Methodology For Rapid Detection Research Articles

Truncated M13 phage for smart detection of E. coli under dark field

BackgroundThe urgent need for affordable and rapid detection methodologies for foodborne pathogens, particularly Escherichia coli (E. coli), highlights the importance of developing efficient and widely accessible diagnostic systems. Dark field microscopy, although effective, requires specific isolation of the target bacteria which can be hindered by the high cost of producing specialized antibodies. Alternatively, M13 bacteriophage, which naturally targets E. coli, offers a cost-efficient option with well-established techniques for its display and modification. Nevertheless, its filamentous structure with a large length-diameter ratio contributes to nonspecific binding and low separation efficiency, posing significant challenges. Consequently, refining M13 phage methodologies and their integration with advanced microscopy techniques stands as a critical pathway to improve detection specificity and efficiency in food safety diagnostics.MethodsWe employed a dual-plasmid strategy to generate a truncated M13 phage (tM13). This engineered tM13 incorporates two key genetic modifications: a partial mutation at the N-terminus of pIII and biotinylation at the hydrophobic end of pVIII. These alterations enable efficient attachment of tM13 to diverse E. coli strains, facilitating rapid magnetic separation. For detection, we additionally implemented a convolutional neural network (CNN)-based algorithm for precise identification and quantification of bacterial cells using dark field microscopy.ResultsThe results obtained from spike-in and clinical sample analyses demonstrated the accuracy, high sensitivity (with a detection limit of 10 CFU/μL), and time-saving nature (30 min) of our tM13-based immunomagnetic enrichment approach combined with AI-enabled analytics, thereby supporting its potential to facilitate the identification of diverse E. coli strains in complex samples.ConclusionThe study established a rapid and accurate detection strategy for E. coli utilizing truncated M13 phages as capture probes, along with a dark field microscopy detection platform that integrates an image processing model and convolutional neural network.Graphical

Accelerated Detection of Foodborne Pathogens and Toxins: Techniques and Applications – A Review

Pathogens associated with food and their corresponding toxins, including mycotoxins, pose a sig¬nificant threat to public health, leading to various health issues worldwide, particularly affecting infants, young children, and the elderly. The rise in foodborne illnesses highlights the crucial importance of food safety in ensuring a reliable food supply and promoting health. Timely detection of foodborne pathogens, toxins, and mycotoxins is essential for reducing the incidence of foodborne diseases. This review aims to provide comprehensive insights into the methodology for rapid detection, detailing the principles, appli¬cations, advantages, and limitations to enhance current knowledge. Conventional culture-based methods for foodborne pathogen identification are selective and are hindered by significant time demands, labor intensity, challenges from complex sample preparation, delayed results, and the need for specialized per¬sonnel. In contrast, the new emerging detection techniques such as immunological, nucleic acid-based, and biosensor-based methods offer time efficiency, reduced labor, improved accuracy, sensitivity, specificity, and reliability, and are user-friendly. Recently, numerous novel techniques have been developed for the rapid identification of foodborne pathogens, toxins, and mycotoxins, transforming food safety practices. Providing rapid and accurate results is crucial for controlling and managing foodborne disease outbreaks while ensuring food safety. As the global food supply chain becomes more complex, advancing rapid and automated detection methods remains essential for safeguarding food safety and quality, as well as enhanc¬ing public health.

Training strategy and intelligent model for in-situ rapid measurement of subgrade compactness

Current methods for inspecting subgrade compaction quality are time-consuming and destructive. This paper presents an in-situ measurement methodology for rapid detection of subgrade compaction quality using Ultrasonic Pulse Velocity (UPV) and Intelligent Compaction (IC). Field compaction tests, field UPV test, and laboratory tests are performed to construct the heterogeneous datasets. A set of intelligent models are established to estimate the subgrade compactness in terms of the in-time measured UPV. The training strategy is proposed by expanding dataset quantity and dataset dimension to enhance the model performance. The model demonstrates high accuracy in compactness estimation and strong generalization in verification. This contribution can be utilized in engineering applications to effectively detect the compaction quality during the construction.

Dispersant-free supra single-walled carbon nanotubes for simultaneous and highly sensitive biomolecule sensing in ex vivo mouse tissues

A simple sensing methodology for rapid detection of multiple biomolecules in human health diagnostics remains a significant challenge. Herein, we developed a novel dispersant-free supra single-walled carbon nanotube (supra SWCNT)-coated glassy carbon electrode (GCE) for simultaneous sensing of biomolecules, such as ascorbic acid (AA), dopamine (DA), and uric acid (UA), in ex vivo mouse tissues. Supra SWCNTs with stable hydrogen bonds were synthesized via a simple chemical modification, after which they were characterized and confirmed using different physicochemical and electrochemical techniques. The supra SWCNT-coated GCE exhibited simultaneous sensing of AA, DA, and UA at linear dynamic ranges with lower detection limits of 12, 0.02, and 0.36 μM, respectively. Furthermore, they demonstrated high selectivity (with co-existing interference) and excellent reproducibility for effective sensing of the target biomolecules in different real samples. The rapid functioning, low cost, scalable preparation, and enhanced applicability for simultaneous sensing of target biomolecules in Parkinson's disease-induced mouse liver, kidney, and brain tissues suggest the suitability of the developed sensor for real-world applications.

STATE: A Test Structure for Rapid and Reliable Prediction of Resistive RAM Endurance

Characterizing and quantifying the endurance of Resistive RAM devices is critical to assess their reliability for integration in electronic systems. This paper proposes a novel characterization methodology for rapid detection of RRAMs reliability issues during endurance tests. A test structure consisting of an array of non-addressable RRAM memory cells with parallel connection of all the memory elements is used for this purpose. The test structure fills the gap between isolated cells and circuit level endurance tests as it combines the test speed of an isolated cell while providing at the same time data from multiple devices. Endurance tests are conducted while monitoring RRAM electrical parameters of interest for each switching cycle. Experimental results show that most studies that claimed high endurance without capturing all switching cycles, or by considering an isolated cell, lead to an overestimation of the endurance. Finally, an endurance failures mitigation scheme based on RRAM current sensing in the RESET direction is presented to improve the endurance.

A Naive and Semantic Approach for Detecting Face Mask Region Based Convolutional Neural Networks (R-CNN)

Due to Covid-19, most of the health care organizations and governments has ordered their citizens to wear face mask to protect themself. This novel research presents a tactical methodology for rapid detection whether a person is wearing a face mask or not. It is entirely different from the existing system; the singular aim of the proposed system is to train the deep learning model with a minimum number of image samples and to operate face mask instance segmentation and along with object box detection. In this work, we proposed a novel and semantic pixel-to-pixel region based deep network which can detect no of face mask instances in different categories pixel wise to organize the segment bounding box and the confidence of the various categories for each pixel. This system experimental output demonstrate that this approach can effectively and precisely detect the face mask with multi-feature combination. It is also reported that our application performance outperforms the existing system.

APPLICATION OF SENTINEL-1 DATA FOR RAPID DETECTION OF LANDSLIDES

The technique of interferometric processing of radar images from the Sentinel-1 satellite on the territory of Iceland, which is affected by landslide processes, was implemented. The possibility of applying the methodology for rapid detection of landslides, as well as monitoring to predict them is shown.

Superomniphobic Papers for On‐Paper pH Sensors

AbstractPaper‐based superomniphobic surfaces are of great interest because paper is flexible, inexpensive, lightweight, breathable, and recyclable. Prior reports on paper‐based superomniphobic surfaces have failed to demonstrate high mobility with low surface tension liquids. In order to overcome this issue, in this work, superomniphobic papers are developed through growth of nanofilaments on inherent microfibers of papers without noticeably altering their microscale features (i.e., diameter and distance of the microfibers). These superomniphobic papers display very low roll‐off angle, indicative of ultra‐high droplet mobility, even with low surface tension liquids. Here, a facile method is also developed to control the motion and adhesion of the droplets on the superomniphobic paper. Utilizing such liquid mobility in a controlled manner on these superomniphobic papers, a simple on‐paper pH sensor is fabricated. It is anticipated that this on‐paper, simple, and rapid detection methodology can also be extended to the colorimetric sensing of protein and chemical assays. Further, these superomniphobic papers have potential applications in water–oil separation and enhanced weight‐bearing capacity.

Inactivation of Escherichia coli Using Nanosecond Electric Fields and Nisin Nanoparticles: A Kinetics Study

Nisin is a recognized bacteriocin widely used in food processing, however, being ineffective against gram-negative bacteria and in complex food systems. As a result, the research of methods that have cell wall–permeabilizing activity is required. In this study, electroporation to trigger sensitization of gram-negative bacteria to nisin-loaded pectin nanoparticles was used. As a model microorganism, bioluminescent strain of E. coli was introduced. Inactivation kinetics using nanosecond pulsed electric fields (PEFs) and nisin nanoparticles have been studied in a broad range (100–900 ns, 10–30 kV/cm) of pulse parameters. As a reference, the microsecond range protocols (100 μs × 8) have been applied. It was determined that the 20–30 kV/cm electric field with pulse duration ranging from 500 to 900 ns was sufficient to cause significant permeabilization of E. coli to trigger a synergistic response with the nisin treatment. The kinetics of the inactivation was studied with a time resolution of 2.5 min, which provided experimental evidence that the efficacy of nisin-based treatment can be effectively controlled in time using PEF. The results and the proposed methodology for rapid detection of bacteria inactivation rate based on bioluminescence may be useful in the development and optimization of protocols for PEF-based treatments.

Catching “butterflies” in the morning: A new methodology for rapid detection of aerially deployed plastic land mines from UAVs

It is estimated that there are at least 100 million military munitions and explosives of concern (MEC) devices in the world of various size, shape, and composition. Millions of these are surface plastic land mines with low-pressure detonation regimes, such as the mass-produced Soviet PFM-1. These aerially deployed land mines are concentrated primarily in postconflict developing countries such as Afghanistan and represent a continued humanitarian threat, while also thwarting economic and social development in impacted regions. Identification of this particular type of MEC category poses a significant geophysical challenge, as these mines contain almost no metal (nonferrous aluminum). As a result, standard MEC detection and remediation methodologies based on geophysical principles of magnetometry and electromagnetic induction prove largely ineffective and possibly dangerous. Low-cost unmanned aerial vehicles can rapidly collect large remotely sensed data sets with no risk to MEC technicians. We present results of an experiment focused on remotely assessing thermal signatures of plastic land mines relative to background geology to show that this type of analysis permits rapid detection of randomly dispersed plastic MECs with a high degree of accuracy and low associated costs.

In-field detection of multiple pathogenic bacteria in food products using a portable fluorescent biosensing system

Foodborne diseases caused by pathogenic bacteria have increasingly become a major public health issue worldwide. Rapid, simple, and accurate detection methods are urgently needed for in-field screening of bacterial pathogens. In our previous work, rapid detection methodologies have been established based on fluorescent nanobiosensors for simultaneous separation and detection of multiple foodborne pathogenic bacteria. In this research, a portable fluorescent biosensing system was designed and built and further assessed for in-field detection of three main types of bacterial pathogens that have been associated with the outbreaks of foodborne illness. Using the developed fluorescent nanobiosensor coupled with nanobead-based immunomagnetic separation, we conducted blind tests with the portable device to simultaneously detect E. coli O157:H7, L. monocytogenes, and S. Typhimurium in different food products in three cities selected from three big agricultural provinces in China. Specificity tests showed low interference of this multiplex biosensor from non-targets in food samples. The detection could be done from sampling to results within 60 min. Limits of detections of this method for E. coli O157:H7, L. monocytogenes, and S. Typhimurium were determined to be 102, 103, and 103 CFU/mL in lettuce, shrimp, and ground beef, respectively. Recovery tests were also investigated and this method was evaluated to be accurate comparing with the gold standard culturing method. Therefore, it is feasible for this portable fluorescence biosensing system to be used in rapid and in-field screening of multiple foodborne pathogenic bacteria in foods, such as vegetables, livestock meat, and sea food. And together with fluorescent nanobiosensors, it provides a promising alternative tool to traditional culturing method, or even conventional ELISA and PCR based methods.

Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.

Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid—locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation.

Prostate Cancer Biomarker Detection with Carbon Nanotubes Modified Screen Printed Electrodes

AbstractDNA hypermethylation is an epigenetic alteration and a promising biomarker for early prostate cancer detection. Simple, sensitive, easy to handle and rapid detection methodologies are imperative for point of care diagnostics especially for cancer. Herein, we describe for the first time a regenerable and compatible electrochemical biosensor for detection of Glutathione S‐Transferase P‐1 (GSTP‐1) gene hypermethylation related to prostate cancer via DNA hybridization onto the disposable Carbon and Multi Walled Carbon Nanotubes (MWCNT) Screen Printed Electrodes (SPEs). In the study, capture probes were adsorbed onto the SPEs by simple passive adsorption and then hybridization was achieved by sending the complementary target onto the probe‐modified electrodes. The selectivity of the biosensor was proved by control studies. Differential Pulse Voltammetry (DPV) technique was used to detect hybridization via guanine oxidation signals changes. The total time of the optimized method was nearly 1h, measurements took for less than 1 min, and the biosensor response was stable up to 40 days of storage period at 4 °C. The main advantages of the biosensor are very low detection limit (picomolar range) and capability of reusing the biosensor for at least 3 times after very simple regeneration process that is a unique property to reduce the cost of the assay. In addition, this is the first study that demonstrates the detection of GSTP‐1 hypermethylation electrochemically by using SPEs in order to create point of care diagnostics. The optimum parameters for the biosensor, as well as its future prospects to enhance the performance of DNA biosensors were also presented.

Rapid detection methodology for inorganic mercury (Hg2+) in seafood samples using conjugated polymer (1,4-bis-(8-(4-phenylthiazole-2-thiol)-octyloxy)-benzene) (PPT) by colorimetric and fluorescence spectroscopy

This paper reports a highly sensitive, selective and rapid detection methodology for inorganic mercury (Hg2+) using conjugated polymer poly (1,4-bis-(8-(4-phenylthiazole-2-thiol)-octyloxy)-benzene) (PPT) by colorimetric and fluorescence spectroscopy “turn-off-turn-on” responses towards I− and Hg2+ in seafood samples. A simple and economical oxidative polymerization reaction method was utilized to synthesize polymer PPT, a derivative of neutral conjugated polymer poly-p-phenylene (PPP). The digested seafood samples were spiked with I− and Hg2+ salts and were analyzed using conjugated polymer PPT. The standard calibration curve was obtained from the plot of fluorescence intensity of PPT-I− complex as a function of the increasing analyte concentration (Hg2+) with an estimated detection limit (LOD) of 1.86nM and (R2=98.86%), respectively. A high specificity for I− and Hg2+ using PPT in a competent environment of several different anions, halides and metal cations were successfully obtained. The recoveries of Hg2+ ranged from 97.92 to 97.43% in fish and shrimp samples, respectively. These results exhibit that this kind of PPT polymer is reliable enough to detect Hg2+ in the seafood samples, providing important support to expand the scope of conjugated polymer application mainly in seafood industry.

Loop-mediated isothermal amplification assays for screening of bacterial integrons.

BackgroundThe occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection.ResultsIn this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 μL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%.ConclusionsThe intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.

Evaluation of Target Sequences for the Polymerase Chain Reaction–Based Detection ofSalmonellain Artificially Contaminated Beef

Salmonella is a major cause of foodborne diseases worldwide, which has fueled the demand for the development and evaluation of sensitive, specific, and rapid detection methodologies, such as polymerase chain reaction (PCR). In this study, six primer pairs for the detection of Salmonella were evaluated by PCR with isolates of Salmonella spp. (115) and other bacteria (104). The primers designed for the sifB gene provided the best performance regarding specificity and sensitivity (100%). These primers were selected and used to develop a PCR assay for Salmonella detection during the enrichment steps of the conventional detection method in spiked beef samples. The enrichment steps were: buffered peptone water (BPW), Rappaport-Vassiliadis soya broth (RVS) and at the Müller-Kauffmann tetrathionate novobiocin broth (MKTTn), after 18 h (BPW) and 24 h (RVS and MKTTn) of incubation. The initial concentrations of the Salmonella inocula were 10¹, 10², and 10³ colony-forming units/25 g. The protocol was able to detect Salmonella at all concentrations in the enrichment steps, but not in the nonenriched samples. These results indicated that the proposed protocol was suitable to detect Salmonella in beef during the intermediate stages of the conventional isolation protocol, substantially reducing the time required to obtain the final results.

A new methodology for rapid detection of Lactobacillus delbrueckii subsp. bulgaricus based on multiplex PCR

In this study we present a novel multiplex PCR assay for rapid and efficient detection of Lactobacillus delbrueckii subsp. bulgaricus. The accuracy of our method was confirmed by the successful identification of L. delbrueckii subsp. bulgaricus in commercial yoghurts and food supplements and it may be readily applied to the food industry.

Spatiotemporal distribution of coastal and oceanic Atlantic cod Gadus morhua sub-groups after escape from a farm

AEI Aquaculture Environment Interactions Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections AEI 1:11-19 (2010) - DOI: https://doi.org/10.3354/aei00003 Spatiotemporal distribution of coastal and oceanic Atlantic cod Gadus morhua sub-groups after escape from a farm Ingebrigt Uglem1,*, Pål Arne Bjørn2, Hiromichi Mitamura3, Rune Nilsen2 1Norwegian Institute of Nature Research, Tungasletta 2, 7485 Trondheim, Norway 2Nofima Marin, Muninbakken 9-13, PO Box 6122, 9291 Tromsø, Norway 3Graduate School of Informatics, Kyoto University, Kyoto 606-8501, Japan *Email: ingebrigt.uglem@nina.no ABSTRACT: Little is known about how escaped farmed Atlantic cod Gadus morhua L. affect the environment, but the potential for negative ecological consequences might be significant. In this study, the movements of adult farmed north-east Arctic cod (NEAC) and Norwegian coastal cod (NCC) were mapped after a simulated escape from an aquaculture farm to evaluate if post-escape movements and potential for recapture differs between these 2 genotypes. There was no major difference between NEAC and NCC equipped with acoustic transmitters with respect to spatiotemporal distribution or swimming depth during a 10 wk period after simulated escape incidents. The tagged cod immediately descended to average depths <20 m, and subsequently were most frequently observed along the shore. A substantial proportion of the farmed cod released at the cod farm left within the first week, while cod released at a nearby mussel farm were attracted to the cod farm. Beyond Week 2, the fish detected at the cod farm consisted of approximately similar proportions of fish released at either the mussel or cod farm. One third of the tagged cod was recaptured in local small-scale commercial and recreational fisheries during the first 3 wk after release. Thus, our results indicate that organised recapture fishery is a realistic option for reducing possible ecological and genetic impacts of escaped farmed cod. Such fishery must, however, be initiated as soon as possible after an escape incident and there is a need for development of a methodology for rapid detection and efficient recapture of escapees. KEY WORDS: Atlantic cod · Gadus morhua · Aquaculture · Fish escape · Recapture Full text in pdf format PreviousNextCite this article as: Uglem I, Bjørn PA, Mitamura H, Nilsen R (2010) Spatiotemporal distribution of coastal and oceanic Atlantic cod Gadus morhua sub-groups after escape from a farm. Aquacult Environ Interact 1:11-19. https://doi.org/10.3354/aei00003 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AEI Vol. 1, No. 1. Online publication date: April 29, 2010 Print ISSN: 1869-215X; Online ISSN: 1869-7534 Copyright © 2010 Inter-Research.

PCR-based detection of Phomopsis azadirachtae in die-back affected neem seeds

Phomopsis azadirachtae Sateesh, Bhat & Devaki is the incitant of die-back disease of neem trees. Delayed appearance of conidia and presence of other microorganisms in the neem tissues are the obstacles in the rapid and accurate identification of P. azadirachtae. This work was carried out to develop a methodology for rapid detection of the pathogen in diseased tissues especially in the neem seeds. rDNA sequences of many Phomopsis spp. were retrieved from the database and were subjected for multiple alignment to select a 179 bp conserved sequence. This was used to design Phomopsis specific primer pair (Forward and Reverse) having the potential to produce a 154 bp product in PCR. The primer pair was utilised to detect the presence of P. azadirachtae in diseased neem seeds and other tissues. This is the first report on the PCR-based detection of P. azadirachtae directly in die-back diseased neem tissues. This method can be employed for rapid and reliable detection of P. azadirachtae in die-back affected neem seeds. Hence it will have very good application in seed health testing laboratories.

Rapid Quantitation of Neuraminidase Inhibitor Drug Resistance in Influenza virus Quasispecies

Emerging resistance of influenza viruses to neuraminidase inhibitors is a concern, both in surveillance of global circulating strains and in treatment of individual patients. Current methodologies to detect resistance rely on the use of cultured virus, thus taking time to complete or lacking the sensitivity to detect mutations in viral quasispecies. Methodology for rapid detection of clinically meaningful resistance is needed to assist individual patient management and to track the transmission of resistant viruses in the community. We have developed a pyrosequencing methodology to detect and quantitate influenza neuraminidase inhibitor resistance mutations in cultured virus and directly in clinical material. Our assays target polymorphisms associated with drug resistance in the neuraminidase genes of human influenza A H1N1 as well as human and avian H5N1 viruses. Quantitation can be achieved using viral RNA extracted directly from respiratory or tissue samples, thus eliminating the need for virus culture and allowing the assay of highly pathogenic viruses such as H5N1 without high containment laboratory facilities. Antiviral-resistant quasispecies are detected and quantitated accurately when present in the total virus population at levels as low as 10%. Pyrosequencing is a real-time assay; therefore, results can be obtained within a clinically relevant timeframe and provide information capable of informing individual patient or outbreak management. Pyrosequencing is ideally suited for early identification of emerging antiviral resistance in human and avian influenza infection and is a useful tool for laboratory surveillance and pandemic preparedness.