Applications In Pathogen Detection Research Articles

Hollow versatile Ag@Pt alloy nanoparticles with nanozyme activity for detection and photothermal sterilization of Helicobacter pylori.

In view of a large number of people infected with Helicobacter pylori (H. pylori) with great harm followed, there is an urgent need to develop a non-invasive, easy-to-operate, and rapid detection method, and to identify effective sterilization strategies. In this study, highly specific nanoprobes with nanozyme activity, Ag@Pt nanoparticles (NPs) with the antibody, were utilized as a novel lateral flow immunoassay (LFIA). The optical label (Ag@Pt NPs) was enhanced by the introduction of the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) and compared with a gold nanoparticles (Au NPs) optical label. Under the optimal condition, Ag@Pt-LFIA and TMB-enhanced Ag@Pt-LFIA for H. pylori were successfully established, two of which were over twofold and 100-fold more sensitive than conventional visual Au NP-based LFIA, respectively. Furthermore, Ag@Pt NPs with the antibody irradiated with NIR laser (808nm) at a power intensity of 550 mW/cm2 for 5min exhibited a remarkable antibacterial effect. Thenanoprobes could close to bacteria through effective interactions between antibodies and bacteria, thereby benefiting photothermal sterilization. Overall, Ag@Pt NPs provide promising applications in pathogen detection and therapeutic applications.

Development of a Gold Nanoparticle-Based ELISA for Detection of PCV2.

In this new methodology, plasmonic ELISA (pELISA) was used to detect Circovirus porcine2 (PCV2) in serum samples without the need for plate reading equipment. This process occurs by adapting the conventional ELISA test with gold nanoparticles (AuNPs) to promote a color change on the plate and quickly identify this difference with the naked eye, generating a dark purple-gray hue when the samples are positive and red when the samples are negative. The technique demonstrated high efficiency in detecting samples with a viral load ≥ 5 log10 copies/mL. Plasmonic ELISA offers user-friendly, cost-effective, and reliable characteristics, making it a valuable tool for PCV2 diagnosis and potentially adaptable for other pathogen detection applications.

The Application of Hybridization Chain Reaction in the Detection of Foodborne Pathogens.

Today, with the globalization of the food trade progressing, food safety continues to warrant widespread attention. Foodborne diseases caused by contaminated food, including foodborne pathogens, seriously threaten public health and the economy. This has led to the development of more sensitive and accurate methods for detecting pathogenic bacteria. Many signal amplification techniques have been used to improve the sensitivity of foodborne pathogen detection. Among them, hybridization chain reaction (HCR), an isothermal nucleic acid hybridization signal amplification technique, has received increasing attention due to its enzyme-free and isothermal characteristics, and pathogenic bacteria detection methods using HCR for signal amplification have experienced rapid development in the last five years. In this review, we first describe the development of detection technologies for food contaminants represented by pathogens and introduce the fundamental principles, classifications, and characteristics of HCR. Furthermore, we highlight the application of various biosensors based on HCR nucleic acid amplification technology in detecting foodborne pathogens. Lastly, we summarize and offer insights into the prospects of HCR technology and its application in pathogen detection.

MAGNETIC FIELD CONTROL IN AN ANALYTIC PLATFORM FOR ASSESSMENT OF PATHOGENIC BACTERIA

The paper presents a conceptual, analytical platform with various pathogen detection applications, based on a hybrid magnetic field source, comprised of permanent magnets and solenoid coils fixed on the same magnetic yoke that acts as a magnetic flux concentrator. The magnetic field creates magnetization forces that guide functionalized paramagnetic microparticles through the volume of an analyte sample, in order to capture targeted pathogens. The presence of bacteria is sensed with an additional electrical impedance spectroscopy (EIS) module attached to the system, at high-frequency electrical fields. The magnetic field control is studied using mathematical modeling and numerical simulation in several geometry setups, coil excitation, and remanent flux density values.

Advances in the application of recombinase-aided amplification combined with CRISPR-Cas technology in quick detection of pathogenic microbes.

The rapid diagnosis of pathogenic infections plays a vital role in disease prevention, control, and public health safety. Recombinase-aided amplification (RAA) is an innovative isothermal nucleic acid amplification technology capable of fast DNA or RNA amplification at low temperatures. RAA offers advantages such as simplicity, speed, precision, energy efficiency, and convenient operation. This technology relies on four essential components: recombinase, single-stranded DNA-binding protein (SSB), DNA polymerase, and deoxyribonucleoside triphosphates, which collectively replace the laborious thermal cycling process of traditional polymerase chain reaction (PCR). In recent years, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated proteins) system, a groundbreaking genome engineering tool, has garnered widespread attention across biotechnology, agriculture, and medicine. Increasingly, researchers have integrated the recombinase polymerase amplification system (or RAA system) with CRISPR technology, enabling more convenient and intuitive determination of detection results. This integration has significantly expanded the application of RAA in pathogen detection. The step-by-step operation of these two systems has been successfully employed for molecular diagnosis of pathogenic microbes, while the single-tube one-step method holds promise for efficient pathogen detection. This paper provides a comprehensive review of RAA combined with CRISPR-Cas and its applications in pathogen detection, aiming to serve as a valuable reference for further research in related fields.

Absorbance biosensors-based hybrid mathbf {MoS_{2}} nanosheets for Escherichia coli detection

Detecting Escherichia coli is essential in biomedical, environmental, and food safety applications. In this paper, we have developed a simple, rapid, sensitive, and selective E. coli DNA sensor based on the novel hybrid-type text {MoS}_text {2} and mathrm {(NH_4)_6Mo_7O_{24}} nanosheets. The sensor uses the absorbance measurement to distinguish among the DNA of E. coli, Vibrio proteolyticus, and Bacillus subtilis when implemented in conjunction with text {NH}_text {2}-probes. Our experiments showed that the absorbance increased when sensors detected E. coli DNA, whereas it decreased when sensors detected V. proteolyticus and B. subtilis DNA. To the best of authors’ knowledge, there are no reports using the novel hybrid-mathrm {MoS_2} and mathrm {(NH_4)_6Mo_7O_{24}} materials for differentiating three types of DNA using cost-effective and rapid absorbance measurements. In addition, the label-free E. coli DNA biosensor exhibited a linear response in the range of 0 fM to 11.65 fM with a limit of detection of 2 fM. The effect of text {NH}_text {2}-probes on our sensors’ working performance is also investigated. Our results will facilitate further research in pathogen detection applications, which have not been fully developed yet.

Evolution of the Probe-Based Loop-Mediated Isothermal Amplification (LAMP) Assays in Pathogen Detection.

Loop-mediated isothermal amplification (LAMP), as the rank one alternative to a polymerase chain reaction (PCR), has been widely applied in point-of-care testing (POCT) due to its rapid, simple, and cost-effective characteristics. However, it is difficult to achieve real-time monitoring and multiplex detection with the traditional LAMP method. In addition, these approaches that use turbidimetry, sequence-independent intercalating dyes, or pH-sensitive indicators to indirectly reflect amplification can result in false-positive results if non-specific amplification occurs. To fulfill the needs of specific target detection and one-pot multiplex detection, a variety of probe-based LAMP assays have been developed. This review focuses on the principles of these assays, summarizes their applications in pathogen detection, and discusses their features and advantages over the traditional LAMP methods.

Closed-Loop Microreactor on PCB for Ultra-Fast DNA Amplification: Design and Thermal Validation.

Polymerase chain reaction (PCR) is the most common method used for nucleic acid (DNA) amplification. The development of PCR-performing microfluidic reactors (μPCRs) has been of major importance, due to their crucial role in pathogen detection applications in medical diagnostics. Closed loop (CL) is an advantageous type of μPCR, which uses a circular microchannel, thus allowing the DNA sample to pass consecutively through the different temperature zones, in order to accomplish a PCR cycle. CL μPCR offers the main advantages of the traditional continuous-flow μPCR, eliminating at the same time most of the disadvantages associated with the long serpentine microchannel. In this work, the performance of three different CL μPCRs designed for fabrication on a printed circuit board (PCB) was evaluated by a computational study in terms of the residence time in each thermal zone. A 3D heat transfer model was used to calculate the temperature distribution in the microreactor, and the residence times were extracted by this distribution. The results of the computational study suggest that for the best-performing microreactor design, a PCR of 30 cycles can be achieved in less than 3 min. Subsequently, a PCB chip was fabricated based on the design that performed best in the computational study. PCB constitutes a great substrate as it allows for integrated microheaters inside the chip, permitting at the same time low-cost, reliable, reproducible, and mass-amenable fabrication. The fabricated chip, which, at the time of this writing, is the first CL μPCR chip fabricated on a PCB, was tested by measuring the temperatures on its surface with a thermal camera. These results were then compared with the ones of the computational study, in order to evaluate the reliability of the latter. The comparison of the calculated temperatures with the measured values verifies the accuracy of the developed model of the microreactor. As a result of that, a total power consumption of 1.521 W was experimentally measured, only ~7.3% larger than the one calculated (1.417 W). Full validation of the realized CL μPCR chip will be demonstrated in future work.

Point‐of‐Care Diagnostic Platforms for Loop‐Mediated Isothermal Amplification

The loop‐mediated isothermal amplification (LAMP) method is one of the Nucleic acid amplification tests (NAATs) that allows for the amplification of target regions without using a thermal cycle. With its unique primer design, LAMP ensures the rapid replication of the targeted DNA region with high specificity and high efficiency. LAMP technology is used for diagnostic purposes in pathogen detection due to its ease of use, low cost, and simplicity without requiring complex equipment. A wide range of LAMP diagnostic platforms have been developed for applications in bacteria, virus, and parasitic pathogen detection. Herein, the methodology of LAMP technology and its applications in pathogen detection and SNP genotyping and mutation detection are discussed. Point‐of‐care (PoC) LAMP platforms designed with the principles of microfluidic chip technology, including LAMP‐on‐a‐chip, paper‐based LAMP, and smartphone‐based LAMP applications have been elaborated. LAMP technology represents a fast, robust, and reliable diagnostic platform for point‐of‐care testing.

Limited-resource preparable chitosan magnetic particles for extracting amplification-ready nucleic acid from complex biofluids.

Extraction and concentration of pure nucleic acid from complex biofluids are the prerequisite for nucleic acid amplification test (NAAT) applications in pathogen detection, biowarfare prevention, and genetic diseases. However, conventional spin-column mediated nucleic acid extraction is constricted by the requirement for costly power-intensive centralized lab infrastructure, making it unsuitable for limited-resource settings. Significant progress in lab-on-a-chip devices or cartridges (e.g., Cepheid GeneXpert®) that integrate nucleic acid extraction and amplification has been made, but these approaches either require additional equipment or are costly. Similarly, their complexities make them difficult to fabricate in low-resource settings by the end-user themselves. The application of magnetic particles such as silica-coated iron oxide beads for nucleic acid extraction is relatively instrument-free, rapid, user-friendly, and amenable to automation. But, they rely on hazardous chaotropic salt chemistry and ethanol desalting that could limit their efficacy for downstream NAATs. Recent advances in several types of novel material (e.g., polyamine) coated magnetic bead-based chaotropic salt-free extraction methods offer a possible solution to this problem. However, these materials also involve multistep synthesis impermissible in limited-resource settings. To offer a possible instrument-free magnetic particle-based nucleic acid extraction doable at limited-resource settings, we investigated the nucleic acid capture ability of two chitosan-coated magnetic particles that are preparable by minimally trained personnel using only a water bath and a magnetic stirrer within 6-8 h. We quantitatively probed the efficiency of the passive (without any electrical shaking or vortex-aided) DNA magnetocapture (i.e., binding to chitosan magnetic particles, physical separation from its sample of origin, and release from the particles) using UV260. To explore their suitability towards clinically relevant sensitive downstream NAATs, 100-1000 copies (i.e., in the order of zeptomole) of Escherichia coli (E. coli) or human genomic DNA from aqueous solution, crude cell lysate, and fetal bovine serum were extracted by them and then successfully detected using quantitative real-time loop-mediated isothermal amplification (LAMP) or real-time polymerase chain reaction (PCR). Alongside, their suitability with gel-based LAMP, colorimetric LAMP, and in situ (on beads) LAMP was also probed. The required optimization of the amplification methods has been discussed. Overall, the turnaround time for the magnetocapture combined with NAAT was 1.5-2 h and is thus expected to aid in rapid clinical decision making. With the ease of preparation, reproducibility, and compatibility with downstream NAATs, we anticipate that these magnetic particles would facilitate the expansion and decentralization of nucleic acid-based diagnosis for limited-resource settings.

Proximity ligation assay: an ultrasensitive method for protein quantification and its applications in pathogen detection.

It is of great significance to establish sensitive and accurate pathogen detection methods, considering the continuous emergence or re-emergence of infectious diseases seriously influences the safety of human and animals. Proximity ligation assay (PLA) is developed for the sensitive protein detection and also can be used for the detection of pathogens. PLA employs aptamer or monoclonal/polyclonal antibody-nucleic acid complexes as proximity probes. When the paired proximity probes bind to the same target protein or protein complex, they will be adjacent to each other and form an amplifiable DNA sequence through ligation. Combining the specificity of enzyme-linked immunosorbent assay (ELISA) and sensitivity of polymerase chain reaction (PCR), PLA transforms the detection of protein into the detection of DNA nucleic acid sequence. Therefore, as an ultrasensitive protein assay, PLA has great potential for quantification, localization of protein, and clinical diagnostics. In this review, we summarize the basic principles of PLA and its applications in pathogen detection. KEY POINTS: • Different forms of proximity ligation assay are introduced. • Applications of proximity ligation assay in pathogen detection are summarized. • Proximity ligation assay is an ultrasensitive method to quantify protein and pathogen.

Simultaneous detection and comprehensive analysis of HPV and microbiome status of a cervical liquid-based cytology sample using Nanopore MinION sequencing

Human papillomavirus (HPV) is a major pathogen that causes cervical cancer and many other related diseases. HPV infection related cervical microbiome could be an induce factor of cervical cancer. However, it is uncommon to find a single test on the market that can simultaneously provide information on both HPV and the microbiome. Herein, a novel method was developed in this study to simultaneously detect HPV infection and microbiota composition promptly and accurately. It provides a new and simple way to detect vaginal pathogen situation and also provide valuable information for clinical diagnose. This approach combined multiplex PCR, which targeted both HPV16 E6E7 and full-length 16S rRNA, and Nanopore sequencing to generate enough information to understand the vagina condition of patients. One HPV positive liquid-based cytology (LBC) sample was sequenced and analyzed. After comparing with Illumina sequencing, the results from Nanopore showed a similar microbiome composition. An instant sequencing evaluation showed that 15 min sequencing is enough to identify the top 10 most abundant bacteria. Moreover, two HPV integration sites were identified and verified by Sanger sequencing. This approach has many potential applications in pathogen detection and can potentially aid in providing a more rapid clinical diagnosis.

A laser-induced breakdown spectroscopy-integrated lateral flow strip (LIBS-LFS) sensor for rapid detection of pathogen

Incorporation of new readout methods with established analytical devices allows methodological innovations in analytical sciences. Herein, we present a new sensing platform by combining an ultrasensitive element analyzer, namely the laser-induced breakdown spectroscopy (LIBS) and a lateral flow strip (LFS). AgxAuy bimetallic nanoparticles (AgxAuyBNPs) are selected as the labels to deliver the optimal quantitative performance by analyzing the Ag (I) signal from the test (T) line of LFS. For prototypical application in pathogen detection, the LIBS-LFS sensor can achieve a detection limit of 1.6 cfu mL−1 of Staphylococcus aureus (S. aureus) within 10 min, which is superior to conventional methods. Importantly, the signals of AgxAuyBNPs for visual and LIBS analysis are stable and still readable after the detection is finished and the test strip is stored for up to 13 days, suggesting a potential for long-term data preservation. This combination of LIBS with LFS provides a new concept toward integrated nano/analytical devices that can benefit various application scenarios.

An improved assay for rapid detection of viable Staphylococcus aureus cells by incorporating surfactant and PMA treatments in qPCR

BackgroundStaphylococcus aureus is an important human pathogen causing a variety of life-threatening diseases. Rapid and accurate detection of Staphylococcus aureus is a necessity for prevention of outbreaks caused by this pathogen. PCR is a useful tool for rapid detection of foodborne pathogens, however, its inability to differentiate DNA from dead cells and live cells in amplification severely limits its application in pathogen detection. The aim of this study was to develop an improved assay was developed by incorporating the sample treatments with a surfactant and propidium monoazide (PMA) in qPCR for detection of viable S. aureus cells.ResultsThe cell toxic effect testing with the two surfactants showed that the viability of S. aureus was virtually not affected by the treatment with 0.5% triton x-100 or 0.025% sarkosyl. Triton x-100 was coupled with PMA for sample treatments for detection of viable S. aureus cells in artificially contaminated milk. The qPCR results indicated that the assay reached high an amplification efficiency of 98.44% and the live S. aureus cells were accurately detected from the triton-treated spiked milk samples by the PMA-qPCR assay.ConclusionsThe qPCR assay combined with treatments of PMA and surfactants offers a sensitive and accurate means for detection of viable S. aureus cells. Cell toxic effect testing with the two surfactants showed that the viability of S. aureus was virtually not affected by the treatment with 0.5% triton x-100 or 0.025% sarkosyl. The information on sample treatment with surfactants to improve the dead cell DNA removal efficiency in qPCR by increasing PMA’s permeability to dead cells can be used for other pathogens, especially for Gram-positive bacteria.

Development of DNAzyme-based PCR signal cascade amplification for visual detection of Listeria monocytogenes in food

Listeria monocytogenes is an important foodborne pathogen, and it can cause severe diseases. Rapid detection of L. monocytogenes is crucial to control this pathogen. A simple and robust strategy based on the cascade of PCR and G-quadruplex DNAzyme catalyzed reaction was used to detect L. monocytogenes. In the presence of hemin and the aptamer formed during PCR, the catalytic horseradish peroxidase-mimicking G-quadruplex DNAzymes allow the colorimetric responses of target DNA from L. monocytogenes. This assay can detect genomic DNA of L. monocytogenes specifically with as low as 50 pg/reaction with the naked eye. Through 20 pork samples assay, visual detection assay had the same results as conventional detection methods, and had a good performance. This is a powerful demonstration of the ability of G-quadruplex DNAzyme to be used for PCR-based assay with significant advantages of high sensitivity, low cost and simple manipulation over existing approaches and offers the opportunity for application in pathogen detection.

XMAP Technology: Applications in Detection of Pathogens.

xMAP technology is applicable for high-throughput, multiplex and simultaneous detection of different analytes within a single complex sample. xMAP multiplex assays are currently available in various nucleic acid and immunoassay formats, enabling simultaneous detection and typing of pathogenic viruses, bacteria, parasites and fungi and also antigen or antibody interception. As an open architecture platform, the xMAP technology is beneficial to end users and therefore it is used in various pharmaceutical, clinical and research laboratories. The main aim of this review is to summarize the latest findings and applications in the field of pathogen detection using microsphere-based multiplex assays.

Aptamer-Based Technologies in Foodborne Pathogen Detection.

Aptamers are single stranded DNA or RNA ligands, which can be selected by a method called systematic evolution of ligands by exponential enrichment (SELEX); and they can specifically recognize and bind to their targets. These unique characteristics of aptamers offer great potentials in applications such as pathogen detection and biomolecular screening. Pathogen detection is the critical means in detecting and identifying the problems related to public health and food safety; and only the rapid, sensitive and efficient detection technologies can enable the users to make the accurate assessments on the risks of infections (humans and animals) or contaminations (foods and other commodities) caused by various pathogens. This article reviews the development in the field of the aptamer-based approaches for pathogen detection, including whole-cell SELEX and Genomic SELEX. Nowadays, a variety of aptamer-based biosensors have been developed for pathogen detection. Thus, in this review, we also cover the development in aptamer-based biosensors including optical biosensors for multiple pathogen detection by multiple-labeling or label-free models such as fluorescence detection and surface plasmon resonance, electrochemical biosensors and lateral chromatography test strips, and their applications in pathogen detection and biomolecular screening. While notable progress has been made in the field in the last decade, challenges or drawbacks in their applications such as pathogen detection and biomolecular screening remain to be overcome.

Future Perspectives on New Approaches in Pathogen Detection

Microbial pathogens are responsible for most of the rapidly-spreading deadly infectious diseases against humans. Thus, there is an urgent need for efficient and rapid detection methods for infectious microorganisms. The detection methods should not only be targeted and specific, but they have to be encompassing of potential changes of the pathogen as it evolves and mutates quickly during an epidemic or pandemic. The existing diagnostics such as the antibody-based ELISA immunoassay and PCR methods are too selective and narrowly focused; they are insufficient to capture newly evolved mutant strains of the pathogen. Here, we introduce a fresh perspective on some new technologies, including aptamers and next generation sequencing for pathogen detection. These technologies are not in their infancy; they are reasonably mature and ready, and they hold great promise for unparalleled applications in pathogen detection.

Aptamer-based detection of Salmonella enteritidis using double signal amplification by Klenow fragment and dual fluorescence

This article describes a sensitive and selective fluorometric method for the determination of Salmonella enteritidis by exploiting the polymerase activity of the Klenow fragment and dual fluorescence. First, one end of a target-selective aptamer was labeled with the fluorophore 6-carboxyfluorescein (FAM). Once the labeled aptamer binds to graphene oxide (GO) via π-stacking interaction, the fluorescence of FAM is quenched. However, the addition of target (16S rRNA) leads to the restoration of fluorescence due to the binding of probe and target which shifts the FAM fluorophore away from the quenching GO. By using the Klenow fragment and by exploiting the synergistic effect of FAM and the DNA probe SYBR Green I (which is strongly fluorescent in presence of dsDNA only), fluorescence is strongly amplified and sensitivity improved. The analyte 16SrRNA can be determined by this method in the 60 pM to 100 nM concentration range, and the detection limit is 60 pM. It is also shown that Salmonella enteritidis can be determined in milk samples by this method in concentrations between 102 to 105 cfu⋅mL‾1, with a detection limit of 300 cfu⋅mL‾1. This assay displays high sensitivity and selectivity and may possess wide applications in pathogen detection.

Biophysically inspired rational design of structured chimeric substrates for DNAzyme cascade engineering.

The development of large-scale molecular computational networks is a promising approach to implementing logical decision making at the nanoscale, analogous to cellular signaling and regulatory cascades. DNA strands with catalytic activity (DNAzymes) are one means of systematically constructing molecular computation networks with inherent signal amplification. Linking multiple DNAzymes into a computational circuit requires the design of substrate molecules that allow a signal to be passed from one DNAzyme to another through programmed biochemical interactions. In this paper, we chronicle an iterative design process guided by biophysical and kinetic constraints on the desired reaction pathways and use the resulting substrate design to implement heterogeneous DNAzyme signaling cascades. A key aspect of our design process is the use of secondary structure in the substrate molecule to sequester a downstream effector sequence prior to cleavage by an upstream DNAzyme. Our goal was to develop a concrete substrate molecule design to achieve efficient signal propagation with maximal activation and minimal leakage. We have previously employed the resulting design to develop high-performance DNAzyme-based signaling systems with applications in pathogen detection and autonomous theranostics.