On-site Pathogen Research Articles

A phosphorylated guanidine chitosan and UiO-66-NH2 modified magnetic nanoparticle platform for enrichment and detection of multiple bacteria

Wastewater-based epidemiology (WBE) is a powerful tool for early warning of infectious disease outbreaks. Hence, a rapid and portable pathogen monitoring system is urgent needed for on-site detection. In this work, we first reported synthesis of an artificial modulated wide-spectrum bacteria capture nanoparticle (Arg-CSP@UiO@Fe3O4). Arginine-modified phosphorylated chitosan (Arg-CSP) coating could provide strongly positive charged guanidinium group for pathogen interaction by electrostatic attraction, and UiO-66-NH2 layer could help Arg-CSP graft onto Fe3O4 magnetic particles. The capture efficiency of Arg-CSP@UiO@Fe3O4 reached 92.2 % and 97.3 % for Escherichia coli (E.coli) and Staphylococcus epidermidis (S.epidermidis)within 40 min, in 10 mL sample. To prevent pathogen degradation in sewage, a portable nucleic acid extraction-free method was also developed. UiO-66-NH2 could disintegrate in buffer with high concentration of PO43− for bacterium desorption, and then nucleic acid of the bacteria was released by heating. The DNA template concentration obtained by this method was 779.28 times higher than that of the direct thermal lysis product and 8.63 times higher than that of the commercial kit. Afterwards, multiple detection of bacteria was realized by loop-mediated isothermal amplification (LAMP). Artificial regulated pathogen desorption could prevent non-specific adsorption of nucleic acid by nanoparticles. The detection limit of Arg-CSP@UiO@Fe3O4-LAMP method was 80 cfu/mL for E.coli and 300 cfu/mL for S.epidermidis. The accuracy and reliability of the method was validated by spiked sewage samples. In conclusion, this bio-monitoring system was able to detect multiple bacteria in environment conveniently and have good potential to become an alternative solution for rapid on-site pathogen detection.

Isothermal Nucleic Acid Amplification-Based Lateral Flow Testing for the Detection of Plant Viruses.

Isothermal nucleic acid amplification-based lateral flow testing (INAA-LFT) has emerged as a robust technique for on-site pathogen detection, providing a visible indication of pathogen nucleic acid amplification that rivals or even surpasses the sensitivity of real-time quantitative PCR. The isothermal nature of INAA-LFT ensures consistent conditions for nucleic acid amplification, establishing it as a crucial technology for rapid on-site pathogen detection. However, despite its considerable promise, the widespread application of isothermal INAA amplification-based lateral flow testing faces several challenges. This review provides an overview of the INAA-LFT procedure, highlighting its advancements in detecting plant viruses. Moreover, the review underscores the imperative of addressing the existing limitations and emphasizes ongoing research efforts dedicated to enhancing the applicability and performance of this technology in the realm of rapid on-site testing.

Nanotopology-Enabled On-Site Pathogen Detection for Managing Atopic Dermatitis.

Atopic dermatitis (AD), a prevalent skin condition often complicated by microbial infection, poses a significant challenge in identifying the responsible pathogen for its effective management. However, a reliable, safe tool for pinpointing the source of these infections remains elusive. In this study, a novel on-site pathogen detection that combines chemically functionalized nanotopology with genetic analysis is proposed to capture and analyze pathogens closely associated with severe atopic dermatitis. The chemically functionalized nanotopology features a 3D hierarchical nanopillar array (HNA) with a functional polymer coating, tailored to isolate target pathogens from infected skin. This innovative nanotopology demonstrates superior pathogenic capture efficiency, favorable entrapment patterns, and non-cytotoxicity. An HNA-assembled stick is utilized to directly retrieve bacteria from infected skin samples, followed by extraction-free quantitative loop-mediated isothermal amplification (direct qLAMP) for validation. To mimic human skin conditions, porcine skin is employed to successfully capture Staphylococcus aureus, a common bacterium exacerbating AD cases. The on-site detection method exhibits an impressive detection limit of 103 cells mL-1. The HNA-assembled stick represents a promising tool for on-site detection of bacteria associated with atopic dermatitis. This innovative approach enables to deepen the understanding of AD pathogenesis and open avenues for more effective management strategies for chronic skin conditions.

Development of Loop-Mediated Isothermal Amplification Assays for the Rapid and Accurate Diagnosis of Exserohilum turcicum for Field Applications.

Northern corn leaf blight (NCLB), caused by Exserohilum turcicum, is one of the most devastating foliar diseases of maize. Rapid and accurate diagnosis for this disease is urgently needed but still limited. Here, we establish a field-deployable diagnostic method to detect E. turcicum based on loop-mediated isothermal amplification (LAMP) assays. A software application called K-mer Elimination by Cross-reference was used to search for the specific sequences belonging to E. turcicum by comparing the whole genome sequence between E. turcicum and other known maize pathogens. Five LAMP primer sets were designed based on specific and single-copy fragments of E. turcicum. Post-LAMP analyses indicated that only the primer set, Et9468_set1, was the most suitable, producing a ladder-like amplification pattern in the agarose gel electrophoresis and a strong fluorescence signal in the presence of SYBR Green I. The LAMP assay using Et9468_set1 primers demonstrated a high level of specificity in distinguishing E. turcicum from six other common fungal pathogens of maize, as well as 12 more fungal and oomycete strains including the epiphytic fungi from maize leaves and other crop pathogens. Moreover, it exhibited remarkable sensitivity by detecting five copies per reaction, which was approximately 104 times more sensitive compared with conventional PCR. The LAMP assay successfully detected E. turcicum in field maize leaves without DNA extraction, demonstrating its suitability for rapid on-spot detection of NCLB. Our study provides a direct LAMP diagnostic method to detect E. turcicum, which enables on-site pathogen detection in the field and the development of preventive strategies for NCLB management.

A LAMP-based hydrogen ion selective electrochemical sensor for highly sensitive detection of Mycoplasma pneumoniae.

A concise and rapid detection method for Mycoplasma pneumoniae is urgently required due to its severe impact on human health. To meet such a need, this study proposed and constructed an innovative point-of-care testing (POCT) platform that consists of a hydrogen ion-selective loop-mediated isothermal amplification (H+-LAMP) sensor and an electrochemical detection device. The H+-LAMP sensor successfully integrated the working and reference electrodes and converted the H+ generated during the LAMP process into an electrochemical signal. High sensitivity and stability for pathogen detection were also achieved by treating the working electrode with an electrodeposited polyaniline solid contact layer and by using an ion-selective membrane. As a result, the sensor shows a sensitivity of 68.26 mV per pH, a response time of less than 2 s, and a potential drift of less than 5 mV within one hour, which well meets the urgent need. The results also demonstrated that the detection limit for Mycoplasma pneumoniae was lowered to 1 copy per μL, the nucleic acid extraction and detection process could be completed in 30 minutes, and the impact of interfering ions on the sensor was negligible. Validation with 20 clinical samples yielded satisfactory results. More importantly, the storage lifespan of such an electrochemical sensor is over seven days, which is a great advantage for on-site pathogen detection. Therefore, the hydrogen ion-selective sensor constructed in this investigation is particularly suitable as a core component for instant pathogen detection platforms.

An intelligent readable and capture-antibody-independent lateral flow immunoassay based on Cu2-xSe nanocrystals for point-of-care detection of Escherichia coli O157:H7.

Escherichia coli (E. coli) O157:H7 is a common foodborne pathogen which can cause serious harm. It is particularly important to establish a simple and portable method to achieve on-site pathogen detection. In this study, a capture-antibody-independent lateral flow immunoassay (LFIA) was constructed based on Cu2-xSe nanocrystals (Cu2-xSe NCs) for rapid detection of E. coli O157:H7. Cu2-xSe NCs can not only be regarded as the "nano-antibody" for the recognition of E. coli O157:H7 through electrostatic adsorption, but also as nanozymes that show good peroxidase-like catalytic activity. The formed compound of E. coli O157:H7 and Cu2-xSe NCs would be captured by a detection antibody on the T line due to the specific recognition of the antibody and E. coli O157:H7. Then, Cu2-xSe NCs could catalyze the oxidation of TMB by H2O2 to generate oxTMB, thereby generating blue bands. Meanwhile, we developed a mobile app for rapid data analysis. Under the optimal reaction conditions, E. coli O157:H7 could be detected within 70 min. The detection limit of this method was 2.65 × 105 CFU mL-1 with good specificity and stability. Additionally, it could achieve on-site rapid detection of E. coli O157:H7 in environmental water samples, providing a promising biosensor for portable pathogen detection.

In-field detection and characterization of B/Victoria lineage deletion variant viruses causing early influenza activity and an outbreak in Louisiana, 2019.

In 2019, the Louisiana Department of Health reported an early influenza B/Victoria (B/VIC) virus outbreak. As it was an atypically large outbreak, we deployed to Louisiana to investigate it using genomics and a triplex real-time RT-PCR assay to detect three antigenically distinct B/VIC lineage variant viruses. The investigation indicated that B/VIC V1A.3 subclade, containing a three amino acid deletion in the hemagglutinin and known to be antigenically distinct to the B/Colorado/06/2017 vaccine virus, was the most prevalent circulating virus within the specimens evaluated (86/88 in real-time RT-PCR). This work underscores the value of portable platforms for rapid, onsite pathogen characterization.

Recombinase Polymerase Amplification-Based Biosensors for Rapid Zoonoses Screening.

Recent, outbreaks of new emergency zoonotic diseases have prompted an urgent need to develop fast, accurate, and portable screening assays for pathogen infections. Recombinase polymerase amplification (RPA) is sensitive and specific and can be conducted at a constant low temperature with a short response time, making it especially suitable for on-site screening and making it a powerful tool for preventing or controlling the spread of zoonoses. This review summarizes the design principles of RPA-based biosensors as well as various signal output or readout technologies involved in fluorescence detection, lateral flow assays, enzymatic catalytic reactions, spectroscopic techniques, electrochemical techniques, chemiluminescence, nanopore sequencing technologies, microfluidic digital RPA, and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems. The current status and prospects of the application of RPA-based biosensors in zoonoses screening are highlighted. RPA-based biosensors demonstrate the advantages of rapid response, easy-to-read result output, and easy implementation for on-site detection, enabling development toward greater portability, automation, and miniaturization. Although there are still problems such as high cost with unstable signal output, RPA-based biosensors are increasingly becoming one of the most important means of on-site pathogen screening in complex samples involving environmental, water, food, animal, and human samples for controlling the spread of zoonotic diseases.

Copper: DNA extraction and solid phase detection agent for all-in-one molecular diagnostic device coupled with isothermal amplification.

In this study, an all-in-one poly(methyl methacrylate) (PMMA) device integrating two novel techniques - DNA extraction employing a CuSO4/H2O2 system and DNA detection utilizing solid phase copper tape - coupled with loop-mediated isothermal amplification (LAMP) is developed for on-site pathogen detection. The CuSO4/H2O2 system, also known as Fenton-like reaction, is used to produce hydroxyl radicals, which can disrupt bacterial membranes via lipid peroxidation and release DNA at room temperature. The released DNA is subsequently amplified by LAMP reaction. The acidic environment resulting from the production of hydrogen ions in the presence of target DNA in the LAMP reaction can stimulate the color change on copper tape due to the corrosion, while the innate alkaline environment in a negative sample not containing target DNA cannot stimulate the corrosion. The fabricated PMMA device integrates all the functionalities necessary for molecular diagnostics such as DNA extraction, amplification, and detection, and a carbon paste-based heater is fabricated for LAMP reaction. Using the PMMA device, Enterococcus faecium was detected as low as 4.67×102CFU/mL within 90min. E. faecium spiked in milk was successfully detected using the all-in-one PMMA device. The equipment-free techniques for decentralized diagnostics and naked-eye readout of results coupled with the portable heater serves as a promising solution for point-of-care testing particularly in a resource-limited environment.

Polyaniline-based 3D network structure promotes entrapment and detection of drug-resistant bacteria

Sensitive and accurate capture, enrichment, and identification of drug-resistant bacteria on human skin are important for early-stage diagnosis and treatment of patients. Herein, we constructed a three-dimensional hierarchically structured polyaniline nanoweb (3D HPN) to capture, enrich, and detect drug-resistant bacteria on-site by rubbing infected skins. These unique hierarchical nanostructures enhance bacteria capture efficiency and help severely deform the surface of the bacteria entrapped on them. Therefore, 3D HPN significantly contributes to the effective and reliable recovery of drug-resistant bacteria from the infected skin and the prevention of potential secondary infection. The recovered bacteria were successfully identified by subsequent real-time polymerase chain reaction (PCR) analysis after the lysis process. The molecular analysis results based on a real-time PCR exhibit excellent sensitivity to detecting target bacteria of concentrations ranging from 102 to 107 CFU/mL without any fluorescent signal interruption. To confirm the field applicability of 3D HPN, it was tested with a drug-resistant model consisting of micropig skin similar to human skin and Klebsiella pneumoniae carbapenemase-producing carbapenem-resistant Enterobacteriaceae (KPC-CRE). The results show that the detection sensitivity of this assay is 102 CFU/mL. Therefore, 3D HPN can be extended to on-site pathogen detection systems, along with rapid molecular diagnostics through a simple method, to recover KPC-CRE from the skin.Graphical

Detection of Phytophthora, Pythium, Globisporangium, Hyaloperonospora, and Plasmopara Species in High-Throughput Sequencing Data by In Silico and In Vitro Analysis Using Microbe Finder (MiFi)

Species identification of plant pathogenic oomycetes based on morphology is challenging because of the limited number of variable characters available. Hence, pure culture isolation followed by internal transcribed spacer sequencing is routinely conducted for diagnostic purposes. E-probe Diagnostic Nucleic acid Analysis (EDNA), a pipeline that couples high-throughput sequencing and metagenomic sequence analysis, is a detection system used to identify targets without relying on often time-consuming serological and molecular approaches. The EDNA pipeline, implemented through the Microbe Finder (MiFi), was used to design short species-specific sequences (e-probes) with sizes from 40 to 80 nucleotides and to detect plant pathogenic oomycetes in metagenomic data. To avoid false positives and increase target specificity, we removed the e-probes with sequences similar to nontarget species in NCBI's nucleotide database. The designed e-probes were tested using in silico simulated metagenomes with different target pathogen and host sequence levels. E-probes of Phytophthora nicotianae were further tested with metagenomes generated using nanopore sequencing. In silico detection with 100% sensitivity was obtained at 0.01 to 1% pathogen read abundances with the three e-probe lengths tested. MiFi also successfully identified Ph. nicotianae in the sequenced metagenomes with nanopore DNA sequencing. Thus, EDNA-MiFi could enable rapid detection (<24 h from sample collection to diagnosis) without requiring post-sequencing processing. EDNA, as implemented in the MiFi web application, is a powerful tool for detecting oomycetes from metagenomic samples. Our study shows the outstanding potential of EDNA-MiFi coupled with nanopore sequencing for plant and possible on-site pathogen diagnosis. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

On-site airborne pathogen detection for infection risk mitigation.

Human-infecting pathogens that transmit through the air pose a significant threat to public health. As a prominent instance, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that caused the COVID-19 pandemic has affected the world in an unprecedented manner over the past few years. Despite the dissipating pandemic gloom, the lessons we have learned in dealing with pathogen-laden aerosols should be thoroughly reviewed because the airborne transmission risk may have been grossly underestimated. From a bioanalytical chemistry perspective, on-site airborne pathogen detection can be an effective non-pharmaceutic intervention (NPI) strategy, with on-site airborne pathogen detection and early-stage infection risk evaluation reducing the spread of disease and enabling life-saving decisions to be made. In light of this, we summarize the recent advances in highly efficient pathogen-laden aerosol sampling approaches, bioanalytical sensing technologies, and the prospects for airborne pathogen exposure measurement and evidence-based transmission interventions. We also discuss open challenges facing general bioaerosols detection, such as handling complex aerosol samples, improving sensitivity for airborne pathogen quantification, and establishing a risk assessment system with high spatiotemporal resolution for mitigating airborne transmission risks. This review provides a multidisciplinary outlook for future opportunities to improve the on-site airborne pathogen detection techniques, thereby enhancing the preparedness for more on-site bioaerosols measurement scenarios, such as monitoring high-risk pathogens on airplanes, weaponized pathogen aerosols, influenza variants at the workplace, and pollutant correlated with sick building syndromes.

On-site marine pathogen (Vibrio parahaemolyticus) rapid colorimetric determination based on modified-free aptamer and metal-organic frameworks with simple washing step

Nowadays, microbial visual sensing platforms based on aptamers and metal-organic frameworks (MOFs) have been reported, but most of them require complex modification processes, which undoubtedly increases the difficulty of detection. Here, we constructed modified-free magnetic-aptamer MOF (Fe3O4-Ap@ZIF-8) and signal-aptamer MOF (Pt-Ap@ZIF-8), which effectively avoided complex biological modification. The rapid sensing strategy was adopted in this method to avoid the complicated washing steps and greatly shorten the detection time. The performance of this method was verified by taking Vibrio parahaemolyticus (VP) as the mode target. The limit of detection (LOD) of VP is 102 CFU/mL. Furthermore, this strategy can achieve rapid visual analysis of other target objects only by changing different aptamers, and has broad application prospects in the fields of rapid detection of food pollutants, rapid medical diagnosis, environmental monitoring, etc.

Rapid and Accurate Campylobacter jejuni Detection With CRISPR-Cas12b Based on Newly Identified Campylobacter jejuni-Specific and -Conserved Genomic Signatures.

Campylobacter jejuni is among the most prevalent foodborne zoonotic pathogens leading to diarrheal diseases. In this study, we developed a CRISPR-Cas12b-based system to rapidly and accurately detect C. jejuni contamination. Identification of C. jejuni-specific and -conserved genomic signatures is a fundamental step in development of the detection system. By comparing C. jejuni genome sequences with those of the closely related Campylobacter coli, followed by comprehensive online BLAST searches, a 20-bp C. jejuni-conserved (identical in 1024 out of 1037 analyzed C. jejuni genome sequences) and -specific (no identical sequence detected in non-C. jejuni strains) sequence was identified and the system was then assembled. In further experiments, strong green fluorescence was observed only when C. jejuni DNA was present in the system, highlighting the specificity of this system. The assay, with a sample-to-answer time of ∼40 min, positively detected chicken samples that were contaminated with a dose of approximately 10 CFU C. jejuni per gram of chicken, which was >10 times more sensitive than the traditional Campylobacter isolation method, suggesting that this method shows promise for onsite C. jejuni detection. This study provides an example of bioinformatics-guided CRISPR-Cas12b-based detection system development for rapid and accurate onsite pathogen detection.

Ultrasensitive isothermal detection of a plant pathogen by using a gold nanoparticle-enhanced microcantilever sensor

• GNP-enhanced dynamic microcantilever biosensor is constructed for pathogen analysis. • Combined with RPA, a LOD as low as one copy genomic DNA could be achieved. • This RPA-MCL could detect 57 ppm pathogen genomic DNA out of oilseed plant. Sensitive and rapid detection of plant pathogens, particularly for on-site diagnosis during custom inspections, is essential to prevent and control the spread of these pathogens. To address the need, a combined assay for detecting plant pathogens was developed by using gold nanoparticle-enhanced dynamic microcantilever (MCL) biosensing and isothermal recombinase polymerase amplification (RPA). In this study, we used Leptosphaeria maculans (L . maculans) , an aggressive fungus responsible for major losses in oilseed rape yield worldwide, as the target pathogen to demonstrate the merits of the combined RPA and MCL strategy (named as the RPA-MCL assay). The results indicated that the sensitivity of the RPA-MCL assay is higher than that of the fluorescence RPA assay, with the detection limit of only one copy of L . maculans DNA. In the practical assay, the newly developed RPA-MCL method was found to detect 57 ppm L . maculans genomic DNA in the oilseed rape seeds genomic DNA sample. Considering the high sensitivity and specificity of this strategy, we envision that the proposed RPA-MCL assay could have wide applications in nucleic acid diagnostics for on-site plant pathogen detection and species identification.

Direct triplex loop-mediated isothermal amplification assay for the point-of-care molecular detection of Salmonella genus, subspecies I, and serovar Typhimurium

In this study, a novel triplex loop-mediated isothermal amplification (LAMP) assay was developed and applied to a model of representative foodborne pathogen, Salmonella, to investigate the performance of the assay with high rapidity, specificity and sensitivity, and its ability to detect a pathogen in contaminated food. Three sets of LAMP primers were designed for target genes in Salmonella genus, subspecies I, and S. Typhimurium. Multiple targets were differentiated by the simultaneous analysis of annealing curves in a single LAMP reaction. In a triplex LAMP assay, each primer amplifies the target genes only, without any cross-reactivity with non-target genes. The limit of detection of this assay was 2.5 pg of S. Typhimurium DNA. The assay was applied to chicken meat artificially inoculated with S. Typhimurium. To decrease the inhibitory effects of the food matrix on the LAMP reaction, proteinase K was used in sample preparation, and the sample was heated and used directly as a DNA template. A combination of the direct extraction method optimized for food with triplex LAMP was able to detect low levels of S. Typhimurium (6.4 × 101 CFU/g) without a pre-enrichment step. This novel triplex multiplex LAMP assay successfully identified Salmonella genus, subspecies I, and S. Typhimurium within 60 min from sampling to results. This is therefore a rapid, reliable, sensitive, specific, and easy molecular diagnostic tool for on-site pathogen detection and food safety testing.

An on-site, highly specific immunosensor for Escherichia coli detection in field milk samples from mastitis-affected dairy cattle

Bovine mastitis is the most economically important infectious disease in dairy industry, and Escherichia coli (E. coli) is one of the major causative pathogens. Rapid identification and quantitative detection of E. coli are of great importance for bovine mastitis control and milk quality monitoring. Capitalizing on dielectrophoresis and interphase capacitance sensing, we have developed an immunosensor for E. coli detection by modifying low cost commercial microelectrodes with an E. coli specific antibody. The limit of detection reaches as low as 775 cells/mL within a 15 s’ response time, which can satisfy the requirement for on-site detection and field diagnosis of bovine mastitis. To demonstrate the sensor's specificity, tests against Staphylococcus aureus and Streptococcus uberis samples are performed showing negligible responses, and the selectivity is calculated to be 3063: 1. Furthermore, a simple pretreatment protocol is developed for on-site testing of raw milk, which only involves incubation, centrifugation and dilution steps. Then correct detection of E. coli is demonstrated for both artificially inoculated and infected field milk samples. This immunosensor and the corresponding protocol have advantages in speed, sensitivity, specificity, operability, and low cost, which make it highly promising for on-site pathogen detection of bovine mastitis.

Rapid Detection of Genetic Engineering, Structural Variation, and Antimicrobial Resistance Markers in Bacterial Biothreat Pathogens by Nanopore Sequencing

Widespread release of Bacillus anthracis (anthrax) or Yersinia pestis (plague) would prompt a public health emergency. During an exposure event, high-quality whole genome sequencing (WGS) can identify genetic engineering, including the introduction of antimicrobial resistance (AMR) genes. Here, we developed rapid WGS laboratory and bioinformatics workflows using a long-read nanopore sequencer (MinION) for Y. pestis (6.5 h) and B. anthracis (8.5 h) and sequenced strains with different AMR profiles. Both salt-precipitation and silica-membrane extracted DNA were suitable for MinION WGS using both rapid and field library preparation methods. In replicate experiments, nanopore quality metrics were defined for genome assembly and mutation analysis. AMR markers were correctly detected and >99% coverage of chromosomes and plasmids was achieved using 100,000 raw sequencing reads. While chromosomes and large and small plasmids were accurately assembled, including novel multimeric forms of the Y. pestis virulence plasmid, pPCP1, MinION reads were error-prone, particularly in homopolymer regions. MinION sequencing holds promise as a practical, front-line strategy for on-site pathogen characterization to speed the public health response during a biothreat emergency.

Development and evaluation of a real-time RT-PCR and a field-deployable RT-insulated isothermal PCR for the detection of Seneca Valley virus

BackgroundSeneca Valley virus (SVV) has emerged in multiple countries in recent years. SVV infection can cause vesicular lesions clinically indistinguishable from those caused by other vesicular disease viruses, such as foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus (VESV). Sensitive and specific RT-PCR assays for the SVV detection is necessary for differential diagnosis. Real-time RT-PCR (rRT-PCR) has been used for the detection of many RNA viruses. The insulated isothermal PCR (iiPCR) on a portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, SVV rRT-PCR and RT-iiPCR were developed and validated.ResultsNeither the SVV rRT-PCR nor the RT-iiPCR cross-reacted with any of the vesicular disease viruses (20 FMDV, two SVDV, six VSV, and two VESV strains), classical swine fever virus (four strains), and 15 other common swine viruses. Analytical sensitivities of the SVV rRT-PCR and RT-iiPCR were determined using serial dilutions of in vitro transcribed RNA as well as viral RNA extracted from a historical SVV isolate and a contemporary SVV isolate. Diagnostic performances were further evaluated using 125 swine samples by two approaches. First, nucleic acids were extracted from the 125 samples using the MagMAX™ kit and then tested by both RT-PCR methods. One sample was negative by the rRT-PCR but positive by the RT-iiPCR, resulting in a 99.20% agreement (124/125; 95% CI: 96.59–100%, κ = 0.98). Second, the 125 samples were tested by the taco™ mini extraction/RT-iiPCR and by the MagMAX™ extraction/rRT-PCR system in parallel. Two samples were positive by the MagMAX™/rRT-PCR system but negative by the taco™ mini/RT-iiPCR system, resulting in a 98.40% agreement (123/125; 95% CI: 95.39–100%, κ = 0.97). The two samples with discrepant results had relatively high CT values.ConclusionsThe SVV rRT-PCR and RT-iiPCR developed in this study are very sensitive and specific and have comparable diagnostic performances for SVV RNA detection. The SVV rRT-PCR can be adopted for SVV detection in laboratories. The SVV RT-iiPCR in a simple field-deployable system could serve as a tool to help diagnose vesicular diseases in swine at points of need.

QuickAnalysis: A Software Designed and Developed for a Portable On-Site Pathogen Detection System.

To develop a software program used for controlling the portable on-site pathogen detection system which is based on real-time PCR, this software is developed by C# over the Microsoft Visual Studio 2013 and used on local computers which are equipped with Windows systems. Taking the actual demand as the guidance, it constructs according to the framework design and unifies the barcode technology, the cloud service, and the Bluetooth technology. Based on the above methods, this software can meet the demands of the system such as free experiment design, auto-control of the hardware and efficient data management. During the operation of this program, the serial port and network communication between it and the device and web server remains stable, no matter it is processing the data collection or data analysis and display. It is feasible and practical to apply this software to the portable on-site pathogen detection system in clinical practices and research work.