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Sensitive detection of pathogens in livestock farms is an integral part of the One Health Action Plan of the European Union (EU). Ensuring this requires on-site testing devices that are compatible with complex matrices such as primary production samples. Among all, faeces are considered the most challenging matrix type that makes it difficult to identify pathogens because of complexity in sample preparation for molecular testing. We have developed a loop-mediated isothermal amplification (LAMP) based veterinary point-of-care (POC) device (VETPOD) and adapted it to detect Salmonella enterica in primary production samples. Three different sampling methods (semi-wet chicken faeces, boot socks collection and dust samples from poultry shed) were iteratively tested to assess their nature of complexity and possibility for adapting them as suitable sampling methods for on-site testing. During the study, the sample preparation method that included a two-step centrifugation combined with washing of the enriched Salmonella cells was found crucial in eliminating amplification inhibitors originating from the faecal matrices. A total of 90 samples were tested that included 60 samples for sensitivity study and 30 samples for relative level of detection (RLOD, a level of detection in comparison to ISO 6579:1 reference method). Overall, the VETPOD had a sensitivity of 90%, 84.62% and 81.82% for boot sock, faecal and dust samples, respectively. The RLOD was 2.23 CFU/25 g which was found to be 1.33 times higher than the ISO 6579:1. Performing with an excellent agreement with ISO 6579:1, the VETPOD proved as a promising alternative to detect Salmonella spp. in primary production and animal husbandry samples., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Sensitive and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a vital goal in the ongoing COVID-19 pandemic. We present in this comprehensive work, for the first time, detailed fabrication and clinical validation of a point of care (PoC) device for rapid, onsite detection of SARS-CoV-2 using a real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) reaction on a polymer cartridge. The PoC system, namely PATHPOD, consisting of a standalone device (weight less than 1.2 kg) and a cartridge, can perform the detection of 10 different samples and two controls in less than 50 min, which is much more rapid than the golden standard real-time reverse-transcription Polymerase Chain Reaction (RT-PCR), typically taking 16-48 h. The novel total internal reflection (TIR) scheme and the reactions inside the cartridge in the PoC device allow monitoring of the diagnostic results in real-time and onsite. The analytical sensitivity and specificity of the PoC test are comparable with the current RT-PCR, with a limit of detection (LOD) down to 30-50 viral genome copies. The robustness of the PATHPOD PoC system has been confirmed by analyzing 398 clinical samples initially examined in two hospitals in Denmark. The clinical sensitivity and specificity of these tests are discussed., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors.)

Accurate and rapid detection of pathogens in foods of animal origin has been a critical part of the One Health Action Plan of the European Union (EU). Biosensors have the potential in bringing required technologies to accomplish this on the field, wherein loop-mediated isothermal amplification (LAMP) and lab-on-a-chip have proven to be ideal. We have developed a LAMP-based point-of-care (POC) device, the VETPOD, as a solution to the contemporary challenges in the rapid detection of Salmonella spp. The core technology in the VETPOD is a ready-to-use cartridge that included an injection-molded polymer chip with pyramid-shaped optical structures embedded within the chip. These pyramid-shaped optical structures direct the incident light, due to total internal reflection (TIR), through the reaction chambers to the phototransistor. The VETPOD was validated against the ISO 6579-1 reference method. A total of 310 samples were tested that included 180 Salmonella spiked samples in 6 different meat categories and 130 strains to determine the specificity. The overall results were satisfactory, wherein the VETPOD had an acceptable sensitivity (96.51%) compared to the reference (98.81%) and near perfect agreement with ISO 6579-1 with an overall Cohen's kappa of 0.94. The relative level of detection (RLOD) for the VETPOD was 1.38 CFU/25 g that was found to be 1.17 times higher than the reference. The VETPOD showed 98% precision for inclusivity and 100% precision for the exclusivity samples. The VETPOD proved as a useful alternative to detect Salmonella spp. that can be adaptable to a broader spectrum of pathogens in future.

Adaptations of new generation molecular techniques for multiplexed detection of pathogens are gaining interest in the field of point-of-care (POC) industry and onsite testing. Loop-mediated isothermal amplification (LAMP), an advanced molecular amplification technique, has proven promising due to its unique features that suits ideal for POC applications. However, application of LAMP for multiplexed detection of pathogens remains challenging because of the difficulty in the identification of specific LAMP amplicons that does not have a well-definite molecular size. In this study, we developed a solid-phase loop-mediated isothermal amplification (SP-LAMP) technique to address the challenge. Integration of LAMP with the supercritical angle fluorescence (SAF) micro-optic structures as a solid support (SS) in an array format enabled spatial separation of LAMP amplicons in a multiplexed configuration. Important parameters such as length of the SS primers, length of the primer-binding region, the effect of surface density of immobilized SS primers, and cross-reactivity among the primers of different targets were iteratively tested and optimized. With the combination of SP-LAMP and SAF techniques, it was possible to detect multiple pathogens that include Salmonella spp, Campylobater spp., Campylobacter coli , Campylobacter jejuni , avian influenza virus (AIV), and pan avian internal control (IC) under singleplex conditions. The multiplexing capacity of the SP-LAMP was demonstrated using AIV and IC with promising results. The success of SP-LAMP has opened a promising direction toward the development of a multiplex POC system for rapid detection of multiple pathogens.

The COVID-19 pandemic emphasized the importance of rapid, portable, and on-site testing technologies necessary for resource-limited settings for effective testing and screening to reduce spreading of the infection. Realizing this, we developed a fluorescence-based point-of-care (fPOC) detection system with real-time reverse transcriptase loop-mediated isothermal amplification for rapid and quantitative detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The system is built based on the Arduino platform compatible with commercially available open-source hardware-software and off-the-shelf electronic components. The fPOC system comprises of three main components: 1) an instrument with integrated heaters, 2) optical detection components, and 3) an injection-molded polymeric cartridge. The system was tested and experimentally proved to be able to use for fast detection of the SARS-CoV-2 virus in real-time in less than 30 min. Preliminary results of testing the performance of the fPOC revealed that the fPOC could detect the SARS-CoV-2 virus at a limit of detection (LOD 50% ) at two to three copies/microliter (15.36 copies/reaction), which was comparable to reactions run on a standard commercial thermocycler. The performance of the fPOC was evaluated with 12 SARS-CoV-2 clinical throat swab samples that included seven positive and five negative samples, as confirmed by reverse transcription-polymerase chain reaction. The fPOC showed 100% agreement with the commercial thermocycler. This simple design of the fPOC system demonstrates the potential to greatly enhance the practical applicability to develop a totally integrated point-of-care system for rapid on-site screening of the SARS-CoV-2 virus in the management of the pandemic., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Van Ngoc, Quyen, Vinayaka, Bang and Wolff.)

Loop-mediated isothermal amplification (LAMP) is being used as a robust rapid diagnostic tool to prevent the transmission of infectious diseases. However, carryover contamination of LAMP-amplified products originating from previous tests has been a problem in LAMP-based bio-analytical assays. In this study, we developed a Cod-uracil-DNA-glycosylase real-time reverse transcriptase LAMP assay (Cod-UNG-rRT-LAMP) for the elimination of carryover contamination and the rapid detection of SARS-CoV-2 in point-of-care (POC) testing. Using the Cod-UNG-rRT-LAMP assay, the SARS-CoV-2 virus could be detected as low as 2 copies/µl (8 copies/reaction) within 45 min of amplification and 2.63 ± 0.17 pg (equivalent to 2.296 × 10 9 copies) of contaminants per reaction could be eliminated. Analysis of clinical SARS-CoV-2 samples using the Cod-UNG-rRT-LAMP assay showed an excellent agreement with a relative accuracy of 98.2%, sensitivity of 97.1%, and specificity of 95.2% in comparison to rRT-PCR. The results obtained in this study clearly demonstrate the feasibility of the use of the Cod-UNG-rRT-LAMP assay for applications toward the POC diagnosis of SARS-CoV-2 and on-site testing of other pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Quyen, Vinayaka, Golabi, Ngoc, Bang and Wolff.)

Invasive non-typhoidal salmonellosis is gaining worldwide attention as an emerging disease cluster among bloodstream infections. The disease has the highest burden among immunocompromised and malnourished children in resource-limited areas due to poor access to reliable and rapid diagnostics. Point-of-care (POC) diagnostics are promising for use in such low infrastructure laboratory settings. However, there still remains a major challenge for POC testing to deal with the complexity of blood matrices in rapid detection of an extremely low concentration of blood-borne pathogens. In this work, the challenges were addressed by combining magnetic bead based pathogen concentration and Loop Mediated Isothermal Amplification (LAMP) technology. Sensitivity and performance of the combined approach were determined and compared with a direct PCR method. A direct visual detection strategy, adapted using SYTO-24 DNA intercalating dye, resulted in a limit of detection (LoD) as low as 14 CFU/mL in blood samples with a total analysis time of less than 2 h, including sample preparation. This approach has the potential for wide application as a high-throughput POC testing method to analyze pathogens in clinical, food, feed and environmental samples., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Avian influenza virus (AIV) outbreaks occur frequently worldwide, causing a potential public health risk and great economic losses to poultry industries. Considering the high mutation rate and frequent genetic reassortment between segments in the genome of AIVs, emerging new strains are a real threat that may infect and spread through the human population, causing a pandemic. Therefore, rapid AIV diagnostic tests are essential tools for surveillance and assessing virus spreading. Real-time reverse transcription PCR (rRT-PCR), targeting the matrix gene, is the main official standard test for AIV detection, but the method requires well-equipped laboratories. Reverse transcription Loop-Mediated Isothermal Amplification (RT-LAMP) has been reported as a rapid method and an alternative to PCR in pathogen detection. The high mutation rate in the AIV genome increases the risk of false negative in nucleic acid amplification methods for detection, such as PCR and LAMP, due to possible mismatched priming. In this study, we analyzed 800 matrix gene sequences of newly isolated AIV in the EU and designed a highly efficient LAMP primer set that covers all AIV subtypes. The designed LAMP primer set was optimized in real-time RT-LAMP (rRT-LAMP) assay. The rRT-LAMP assay detected AIV samples belonging to nine various subtypes with the specificity and sensitivity comparable to the official standard rRT-PCR assay. Further, a two-color visual detection RT-LAMP assay protocol was adapted with the aim to develop on-site diagnostic tests. The on-site testing successfully detected spiked AIV in birds oropharyngeal and cloacal swabs samples at a concentration as low as 10 0.8 EID 50 per reaction within 30 minutes including sample preparation. The results revealed a potential of this newly developed rRT-LAMP assay to detect AIV in complex samples using a simple heat treatment step without the need for RNA extraction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Golabi, Flodrops, Grasland, Vinayaka, Quyen, Nguyen, Bang and Wolff.)

Bloodstream infections and invasive nontyphoidal Salmonellosis in particular remain a major health and economic burden worldwide. The complexity of blood matrixes along with extremely low concentration of pathogens in blood poses a great challenge for rapid and ultrasensitive detection. Sample preparation has been the critical step that should provide blood-matrix-free sample with the targeted pathogen in the highest possible concentration. In this work, we addressed this challenge by combining magnetic-bead-based pathogen concentration and solid-phase PCR (SP-PCR). The SP-PCR performed on a supercritical angle fluorescence (SAF) microlens array embedded in a microchip enabled quick and accurate detection of low levels of Salmonella enterica serovar typhimurium and enteritidis in blood samples without culture enrichment. Protein AG-magnetic beads immobilized with antisalmonella antibody could efficiently concentrate both Salmonella serovars with a capturing efficiency >95%. Higher tolerance of Phusion hot start DNA polymerase to PCR inhibitors and its compatibility with protein AG-magnetic beads allowed the integration of SP-PCR. Analysis of Salmonella -spiked blood samples with the SP-PCR resulted in a limit of detection (LoD) as low as 86 CFU/mL and 94 CFU/mL for S . typhimurium and S . enteritidis , respectively, that could be attributed to the high fluorescence collection efficiency of the SAF microlens array. These combinations reduced the duration of analysis to less than 3 h including sample preparation. This platform has the potential for wide application as a high-throughput biosensor to analyze pathogens in clinical, food, and environmental samples.

Campylobacteriosis is one of the most common foodborne diseases worldwide. Two Campylobacter species - C. jejuni and C. coli in poultry and poultry products are considered to be the main source of human campylobacteriosis. Therefore, studying Campylobacter status in poultry flocks is needed to prevent transmission of disease and reduce human risk, health cost, and economic losses. In this study, we adapted and used a Loop-Mediated Isothermal Amplification (LAMP) assay for specific, sensitive, simple and cost-effective rapid detection of C. jejuni and C. coli in the poultry production chain. Amplified LAMP products were detected using a small, low-cost portable commercial blue LED transilluminator and a direct visual detection strategy was demonstrated. By using optimized conditions for amplification a limit of detection (LOD) of 50 CFU/ml was achieved for testing of C. jejuni and C. coli in spiked chicken feces without enrichment. The method took 60-70 min from receiving the samples to the final results (including 30 min for amplification). The optimized LAMP showed a relative accuracy of 98.4%, a specificity of 97.9%, and a sensitivity of 100% in comparison to real-time PCR method. Cohen's kappa index also showed an excellent agreement (0.94) between the two methods. The results showed that the method is specific, sensitive and is suitable to develop for rapid detection of Campylobacter spp. at poultry production., (Copyright © 2019 Quyen, Nordentoft, Vinayaka, Ngo, Engelsmenn, Sun, Madsen, Bang and Wolff.)

LAMP has received great interest and is widely utilized in life sciences for nucleic acid analysis. To monitor a real-time LAMP assay, a fluorescence DNA dye is an indispensable component and therefore the selection of a suitable dye for real-time LAMP is a need. To aid this selection, we investigated the inhibition effects of twenty-three DNA dyes on real-time LAMP. Threshold time (T t ) values of each real-time LAMP were determined and used as an indicator of the inhibition effect. Based on the inhibition effects, the dyes were classified into four groups: (1) non-inhibition effect, (2) medium inhibition effect, (3) high inhibition effect, and (4) very high inhibition effect. The signal to noise ratio (SNR) and the limit of detection (LOD) of the dyes in groups 1, 2, and 3 were further investigated, and possible inhibition mechanisms of the DNA dyes on the real-time LAMP are suggested and discussed. Furthermore, a comparison of SYTO 9 in different LAMP reactions and different systems is presented. Of the 23 dyes tested, SYTO 9, SYTO 82, SYTO 16, SYTO 13, and Miami Yellow were the best dyes with no inhibitory effect, low LOD and high SNR in the real-time LAMP reactions. The present classification of the dyes will simplify the selection of fluorescence dye for real-time LAMP assays in point of care setting., (Copyright © 2019 Quyen, Ngo, Bang, Madsen and Wolff.)

The volume of point of care (POC) testing continues to grow steadily due to the increased availability of easy-to-use devices, thus making it possible to deliver less costly care closer to the patient site in a shorter time relative to the central laboratory services. A novel class of molecules called microRNAs have recently gained attention in healthcare management for their potential as biomarkers for human diseases. The increasing interest of miRNAs in clinical practice has led to an unmet need for assays that can rapidly and accurately measure miRNAs at the POC. However, the most widely used methods for analyzing miRNAs, including Northern blot-based platforms, in situ hybridization, reverse transcription qPCR, microarray, and next-generation sequencing, are still far from being used as ideal POC diagnostic tools, due to considerable time, expertize required for sample preparation, and in terms of miniaturizations making them suitable platforms for centralized labs. In this review, we highlight various existing and upcoming technologies for miRNA amplification and detection with a particular emphasis on the POC testing industries. The review summarizes different miRNA targets and signals amplification-based assays, from conventional methods to alternative technologies, such as isothermal amplification, paper-based, oligonucleotide-templated reaction, nanobead-based, electrochemical signaling- based, and microfluidic chip-based strategies. Based on critical analysis of these technologies, the possibilities and feasibilities for further development of POC testing for miRNA diagnostics are addressed and discussed.

Foodborne salmonellosis remains a major economic burden worldwide and particularly for food industries. The diverse and complexity of food matrices pose great challenges for rapid and ultra-sensitive detection of Salmonella in food samples. In this study, combination of pathogen pre-concentration with rapid molecular identification is presented to overcome these challenges. This combination enabled effective real-time PCR detection of low levels of Salmonella enterica serovar Typhimurium without culture enrichment. Anti-salmonella antibody, immobilized on protein AG-magnetic beads, could efficiently concentrate Salmonella Typhimurium with a capturing efficiency of 95%. In the direct PCR, a strong linear relationship between bacteria concentration and the number of cycles was observed with a relative PCR efficiency of ∼92% resulting in a limit of detection (LoD) of ∼2 CFU/mL. Analysis of spiked food samples that include vegetable salad, egg yolk, egg white, whole egg and minced pork meat has validated the precision of the method. A relative accuracy of 98.3% with a sensitivity of 91.6% and specificity of 100% was achieved in the Salmonella spiked food samples. The use of a Phusion hot start DNA polymerase with a high tolerance to possible PCR inhibitors allowed the integration of direct PCR, and thereby reducing the duration of analysis to less than 3 h. The Cohen's kappa index showed excellent agreement (0.88) signifying the capability of this method to overcome the food matrix effects in rapid and ultra-sensitive detection of Salmonella in food. This approach may lay a future platform for the integration into a Lab-on-a-chip system for online monitoring of foodborne pathogens., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The genus Arcobacter (Vandamme et al., 1991), comprised of Campylobacter -related species, are considered zoonotic emergent pathogens. The presence of Arcobacter in food products like shellfish, has an elevated incidence worldwide. In this study, we developed a specific viable quantitative PCR (v-qPCR), using the dye propidium monoazide (PMA), for quantification of the viable Arcobacter spp. cells in raw oysters and mussels. The high selectivity of primers was demonstrated by using purified DNA from 38 different species, 20 of them from the genus Arcobacter . The optimization of PMA concentration showed that 20 μM was considered as an optimal concentration that inhibits the signal from dead cells at different concentrations (OD 550 from 0.2 to 0.8) and at different ratios of live: dead cells (50:50 and 90:10). The v-qPCR results from shellfish samples were compared with those obtained in parallel using several culture isolation approaches (i.e., direct plating on marine and blood agar and by post-enrichment culturing in both media). The enrichment was performed in parallel in Arcobacter-CAT broth with and without adding NaCl. Additionally, the v-qPCR results were compared to those obtained with traditional quantitative (qPCR). The v-qPCR and the qPCR resulted in c.a. 94% of positive detection of Arcobacter vs. 41% obtained by culture approaches. When examining the reduction effect resulting from the use of v-qPCR, samples pre-enriched in Arcobacter-CAT broth supplemented with 2.5% NaCl showed a higher reduction (3.27 log copies) than that of samples obtained directly and those pre-enriched in Arcobacter-CAT broth isolation (1.05 and 1.04). When the v-qPCR was applied to detect arcobacter from real shellfish samples, 15/17 samples tested positive for viable Arcobacter with 3.41 to 8.70 log copies 1g -1 . This study offers a new tool for Arcobacter surveillance in seafood.

Rapid detection of foodborne pathogens at an early stage is imperative for preventing the outbreak of foodborne diseases, known as serious threats to human health. Conventional bacterial culturing methods for foodborne pathogen detection are time consuming, laborious, and with poor pathogen diagnosis competences. This has prompted researchers to call the current status of detection approaches into question and leverage new technologies for superior pathogen sensing outcomes. Novel strategies mainly rely on incorporating all the steps from sample preparation to detection in miniaturized devices for online monitoring of pathogens with high accuracy and sensitivity in a time-saving and cost effective manner. Lab on chip is a blooming area in diagnosis, which exploits different mechanical and biological techniques to detect very low concentrations of pathogens in food samples. This is achieved through streamlining the sample handling and concentrating procedures, which will subsequently reduce human errors and enhance the accuracy of the sensing methods. Integration of sample preparation techniques into these devices can effectively minimize the impact of complex food matrix on pathogen diagnosis and improve the limit of detections. Integration of pathogen capturing bio-receptors on microfluidic devices is a crucial step, which can facilitate recognition abilities in harsh chemical and physical conditions, offering a great commercial benefit to the food-manufacturing sector. This article reviews recent advances in current state-of-the-art of sample preparation and concentration from food matrices with focus on bacterial capturing methods and sensing technologies, along with their advantages and limitations when integrated into microfluidic devices for online rapid detection of pathogens in foods and food production line., (Copyright © 2018. Published by Elsevier Inc.)

Molecularly imprinted polymers (MIPs) are biomimetics which can selectively bind to analytes of interest. One of the most interesting areas where MIPs have shown the biggest potential is food analysis. MIPs have found use as sorbents in sample preparation attributed to the high selectivity and high loading capacity. MIPs have been intensively employed in classical solid-phase extraction and solid-phase microextraction. More recently, MIPs have been combined with magnetic bead extraction, which greatly simplifies sample handling procedures. Studies have consistently shown that MIPs can effectively minimize complex food matrix effects, and improve recoveries and detection limits. In addition to sample preparation, MIPs have also been viewed as promising alternatives to bio-receptors due to the inherent molecular recognition abilities and the high stability in harsh chemical and physical conditions. MIPs have been utilized as receptors in biosensing platforms such as electrochemical, optical and mass biosensors to detect various analytes in food. In this review, we will discuss the current state-of-the-art of MIP synthesis and applications in the context of food analysis. We will highlight the imprinting methods which are applicable for imprinting food templates, summarize the recent progress in using MIPs for preparing and analysing food samples, and discuss the current limitations in the commercialisation of MIPs technology. Finally, future perspectives will be given., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Solid-phase PCR (SP-PCR) has become increasingly popular for molecular diagnosis and there have been a few attempts to incorporate SP-PCR into lab-on-a-chip (LOC) devices. However, their applicability for on-line diagnosis is hindered by the lack of sensitive and portable on-chip optical detection technology. In this paper, we addressed this challenge by combining the SP-PCR with super critical angle fluorescence (SAF) microlens array embedded in a microchip. We fabricated miniaturized SAF microlens array as part of a microfluidic chamber in thermoplastic material and performed multiplexed SP-PCR directly on top of the SAF microlens array. Attribute to the high fluorescence collection efficiency of the SAF microlens array, the SP-PCR assay on the LOC platform demonstrated a high sensitivity of 1.6 copies/µL, comparable to off-chip detection using conventional laser scanner. The combination of SP-PCR and SAF microlens array allows for on-chip highly sensitive and multiplexed pathogen detection with low-cost and compact optical components. The LOC platform would be widely used as a high-throughput biosensor to analyze food, clinical and environmental samples., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture method is time consuming. In response to the demand for rapid on line or at site detection of pathogens, in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair of newly designed primers targeting Salmonella spp. at subtype level were incorporated in the multiplex Direct PCR. To maximize the efficiency of the Direct PCR, the ratio between sample and dilution buffer was optimized. The sensitivity and specificity of the multiplex Direct PCR were tested using naturally contaminated pork meat samples for detecting and subtyping of Salmonella spp. Conventional bacterial culture methods were used as reference to evaluate the performance of the multiplex Direct PCR. Relative accuracy, sensitivity and specificity of 98.8%; 97.6% and 100%, respectively, were achieved by the method. Application of the multiplex Direct PCR to detect Salmonella in pork meat at slaughter reduces the time of detection from 5 to 6 days by conventional bacterial culture and serotyping methods to 14 h (including 12 h enrichment time). Furthermore, the method poses a possibility of miniaturization and integration into a point-of-need Lab-on-a-chip system for rapid online pathogen detection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Solid-phase PCR (SP-PCR) has attracted considerable interest in different research fields since it allows parallel DNA amplification on the surface of a solid substrate. However, the applications of SP-PCR have been hampered by the low efficiency of the solid-phase amplification. In order to increase the yield of the solid-phase amplification, we studied various parameters including the length, the density, as well as the annealing position of the solid support primer. A dramatic increase in the signal-to-noise (S/N) ratio was observed when increasing the length of solid support primers from 45 to 80 bp. The density of the primer on the surface was found to be important for the S/N ratio of the SP-PCR, and the optimal S/N was obtained with a density of 1.49 × 10 11  molecules/mm 2 . In addition, the use of solid support primers with a short overhang at the 5' end would help improve the S/N ratio of the SP-PCR. With optimized conditions, SP-PCR can achieve amplification efficiency comparable to conventional PCR, with a limit of detection of 1.5 copies/μl (37.5 copies/reaction). These improvements will pave the way for wider applications of SP-PCR in various fields such as clinical diagnosis, high-throughput DNA sequencing, and single-nucleotide polymorphism analysis. Graphical abstract Schematic representation of solid-phase PCR.

A miniaturised array of supercritical angle fluorescence (SAF) micro-optics embedded in a microfluidic chamber was fabricated by injection moulding. The fabricated chip could enhance the fluorescence signal around 46 times compared to a conventional microscope. Collection of the fluorescence signal from the SAF array is almost independent of the numerical aperture, and the limit of detection was improved 36-fold using a simple and inexpensive optical detection system.

Foodborne disease is a major public health threat worldwide. Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture or molecular-based methods are time consuming and usually take a few hours to days to complete. In response to the demand for rapid on line or on site detection of pathogens, in this study, we describe for the first time an eight-chamber lab-on-a-chip (LOC) system with integrated magnetic bead-based sample preparation and loop-mediated isothermal amplification (LAMP) for rapid and quantitative detection of Salmonella spp. in food samples. The whole diagnostic procedures including DNA isolation, isothermal amplification, and real-time detection were accomplished in a single chamber. Up to eight samples could be handled simultaneously and the system was capable to detect Salmonella at concentration of 50 cells per test within 40 min. The simple design, together with high level of integration, isothermal amplification, and quantitative analysis of multiple samples in short time, will greatly enhance the practical applicability of the LOC system for rapid on-site screening of Salmonella for applications in food safety control, environmental surveillance, and clinical diagnostics.

Weissella ceti, a Gram-positive lactic acid bacterium, has emerged as a novel pathogen causing Weissellosis in rainbow trout (Oncorhynchus mykiss). Until now, it has not been easy to distinguish W. ceti from other pathogens by the symptoms of the infected rainbow trout, especially at the primary stage of infection. Thus, a convenient and rapid identification method is needed to determine this pathogen. The current study demonstrated loop-mediated isothermal amplification (LAMP) superior specificity and sensitivity for W. ceti. Primers were designed targeting the hemolysin A gene, and LAMP was accomplished within 30 min at 63. The minimum concentration of LAMP for W. ceti DNA was 5.9 x 10-6 ng/µl, which was 100 times more sensitive than traditional PCR. LAMP confirmed the high specificity for W. ceti, whereas the other six pathogens were negative reactions. Furthermore, the effectiveness of LAMP was also demonstrated in simulated clinical samples. Based on the results, LAMP was rapid and convenient for point-of-care testing to diagnose W. ceti infections. [ABSTRACT FROM AUTHOR]

Fecal microbiota transplantation (FMT) is a therapeutic modality for treating neonatal calf diarrhea. Several practical barriers, including donor selection, fecal collection, and a limited timeframe for FMT, are the main constraints to using fresh feces for implementing on-farm FMT. We report the utility of FMT with pretreated ready-to-use frozen (F) or freeze-dried (FD) microorganisms for treating calf diarrhea. In total, 19 FMT (F-FMT, n = 10 and FD-FMT, n = 9) treatments were conducted. Both FMT treatments were 100% clinically effective; however, multi-omics analysis showed that FD-FMT was superior to F-FMT. Machine learning analysis with SourceTracker confirmed that donor microbiota was retained four times better in the recipient calves treated with FD-FMT than F-FMT. A predictive model based on receiver operating characteristic curve analysis and area under the curve showed that FD-FMT was more discriminative than F-FMT of the observed changes in microbiota and metabolites during disease recovery. These results provide new insights into establishing methods for preparing fecal microorganisms to increase the quality of FMT in animals and may contribute to FMT in humans. [ABSTRACT FROM AUTHOR]

A molecularly imprinted polymer-based dispersive micro-solid-phase extraction method has been developed for the efficient extraction and preconcentration of histamine from tuna fish samples prior to its determination by gas chromatography–flame ionization detector. In this approach, a molecularly imprinted polymer was prepared by chemical oxidation of pyrrole using iron (III) chloride, and then, it was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy and used in the extraction procedure. The method was validated using the International Council Research Protocol, and the results showed low limits of detection (0.06 mg kg−1) and quantification (0.21 mg kg−1), good precision (relative standard deviation = 3.2%), linearity (r2 ≥ 0.9969) and acceptable extraction recovery (98%). The method was successfully done on various tuna fish samples, and histamine was determined in them. [ABSTRACT FROM AUTHOR]

Indoxyl sulfate (IS), a uremic toxin, is a harmful factor that damages kidneys. Chronic heat stress in laying hens causes renal injury; however, whether IS accumulation is involved in this injury remains unknown. We selected 20 Boris brown laying hens (27 weeks old) and randomly assigned them to two groups (n = 10), one group was exposed to chronic heat stress (32 °C for 4 weeks), whereas the other was maintained at 24 °C. Chronic heat exposure significantly increased plasma and renal IS concentrations (P < 0.05). Exposure to heat also increased renal expression of the aryl hydrocarbon receptor (AhR) and its target genes (CYP1A4 and CYP1B1). Furthermore, chronic heat exposure tended to increase the 2-thiobarbituric acid reactive substances content (P = 0.08) and significantly decreased the antioxidant capacity in the kidney, while increasing the transcription levels of nuclear factor κB and fibrosis-related genes (COLA1A1, αSMA, TGF-β, Smad3, and VCAM-1) and the area of renal fibrosis. Our results indicate that chronic heat exposure induces systemic and renal IS accumulation in laying hens. This accumulated IS may activate the AhR pathway and chronically disrupt the oxidative stress status and antioxidant activity, thus promoting renal fibrosis and dysfunction in laying hens. [ABSTRACT FROM AUTHOR]

Loop-Mediated Isothermal Amplification (LAMP) represents a valuable technique for DNA/RNA detection, known for its exceptional sensitivity, specificity, speed, accuracy, and affordability. This study focused on optimizing a LAMP-based method to detect early signs of Plasmopara halstedii, the casual pathogen of sunflower downy mildew, a severe threat to sunflower crops. Specifically, a set of six LAMP primers (two outer, two inner, and two loop) were designed from P. halstedii genomic DNA, targeting the ribosomal Large Subunit (LSU). These primers were verified by in silico analysis and experimental validation using both target and non-target species' DNAs. Optimizations encompassing reaction conditions (temperature, time) and component concentrations (magnesium, Bst DNA polymerase, primers, and dNTP) were determined. Validation of these optimizations was performed by agarose gel electrophoresis. Furthermore, various colorimetric chemicals (Neutral Red, Hydroxynaphthol Blue, SYBR Safe, Thiazole Green) were evaluated to facilitate method analysis, and the real-time analysis has been optimized, presenting multiple approaches for detecting sunflower downy mildew using the LAMP technique. The analytical sensitivity of the method was confirmed by detecting P. halstedii DNA concentrations as low as 0.5 pg/μl. This pioneering study, establishing P. halstedii detection through the LAMP method, stands as unique in its field. The precision, robustness, and practicality of the LAMP protocol make it an ideal choice for studies focusing on sunflower mildew, emphasizing its recommended use due to its operational ease and reliability. [ABSTRACT FROM AUTHOR]

Cell cultures are models in biological and medical research to understand physiological and pathological processes. Cell lines are not always available depending on cell type and required species. In addition, the immortalization process often affects cell biology. Primary cells generally maintain a greater degree of similarity in short-term culture to the cells in tissue. Goal of this study was to verify the suitability of chicken primary epithelial caecal cells (PECCs) for in vitro investigations of host‒pathogen interactions. Epithelial nature of PECCs was confirmed by detection of tight and adherens junctions and cobblestone-like cell morphology. Sialic acids distribution was similar to that in caecal cyrosections. To understand the capacity of PECCs to respond to microbial challenges, the Toll-like receptors (TLRs) repertoire was determined. Exposure of PECCs to polyinosinic-polycytidylic acid (poly(I:C)) or lipopolysaccharide (LPS) led to upregulation of type I and III interferon (IFN) as well as interleukin (IL-) 1β, IL-6 and IL-8 mRNA expression. Overall, the PECCs showed properties of polarized epithelial cells. The presence of TLRs, their differential expression, as well as pattern recognition receptor dependent immune responses enable PECCs to act as suitable in vitro model for host‒pathogen interaction studies, which are difficult to conduct under in vivo conditions. [ABSTRACT FROM AUTHOR]

Campylobacteriosis outbreaks have previously been linked to dairy foods. While the genetic diversity of Campylobacter is well understood in high-income countries, it is largely unknown in low-income countries, such as Ethiopia. This study therefore aimed to conduct the first genomic characterization of Campylobacter isolates from the Ethiopian dairy supply chain to aid in future epidemiological studies. Fourteen C. jejuni and four C. coli isolates were whole genome sequenced using an Illumina platform. Sequences were analyzed using the bioinformatics tools in the GalaxyTrakr platform to identify MLST types, and single nucleotide polymorphisms, and infer phylogenetic relationships among the studied isolates. Assembled genomes were further screened to detect antimicrobial resistance and virulence gene sequences. Among 14 C. jejuni, ST 2084 and ST 51, which belong to the clonal complexes ST-353 and ST-443, respectively, were identified. Among the 4 sequenced C. coli isolates, two isolates belonged to ST 1628 and two to ST 830 from the clonal complex ST-828. The isolates of C. jejuni ST 2084 and ST 51 carried β-lactam resistance gene blaOXA-605, a fluoroquinolone resistance-associated mutation T86I in the gryA gene, and a macrolide resistance-associated mutation A103V in 50S L22. Only ST 2084 isolates carried the tetracycline resistance gene tetO. Conversely, all four C. coli ST 830 and ST 1628 isolates carried tetO, but only ST 1628 isolates also carried blaOXA-605. Lastly, C. jejuni ST 2084 isolates carried a total of 89 virulence genes, and ST 51 isolates carried up to 88 virulence genes. Among C. coli, ST 830 isolates carried 71 genes involved in virulence, whereas two ST 1628 isolates carried up to 82 genes involved in virulence. Isolates from all identified STs have previously been isolated from human clinical cases, demonstrating a potential food safety concern. This finding warrants further monitoring of Campylobacter in dairy foods in Ethiopia to better understand and manage the risks associated with Campylobacter contamination and transmission. [ABSTRACT FROM AUTHOR]

Salmonella, the prevailing zoonotic pathogen within the Enterobacteriaceae family, holds the foremost position in global bacterial poisoning incidents, thereby signifying its paramount importance in public health. Consequently, the imperative for expeditious and uncomplicated detection techniques for Salmonella in food is underscored. After more than two decades of development, loop-mediated isothermal amplification (LAMP) has emerged as a potent adjunct to the polymerase chain reaction, demonstrating significant advantages in the realm of isothermal amplification. Its growing prominence is evident in the increasing number of reports on its application in the rapid detection of Salmonella. This paper provides a systematic exposition of the technical principles and characteristics of LAMP, along with an overview of the research progress made in the rapid detection of Salmonella using LAMP and its derivatives. Additionally, the target genes reported in various levels, including Salmonella genus, species, serogroup, and serotype, are summarized, aiming to offer a valuable reference for the advancement of LAMP application in Salmonella detection. Finally, we look forward to the development direction of LAMP and expect more competitive methods to provide strong support for food safety applications. [ABSTRACT FROM AUTHOR]

Overview: The roadmap adopted by the World Health Organization (WHO) for eliminating neglected tropical diseases aims to eliminate schistosomiasis, as a public health concern, by 2030. While progress has been made towards reducing schistosomiasis morbidity control in several sub-Saharan African countries, there is still more that needs to be done. Proper surveillance using accurate diagnostics with acceptable sensitivity and specificity is essential for evaluating the success of all efforts against schistosomiasis. Microscopy, despite its low sensitivity, remains the gold standard approach for diagnosing the disease. Although many efforts have been made to develop new diagnostics based on circulating parasite proteins, genetic markers, schistosome egg morphology, and their paramagnetic properties, none has been robust enough to replace microscopy. This review highlights common diagnostic approaches for detecting schistosomiasis in field and clinical settings, major challenges, and provides new and novel opportunities and diagnosis pathways that will be critical in supporting elimination of schistosomiasis. Methods: We searched for relevant and reliable published literature from PubMed, Scopus, google scholar, and Web of science. The search strategies were primarily determined by subtopic, and hence the following words were used (schistosom*, diagnosis, Kato–Katz, antibody test, circulating antigen, POC-CCA, UCP-LF-CAA, molecular diagnostics, nucleic acid amplification test, microfluidics, lab-on a disk, lab-on chip, recombinase polymerase amplification (RPA), LAMP, portable sequencer, nanobody test, identical multi-repeat sequences, diagnostic TPPs, REASSURED, extraction free), and Boolean operators AND and/OR were used to refine the searching capacity. Due to the global public health nature of schistosomiasis, we also searched for reliable documents, reports, and research papers published by international health organizations, World Health Organization (WHO), and Center for Disease control and Elimination. [ABSTRACT FROM AUTHOR]

The expanding use of phytopharmaceuticals, as a more safe and efficient remedies for different pharmacological activities, and as dietary and nutritional supplements, has created a need for having efficient, eco-friendly, and selective extraction techniques to enhance the economic and environmental greenness of the applied traditional extraction methods. Recently, the application of molecularly imprinted polymers (MIPs), has grasped the attention in the field of natural products due to their ease of design and preparation with specific recognition cavities that can selectively bind to a target analyte. The present review aims not only to discuss the application of MIPs with the past ten years in the field solid phase extraction of different classes of natural products (NPs) but also their impact on the effectiveness of chromatographic separation and detection will be elaborated. Based on their performance, MIPs represent a promising class of artificial adsorbents that can be commercialized for beneficiary extraction of natural products with much less consumption of solvents and higher selectivity when compared to the traditional types of chromatographic adsorbents besides the possibility of their 3D printing to create more optimized and uniform manifolds to match the different types of extraction and detection applications of natural products (NPs). [ABSTRACT FROM AUTHOR]

Background: Campylobacter (C.) jejuni is one of the most important bacterial foodborne pathogens worldwide. Probiotics such as Lactobacillus or Bacillus species are considered one option for reducing the colonization rate and magnitude in poultry, the most frequent source of human infections. Due to the lack of suitable avian in vitro models such as chicken intestinal cell lines, especially those derived from the cecum, most in vitro studies on C. jejuni host interaction have been conducted with human intestinal cell lines. In this study, we compared C. jejuni-cell interactions between primary chicken cecal cells and the human intestinal cell line Caco-2, which is derived from colorectal adenocarcinoma, and investigated possible interfering effects of selected probiotic candidates. Results: We detected differences in adhesion and invasion between the two tested gut cell types and between different C. jejuni strains. The probiotic inhibition of C. jejuni adhesion and invasion of human and avian gut cells was affected by host cell type, investigated C. jejuni strain and time points of probiotic treatment. Additionally, our results suggest a possible correlation between C. jejuni invasion and the detected increase in IL-6 mRNA expression. Conclusions: Our results indicate distinct differences between avian and human gut cells in their interaction with C. jejuni. Therefore, data obtained in one host species on C. jejuni-host interaction may not easily be transferrable to another one. The factors influencing the variable efficacy of probiotic intervention in chicken and human derived cells should be investigated further. [ABSTRACT FROM AUTHOR]

Nipah and Hendra viruses belong to the Paramyxoviridae family, which pose a significant threat to human health, with sporadic outbreaks causing severe morbidity and mortality. Early symptoms include fever, cough, sore throat, and headache, which offer little in terms of differential diagnosis. There are no specific therapeutics and vaccines for these viruses. This review comprehensively covers a spectrum of diagnostic techniques for Nipah and Hendra virus infections, discussed in conjunction with appropriate type of samples during the progression of infection. Serological assays, reverse transcriptase Real-Time PCR assays, and isothermal amplification assays are discussed in detail, along with a listing of few commercially available detection kits. Patents protecting inventions in Nipah and Hendra virus detection are also covered. Despite several outbreaks of Nipah and Hendra infections in the past decade, in-depth research into their pathogenesis, Point-of-Care diagnostics, specific therapies, and human vaccines is lacking. A prompt and accurate diagnosis is pivotal for efficient outbreak management, patient treatment, and the adoption of preventative measures. The emergence of rapid point-of-care tests holds promise in enhancing diagnostic capabilities in real-world settings. The patent landscape emphasizes the importance of innovation and collaboration within the legal and business realms. [ABSTRACT FROM AUTHOR]

Background: Avian influenza virus (AIV) not only causes huge economic losses to the poultry industry, but also threatens human health. Reverse transcription recombinase-aided amplification (RT-RAA) is a novel isothermal nucleic acid amplification technology. This study aimed to improve the detection efficiency of H5, H7, and H9 subtypes of AIV and detect the disease in time. This study established RT-RAA-LFD and real-time fluorescence RT-RAA (RF-RT-RAA) detection methods, which combined RT-RAA with lateral flow dipstick (LFD) and exo probe respectively, while primers and probes were designed based on the reaction principle of RT-RAA. Results: The results showed that RT-RAA-LFD could specifically amplify H5, H7, and H9 subtypes of AIV at 37 °C, 18 min, 39 °C, 20 min, and 38 °C, 18 min, respectively. The sensitivity of all three subtypes for RT-RAA-LFD was 102 copies/µL, which was 10 ∼100 times higher than that of reverse transcription polymerase chain reaction (RT-PCR) agarose electrophoresis method. RF-RT-RAA could specifically amplify H5, H7, and H9 subtypes of AIV at 40 °C, 20 min, 38 °C, 16 min, and 39 °C, 17 min, respectively. The sensitivity of all three subtypes for RF-RT-RAA was 101 copies/µL, which was consistent with the results of real-time fluorescence quantification RT-PCR, and 100 ∼1000 times higher than that of RT-PCR-agarose electrophoresis method. The total coincidence rate of the two methods and RT-PCR-agarose electrophoresis in the detection of clinical samples was higher than 95%. Conclusions: RT-RAA-LFD and RF-RT-RAA were successfully established in this experiment, with quick response, simple operation, strong specificity, high sensitivity, good repeatability, and stability. They are suitable for the early and rapid diagnosis of Avian influenza and they have positive significance for the prevention, control of the disease, and public health safety. [ABSTRACT FROM AUTHOR]

The properties of six commonly used, commercially available, fluorescent dyes were compared in staining right-handed B-DNA and left-handed Z-DNA. All showed different degree of fluorescence turn-on in the presence of B-DNA, but very little in the presence of Z-DNA. The optimal range of dye-DNA ratios of DNA was determined. While these dyes do not provide a turn-on type probe for Z-DNA, staining between B- and Z-DNA using dyes such as SYBR Green I was shown to be useful in tracking the kinetics of conformational changes between these two forms of DNA. Finally, SYBR Green I showed unique circular dichroism patterns in 4 M NaCl that change in the presence of double stranded DNA, both in the visible and UV range. [ABSTRACT FROM AUTHOR]

This work investigates a digital microfluidic chip and presents an advancement to microfluidic optical annealing methods through the application of whispering gallery mode (WGM) with near infrared excitation. Establishing a microfluidic chip with point‐of‐care capabilities, including actuation and annealing, has proven to be important. Unfortunately, poor heat absorption due to the long optical penetration depth of near infrared light creates scaling limitations for applications in optical‐based microfluidics. Through the application of WGM, the interaction length between the droplet and light is increased beyond the droplet diameter to improve heating and optical absorption. This is supported by finite‐difference time‐domain electromagnetic simulations and experimental results showing a greatly improved temperature change. Such a system is implemented in an open system digital microfluidic chip, to facilitate annealing via side illumination of droplets. The open system digital microfluidic chip is programmable for droplet actuation. The fundamental experiment of preprogrammed actuation of microdroplets is demonstrated in a 36 electrode grid. The results of annealing and actuation show potential for implementation in point‐of‐care microfluidic devices. [ABSTRACT FROM AUTHOR]

Background: Some epidemiologic studies associate traumatic brain injury (TBI) with Alzheimer's disease (AD). Objective: To test whether a TBI-induced acceleration of age-related mitochondrial change could potentially mediate the reported TBI-AD association. Methods: We administered unilateral controlled cortical impact (CCI) or sham injuries to 5-month-old C57BL/6J and tau transgenic rTg4510 mice. In the non-transgenics, we assessed behavior (1–5 days, 1 month, and 15 months), lesion size (1 and 15 months), respiratory chain enzymes (1 and 15 months), and mitochondrial DNA copy number (mtDNAcn) (1 and 15 months) after CCI/sham. In the transgenics we quantified post-injury mtDNAcn and tangle burden. Results: In the non-transgenics CCI caused acute behavioral deficits that improved or resolved by 1-month post-injury. Protein-normalized complex I and cytochrome oxidase activities were not significantly altered at 1 or 15 months, although complex I activity in the CCI ipsilesional cortex declined during that period. Hippocampal mtDNAcn was not altered by injury at 1 month, increased with age, and rose to the greatest extent in the CCI contralesional hippocampus. In the injured then aged transgenics, the ipsilesional hippocampus contained less mtDNA and fewer tangles than the contralesional hippocampus; mtDNAcn and tangle counts did not correlate. Conclusions: As mice age their brains increase mtDNAcn as part of a compensatory response that preserves mitochondrial function, and TBI enhances this response. TBI may, therefore, increase the amount of compensation required to preserve late-life mitochondrial function. If TBI does modify AD risk, altering the trajectory or biology of aging-related mitochondrial changes could mediate the effect. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This paper gives an account of the history of veterinary bacteriology including clinical veterinary bacteriology as well as the area of veterinary public health in Denmark from the 1880s to 2022. We describe key persons, including B. Bang, C.O. Jensen, K.A. Jensen and others who made important contributions to the development of these areas of microbiological expertise, and we discuss how challenges ranging from bovine tuberculosis to bacterial antimicrobial resistance have been met. Further, we describe progress in research on important bacterial pathogens both with regard to animal clinical aspects and zoonotic food‐related aspects. Finally, we describe current issues in relation to One Health and research organization. [ABSTRACT FROM AUTHOR]

Background: The Northern bobwhite (Colinus virginianus) is an economically important, and popular game bird in North America. Northern bobwhites have experiencing declines of > 3.5% annually in recent decades due to several factors. The eyeworm Oxyspirura petrowi is a nematode parasite frequently found in the eyes of bobwhites. Although reported frequently in wild bobwhites, there is no research to understand the host-parasite mechanism. Hence, it is important to investigate mechanisms of eyeworm invasion and immune modulation in bobwhite. Cytokine gene expression using RT-PCR is widely used to identify the innate immune response of a host to an infection. Methodology: In this study, we evaluated ten reference genes (HMBS, RPL19, RPL32, RPS7, RPS8, TATA, SDHA, YWHAZ, GAPDH, and ACTB) for their stability across three tissues (liver, spleen, and caecal tonsils) of control and O. petrowi infected Northern bobwhites. Primer efficiency and reference genes stability were assessed using GeNorm, NormFinder, and BestKeeper. Results: Expression of these reference genes with respect to O. petrowi infection in bobwhites showed RPL32 and HMBS were the most stable genes in the liver, HMBS and SDHA were the most stable genes in the spleen, and HMBS and YWHAZ were equally stable reference genes in the caecal tonsils. Conclusion: Based on the geometric mean of all three analyses, our results indicate that the combination of RPL32 and HMBS for the liver, HMBS and SDHA for the spleen, and YWHAZ and HMBS for caecal tonsils might be used as reference genes for normalization in gene expression investigations on Northern bobwhites. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the effect of age, breed, and sex of broilers, as well as a probiotic or phytobiotic product on mucosal morphology, bacterial metabolites, and immune traits in the ileum of broilers. A total of 2,880 one-day-old male and female broiler chicks from two breeds (Ross308® and Cobb500®) were randomly assigned to 72 pens. Broilers were offered a wheat-soybean diet without (CO), or with either a probiotic (PO; 2.4 × 109 CFU/kg of Bacillus subtilis DSM32324 and DSM32325 and B. amyloliquefaciens DSM25840) or a phytobiotic (PY; grape extract, 165 ppm procyanidin and 585 ppm polyphenols of the diet) product. The trial was conducted with a 3 × 2 × 2 factorial arrangement of diet, breed, and sex in a completely randomized design (6 replicate-pens per treatment). At day 7, 21, and 35, one chicken per pen was slaughtered for collecting ileal tissue to evaluate of histomorphology and mRNA expression, as well as ileal digesta to measure bacterial metabolites. Data were subjected to ANOVA (the main factors; age, diet, breed, and sex) and Four-Way ANOVA (interactions) using GLM procedure. Overall, the concentration of acetate and total short chain fatty acids reached the peak and lactate decreased to its lowest on day 21, but their concentrations at day 7 and 35 were similar (p > 0.05). Spermine, spermidine, and ammonia decreased after day 7, while putrescine and cadaverine increased after day 21 (p < 0.05). mRNA expression of cytokines, mucin 2 (MUC2) and claudin 5 (CLDN5) was similar; increased from day 7 to 21 and decreased afterward (p < 0.05). Villus height, crypt depth and villus surface area increased with age (p < 0.05). Acidic goblet cells (GC) number and density increased after day 21 (p < 0.05). Ross broilers showed higher D-lactate concentration and IFN-γ expression, while Cobb broilers had greater IL-4, IL-6 and TNF-α expression and higher total GC number (p < 0.05). Female displayed higher villus height and GC number and density (mixed and total GC) than male (p < 0.05). The effect of dietary treatment was not found on any investigated variables (p > 0.05). In conclusion, aging of broilers affected ileal histomorphology, cytokine expression, and barrier integrity, as well as bacterial activity. These observed impacts could be attributed to host-microbiota interaction and the direct effects of bacterial metabolites on intestinal cells and immune system. [ABSTRACT FROM AUTHOR]

In the era of liquid biopsy, microRNAs emerge as promising candidates for the early diagnosis and prognosis of cancer, offering valuable insights into the disease's development. Among all the existing analytical approaches, even if traditional approaches such as the nucleic acid amplification ones have the advantages to be highly sensitive, they cannot be used at the point-of-care, while sensors might be poorly sensitive despite their portability. In order to improve the analytical performance of existing electroanalytical systems, we demonstrate how a simple chromatographic paper-based disk might be useful to rationally improve the sensitivity, depending on the number of preconcentration cycles. A paper-based electrochemical platform for miRNA detection has been developed by modifying a paper-based electrode with a methylene blue (MB)-modified single-stranded sequence (ssDNA) complementary to the chosen miRNA, namely miR-224 that is associated with lung cancer. A detection limit of ca. 0.6 nM has been obtained in spiked human serum samples. To further enhance the sensitivity, an external chromatographic wax-patterned paper-based disk has been adopted to preconcentrate the sample, and this has been demonstrated both in standard and in serum solutions. For each solution, three miR-224 levels have been preconcentrated, obtaining a satisfactory lowering detection limit of ca. 50 pM using a simple and sustainable procedure. This approach opens wide possibilities in the field of analytical and bioanalytical chemistry, being useful not only for electrochemistry but also for other architectures of detection and transduction., (© 2024. The Author(s).)

Background: Malaria is a globally distributed infectious disease. According to the World Health Organization, Angola is one of the six countries that account for over half the global malaria burden in terms of both malaria cases and deaths. Diagnosis of malaria still depends on microscopic examination of thin and thick blood smears and rapid diagnostic tests (RDTs), which often lack analytical and clinical sensitivity. Molecular methods could overcome these disadvantages. The aim of this study was to evaluate, for the first time to our knowledge, the performance of a loop-mediated isothermal amplification (LAMP) for the diagnosis of malaria in an endemic area in Cubal, Angola, and to assess the reproducibility at a reference laboratory. Methods: A total of 200 blood samples from patients attended at Hospital Nossa Senhora da Paz, Cubal, Angola, were analysed for Plasmodium spp. detection by microscopy, RDTs, and LAMP. LAMP assay was easily performed in a portable heating block, and the results were visualized by a simple colour change. Subsequently, the samples were sent to a reference laboratory in Spain to be reanalysed by the same colorimetric LAMP assay and also in real-time LAMP format. Results: In field tests, a total of 67/200 (33.5%) blood samples were microscopy-positive for Plasmodium spp., 98/200 RDT positive, and 112/200 (56%) LAMP positive. Using microscopy as reference standard, field LAMP detected more microscopy-positive samples than RDTs (66/67; 98% vs. 62/67; 92.5%). When samples were reanalysed at a reference laboratory in Spain using both colorimetric and real-time assays, the overall reproducibility achieved 84.5%. Conclusions: This is the first study to our knowledge in which LAMP has been clinically evaluated on blood samples in a resource-poor malaria-endemic area. The colorimetric LAMP proved to be more sensitive than microscopy and RDTs for malaria diagnosis in field conditions. Furthermore, LAMP showed an acceptable level of reproducibility in a reference laboratory. The possibility to use LAMP in a real-time format in a portable device reinforces the reliability of the assay for molecular diagnosis of malaria in resource-poor laboratories in endemic areas. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Faculty of Veterinary Medicine, Erciyes University / Erciyes Üniversitesi Veteriner Fakültesi Dergisi is the property of Erciyes University, Faculty of Veterinary Medicine and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

l‐Malate is a key flavor enhancer and acidulant in the food and beverage industry, particularly winemaking. Enzyme‐based amperometric biosensors offer convenience for monitoring its concentration. However, only a small number of off‐the‐shelf malate‐oxidizing enzymes have been used in previous devices. These typically have linear ranges poorly suited for the l‐malate concentrations found in fruit processing and winemaking, making it necessary to use precisely diluted samples. Here, we describe a pipeline of database‐mining, gene synthesis, recombinant expression, and spectrophotometric assays to characterize previously untested enzymes for their suitability in biosensors. The pipeline yielded a bespoke biocatalyst—the Ascaris suum malic enzyme carrying mutation R181Q [AsME(R181Q)]. Our first prototype with AsME(R181Q) had an ultra‐wide linear range of 50–200 mM l‐malate, corresponding to concentrations found in undiluted fruit juices (including grape). Changing the dication from Mg2+ to Mn2+ increased sensitivity five‐fold and adding citrate (100 mM) increased it another six‐fold, albeit decreasing the linear range to 1–10 mM. To our knowledge, this is the first time an l‐malate biosensor with a tuneable combination of sensitivity and linear range has been described. The sensor response was also tested in the presence of various molecules abundant in juices and wines, with ascorbate shown to be a potent interferent. Interference was mitigated by the addition of ascorbate oxidase, allowing for differential measurements on an undiluted, untreated wine sample that corresponded well with commercial l‐malate testing kits. Overall, this work demonstrates the power of an enzyme‐centric approach for designing electrochemical biosensors with improved operational parameters and novel functionality. [ABSTRACT FROM AUTHOR]