Detection Of Various Pathogens Research Articles

Dynamics of Amphibian Pathogen Detection Using Extended Museum Specimens.

Natural history collections have long served as the foundation for understanding our planet's biodiversity, yet they remain a largely untapped resource for wildlife disease studies. Extended specimens include multiple data types and specimen preparations that capture the phenotype and genotype of an organism and its symbionts-but preserved tissues may not always be optimized for downstream detection of various pathogens. Frogs are infected by an array of pathogens including Batrachochytrium dendrobatidis (Bd), Ranavirus (Rv), and Amphibian Perkinsea (Pr), which provides the opportunity to study differences in detection dynamics across tissue types. We used quantitative PCR protocols to screen two tissue types commonly deposited in museum collections, toe clips and liver, from two closely related host species, Rana catesbeiana and Rana clamitans. We compared Bd, Rv, and Pr infection prevalence and intensity between species and tissue types and found no significant difference in prevalence between species, but Bd intensity was higher in R. clamitans than R. catesbeiana. Toe tissue exhibited significantly higher Bd infection loads and was more useful than liver for detecting Bd infections. In contrast, Rv was detected from more liver than toe tissues, but the difference was not statistically significant. Our results support the use of extended specimen collections in amphibian disease studies and demonstrate that broader tissue sampling at the time of specimen preparation can maximize their utility for downstream multipathogen detection.

Ov-RPA-CRISPR/Cas12a assay for the detection of Opisthorchis viverrini infection in field-collected human feces.

Opisthorchis viverrini infection is traditionally diagnosed using the Kato-Katz method and formalin ethyl-acetate concentration technique. However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect O.viverrini infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (RPA-CRISPR/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and Mycobacterium tuberculosis. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA-CRISPR/Cas assay to detect O.viverrini infection in field-collected human feces. To detect O.viverrini infection in fecal samples, we developed a CRISPR/Cas12a (RNA-guided endonuclease) system combined with RPA (Ov-RPA-CRISPR/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR/Cas detection conditions, detection limits, and specificity of the RPA-CRISPR/Cas12a assay for detecting O.viverrini infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the Ov-RPA-CRISPR/Cas12a assay was evaluated on the basis of comparison with standard methods. The Ov-RPA-CRISPR/Cas12a assay exhibited high specificity for detecting O.viverrini DNA. On the basis of the detection limit, the assay could detect O.viverrini DNA at concentrations as low as 10-1ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1ng. To validate the Ov-RPA-CRISPR/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for O.viverrini-like eggs. Of these, 18 were confirmed as true positives on the basis of the Ov-RPA-CRISPR/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections. The Ov-RPA-CRISPR/Cas12a assay developed in this study can successfully detect O.viverrini infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm O.viverrini infection, marking an initial step in the development of point-of-care diagnosis.

Ultraspecific One-Pot CRISPR-Based "Green-Yellow-Red" Multiplex Detection Strategy Integrated with Portable Cartridge for Point-of-Care Diagnosis.

Versatile, informative, sensitive, and specific nucleic acid detection plays a crucial role in point-of-care pathogen testing, genotyping, and disease monitoring. In this study, we present a novel one-pot Cas12b-based method coupled with the "Green-Yellow-Red" strategy for multiplex detection. By integrating RT-LAMP amplification and Cas12b cleavage in a single tube, the entire detection process can be completed within 1 h. Our proposed method exhibits high specificity, enabling the discrimination of single-base mutations with detection sensitivity approaching single molecule levels. Additionally, the fluorescent results can be directly observed by the naked eye or automatically analyzed using our custom-designed software Result Analyzer. To realize point-of-care detection, we developed a portable cartridge capable of both heating and fluorescence excitation. In a clinical evaluation involving 20 potentially SARS-CoV-2-infected samples, our method achieved a 100% positive detection rate when compared to standard RT-PCR. Furthermore, the identification of SARS-CoV-2 variants using our method yielded results that were consistent with the sequencing results. Notably, our proposed method demonstrates excellent transferability, allowing for the simultaneous detection of various pathogens and the identification of mutations as low as 0.5% amidst a high background interference. These findings highlight the tremendous potential of our developed method for molecular diagnostics.

A simple, effective and inexpensive method to isolate the nucleic acid (DNA/RNA) from a single tick for molecular detection of various pathogens

Objectives: Ticks are vectors of a number of pathogens, which cause diseases with fatal consequences, therefore it is essential to detect such pathogens from single tick. Molecular methods like polymerase chain reaction (PCR) are offering such possibilities. At present, cumbersome methods involving liquid nitrogen, cutting ticks with scalpels as well as pooled ticks are being used worldwide. Our goal is to develop a reliable and fast method to obtain nucleic acid (DNA/RNA) from the single tick shipped at room temperature to detect various pathogens. Methods: We developed a mechanical crushing method with mini column nucleic acid isolation from a single tick shipped at room temperature with postal or currier service in a letter. PCR detection was done for Borrelia burgdorferi and tick-borne encephalitis virus as examples. Results: This method was used successfully for the isolation of nucleic acid from single tick and later used for PCR detection of B. burgdorferi and tick-borne encephalitis virus on 17 single tick samples as examples, but for last 18 years, this method was used on more than 250 ticks from Germany. Spectrometric values indicate the presence of sufficient yield of DNA and RNA (up to 900 µg/mL per tick) during the isolation. Conclusions: This may be the first report about a number of one single tick cases, which were sent at room temperature in letters with postal services for isolation of the nucleic acid with mini column kit and used later on for PCR detection of various pathogens. This inexpensive and simple method may be used in any laboratory worldwide for monitoring the presence of tick-borne pathogens.

Establishing environmental DNA and RNA protocols for the simultaneous detection of fish viruses from seawater

AbstractAquatic viruses are major threats to global aquacultural productivity. While conventional diagnostic methods for disease investigation are laborious, time‐consuming, and often involve the sacrifice of animals, environmental DNA and RNA (eDNA/eRNA) tools have the potential in being non‐invasive alternatives for the effective and early detection of various pathogens simultaneously. In this study, three seawater filtration methods—Sterivex syringe filtration, centrifugal ultrafiltration, and vacuum pump filtration with iron flocculation—were assessed for the recovery rates in co‐detecting fish virus eDNA/eRNA from natural seawater that was spiked with fish red seabream iridovirus (RSIV, DNA virus) and nervous necrosis virus (NNV, RNA virus). The centrifugal ultrafiltration method was the most effective for the capture of small‐sized viruses like NNV with a recovery rate of 63.23%, while the method of vacuum pump filtration with iron flocculation and chloroform disintegration of filter membranes had the highest RSIV recovery rate of 32.61%. We also optimized both automated and manual nucleic acid extraction methods and found comparable eDNA/eRNA extraction efficiencies. Our findings from the systematic comparison of seawater filtration and extraction methods suggest that each seawater filtration/nucleic acid extraction method can cater to different aquatic animal virus surveillance and disease investigation scenarios. These highlight the potential of virus eDNA/eRNA approaches for advancing the field of disease ecology and safeguarding aquatic animal health.

Reference material development for detection of human respiratory syncytial virus using digital PCR.

Nucleic acid testing is a powerful tool for the detection of various pathogens. Respiratory syncytial virus (RSV) is a major cause of acute respiratory infection, especially in young children and infants. To improve the confidence and reliability of nucleic acid testing results for RSV, reference materials (RMs) of both type A and B of RSV were developed by the National Institute of Metrology, China, code numbers NIM-RM 4057 and 4058. The reference material was composed of in vitro transcribed RNA containing the nucleocapsid (N) gene, matrix (M) gene, and partial polymerase (L) gene of RSV. A duplex reverse transcription digital PCR method was established with limit of blank (LoB), limit of detection (LoD) and limit of quantification (LoQ) of 2, 5, and 23 copies per reaction for RSV-A and 4, 8, and 20 copies per reaction for RSV-B. The certified value and expanded uncertainty (U, k = 2) of the two RMs were determined to be (6.1 ± 1.4) × 104 copies/μL for RSV-A and (5.3 ± 1.2) × 104 copies/μL for RSV-B. The developed RMs can be used as standards to evaluate the performance of RSV detection assays.

Multipurpose advanced split T7 promoter-based transcription amplification for ultrasensitive molecular diagnostics

An accurate, convenient, and rapid diagnostic platform, which can be applied in facility-limited or point-of-care (POC) settings, is essential to help prevent the spread of infectious diseases and enable the most effective treatment to be selected. In this study, we describe the development of a new isothermal molecular diagnostic system named multipurpose advanced split T7 promoter-based transcription amplification (MASTER) for the rapid and ultrasensitive detection of various pathogens containing single-stranded RNA and double-stranded DNA. MASTER produces a large number of RNA amplicons in the presence of target pathogens, which generate fluorescence or colorimetric signals based on light-up RNA aptamers or lateral flow assays. Implementing MASTER at 37 °C for<1 h achieved the detection of a single copy per reaction without cross-reactivity. Moreover, the testing of 40 clinical samples revealed that MASTER exhibited excellent accuracy with 100% sensitivity and specificity for SARS-CoV-2 diagnosis. Furthermore, a one-pot MASTER system capable of accelerating practical applications was demonstrated, indicating that the MASTER system is a promising platform for the effective surveillance of various pathogens.

Modification of amphiphilic block copolymers for responsive and biologically active surfactants in complex droplets

Concerns regarding the speed and portability of sensing devices have spurred development of numerous novel platforms. Complex emulsions with responsive surfactants have emerged as a promising class of materials for the detection of various pathogens and environmental toxins. Herein, we report a study of the amphiphilic block copolymer surfactant (BCP), polystyrene-block-poly(acrylic acid), and its use as a functional surfactant. We observe that the composition and molecular weight of BCPs affect the interfacial properties, specifically, more amphiphilic BCPs lead to greater reductions in interfacial tension at the water/oil interface. We further demonstrate that conformational change of poly(acrylic acid) leads to changes in the interfacial tension reductions at these interfaces. Next, we present modification of BCPs with trypsin through carbodiimide mediated amidation to produce functional BCP-trypsin. The modified polymers retain their surfactant capabilities, as well as the functionality of the initial trypsin. Furthermore, we successfully demonstrate the use of the modified polymers within the active complex emulsion framework and the ability of the emulsion framework to turn “on” and “off” functionality through shielding of the active compounds. These responses to chemical changes in their surrounding environment illustrate the potential use of amphiphilic block copolymers as the key component of a complex emulsion systems for sensing device that is rapid, portable, and produces results in real-time.

Solvent engineering studies on recombinase polymerase amplification

Recombinase polymerase amplification (RPA) is a technique that is used to specifically amplify a target nucleic acid sequence. Unlike the polymerase chain reaction (PCR), RPA is performed at a constant temperature between 37 and 42°C. Therefore, it can be potentially used for the onsite detection of various pathogens when combined with DNA extraction and amplicon detection techniques. In this study, we prepared recombinant recombinase and single-stranded DNA-binding protein from T4 phage and used them to examine the effects of reaction conditions and additives on the efficiency of RPA. The results revealed that the optimal pH was 7.5-8.0, optimal potassium acetate concentration was 40-80mM, and optimal reaction temperature was 37-45°C although dimethyl sulfoxide at 5% v/v and formamide at 5% v/v inhibited the reaction. Our results suggest that RPA could be conducted using a wider range of optimal reaction conditions than those required for PCR and that RPA is highly suitable for point-of-care use.

Glycoprotein- and Lectin-Based Approaches for Detection of Pathogens.

Infectious diseases alone are estimated to result in approximately 40% of the 50 million total annual deaths globally. The importance of basic research in the control of emerging and re-emerging diseases cannot be overemphasized. However, new nanotechnology-based methodologies exploiting unique surface-located glycoproteins or their patterns can be exploited to detect pathogens at the point of use or on-site with high specificity and sensitivity. These technologies will, therefore, affect our ability in the future to more accurately assess risk. The critical challenge is making these new methodologies cost-effective, as well as simple to use, for the diagnostics industry and public healthcare providers. Miniaturization of biochemical assays in lab-on-a-chip devices has emerged as a promising tool. Miniaturization has the potential to shape modern biotechnology and how point-of-care testing of infectious diseases will be performed by developing smart microdevices that require minute amounts of sample and reagents and are cost-effective, robust, and sensitive and specific. The current review provides a short overview of some of the futuristic approaches using simple molecular interactions between glycoproteins and glycoprotein-binding molecules for the efficient and rapid detection of various pathogens at the point of use, advancing the emerging field of glyconanodiagnostics.

Immuno-HCR based on contact quenching and fluorescence resonance energy transfer for sensitive and low background detection of Escherichia coli O157:H7

Escherichia coli O157:H7 makes a major threat to human health. Aiming to detect Escherichia coli O157:H7 sensitively, hybridization chain reaction signal amplified immunoassay (immuno-HCR) based on contact quenching (CQ) and fluorescence resonance energy transfer (FRET) was developed. The background of the new designed HCR hairpins (CQ-FRET hairpins) was reduced by contact-quenching fluorescein (FAM) and breaking FRET from donor (FAM) to acceptor (Cy5). The F/F0 ratio of CQ-FRET hairpins (37.02) was obviously higher than that of two other common HCR fluorescent hairpins (CQ hairpins, 21.45; FRET hairpins, 4.61). The limit of detection of the assay was 3.5 × 101 CFU/mL and obviously lower than that of CQ hairpins based immuno-HCR (3.28 × 103 CFU/mL) and FRET hairpins based immuno-HCR (6.49 × 104 CFU/mL). The proposed low fluorescent background immuno-HCR with high sensitivity which was verified in contaminated milk samples could be potentially used in the detection of various pathogens.

Influenza species and subtypes circulation among hospitalized patients in Laleh hospital during two influenza seasonal (2016-2017 and 2017-2018) using a multiplex Real Time-Polymerase Chain Reaction.

The introduction of polymerase chain reaction (PCR) techniques has improved the detection of respiratory viruses, particularly with the use of multiplex real-time technique with the capability of simultaneous detection of various pathogens in a single reaction. The aim of this study was to apply the above technology for the diagnosis of influenza infections and at the same time to differentiate between common flu species between hospitalized patients in Laleh hospital (Iran) between two flu seasons (2016-2017 and 2017-2018). Different respiratory specimens were collected from 540 patients from a period of December 2016 to May 2018 and were sent to the laboratory for molecular diagnosis. RNAs were extracted and subsequently, a multiplex real time PCR identifying flu A, flu B and typing flu A (H1N1) was carried out. The mean age of patients was 47.54±23.96. 216 (40%) and 321 (60%) of subjects were male and female, respectively. 219 out of 540 (40.5%) were positive for influenza infection including flu A (n=97, 44.3%), flu A (H1N1) (n=45, 20.7%) and flu B (n=77, 35%). Flu A was the dominant species on 2016-2017 and flu B was the major species on 2017-2018. Flu A (H1N1) was comparable in both time periods. Flu infections were most frequently diagnosed in age groups 21-40. Flu-positive patients suffered more from body pain and sore throat than flunegative patients with significant statistical difference (P values <0.001). The mean duration of hospitalization was shorter for flu-positive patients (P value = 0.016). Application of multiplex real time PCR could facilitate the influenza diagnosis in a short period of time, benefiting patients from exclusion of bacterial infections and avoiding unnecessary antibiotic therapy. Influenza diagnosis was not achieved in up to 60% of flu-like respiratory infections, suggesting the potential benefit of adopting the same methodology for assessing the involvement of other viral or/and bacterial pathogens in those patients.

Calorimetric lateral flow immunoassay detection platform based on the photothermal effect of gold nanocages with high sensitivity, specificity, and accuracy.

BackgroundLateral flow assays (LFA) play an increasingly important role in the rapid detection of various pathogens, pollutants, and toxins.PurposeTo overcome the drawbacks of low sensitivity and poor quantification in LFA, we developed a new calorimetric LFA (CLFA) using gold nanocages (GNCs) due to their high photothermal conversion efficiency, good stability of photophysical properties, and stronger penetrating ability of NIR light.MethodsThiol-polyethylene glycol-succinyl imide ester (HS-PEG-NHS) was modified onto GNCs, and the complex was conjugated with an antibody. Subsequently, the antibody-conjugated GNCs were analyzed by UV/Vis spectrophotometer, transmission electron microscope, high-resolution transmission electron microscope with energy dispersive spectrometer, dynamic light scattering instrument, and Atom force microscope. The GNC-based CLFA of alpha-fetoprotein (AFP) and zearalenone (ZEN), a food toxin, required nitrocellulose strips, a NIR laser source, and an infrared camera.ResultsThe GNC-labeled CLFA platform technique exhibited detection sensitivity, qualitative specificity, and quantitative accuracy. The superior performance of the technique was evident both in sandwich format detection of biomacromolecules (eg, AFP protein) or competitive format detection of small molecules (eg, ZEN). After optimizing various test parameters, GNC-labeled CLFA provided ca. 5-6-fold enhanced sensitivity, higher correlativity (R2>0.99), and more favorable recovery (82–115%) when compared with visual LFA.ConclusionGNC-labeled CLFA may be a promising detection platform with high sensitivity, specificity, and precision.

Colorimetric biosensor using dual-amplification of enzyme-free reaction through universal hybridization chain reaction system.

On-site genetic detection needs to develop a sensitive and straightforward biosensor without special equipment, which can detect various genetic biomarkers. Hybridization chain reaction(HCR) amplifying signal isothermally could be considered as a good candidate for on-site detection. Here, we developed a novel genetic biosensor on the basis of enzyme-free dual-amplification of universal hybridization chain reaction (uHCR) and hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme. The uHCR is the strategy which enables simple design for multiple target detection by the introduction of target-specific trigger hairpin without changing the whole system according to a target change. Also, HRP-mimicking DNAzyme could produce a sensitive and quantitative colorimetric signal with increased stability with a limit of detection (LOD) of 5.67 nM. The universality of the uHCR biosensor was proven by the detection of four different targets (miR-21, miR-125b, KRAS-Q61K, and BRAF-V600E) for cancer diagnosis. The uHCR biosensor showed specificity that could discriminate single-nucleotide polymorphism. Moreover, the uHCR biosensor could detect targets in the diluted serum sample. Overall, the uHCR biosensor demonstrated the potential for field testing with a simple redesign without complicated steps or special equipment using a universal hairpin system and enzyme-free amplification. This strategy could enable stable and sensitive detection of a variety of targets. Therefore, it could be applied to urgent detection of various pathogens, remote diagnosis, and self-screening of diseases.

CLINICAL INFORMATIVE VALUE IN THE DIAGNOSTIC TESTING FOR DETECTION OF ROTAVIRUS INFECTIONS IN ADULT PATIENTS WITH ACUTE DIARRHEA

The paper suggests the results of retrospective cohort study. We analyzed the results of clinical and laboratory examination of 102 hospitalized adult patients with acute diarrhea (AD) of moderate severity and positive feces test for rotavirus. Specific examination for the detection of various pathogens of AD along with standard cultural, serologic (based on the determination of specific antibodies) and ELISA methods included the study of the patient’s feces by PCR. It was found that the use of a complex of modern methods of specific diagnosis allows in 67.2% of individuals along with markers of rotavirus simultaneously identify markers of other pathogens, including bacterial – 51% of patients. Among the examined persons hospitalized on the 4th day of the disease and later, there are 2 times more patients with concomitant diseases of the gastrointestinal tract and 2.4 times more patients taking antimicrobial drugs at the prehospital stage than among those who admitted for the 1st three days of the disease. The cases in adult patients with concomitant non-infectious pathology of the gastrointestinal tract compared to those without it turned to be significantly rarer simultaneously positive for rotavirus and Campylobacter spp. In the examined patients with colitis syndrome there is an apparent trend of more frequent detection of a positive cultural test for Salmonella spp. In the absence of markers of bacterial enteropathogens in the clinical material in the group of adult patients with only detected rotavirus, colitis syndrome is documented 2 times more often than in patients with a positive combined reaction to rota- and norovirus.

Some aspects on tick species in Mongolia and their potential role in the transmission of equine piroplasms, Anaplasma phagocytophilum and Borrelia burgdorferi L.

Ticks are cosmopolitan vectors of numerous diseases, and detection of various pathogens in ticks can help to assess their distribution. In the current study, 528 adult ticks were collected from grazing animals or the ground in ten different Mongolian provinces. Dermacentor nuttalli constituted 76.1% of them and was found in all ecozones except the eastern desert. Dermacentor marginatus (8.3%), Dermacentor silvarum (1.1%) and Ixodes persulcatus (3.0%) were found in the northern forest areas and Hyalomma asiaticum (11.4%) only in the southern (semi-)desert. Of these, 359 ticks were subjected to DNA extraction and PCR was carried out to detect various pathogens. Anaplasma spp. was found in D. marginatus and D. nuttalli (2.5% positive each), including flagged specimen and identified as Anaplasma phagocytophilum. Borrelia spp. were found in 2.5% of the ticks (mostly in I. persulcatus) and identified as Borrelia garinii. Babesia spp. (40%) identified as Babesia caballi were detected in all five tick species including flagged Dermacentor spp. and I. persulcatus, and 3.5% of the ticks (all species except D. silvarum) were positive for Theileria spp. identified as Theileria equi. The piroplasms were found in all provinces. Tick-borne encephalitis virus was not detected. The results highlight the high risk of equine piroplasmosis in Mongolia, which is a concern for both the nomadic population who rely on horses for transport and for conservation of Przewalski’s horses in Mongolia. In addition, zoonotic agents such as the avian B. garinii and A. phagocytophilum were also detected, outlining a high risk for exposed humans.

Multiplexed Instrument-Free Bar-Chart SpinChip Integrated with Nanoparticle-Mediated Magnetic Aptasensors for Visual Quantitative Detection of Multiple Pathogens.

A portable multiplexed bar-chart SpinChip (MB-SpinChip) integrated with nanoparticle-mediated magnetic aptasensors was developed for visual quantitative instrument-free detection of multiple pathogens. This versatile multiplexed SpinChip combines aptamer-specific recognition and nanoparticle-catalyzed pressure amplification to achieve a sample-to-answer output for sensitive point-of-care testing (POCT). This is the first report of pathogen detection using a volumetric bar-chart chip, and it is also the first bar-chart chip using a "spinning" mechanism to achieve multiplexed bar-chart detection. Additionally, the introduction of the spin unit not only enabled convenient sample introduction from one inlet to multiple separate channels in the multiplexed detection, but also elegantly solved the pressure cross-interference problem in the multiplexed volumetric bar-chart chip. This user-friendly MB-SpinChip allows visual quantitative detection of multiple pathogens simultaneously with high sensitivity but without utilizing any specialized instruments. Using this MB-SpinChip, three major foodborne pathogens including Salmonella enterica, Escherichia coli, and Listeria monocytogenes were specifically quantified in apple juice with limits of detection of about 10 CFU/mL. This MB-SpinChip with a bar-chart-based visual quantitative readout has great potential for the rapid simultaneous detection of various pathogens at the point of care and wide applications in food safety, environmental surveillance, and infectious disease diagnosis.

Nanozyme-based lateral flow assay for the sensitive detection of Escherichia coli O157:H7 in milk

Lateral flow assay (LFA) has been applied in many fields due to its relative ease of use and cost-effectiveness. However, it has low sensitivity and its applications are limited. Probe materials play a significant role in improving the detection efficiency and sensitivity of LFA. In this study, by using concave palladium-platinum (Pd-Pt) nanoparticles as a nanozyme probe, we developed a sensitive LFA based on the sandwich format for qualitative and quantitative detection of Escherichia coli O157:H7. The sensitivity of the LFA was improved by applying the 3,3',5,5'-tetramethylbenzidine (TMB) substrate onto the test line where the nanozyme was accumulated in the presence of analytes. The nanozyme showed high catalytic performance toward TMB and greatly enhanced the signal intensity of the test line. The sensitivity of the nanozyme-based LFA was 9.0 × 102 cfu/mL in milk, which was 111-fold higher than that of traditional colloidal gold-based LFA. The proposed method has remarkable potential in the detection of various pathogens in real samples.

Optical Immunosensors for the Efficient Detection of Target Biomolecules

Recently, immunosensors have attracted attention because they are widely applied for the detection of various pathogens. Among the commonly used immunosensors, the optical immunosensor features prominently as an effective tool for the quantification of the amount of antibodies, antigens, or haptens in complex samples with high sensitivity and specificity. However, very few studies provide comprehensive overviews of optical immunosensors. In this review, we present various methods and applications of optical immunosensors in pathogen detection. We introduced a concise definition of optical immunosensors and the principle of using them for detection. We subsequently discuss the main categories of optical immunosensors and their application to the detection of pathogens, as well as their advantages and limitations. Recent publications from 2006 to 2015 on variously designed optical immunosensors have also been updated. We conclude the review with a brief summary and discuss future directions of optical immunosensors.

Detection of Salmonella Enterica with Magnetoelastic Biosensors in Wash Water Containing Clorox and Chlorine Dioxide

Magnetoelastic (ME) biosensors with phage-displayed oligopeptide probes have been demonstrated to be highly successful in rapid detection of various pathogens, including Salmonella enterica, on the fruit and vegetable surfaces. However, in lieu of testing each produce individually, it is advantageous to detect pathogens in the produce wash water for high-throughput analysis. The sanitizing guidelines of the United States Food and Drug Administration (FDA) suggest adding Clorox and chlorine dioxide to the produce wash water in processing plants to disinfect harmful food-borne pathogens on fruit and vegetable surfaces. Therefore, to determine the efficacy of our ME biosensors in testing for food-borne pathogens in produce wash water, we assessed the stability of our biosensors in the presence of Clorox and chlorine dioxide. Specifically, Enzyme Linked Immunosorbent Assay (ELISA) was used to study the potential effect of Clorox and chlorine dioxide on the S. Typhimurium capturing ability of our phage probe. At concentrations recommended by the FDA for Clorox (100 ppm free chlorine content) and by the United States Environmental Protection Agency for chlorine dioxide (4 ppm), we observed no negative effect of these chemicals on the stability and S. Typhimurium capturing ability of our phage-displayed oligopeptide probe. These data demonstrate the potential efficacy of using our ME biosensors with phage-displayed oligopeptide probes in produce wash waters to determine the presence of food-borne pathogens.