Endotoxin, a novel biomarker for the rapid risk assessment of faecal contamination of coastal and transitional waters.

Current methods for testing water for faecal contamination rely on the culture of faecal indicator bacteria (FIB; Escherichia coli and Enterococci) that take 24-48 h, which leads to delays in taking proactive measures and poses a risk to public health. More rapid methods are therefore required. Here, we have tested a rapid, portable assay (Bacterisk) that detects the bacterial biomarker endotoxin in 30 min to quantify the bacterial biomass present, to evaluate 159 coastal water samples and to compare the results with the traditional culture of FIB. There was a significant correlation between the Bacterisk data given in endotoxin risk (ER) units and FIB culture that could accurately distinguish between poor and sufficient or good quality bathing water using the EU bathing directive values. Receiver operating characteristic analysis was used to determine the optimal ER threshold for coastal water samples, and the area under the curve was 0.9176 with a p-value of <0.0001. The optimal threshold was 7,300 ER units with a sensitivity of 95.45% and a specificity of 83.48%. In conclusion, we have shown that the Bacterisk assay provides a rapid and easy-to-use in situ method to assess bathing water quality.

Development of an online optical prototype for the simultaneous analysis of defects or gels in industrially synthesized polypropylene films. Part 1: Comparison with ASTM D 3351-93 methods.

Polypropylene (PP) films are crucial in various industrial applications, from packaging to medical products. However, a common challenge in PP manufacturing is the presence of gel-like defects. These gels are minor defects on the surface of the films that significantly affect the physicochemical, mechanical, and organoleptic properties of the films, compromising the quality of the final product. This first research focuses on developing and validating an in-line optical method to replace the international method ASTM D 3351-93. The main objective was to create a methodology that has the same scope and analytical performance as those reported by ASTM D 3351-93 in such a way that it can compete with it in terms of precision and accuracy, thus allowing end users to this ASTM, such as PP producers, PP marketers, PP film producers, among others internationally, can use this new methodology with necessary analytical support. This analytical methodology integrates the PP extrusion zones, the film processing stages, and the optical zone for reading and processing analytical data. Additionally, it has the advantage of working with a sample size that is even more representative of the population and has less human error since only one operator is required to carry out the test; this method also has much shorter response times. The developed prototype had 14 online stages that allowed representative quantities of samples to be taken and processed thermally and mechanically for ideal optical measurement. For the online method, a 6-point calibration curve is carried out at concentrations of 40, 10, 5, 2, 1 and 0 ppm for the gel or defect sizes of 200, 400, 500, 600, 700, 800 and 900 µm, showing excellent linearity where the correlation coefficient varied between 0.997 and 0.999, the limits of detection (LOD) varied between 0.85 and 2.61 and the limits of quantification (LOQ) ranged between 2.82 and 8.71. The statistical analyzes by ANOVA of the comparison between the ASTM D 3351-93 method and the proposed simultaneous method indicate that the p value of the evaluation of the means was 0.946, which suggests that the means are not statistically different. To complement, the Tukey test was carried out at 95 %, indicating that the methods have statistical equivalence.•Process optimization•Determination of defects or imperfections in PP films.

Vis-NIR spectroscopy based rapid and non-destructive method to quantitate microplastics: An emerging contaminant in farm soil.

Microplastics (MPs) is the second most important environmental issue and can potentially enter into food chain through farmland contamination and other means. There are no standardized extraction methods for quantification of MPs in soil. The embedded errors and biases generated serious problems regarding the comparability of different studies and leading to erroneous estimation. To address this gap, present study was formulated to develop an efficient method for MPs analysis suitable for a wide range of soil and organic matrices. A method based on Vis-NIR (Visible-Near Infra Red) spectroscopy is developed for four different soil belonging to Alfisol, Inceptisol, Mollisol and Vertisol and two organic matter matrices (FYM and Sludge). The developed method was found as rapid, reproducible, non-destructive and accurate method for estimation of all three-density groups of MPs (Low, Medium and High) with a prediction accuracy ranging from 1.9 g MPs/kg soil (Vertisol) to 3.7 g MPs/kg soil (Alfisol). Two different regression models [Partial Least Square Regression (PLSR) and Principal Component Regression (PCR)] were assessed and PLSR was found to provide better information in terms of prediction accuracy and minimum quantification limit (MQL). However, PCR performed better for organic matter matrices than PLSR. The method avoids any complicated sample preparation steps except drying and sieving thus saving time and acquisition of reflectance spectrum for single sample is possible within 18 s. Owing to have the minimum quantification limit ranging from 1.9-3.7 g/kg soil, the vis-NIR based method is perfectly suitable for estimation of MPs in soil samples collected from plastic pollution hotspots like landfill sites, regular based sludge amended farm soils. Additionally, the method can be adapted by small scale compost industries for assessing MPs load in product like city compost which are applied at agricultural fields and will be helpful in quantifying possible MPs at the sources itself.

DNA Aptamer-Based Staining and Fluorescence Microscopy for Rapid Detection of Cyclospora Cayetanensis Oocysts.

There are no commercial antibodies for detection of Cyclospora cayetanensis, only a relatively slow polymerase chain reaction (PCR) test developed by the U.S. Food and Drug Administration (FDA). However, DNA aptamers have recently been developed by our group against known proteins and whole oocysts of C. cayetanensis and shown to specifically detect the oocysts when attached on their 5' ends to red-emitting fluorophores and used as probes for fluorescence microscopy. Aptamers developed against recombinant wall protein 2 and TA4 antigen-like protein as well as whole oocysts specifically stained C. cayetanensis oocysts while exhibiting little, if any, staining of numerous other waterborne parasite species. Interestingly, the aptamers stained both exterior cell wall moieties and internal structures, suggesting that the aptamers penetrate the oocysts even without added detergents.

Quantitative characterizations of visual fibrousness in meat analogues using automated image analysis.

A desirable quality of plant-based meat analogues is to resemble the fibrous structure of cooked muscle meat. While texture analysis can characterize fibrous structures mechanically, assessment of visual fibrous structures remains subjective. Quantitative assessment of visual fibrous structures of meat analogues relies on expert knowledge, is resource-intensive, and time-consuming. In this study, a novel image-based method (Fiberlyzer) is developed to provide automated, quantitative, and standardized assessment of visual fibrousness of meat analogues. The Fiberlyzer method segments fibrous regions from 2D images and extracts fiber shape features to characterize the fibrous structure of meat analogues made from mung bean, soy, and pea protein. The computed fiber scores (the ratio between fiber length and width) demonstrate a strong correlation with expert panel evaluations, particularly on a per-formulation basis (r2 = 0.93). Additionally, the Fiberlyzer method generates fiber shape features including fiber score, fiber area, and the number of fiber branches, facilitating comparisons of structural similarity between meat analogue samples and cooked chicken meat as a benchmark. With a simple measurement setup and user-friendly interface, the Fiberlyzer method can become a standard tool integrated into formulation development, quality control, and production routines of plant-based meat analogue. This method offers rapid, cheap, and standardized quantification of visual fibrousness, minimizing the need for expert knowledge in the process of quality control.

Enhanced batch sorting and rapid sensory analysis of Mackerel products using YOLOv5s algorithm and CBAM: Validation through TPA, colorimeter, and PLSR analysis.

This study employed the YOLOv5s algorithm to establish a rapid quality identification model for Pacific chub mackerel (S. japonicus) and Spanish mackerel (S. niphonius). Data augmentation was conducted using the copy-paste augmentation within the YOLOv5s network. Furthermore, a small object detection layer was integrated into the network structure's neck, while the convolutional block attention module (CBAM) was incorporated into the convolutional module to optimize the model. The model's accuracy was assessed through sensory evaluation, texture profile analysis, and colorimeter analysis. The findings indicated that the enhanced model achieved a mAP@0.5 score of 0.966, surpassing the original version's score of 0.953. Moreover, the improved model's params was only 7.848 M, and an average detection time of 115 ms/image (image resolution 2400 × 3200). Furthermore, sensory and physicochemical indicators are reliably distinguished between qualified and unqualified samples. The PLSR model exhibited R2X, R2Y, and Q2 values of 0.977, 0.956, and 0.663, respectively.

Rapid method for plasmid DNA recombination (Murakami-system).

DNA recombination is a useful technology for cloning and subsequent functional analysis, while standard techniques for plasmid DNA recombination have remained unchanged. In the present study, we introduced rapid method for plasmid DNA recombination, which we named "Murakami-system", to complete the experiments in under 33 h. For this purpose, we selected the following: PCR amplification with 25 cycles and E. coli strain with rapid growth (incubation time of 6-8 h). In addition, we selected rapid plasmid DNA purification (mini-prep; ∼10 min) and rapid restriction enzyme incubation (20 min). This recombination system enabled rapid plasmid DNA recombination within 24-33 h, which could be useful in various fields. We also established a 1-day method for competent cell preparation. Our rapid recombination system allowed several sessions of plasmid DNA recombination to be performed every week, which improves the functional analysis of various genes.•"Rapid method for plasmid DNA recombination (Murakami-system).•E. coli strain with rapid growth (incubation time of 6-8 h).•Combination of rapid protocols (PCR, electrophoresis, DNA purification, ligation, and mini-prep) enabled plasmid DNA recombination within 24-33 h.

A simple double differential centrifugation-wash procedure to rapidly obtain bacterial identification and direct antimicrobial susceptibility testing from positive blood cultures / Método sencillo basado en una doble centrifugación diferencial para obtener una identificación bacteriana y sensibilidad a antibióticos rápida a partir de hemocultivos positivos

Introduction: This study proposes a simple and rapid method for both bacterial identification and direct antimicrobial susceptibility testing (AST) by using MALDI-TOF and a double differential centrifugation-wash procedure from positive blood cultures. Methods: Fifty-two positive blood cultures (37 gramnegative bacilli and 15 grampositive cocci) were studied by two methods for identification and AST: a reference method, and the rapid MALDI-TOF method obtaining a purified pellet by using a double differential centrifugation procedure. Results : A total of 1101 MIC values (mg/l) were interpreted according to EUCAST clinical breakpoints and compared using the two methods simultaneously. Discrepancies in 81 MIC values (7.35%) were detected. By analyzing standard parameters, we obtained 98.28% essential agreement and 92.65% categorical agreement considering all isolates tested. Conclusion: This method provides rapid bacterial identification and AST, offering definitive results 24–48h earlier than the conventional method (p<0.001) and improving the turnaround time in blood culture diagnostics, especially in laboratories without 24-h on-call.(AU)

Introducción: Este trabajo propone un método sencillo, rápido y barato de identificación bacteriana y sensibilidad antibiótica utilizando MALDI-TOF y una doble centrifugación diferencial a partir de hemocultivo positivo. Métodos: Se estudiaron 52 hemocultivos positivos (37 bacilos gramnegativos y 15 cocos grampositivos). Se compararon 2 métodos: un método convencional de identificación y determinación de sensibilidad a antibióticos automatizada partiendo de colonia crecida, y un método rápido utilizando MALDI-TOF, caracterizado por la obtención de un pellet purificado procedente de un hemocultivo positivo, mediante un procedimiento basado en una doble centrifugación diferencial. Resultados: Se analizaron y compararon 1.101 valores de CMI (mg/l) de acuerdo con los criterios establecidos por EUCAST y obtenidos por ambos métodos. Se detectaron discrepancias en 81 valores de CMI (7,35%). Considerando todos los aislados, la concordancia esencial fue del 98,28% y la concordancia categórica del 92,65%. Conclusión: Este método proporciona resultados de identificación y sensibilidad a antibióticos definitivos 24-48h antes que un método convencional (p<0,001), mejorando el tiempo de respuesta en el diagnóstico microbiológico de bacteriemias, especialmente en laboratorios sin servicio de guardias de 24h.(AU)

A simple double differential centrifugation-wash procedure to rapidly obtain bacterial identification and direct antimicrobial susceptibility testing from positive blood cultures.

INTRODUCTION: This study proposes a simple and rapid method for both bacterial identification and direct antimicrobial susceptibility testing (AST) by using MALDI-TOF and a double differential centrifugation-wash procedure from positive blood cultures. METHODS: Fifty-two positive blood cultures (37 gramnegative bacilli and 15 grampositive cocci) were studied by two methods for identification and AST: a reference method, and the rapid MALDI-TOF method obtaining a purified pellet by using a double differential centrifugation procedure. RESULTS: A total of 1101 MIC values (mg/l) were interpreted according to EUCAST clinical breakpoints and compared using the two methods simultaneously. Discrepancies in 81 MIC values (7.35%) were detected. By analyzing standard parameters, we obtained 98.28% essential agreement and 92.65% categorical agreement considering all isolates tested. CONCLUSION: This method provides rapid bacterial identification and AST, offering definitive results 24-48h earlier than the conventional method (p<0.001) and improving the turnaround time in blood culture diagnostics, especially in laboratories without 24-h on-call.

Development and validation of a prototype for the on-line simultaneous analysis of quality caprolactam synthesized on an industrial scale.

Caprolactam is a highly useful monomer obtained through the Beckmann arrangement, which generates large profits worldwide and is widely used in different industries. During the synthesis process, various components can be generated that weaken the quality of the final product, to have control of the monomer, monitoring is carried out during the synthesis and characterization of the final product. These characterizations generally take time due to the different techniques that must be performed to obtain the data. In this work, a method is designed that associates different techniques to reduce the number of steps carried out in the tests to determine the quality of the material, optimize the times and generate a quality and efficient process in a shorter time, in addition, it is due to a semi-automated system for the simultaneous characterization of caprolactam, which, according to the statistical data obtained for sodium, iron, volatile bases, and moisture analysis were reproducible. The developed prototype had 21 on-line valves that allowed taking the representative volumes of samples and reagents necessary for each measurement. There is excellent linearity where the correlation coefficient has values between 0,9992 and 1. The values obtained for the relative error are between 0.18 and 2.24% for laboratory tests using the traditional method and between 0.21 and 3.83% for tests carried out using the prototype. The P value of the evaluation of the means was 0.997, indicating that the means are not statistically different.•Caprolactam analysis•Process optimization•Determination of impurities.

Assessment of developed paper strip based sensor with pesticide residues in different dairy environmental samples.

According to the results of this study, the paper strip biosensor can detect pesticide at very low concentration like fungicide, organochlorine, organophosphate, carbamate, and herbicide group ranges from 1 to 10, 1-50, 250-500, 1-50, and 1 µg/L, respectively in animal feed, water, milk and soil. This is a significant improvement from the previous study, which found that the paper strip biosensor could only detect pesticide levels of up to 500 or 1000 µg/L. A total of 436 samples were collected from the dairy farm, including 58 samples of green feed, 54 samples of dry feed, 45 samples of concentrated feed, 41 samples of fermented feed, 49 samples of manure, 54 samples of soil, and 86 samples of milk. PSA (Primary Secondary Amine) and MgSO4 (1:2 ratio) were used to remove pigments from dairy farm samples to prevent the enzyme-pesticide interaction leading to colour development on the strip, which was successfully achieved. Using a strip-based test and an optimized extraction protocol, pesticides were detected in 38.49% in the samples. Limit of Detection of 15 pesticides from the organochlorine, organophosphate, carbamate, neonicotinoid, pyrethroid, ryanoid, strobilurins, and triazole groups recommended for use in dairy farms were evaluated in feed/fodder. Pesticides were being detected in various dairy farm matrices using the newly developed test. The developed technology can be used as a semi-quantitative test for pesticides monitoring in the dairy farm as well as for screening of primary produce under field condition for organic certification of various food/feed commodities.

A One-Step Multiplex PCR Method to Rapidly Distinguish Two Strains of Diglyphus wani (Hymenoptera: Eulophidae) Against Agromyzid Leafminers.

Hymenopteran parasitoids generally show a haplo-diploid sex determination system. Haploid males are produced from unfertilized eggs, whereas diploid females develop from fertilized eggs (arrhenotokous). In some cases, diploid females develop from unfertilized eggs (thelytokous). Diglyphus wani (Hymenoptera: Eulophidae) is a biological control agent for agromyzid leafminers and have arrhenotokous and thelytokous strains. However, the morphological characteristics of two strains of D. wani are so similar that it is difficult to accurately distinguish them based on morphology. Here, a rapid molecular identification method was developed based on the mitochondrial gene cytochrome c oxidase I (COI) and one-step multiplex PCR. Two primer combinations, PC1 (Ar-F1/Th-F1/WR2) and PC2 (Ar-F1/Th-F4/WR2), were designed and repeatedly screened to distinguish two strains simultaneously, of which two special forward primers Th-F1/Th-F4 were used for the thelytokous strain and one special forward primer Ar-F1 was used for the arrhenotokous strain. In addition, a common reverse primer, WR2, was used for both strains. The PC1 and PC2 PCR assays were effective in distinguishing the two strains at different developmental stages and field colonies. This method provides a reliable, highly sensitive, and cost-effective tool for the rapid identification of the two strains of D. wani.

Recent advancements in NIR spectroscopy for assessing the quality and safety of horticultural products: A comprehensive review.

The qualitative and quantitative evaluation of agricultural products has often been carried out using traditional, i.e., destructive, techniques. Due to their inherent disadvantages, non-destructive methods that use near-infrared spectroscopy (NIRS) coupled with chemometrics could be useful for evaluating various agricultural products. Advancements in computational power, machine learning, regression models, artificial neural networks (ANN), and other predictive tools have made their way into NIRS, improving its potential to be a feasible alternative to destructive measurements. Moreover, the incorporation of suitable preprocessing techniques and wavelength selection methods has arguably proven its practical feasibility. This review focuses on the various computation methods used for processing the spectral data collected and discusses the potential applications of NIRS for evaluating the quality and safety of agricultural products. The challenges associated with this technology are also discussed, as well as potential future perspectives. We conclude that NIRS is a potentially useful tool for the rapid assessment of the quality and safety of agricultural products.

Rapid and inexpensive method of PCR ready DNA isolation from human peripheral blood and saliva.

BACKGROUND: The isolation of nucleic acids is a frequently performed procedure in the molecular biology area. Although several rapid DNA isolation techniques from human peripheral blood and saliva have been developed, there are still some disadvantages - volume, time, cost, and yield are a few notable ones. OBJECTIVE: We aim to develop a rapid and inexpensive method to isolate high-molecular-weight genomic DNA from human peripheral blood and saliva that can be used for molecular biology experiments. METHODS: Five DNA isolation methods with slightly varying protocols were used. High-quality DNA obtained from one specific method was further amplified by PCR and the template with good amplification was further used for performing RFLP and sequencing. RESULTS: Out of 5 different isolation methods (R1 to R5), DNA obtained from the R4 was of good quality (molecular weight is > 10 kb and 260/280 ratio is 1.89 ± 0.2), which allows successful PCR amplification and good separation in Restriction Fragment Length Polymorphism analysis. Sequencing by the Sanger Sequencing produced a good readable sequence of an amplified fragment from Method R4 DNA. CONCLUSION: In the present study we have developed a simple, rapid, and cost-effective DNA isolation method, which uses low sample volume and yields good quantity and high-quality product. The DNA obtained is highly fit for molecular genetics research applications.

Determination of Moisture and Protein Content in Living Mealworm Larvae (Tenebrio molitor L.) Using Near-Infrared Reflectance Spectroscopy (NIRS).

Yellow mealworm larvae (Tenebrio molitor L.) are a sustainable source of protein for food and feed. This study represents a new approach in analyzing changes in the nutritional composition of mealworm larvae using near-infrared reflectance spectroscopy (NIRS) combined with multivariate analysis. The moisture and protein content of living larvae were scanned with a near-infrared spectrometer using wavelengths from 1100 to 2100 nm. Different feeding groups with varying moisture sources and amount and the difference between low (50%) and high (75%) humidity were tested, and the influence on larval moisture and protein content was measured. A calibration was developed, with modified partial least squares as the regression method. The NIR spectra were influenced by the moisture and protein content of the larvae, because the absorbance values of the larval groups differed greatly. The coefficient of the determination of calibration (R2c) and prediction (R2p) were over 0.98 for moisture and over 0.94 for protein content. The moisture source and content also had a significant influence on the weight gain of the larvae. Consequently, significant differences in protein content could be determined, depending on the water supply available. With respect to wet weight, the larvae moisture content varied from 60 to 74% and protein content from 16 to 24%. This investigation revealed that with non-invasive NIRS online monitoring, the composition of insects can be continuously recorded and evaluated so that specific feeding can be carried out in the course of larval development and composition.

A compact, dual-zone vertical tube furnace for the determination of tritium and carbon-14 in decommissioning wastes.

A compact dual zone, two work-tube, vertical tube furnace system (Raddec Pyrolyser-Mini) has been designed for the determination of H-3 and C-14 in decommissioning wastes. An optimised methodology was developed following improvements to sample holder and bubbler trap design, sample loading and loading temperature, as well as length and style of heating programmes. A significant efficiency enhancement was obtained through 'hot-loading' of the sample into the furnace at 600 °C before finally ramping to 900 °C. Direct trapping of H-3 and C-14 in a scintillation vial located in a special anti-suck-back bubbler further improved operations, leading to a reduction in analysis time and measurement sensitivity. Co-trapping of the analytes and dual-label liquid scintillation counting also proved effective. Overall, the developed methodology led to a reduced analyte extraction/trapping time of 150 min whilst achieving limits of detection of <1 Bq/g. Validation of the procedure was assessed using a range of spiked matrices relevant to nuclear site decommissioning, reference materials and operationally-exposed materials. The compact size of this thermal extraction system is such that it allows for deployment in fume cupboards, gloveboxes and a mobile laboratory.

Check-All-That-Apply (CATA) Test to Investigate the Consumers' Perception of Olive Oil Sensory Properties: Effect of Storage Time and Packaging Material.

Sensory changes during shelf-life of oils have been mostly studied by descriptive methods, while consumer-based approaches have been poorly explored. This study assessed the variations in consumers' liking and sensory perception of extra virgin olive oil (EVOO) and olive oil (OO) packaged in glass, polyethylene terephthalate and tinplate. After 2, 10 and 19 months of storage, oil perception was investigated with consumers (n = 50) performing both a liking test and a check-all-that-apply (CATA) test. No significant effect of the packaging material on consumers' response was found, whereas storage time negatively affected the sensory properties of and acceptability of OOs and EVOOs from the 10th month of storage. The CATA test results revealed the sensory changes in oils over 19 months, mainly described as a decrease in pungency for EVOO and a decrease in herbaceous and ripe fruitiness in OO. The CATA technique combined with the liking test allowed the drivers of liking ("olive" for OO and "green fruitiness" for EVOO) and disliking ("bitter" and "pungent" for EVOO) to be identified. In conclusion, the sensory approach based both on CATA technique and liking test seems promising as a rapid tool to evaluate the changes in sensory properties perceivable during the shelf-life of oils.

Rapid Cell Transfer by Means of Nylon Mesh to Improve Cellular Diagnosis: The Role of Immunocytochemistry.

Immunocytochemistry (ICC) is an important ancillary technique in clinical cytology for not only identifying and characterizing tumor cells but also gaining prognostic or therapeutic information. Although cell blocks are often prepared for immunocytochemical evaluation of body cavity fluid and fine-needle aspiration specimens, they are not suitable for hypocellular samples. Liquid-based cytology can help prepare additional smears from residual cytological specimens. However, since conventional methods are used for nongynecological specimens in most laboratories, ICC is often limited by the number of cytological smears. Cell transfer methods permit to evaluate several immunocytochemical markers in a single cytological smear. Yet, these methods have some limitations; for example, they are time-consuming (about 3-40 h) and medium membranes with their attached cells are occasionally stretched or torn when peeled off the slides. Therefore, in an attempt to solve these problems, we developed a rapid and reliable cell transfer method using a nylon mesh. Our method requires no special equipment or reagent and can significantly reduce the turnaround time, as compared to previous methods.

A rapid competitive method for bacteriophage genomic DNA extraction.

The bacteriophage (phage) DNA extraction methods for genomics analysis is a critical and time-consuming process. Hence, a rapid and cost-effective method for DNA extraction of phages is favorable for phage biologists. In the present study, a cost-effective, simple and rapid procedure for phage genome extraction in less than 10 min is introduced. Highly concentrated phage lysates were prepared using acetone precipitation followed by extraction using various methods such as commercial kits, TES lysis buffer, potassium iodide, and sodium iodide. The quality of the extracted DNA was analyzed by agarose gel electrophoresis and UV absorbance of DNA at 260 and 280 nm. Finally, the extracted DNA was subjected to restriction digestion and next-generation sequencing to approve the efficiency of the method. Based on the time, cost, and quality of obtained DNA, the acetone precipitation of phages and extraction by potassium iodide or sodium iodide method was determined to be the best method for phage DNA extraction tested in this study. Moreover, the extracted genomic DNA using this method is suitable for phage genomic analysis such as restriction enzyme studies, preparation of DNA library, and also next-generation sequencing.