Sample Incubation Time Research Articles

Label-free and sensitive detection of Citrus Bark Cracking Viroid in hop using Ti3C2Tx MXene-modified genosensor

Since the discovery of Citrus bark cracking viroid (CBCVd) in hops in 2007, affected hop-growing countries such as Slovenia and Germany have been actively pursuing efficient and easily accessible diagnostic tools that could contribute to the early detection of CBCVd in the field. In the early stages of CBCVd infection, typical symptoms or subtle signs of spread may not be evident. Detection becomes feasible only when the plant begins to display symptoms. Unfortunately, there is currently no treatment available, which requires the removal of infected plants as the only viable solution. However, this approach leads to significant economic losses on a large scale. This work demonstrates the development and study of a sensitive, selective, and label-free impedimetric genosensor for the detection of CBCVd in total RNA hop samples. The genosensor is based on a supporting glassy carbon electrode modified with streptavidin-agarose beads, which serve as an effective immobilization layer for a biotinylated single-stranded DNA capture probe. The integration of a 2D-layered Ti3C2Tx MXene into the sensing architecture resulted in a significantly improved electroanalytical performance of the genosensor. Several fabrication and operational parameters were optimized, such as the streptavidin-agarose beads deposition time, capture probe immobilization time and concentration, and sample incubation time. The optimized genosensor exhibited a limit of detection of only 0.5 fg μL⁻¹ (5.5 fmol L−1) in combination with a one-hour incubation with the denatured total RNA hop extract, thus eliminating the need for an additional and laborious amplification step.

Establishment of enzyme-linked immunosorbent assay for beef and lamb contents in cooked meat

In this study, an enzyme-linked immunosorbent assay (ELISA) was established to detect beef and lamb components, and its performance was tested. Double-antibody sandwich ELISA was adopted and determined a coating concentration of capture antibody 3G5 of 1:4 000, a working concentration of enzyme-labeled antibody 2E7-horseradish peroxidase (HRP) of 1:1 000, a sample incubation time of 60 min and a detection antibody reaction time of 60 min. The specificity, sensitivity, repeatability and stability of this assay were determined. The limit of detection for beef and lamb skeletal muscle troponin I was 45 mg/kg, the inter-assay and intra-assay recovery rates ranged from 80.4% to 115.7%, the coefficients of variation were below 13.6%, and the cross reaction rates of the tissue components of chicken, duck and fish were below 13.4%. The sandwich ELISA method established in this study is stable and has high accuracy. The test results were consistent with the polymerase chain reaction (PCR) method at 50 and 100 g/kg. Therefore, this ELISA method can be used to quantitatively detect beef and lamb components in meat products.

High sensitivity and automatic chemiluminescence detection of glucose and lactate using a spin-disc paper-based device.

This paper reports a spin-disc paper-based device with 10 individual detection units containing electromagnetic modules controlling the sample incubation time before chemiluminescence (CL) signal detection. After the sample was added to the top paper chip and incubated with the enzyme, the electromagnet was turned off to allow contact between the top and bottom paper. The H2O2 generated by the sample flowed vertically to the bottom paper and initiated the oxidase of the luminol to generate the CL signal. After one detection the disc was automatically rotated to the next position to repeat the above detection. The advantage of using the device over the lateral flow and the in situ detection was firstly proved using the detection of H2O2 and the glucose/lactate sample with 5 minute incubation. The CL intensity was increased 300 times/1000 times as the glucose/lactate was incubated for 5 minutes compared to the non-incubated samples. Afterward, the device was employed to separately detect glucose and lactate diluted in PBS, artificial sweat, artificial saliva, and fresh cell culture media. Finally, the device was employed to detect the glucose and lactate in the media collected over the 24 hour culture of PC3 cells. The uptake and production rates of glucose and lactate were correspondingly determined as 0.328 ± 0.015 pmol h-1 per cell and 1.254 ± 0.053 pmol h-1 per cell, respectively. The reported device has wide application potential due to its capabilities in automatic detection of multiple samples with very high sensitivity and small sample volume (down to 0.5 μL).

Displacement of PAMAM-Au via acoustic streaming on an electrochemical immunosensing platform

The displacement of an electroactive monitoring agent, i.e., polyamidoamine dendrimers encapsulated gold nanoparticles (PAMAM-Au) upon the presence of a target antibody via acoustic streaming has been studied. Acoustic streaming has been used to improve the mass transfer and reduce the sample incubation rate, thus this study investigated its ability in enhancing the PAMAM-Au displacement efficiency of our immunosensor. For this purpose, the bio-nanogate components of maltose-binding protein carrying the antigenic determinant (MBP-aD) of hepatitis B surface antigen (HBsAg) as a bioreceptor was functionalized, followed by the monitoring agent i.e. PAMAM-Au on the electrode prior to the incubation with targeted anti-hepatitis B surface antigen (anti-HBsAg) antibody. The modified electrode was then coupled with a piezotransducer and connected to the signal transducer to induce acoustic streaming upon sample incubation. Under optimal acoustic actuation, the sample incubation time has been reduced from 20 min to 8 min via the enhancement of PAMAM-Au displacement induced by acoustic streaming. The result also demonstrated that the specificity and selectivity of the sensing platform under acoustic actuation are comparable to the static incubation in detecting the targeted antibody.

Screening and identification of neuraminidase inhibitors from Baphicacanthus cusia by a combination of affinity ultrafiltration, HPLC-MS/MS, molecular docking, and fluorescent techniques

Natural products provide a new opportunity for the discovery of neuraminidase (NA)inhibitors. In this study, an affinity ultrafiltration (AUF) coupled with HPLC-MS/MS method was firstly developed and optimized for screening of NA inhibitors from natural products. The critical factors influencing the interaction of enzyme-ligand (including sample concentration, enzyme concentration, incubation time and temperature, pH of the buffer, and dissociation solvents and time) were investigated and optimized by a one-factor-at-a-time design. The method was then applied to discover NA inhibitory compounds in stems and leaves of Baphicacanthus cusia. As a result, five active alkaloids were screened out and identifiedas 2,4(1H,3H)-quinazolinedione (1), 4(3H)-quinazolinone (2), 2(3H)-benzoxazolone (3), tryptanthrin (4), and indirubin (5) through analysis of their DAD profiles, MS/MS fragments, and comparison with reference substances. These active compounds were further evaluated for their NA inhibitory activity using a fluorescence-based NA inhibition assay. The result from the fluorescent assay revealed that all the five compounds(1–5) showed pronounced NA inhibitory activities with IC50values of 98.98, 64.69, 40.16, 69.44, and 144.73 μM, respectively. Finally, molecular docking of these five alkaloids with NA showed that hydrogen bond and π-cation interactions dominated within the binding sites with binding energies ranging between −5.7 to −7.9 kcal/mol, which was supported by the results of the AUF and the fluorescence-based enzyme assay. The developed AUF method is simple and efficient for screening potential NA inhibitors from stems and leaves of B. cusia.

Automated solid phase DNA extraction on a lab-on-a-disc with two-degrees of freedom instrumentation

BackgroundLab-on-a-disc (LoaD) technology has emerged as a transformative approach for point-of-care diagnostics and high-throughput testing. The promise of integrating multiple laboratory functions onto a single integrated platform has significant implications for healthcare, especially in resource-limited settings. However, one of the primary challenges faced in the design and manufacture of LoaD devices is the integration of effective valving mechanisms. These valves are essential for fluid control and routing, but their intricacy often leads to complexities in design and increased vulnerability to failure. This emphasizes the need for improved designs and manufacturing processes without complex, integrated valving mechanisms. (96) ResultsWe describe a fully automated biological workflow and reagent actuation on a LoaD device without an integrated valving system. The Two Degrees-of-Freedom (2DoF) custom centrifuge alters the centre of rotation, facilitating fluid flow direction changes on the microfluidic platform through a custom programmed interface. A novel 360-degree fluid manipulation approach via secondary planetary gear motion enabled sequential assay reagent actuation without embedded valve triggering, with the addition of infinite incubation times and efficient use of platform realty. The simplified LoaD platform uses clever design, with intermediate flow chambers to avoid cross contamination between reagent steps. Notably, the optimized LoaD platform demonstrated a two-fold DNA yield at higher HEK-293 cell concentrations compared to commercially available spin-column kits. This significantly simplified LoaD platform successfully automated a common, complex workflow without inhibiting DNA purification. (129) SignificanceThis system exhibits the clever coupling of both 2DoF and centrifugal microfluidics to create an autonomous testing package capable of eradicating the need for complex valving systems to automate biological workflows on LoaDs. This automated system has outperformed commercially available DNA extraction kits for higher cell counts. The platform's elimination of valve requirements ensures unlimited sample incubation times and enhances reliability, making it a straightforward option for automated biological workflows, particularly in diagnostics. (73)

Validation of the CompactDry "Nissui" BC for Enumeration of Bacillus cereus in a Variety of Foods: AOAC Performance Tested MethodSM 092201.

The CompactDry "Nissui" BC is a ready-to-use dry media sheet using a chromogenic medium with selective agents for the detection and enumeration of Bacillus cereus in products after incubation at 30 ± 1°C for 24 ± 2 h. The CompactDry "Nissui" BC method was validated to achieve AOAC Performance Tested MethodsSM certification. The performance of the CompactDry "Nissui" BC was compared to that of ISO 7932:2004 for 10 matrixes, including panna cotta, double cream, dried baby food, dried vegetable soup mix, seafood sticks, salmon pâté, sliced ham, pork liver pâté, ham and cheese sandwich, and Caesar pasta salad with chicken and bacon. Performance indicators included repeatability, difference of means (DOM), and inclusivity/exclusivity. After log10 transformation of the data, the relative standard deviation of repeatability (RSDr) was ≤9.2% for 28 of the 30 materials (10 matrixes each at three contamination levels) analyzed by the CompactDry "Nissui" BC method and ≤13% for 27 of the 30 matrix/level combinations analyzed by the reference method. Method equivalence was demonstrated in 28 of the 30 matrix/level combinations based on the 90% confidence interval of the DOM being within (-0.5, 0.5). For inclusivity, 47 of 50 strains tested showed typical colonies and confirmed positive. For exclusivity, 28 of 33 strains tested resulted in no growth or were negative, and five were positive. Inclusivity and exclusivity results were similar on the reference method agar. The method was shown to be robust to changes in sample volume, incubation temperature, and incubation time, and data are presented supporting product consistency and 18-month shelf life. The CompactDry "Nissui" BC method is validated for the determination of Bacillus cereus in a variety of matrixes. The CompactDry "Nissui" BC method is equivalent to the ISO 7932:2004 reference method and is suitable for Performance Tested MethodsSM certification for the matrixes tested.

Mitigation of phytotoxic effect of compost by application of optimized aqueous extraction protocols

The abuse of chemical fertilizers in recent decades has led the promotion of less harmful alternatives, such as compost or aqueous extracts obtained from it. Therefore, it is essential to develop liquid biofertilizers, which in addition of being stable and useful for fertigation and foliar application in intensive agriculture had a remarkable phytostimulant extracts. For this purpose, a collection of aqueous extracts was obtained by applying four different Compost Extraction Protocols (CEP1, CEP2, CEP3, CEP4) in terms of incubation time, temperature and agitation of compost samples from agri-food waste, olive mill waste, sewage sludge and vegetable waste. Subsequently, a physicochemical characterization of the obtained set was performed in which pH, electrical conductivity and Total Organic Carbon (TOC) were measured. In addition, a biological characterization was also carried out by calculating the Germination Index (GI) and determining the Biological Oxygen Demand (BOD5). Furthermore, functional diversity was studied using the Biolog EcoPlates technique. The results obtained confirmed the great heterogeneity of the selected raw materials. However, it was observed that the less aggressive treatments in terms of temperature and incubation time, such as CEP1 (48 h, room temperature (RT)) or CEP4 (14 days, RT), provided aqueous compost extracts with better phytostimulant characteristics than the starting composts. It was even possible to find a compost extraction protocol that maximize the beneficial effects of compost. This was the case of CEP1, which improved the GI and reduced the phytotoxicity in most of the raw materials analyzed. Therefore, the use of this type of liquid organic amendment could mitigate the phytotoxic effect of several composts being a good alternative to the use of chemical fertilizers.

The peroxidase-like activity of papain colorimetrically detects H2O2 and glucose with high sensitivity

To date, several glucometer methods are available for patient-guided glycemic control, which diminishes the onset and severity of diabetic complications. In a dipstick urine glucose test, horseradish peroxidase (HRP) oxidizes tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2) to quantitatively produce a colorimetric signal and predict glucose concentration. Papain, a sulfhydryl protease secreted by the latex of Carica papaya, displays HRP-like activity with high selectivity. However, the current papain-based glucometer method lacks sensitivity. We hypothesized that catalyzing the oxidation of papain under varying buffer conditions (e.g., papain, TMB, and acetic acid concentrations) would improve glucose detection sensitivity. To test this, we performed glucose oxidase (GOx)-mediated glucose oxidation and papain-mediated TMB oxidation under varying sample conditions in vitro and spontaneously coupled them. The aims of this study were to validate the coupling of GOx-mediated glucose oxidation and papain-mediated TMB oxidation and identify the optimized conditions that maximize catalytic activity. We confirmed that glucose oxidase quantitatively produces H2O2 (in a glucose concentration-dependent manner), the presence of which catalyzes papain-mediated production of a colorimetric signal. When two oxidation reactions were coupled, we identified that the sensitivity of papain’s glucose detection increased by ten-fold compared to previous studies with a markedly high correlation (R2 = 0.99). In particular, substituting sodium acetate-acetic acid (HAc-NaAc) buffer with acetic acid and increasing the sample incubation time and papain concentration markedly increased the sensitivity of colorimetric signals. The optimized protocols can be integrated into emerging hydrogel technology to develop a non-invasive glucometer patch predicting blood glucose from the patient’s produced sweat.

Zymography: developing of the enzyme soil activity visualization method

The enzymes produced by the soil biota are a key link in the regulation of biochemical processes. The soil enzyme activity can be visualized with zymography, a method based on using fluorescent substrates and obtaining two-dimensional images (zymograms). A variant of a zymographic measuring system has been proposed. Characteristics of lighting, photographic equipment and shooting modes, reagents preparation and calibration are presented. Preparing and analyzing soil samples of different texture (sand and clay loam) and processing the study results have been described. The ways of introducing the substrate are considered in this study, namely pipetting, short-time dipping, and saturation. An analysis of the kinetics of incubation of samples was carried out. The possibilities and disadvantages of the method were also considered and options for solving possible methodological problems during the analysis were proposed. The zymography is a promising method that allows comparing data with the results of other methods. The use of neural network technologies makes it possible to obtain the volumetric distribution of soil enzymes with high reliability. The soil zymography requires qualitative preparatory work and extreme accuracy during the analysis. It is necessary to ensure maximum contact between the substrate and the soil, as this is one of the key factors determining the quality of the results. The most optimal way to introduce the substrate is to saturate the membranes with substrate solution for 60 minutes. At this stage of the development of the method, it is not possible to establish a universal sample incubation time, since this depends on characteristics of both the studied soils and the experiment conditions. Also, it is necessary to document the conditions in detail for discussion the study results.

A Review on the In Vitro Evaluation of the Anticholinesterase Activity Based on Ellman's Method.

Inhibition of cholinesterases is a common strategy for the treatment of several disorders, especially Alzheimer´s disease. In vitro assays represent a critical step towards identifying molecules with potential anticholinesterase effect. This study aimed at providing a comprehensive review of the methodologies used in vitro for the anticholinesterase activity based on the spectrophotometry of Ellman's method. This work used two databases (PubMed and ScienceDirect) to search for original articles and selected publications between 1961 and 2019, which reported in vitro spectrophotometry assays for anticholinesterase activity. After the search process and the selection of publications, the final sample consisted of 146 articles published in several journals submitted by researchers from different countries. Although the studies analyzed in this work are all within the same conception of in vitro tests based on Ellman's method, one can observe a wide divergence in the origin and concentration of enzyme, the choice and pH of the buffer, the concentration of the substrate, the sample diluent, incubation time, temperature, and time of the spectrophotometric reading interval. There is no consensus in the methodology of studies with in vitro tests for anticholinesterase assessment. The methodological variations related to kinetic parameters may interfere in the characterization of cholinesterase inhibitors.

Validation of the CompactDry "Nissui" YMR for Enumeration of Yeasts and Molds in a Variety of Foods: AOAC Performance Tested MethodSM 092002.

The CompactDry "Nissui" YMR is a ready-to-use dry media sheet using a chromogenic medium with selective agents for the detection and enumeration of yeasts and molds in products after incubation at 25 ± 1°C for 3 days. The CompactDry "Nissui" YMR method was validated in order to achieve AOAC Performance Tested MethodsSM certification. The performance of the CompactDry "Nissui" YMR was compared to that of ISO 21527-1:2008 for 10 matrixes including cooked prawns, deli vegetable salad, tuna pâté, fermented yogurt drink, spinach and ricotta quiche, egg custard tarts, fruit and vegetable smoothie, cream cheese, egg salad sandwich, and deli pasta salad. Performance indicators included repeatability, difference of means (DOM), and inclusivity/exclusivity. After log10 transformation of the data, the relative standard deviation of repeatability (RSDr) was <10% for all 30 materials (10 matrixes each at 3 levels) analyzed by the CompactDry "Nissui" YMR method and for 29 of the 30 matrix/level combinations analyzed by the reference method. The DOM ranged from -0.284 (-0.310, -0.257) log10 CFU/g to 0.307 (-0.013, 0.627) log10 CFU/g. Method equivalence was demonstrated in 29 of the 30 matrix/level combinations based on the 90% confidence interval of the DOM being within (-0.5, 0.5). All 51 inclusivity strains showed expected or atypical results, and all 32 exclusivity organisms showed no growth on the medium. The method was shown to be robust to changes in sample volume, incubation temperature, and incubation time, and data are presented supporting product consistency and a 24-month shelf life. The CompactDry "Nissui" YMR method is validated for the determination of yeasts and molds in a variety of matrixes. The CompactDry "Nissui" YMR method is equivalent to the ISO 21527-1:2008 reference method and is suitable for Performance Tested MethodsSM certification for the matrixes tested.

SARS-CoV-2 Aptasensors Based on Electrochemical Impedance Spectroscopy and Low-Cost Gold Electrode Substrates.

SARS-CoV-2 diagnostic practices broadly involve either quantitative polymerase chain reaction (qPCR)-based nucleic amplification of viral sequences or antigen-based tests such as lateral flow assays (LFAs). Reverse transcriptase-qPCR can detect viral RNA and is the gold standard for sensitivity. However, the technique is time-consuming and requires expensive laboratory infrastructure and trained staff. LFAs are lower in cost and near real time, and because they are antigen-based, they have the potential to provide a more accurate indication of a disease state. However, LFAs are reported to have low real-world sensitivity and in most cases are only qualitative. Here, an antigen-based electrochemical aptamer sensor is presented, which has the potential to address some of these shortfalls. An aptamer, raised to the SARS-CoV-2 spike protein, was immobilized on a low-cost gold-coated polyester substrate adapted from the blood glucose testing industry. Clinically relevant detection levels for SARS-CoV-2 are achieved in a simple, label-free measurement format using sample incubation times as short as 15 min on nasopharyngeal swab samples. This assay can readily be optimized for mass manufacture and is compatible with a low-cost meter.

Reactivity of the RSID™-Saliva test to α-amylase present in vaginal secretions

ABSTRACT Presumptive identification of saliva on exhibits collected in relation to sexual offences can provide pivotal evidence regarding specific activities which may have occurred. The immunochromatographic test, RSID™-Saliva, is often employed for this purpose and is reported to be highly specific for α-amylase present at high levels in human saliva. However, a concerning level of positive RSID-Saliva tests to vaginal secretions on the inner crotch of worn underwear was recently reported. Our study aimed to investigate this further using internal vaginal swabs and labial wipes from 30 female donors. The effect of a 1 and 2 hour sample incubation time on the number of positive reactions was also investigated. Positive reactions were observed in 3% of samples which was much lower than previously reported. Positive reactions were not detected for internal vaginal swabs when the incubation time was capped at 1 h and the RSID-Saliva test appeared suited for this application. Caution should be exercised when using the RSID-Saliva test for external samples as the risk of positive reactions appeared higher and was possibly due to cross-contamination with urine and faeces. The effect of shorter sample incubation times on detection of non-saliva biological fluids with the RSID-Saliva test should be investigated.

Antibody Surface Coverage Drives Matrix Interference in Microfluidic Capillary Immunoassays.

The performance of biosensors is often optimized in buffers, which brings inconsistencies during applications with biological samples. Current strategies for minimizing sample (matrix) interference are complex to automate and miniaturize, involving, e.g., sample dilution or recovery of serum/plasma. This study shows the first systematic analysis using hundreds of actual microfluidic immunoassay fluoropolymer strips to understand matrix interference in microflow systems. As many interfering factors are assay-specific, we have explored matrix interference for a range of enzymatic immunoassays, including a direct mIgG/anti-mIgG, a sandwich cancer biomarker PSA, and a sandwich inflammatory cytokine IL-1β. Serum matrix interference was significantly affected by capillary antibody surface coverage, suggesting for the first time that the main cause of the serum matrix effect is low-affinity serum components (e.g., autoantibodies) competing with high-affinity antigens for the immobilized antibody. Additional experiments carried out with different capillary diameters confirmed the importance of antibody surface coverage in managing matrix interference. Building on these findings, we propose a novel analytical approach where antibody surface coverage and sample incubation times are key for eliminating and/or minimizing serum matrix interference, consisting in bioassay optimization carried out in serum instead of buffer, without compromising the performance of the bioassay or adding extra cost or steps. This will help establishing a new route toward faster development of modern point-of-care tests and effective biosensor development.

Comparison of Four Streptococcus pneumoniae Urinary Antigen Tests Using Automated Readers.

Streptococcus pneumoniae urinary antigen tests (UATs) may be interpreted using automatic readers to potentially automate sample incubation and provide standardized results reading. Here, we evaluated four UATs the BinaxNOW S. pneumoniae Antigen Card (Abbott, Chicago, IL, USA), ImmuView S. pneumoniae and Legionella (SSI Diagnostica, Hillerød, Denmark), STANDARD F S. pneumoniae Ag FIA (SD Biosensor, Gyeonggi, South Korea), and Sofia S. pneumoniae FIA (Quidel Corporation, San Diego, CA, USA) with their respective benchtop readers for their ability to detect S. pneumoniae urinary antigen. We found that these assays had a sensitivity of 76.9–86.5%, and specificity of 84.2–89.7%, with no significant difference found among the four UATs. The assays had a high level of agreement with each other, with 84.5% of samples testing consistently across all four assays. The automatically and visually read test results from the two immunochromatographic assays, BinaxNOW and ImmuView, were compared and showed excellent agreement between the two types of reading. Immunofluorescent-based assays, Sofia and STANDARD F, had significantly less time to detect compared to the two immunochromatographic assays due to having less assay setup procedures and shorter sample incubation times. In conclusion, the four UATs performed similarly in the detection of S. pneumoniae urinary antigen, and readers can bring increased flexibility to running UATs in the clinical routine.

The Effect of Weight and Incubation Time in DNA Quality of Kepel (Stelechocarpus burahol) Leaves

Kepel (Stelechocarpus burahol) is a rare fruit plant which becomes the identity plant of the Yogyakarta Region. Kepel plants need to be conserved so they don’t become extinct Germplasm of kepel must be collected to get the genetic diversity of Kepel. Research on the genetic diversity of Kepel plants has never been done, so it needs the right method to obtain good quality DNA for molecular studies for future research. This study aims to obtain the best sample weight and incubation time to produce high-quality DNA for future research. This study used samples from young green leaves of kepel. DNA was extracted and purified from the leaves using a modified CTAB method to obtain DNA of kepel with high-purity and high-concentration. The result of DNA isolation and purification was analyzed by spectrophotometer and agarose gel electrophoresis method. The yield of DNA isolation on young kepel leaves had higher quality and quantity than DNA isolation on mature kepel leaves The results demonstrated that DNA isolation from young green leaves of kepel had a purity level of 1.64 – 2.01. In addition, it also showed that DNA isolation from young green leaves in weight 0.2 g with 30 minutes incubation gave the highest DNA concentration (880 ng/µl).

Determination of the appropriate ratio of sample size to nylon bag area for in situ nylon bag technique evaluation of rumen digestibility of feedstuffs in sheep

The in situ nylon bag technique (ISNBT) is traditional and necessary for the evaluation of ruminant feed, but there are exists some disunity operation schemes. Therefor, we select one way that varying SS:SA to determine the effects of it on feed degradation in the rumen. Eight Yunnan semi-fine wool sheep fitted with permanent ruminal cannul as were used and the nylon bags containing different sample size with fixed surface area. The results demonstrated that increasing the ratio of sample size to nylon bag area (SS:SA) lowered the effective dry matter (DM) degradability of rye straw. There was no difference in degradability with SS:SA ratios of 12.5 and 20.85 mg/cm2 but DM degradability was reduced (P < 0.05) at SS:SA ratios of 29.17 and 37.5 mg/cm2. At these higher SS:SA ratios DM degradability was also reduced (P < 0.05), at incubation durations of less than 36 h. A similar picture was obtained for rye straw crude protein (CP) degradability, although the effects of sample size and incubation time were less marked. In the case of the concentrated feedstuff, soybean meal, ruminal degradation was almost complete at 36 h and there was a trend for higher effective degradability (ED) at the lower SS:SA ratios. These results show that with a standard nylon bag with external dimensions of 6 × 10 cm, increasing the amount of sample, thus raising the ratio of sample size to bag surface area (SS:SA ratio) reduces feed degradation. It is recommended that sample size influenced feed degradation in the nylon bag technique and a sample weight of 2.5 g (SS:SA about 20 mg/cm2) should be used for evaluation of roughage and 3 g (SS:SA about 25 mg/cm2) for evaluation of concentrated diets for sheep using this technique.

The effect of pH and ageing on the fate of CuO and ZnO nanoparticles in soils

The objective of this study was to evaluate the fractionation of ZnO and CuO engineered nanoparticles (ENPs) in soils with a pH adjusted to 4.0, 6.5, and 9.0 after 1 day and 30 days of incubation. Based on the multi-stage extraction, 5 fractions of metals were determined. Moreover, the effect of ENPs on the activity of acid, neutral and alkaline phosphatase was determined. The results of the study revealed that pH had a dominant effect on the metal participation in soils. The levels of those fractions of metals differed between nano-ZnO and nano-CuO, which could have resulted from differences in the dissolution of the ENPs. After 1 day, the concentration of Zn2+ (0.02–7.4 mg L−1) was 10 times higher than that of Cu2+. The metal fractionation in soil treated with ENPs and metal salts may also confirm the role of ENP dissolution. The concentration of potentially bioavailable fraction of Zn increased with a drop in pH. At a 4 pH concentration of Zn in the treatment with nano-ZnO and ZnCl2 was at a similar level (42.1–45 mg kg−1), whereas the addition of nano-CuO resulted in a lower content of Cu (24.7 mg kg−1) than CuCl2 (36.5 mg kg−1). On the other hand, the concentration of fraction exchangeable of both metals in the alkaline soil did not exceed the level of 5.0 mg kg−1. Sample incubation time was especially important for metal participation in samples with a pH of 6.5. The greatest differentiation of metal fractionation between the soils was also noted at a pH of 6.5, which could also have been a result of other properties of the soils. The strong effect of pH on the lability of ENPs in soils confirmed a need to trace the fate of ENPs in extreme soil conditions as well as in changing environment.

Suitability of individual and bulk milk samples to investigate the humoral immune response to lumpy skin disease vaccination by ELISA

BackgroundThe detection of antibodies against capripoxvirus has become easier with a commercially available ELISA validated for serum and plasma. In order to explore its suitability for immunological investigations on alternative samples, this study targeted milk as sample matrix available through non-invasive sampling.MethodsSamples for this study were collected from dairy cows vaccinated against LSD in an area without reported LSD virus circulation. Paired serum and milk (individual and bulk) samples were tested by ELISA without and with modifications of the sample incubation time for the milk samples. For the evaluation of the test specificity, 352 milk samples from a milk repository in Germany were used as negative control. Receiver operating characteristic analysis was performed for determination of the Youden index and determination of the most suitable cut-off value for maximum specificity.ResultsFrom 154 analyzed serum samples from Serbia, 75 were detected as positive in the ELISA. Sensitivity and specificity of the ELISA test for milk samples reached values of 88 to 91% using Youden criteria. A cut-off of 10 was determined aiming for maximum specificity. This cut-off value was used for further analysis. Using the protocol for serum, out of 154 milk samples, 38 were detected as positive, number of positive detected milk samples increase up to 48 with modified protocol. Milk samples from Germany reacted negative, except two samples that had borderline results using modified protocol. Significant statistical difference (p < 0.05) was observed between two incubation protocols. The detection of LSD-specific antibodies from bulk milk samples (pools of 2–10 individuals) came along with a reduced sensitivity over the sample of individual animals.ConclusionsResults show that the detection of capripoxvirus specific antibodies in milk samples using the commercially available ELISA from IDvet is feasible and can represent a helpful tool for LSDV monitoring programs.