CD) 3.3, and identified features were plotted using Kendrick mass defect plot and Van Krevelen diagrams to show unique patterns in different samples and regions of anthropogenic compound classifications.ResultsThe performance of the NTA workflow was evaluated using quality control standards in terms of accuracy, precision, selectivity, and sensitivity, with an average of 98.2%, 20.3%, 98.4% and 71.1%, respectively. Sample preparation was successfully optimized for soil, dust, water, food, and urine. A total of 30, 78, 103, 20 and 265 annotated features were frequently identified (detection frequency >80%) in the food, dust, soil, water, and urine samples, respectively. Common features detected in each matrix were prioritized and classified, providing insight on children's exposure to organic contaminants of concern and their potential toxicities.Impact statementCurrent methods to assess the ingestion of chemicals by children have limitations and are generally restricted by specific classes of targeted organic contaminants of interest. This study offers an innovative approach using non-targeted analysis for the comprehensive screening of organic contaminants that children are exposed to through dust, soil, and diet (drinking water and food).

Dataset Information

A suitable enzymatic method for starch quantification in different organic matrices.

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A suitable enzymatic method for starch quantification in different organic matrices.

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