On-site Detection Of Pathogens Research Articles

Rapid On-Site Detection and Quantification of Foodborne Pathogens Using Microfluidic Devices.

The potential for foodborne infectious disease outbreaks has increased not only on a local scale but also on a regional and international scale. Simple, rapid, and accurate methods to enumerate pathogenic bacteria in food and drink are required to prevent the spread of these bacteria. Here, I describe applications of a microfluidic device for on-chip fluorescent staining and semiautomated counting of target bacteria in food samples.

Plasmonic enhancement in lateral flow sensors for improved sensing of E. coli O157:H7

We propose a plasmonic enhanced lateral flow sensor (pLFS) concept with an enhanced colorimetric signal by utilizing liposome encapsulating reagent to trigger the aggregation of gold nanoparticles (GNPs). Our signal enhancement strategy incorporates the simplicity of lateral flow immunoassays (LFIA) utilizing plasmonic enhancement. The conceptualized hybrid pLFS for onsite rapid detection of pathogens in low numbers in a user friendly format requiring simple steps is the first step in the translation of plasmonic enhancement sensing to a practical regime. The pLFS was carried out with a biotinylated liposome label ruptured to release branched polyethylenimine (BPEI) to trigger the aggregation of GNPs for colorimetric signal generation. BPEI has multiple amino groups and more positive charges in PBS buffer, therefore few of the BPEI groups could induce the aggregation of GNPs, resulting in an enhanced colorimetric signal to detect E. coli O157:H7. Compared with the reported conventional LFIA, the proposed pLFS demonstrated more than 1000-fold improvement in sensitivity. The pLFS could detect as low as 100 CFU/ml of E. coli O157:H7 in buffer and 600 CFU/ml E. coli O157:H7 in liquid food systems.

Ultra-Fast On-Site Molecular Detection of Foodborne Pathogens Using a Combination of Convection Polymerase Chain Reaction and Nucleic Acid Lateral Flow Immunoassay.

Rapid detection and timely treatment of diseases caused by foodborne pathogens is important for improving the curative efficiency and preventing the spread of disease. In this study, we developed an assay utilizing a recently introduced ultra-fast convection polymerase chain reaction (cPCR) in conjunction with a simple nucleic acid lateral flow (NALF) immunoassay for ultra-fast on-site molecular detection of foodborne pathogens. Two Salmonella enterica serovars, Salmonella Enteritidis and Salmonella Typhimurium, and Escherichia coli O157:H7 were used as the target pathogens. We confirmed the specific amplification of the target species with specifically designed modified primer sets for cPCR in singleplex and duplex modes. After cPCR amplification, we compared the detection specificity and sensitivity using agarose gel electrophoresis and NALF assays with one or two test lines. The cPCR amplicons were readily and sensitively detected using the NALF assay, and the sensitivity was comparable with that of agarose gel electrophoresis. To confirm the application of the assay in real-life samples, the assay was used to test artificially contaminated milk. Without sample pre-enrichment, the limit of detection (LOD) was 4.5 × 103 colony-forming units (CFU)/mL for Salmonella species; 4.5 × 104 CFU/mL to differentiate Salmonella Enteritidis and Salmonella Typhimurium; and 2.3 × 103 CFU/mL for E. coli O157:H7, in a duplex assay. With a 6 h pre-enrichment, the LOD was 4.5 CFU/mL for Salmonella Enteritidis and Salmonella Typhimurium, and 2.3 CFU/mL for E. coli O157:H7. The cPCR amplification took only 14 min, and the NALF assay took ca. 5 min. The total analysis time was less than 20 min. Based on these observations, we propose that the developed assay is simple, ultra-fast, and applicable for on-site detection of foodborne pathogens.

A Disposable and Multi-Chamber Film-Based PCR Chip for Detection of Foodborne Pathogen.

Since the increment of the threat to public health caused by foodborne pathogens, researches have been widely studied on developing the miniaturized detection system for the on-site pathogen detection. In the study, we focused on the development of portable, robust, and disposable film-based polymerase chain reaction (PCR) chip containing a multiplex chamber for simultaneous gene amplification. In order to simply fabricate and operate a film-based PCR chip, different kinds of PCR chambers were designed and fabricated using polyethylene terephthalate (PET) and polyvinyl chloride (PVC) adhesive film, in comparison with commercial PCR, which employs a stereotyped system at a bench-top scale. No reagent leakage was confirmed during the PCR thermal cycling using the film PCR chip, which indicates that the film PCR chip is structurally stable for rapid heat cycling for DNA amplification. Owing to use of the thin film to fabricate the PCR chip, we are able to realize fast thermal transfer from the heat block that leads to short PCR amplification time. Moreover, using the film PCR chip, we could even amplify the target pathogen with 10 CFU mL−1. The artificially infected milk with various concentration of Bacillus cereus was successfully amplified on a single film PCR chip. On the basis of the reliable results, the developed film PCR chip could be a useful tool as a POCT device to detect foodborne pathogens via genetic analysis.

Universal and reusable hapten/antibody-mediated portable optofluidic immunosensing platform for rapid on-site detection of pathogens

A universal and reusable hapten–antibody-mediated portable optofluidic immunosensing platform (OIP) was developed for rapid on-site detection of pathogens. By using Escherichia coli O157:H7 (E. coli O157:H7) and bisphenol A-Bovine serum albumin (BPA-BSA)/anti-BPA antibody as a model pathogen and a mediated hapten–antibody, respectively, a novel immunoassay mechanism was proposed to detect pathogens. The BPA-BSA-modified immunosensor and E. coli O157:H7 were initially saturated with anti-BPA antibodies (mouse IgG) and anti-E. coli O157:H7 antibodies (mouse IgG), respectively. Then, the fluorescence-labeled secondary antibodies (goat anti-mouse IgG antibody) were incubated with E. coli O157:H7 with their antibodies. Next, the mixture was introduced into the immunosensor surface bound to the anti-BPA antibodies. A high concentration of E. coli O157:H7 in the sample reduced the number of fluorescence-labeled secondary antibodies bound to the immunosensor surface, thus resulting in the detection of low fluorescence signals. Under optimized conditions, the hapten–antibody-mediated OIP system exhibited a detection limit of 8 cfu/mL E. coli O157:H7 after concentrating 100 times by using centrifugation, and a test cycle, including prereaction, detection, and regeneration, was less than 1 h. The robustness of the hapten–carrier protein-modified immunosensor surface allowed multiple pathogen immunoassays. The proposed strategy demonstrated good recovery, precision, and accuracy through the evaluation of the spiked water samples. We expect that the new platform can be readily used for the detection of other pathogens in a variety of application fields ranging from environmental monitoring and food safety to medical diagnosis.

A review on microscale polymerase chain reaction based methods in molecular diagnosis, and future prospects for the fabrication of fully integrated portable biomedical devices.

Since the advent of microfabrication technology and soft lithography, the lab-on-a-chip concept has emerged as a state-of-the-art miniaturized tool for conducting the multiple functions associated with micro total analyses of nucleic acids, in series, in a seamless manner with a miniscule volume of sample. The enhanced surface-to-volume ratio inside a microchannel enables fast reactions owing to increased heat dissipation, allowing rapid amplification. For this reason, PCR has been one of the first applications to be miniaturized in a portable format. However, the nature of the basic working principle for microscale PCR, such as the complicated temperature controls and use of a thermal cycler, has hindered its total integration with other components into a micro total analyses systems (μTAS). This review (with 179 references) surveys the diverse forms of PCR microdevices constructed on the basis of different working principles and evaluates their performances. The first two main sections cover the state-of-the-art in chamber-type PCR microdevices and in continuous-flow PCR microdevices. Methods are then discussed that lead to microdevices with upstream sample purification and downstream detection schemes, with a particular focus on rapid on-site detection of foodborne pathogens. Next, the potential for miniaturizing and automating heaters and pumps is examined. The review concludes with sections on aspects of complete functional integration in conjunction with nanomaterial based sensing, a discussion on future prospects, and with conclusions. Graphical abstract In recent years, thermocycler-based PCR systems have been miniaturized to palm-sized, disposable polymer platforms. In addition, operational accessories such as heaters and mechanical pumps have been simplified to realize semi-automatted stand-alone portable biomedical diagnostic microdevices that are directly applicable in the field. This review summarizes the progress made and the current state of this field.

Janus Emulsions for the Detection of Bacteria.

Janus emulsion assays that rely on carbohydrate–lectin binding for the detection of Escherichia coli bacteria are described. Surfactants containing mannose are self-assembled at the surface of Janus droplets to produce particles with lectin binding sites. Janus droplets orient in a vertical direction as a result of the difference in densities between the hydrocarbon and fluorocarbon solvents. Binding of lectin to mannose(s) causes agglutination and a tilted geometry. The distinct optical difference between naturally aligned and agglutinated Janus droplets produces signals that can be detected quantitatively. The Janus emulsion assay sensitively and selectively binds to E. coli at 104 cfu/mL and can be easily prepared with long-time stability. It provides the basis for the development of inexpensive portable devices for fast, on-site pathogen detection.

Design and Elementary Evaluation of a Highly-Automated Fluorescence-Based Instrument System for On-Site Detection of Food-Borne Pathogens.

A simple, highly-automated instrument system used for on-site detection of foodborne pathogens based on fluorescence was designed, fabricated, and preliminarily tested in this paper. A corresponding method has been proved effective in our previous studies. This system utilizes a light-emitting diode (LED) to excite fluorescent labels and a spectrometer to record the fluorescence signal from samples. A rotation stage for positioning and switching samples was innovatively designed for high-throughput detection, ten at most in one single run. We also developed software based on LabVIEW for data receiving, processing, and the control of the whole system. In the test of using a pure quantum dot (QD) solution as a standard sample, detection results from this home-made system were highly-relevant with that from a well-commercialized product and even slightly better reproducibility was found. And in the test of three typical kinds of food-borne pathogens, fluorescence signals recorded by this system are highly proportional to the variation of the sample concentration, with a satisfied limit of detection (LOD) (nearly 102–103 CFU·mL−1 in food samples). Additionally, this instrument system is low-cost and easy-to-use, showing a promising potential for on-site rapid detection of food-borne pathogens.

Short communication: A novel method using immunomagnetic separation with a fluorescent nanobeads lateral flow assay for the rapid detection of low-concentration Escherichia coli O157:H7 in raw milk.

Escherichia coli O157:H7 is an important serotype of enterohemorrhagic E. coli that was first identified as a human pathogen in 1982. This pathogen causes several serious diseases. In this study, immunomagnetic separation was coupled with a fluorescent nanobeads lateral flow assay to establish a sensitive and rapid detection method for Escherichia coli O157:H7 in raw milk. The pathogen was captured from raw milk by immunomagnetic separation with immunomagnetic nanobeads and then detected using a fluorescent nanobeads lateral flow assay. A fluorescent line was formed in the test line of the test strip and quantitatively detected using a fluorescent reader. Screening times, which included immunomagnetic separation and the fluorescent nanobeads lateral flow assay, were 8, 7, 6, and 5h when 1, 5, 25, and 125 cfu of E. coli O157:H7, respectively, were inoculated into 25mL of raw milk. The established method could be widely applied to the rapid onsite detection of other pathogens to ensure food safety.

Ion-Mediated Polymerase Chain Reactions Performed with an Electronically Driven Microfluidic Device.

The polymerase chain reaction (PCR) is a powerful method for exponentially amplifying very low amounts of target DNA from genetic, clinical, and forensic samples. However, the heating and cooling steps in PCR largely hamper the miniaturization of thermocyclers for on-site detection of pathogens and point-of-care tests. Herein, we devise an ion-mediated PCR (IM-PCR) strategy by exploiting ion-induced DNA denaturation/renaturation cycles. DNA duplexes are effectively denatured in alkaline solutions; whereas, the denatured single-stranded DNA strands readily reform duplexes at neutral pH. By using an integrated microchip that can programmably control the solution pH simply switching the potential in a range of several hundred millivolts, we can trigger IM-PCR at a constant temperature. Analogously to thermal cycling, 30 cycles of pH-induced denaturation/renaturation were used to amplify protein DNA fragments as confirmed by DNA sequencing. We anticipate that this portable, low-cost, and scalable IM-PCR holds great promise for widespread biological, clinical, and environmental applications.

Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus

Recent outbreaks of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) in multiple countries have caused significant economic losses and remain a serious challenge to the swine industry. Rapid diagnosis is critical for the implementation of efficient control strategies before and during PEDV and PDCoV outbreaks. Insulated isothermal PCR (iiPCR) on the portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, a singleplex PEDV RT-iiPCR, a singleplex PDCoV RT-iiPCR, and a duplex PEDV/PDCoV real-time RT-PCR (rRT-PCR) commercial reagents targeting the M gene were compared to an N gene-based PEDV rRT-PCR and an M gene-based PDCoV rRT-PCR that were previously published and used as reference PCRs. All PCR assays were highly specific and did not cross react with other porcine enteric pathogens. Analytical sensitivities of the PEDV RT-iiPCR, PDCoV RT-iiPCR and duplex PEDV/PDCoV rRT-PCR were determined using in vitro transcribed RNA as well as viral RNA extracted from ten-fold serial dilutions of PEDV and PDCoV cell culture isolates. Performance of each PCR assay was further evaluated using 170 clinical samples (86 fecal swabs, 24 feces, 19 intestines, and 41 oral fluids). Compared to the reference PEDV rRT-PCR, the sensitivity, specificity and accuracy of the PEDV RT-iiPCR were 97.73%, 98.78%, and 98.24%, respectively, and those of the duplex PEDV/PDCoV rRT-PCR were 98.86%, 96.34%, and 97.65%, respectively. Compared to the reference PDCoV rRT-PCR, the sensitivity, specificity and accuracy of the PDCoV RT-iiPCR were 100%, 100%, and 100%, respectively, and those of the PEDV/PDCoV duplex rRT-PCR were 96.34%, 100%, and 98.24%, respectively. Overall, all three new PCR assays were comparable to the reference rRT-PCRs for detection of PEDV and/or PDCoV. The PEDV and PDCoV RT-iiPCRs are potentially useful tools for on-site detection and the duplex PEDV/PDCoV rRT-PCR provides a convenient method to simultaneously detect the two viruses and differentiate PEDV from PDCoV.

Engineered nanoconstructs for the multiplexed and sensitive detection of high-risk pathogens.

Many countries categorize the causative agents of severe infectious diseases as high-risk pathogens. Given their extreme infectivity and potential to be used as biological weapons, a rapid and sensitive method for detection of high-risk pathogens (e.g., Bacillus anthracis, Francisella tularensis, Yersinia pestis, and Vaccinia virus) is highly desirable. Here, we report the construction of a novel detection platform comprising two units: (1) magnetic beads separately conjugated with multiple capturing antibodies against four different high-risk pathogens for simple and rapid isolation, and (2) genetically engineered apoferritin nanoparticles conjugated with multiple quantum dots and detection antibodies against four different high-risk pathogens for signal amplification. For each high-risk pathogen, we demonstrated at least 10-fold increase in sensitivity compared to traditional lateral flow devices that utilize enzyme-based detection methods. Multiplexed detection of high-risk pathogens in a sample was also successful by using the nanoconstructs harboring the dye molecules with fluorescence at different wavelengths. We ultimately envision the use of this novel nanoprobe detection platform in future applications that require highly sensitive on-site detection of high-risk pathogens.

The Latest Progress of On-Site Pathogens Detection Techniques and Instruments Based on Nucleic Acid.

Infectious diseases are a major threat to the survival and development of humanity around the world. Timely and accurate diagnosis is proved to be an essential prerequisite for the prevention and containment of infectious diseases prevalence. Nucleic acid based techniques are the most common used methods for the detection of infectious pathogens, and demonstrate a great potential for developing highly reliable, sensitive and specific diagnostic platforms for on-site pathogens detection. This review focused on some of the most promising nucleic acid testing techniques and commercial devices for the on-site detection of infectious pathogens. Additionally, some emerging technologies that may contribute to the development of robust and reliable diagnostic instruments for infectious pathogens detection have been presented. Finally, personal perspectives with respect to the future trends of diagnostic techniques were given.

Real-Time Detection of Salmonella on Fresh Apples Using Magnetoelastic Biosensors Operating Under Multi-Harmonic Resonance Modes

This paper investigates real-time detection of Salmonella on fresh apples using phage-coated magnetoelastic (ME) biosensors that operate under multi-harmonic resonance modes. The ME biosensor is a class of mass-sensitive biosensors that is being developed for rapid, on-site pathogen detection in the food system. The ME biosensor used in this work consists of a freestanding, strip-shaped ME resonator (1 mm x 0.2 mm x 30 um) as the signal transducer and E2-phage that specifically binds with the pathogen of interest. The ME resonator is made of an amorphous, ferromagnetic alloy with magnetostrictive properties and hence can be placed into mechanical resonance by an externally applied time-varying magnetic field. When this phage-coated ME biosensor comes into contact and binds specifically with the target pathogen, the mass of the biosensor increases, resulting in a decrease in the biosensor's resonant frequency. The conventional theory describes that the mass sensitivity of the ME biosensor (i.e., the ratio of the resonant frequency change to the mass change) is proportional to the harmonic, n, defined as an integer multiple of the fundamental resonant frequency (n = 1, 2, 3, ···). Hence, improved mass sensitivity can be obtained by measuring resonant frequency changes of higher harmonic modes. Additionally, measuring and analyzing multi-harmonic resonant frequencies can reduce possible false negative detection resulting from the Salmonella that are attached to the nodes of the vibrating sensors. As a proof-in-concept experiment, in-situ Salmonella detection on fresh apples was conducted. Both measurement (with phage) and control (without phage) sensors were prepared and placed directly on apples spiked with Salmonella. The sensors’ resonant frequency shifts were then measured in situ using a surface-scanning coil detector as a function of time. While the resonant frequency shifts of the control sensors remained negligible, those of the measurement sensors decreased largely as time progressed, indicating that specific binding of the Salmonella on the measurement sensors had occurred. In addition, the mass sensitivity was found to improve for higher harmonic modes (the 2nd and 3rd longitudinal modes) compared to the fundamental mode. False negative detection was also reduced, which was confirmed by inspecting the sensor surface using SEM and comparing the number of bound Salmonella to the measured frequency shift data. Hence, enhanced detection of Salmonella has been demonstrated using ME biosensors operating in multi-harmonic resonance modes.

A novel nanoprobe for the sensitive detection of Francisella tularensis

Francisella tularensis is a human zoonotic pathogen and the causative agent of tularemia, a severe infectious disease. Given the extreme infectivity of F. tularensis and its potential to be used as a biological warfare agent, a fast and sensitive detection method is highly desirable. Herein, we construct a novel detection platform composed of two units: (1) Magnetic beads conjugated with multiple capturing antibodies against F. tularensis for its simple and rapid separation and (2) Genetically-engineered apoferritin protein constructs conjugated with multiple quantum dots and a detection antibody against F. tularensis for the amplification of signal. We demonstrate a 10-fold increase in the sensitivity relative to traditional lateral flow devices that utilize enzyme-based detection methods. We ultimately envision the use of our novel nanoprobe detection platform in future applications that require the highly-sensitive on-site detection of high-risk pathogens.

Quartz-Crystal Microbalance (QCM) for Public Health: An Overview of Its Applications.

Nanobiotechnologies, from the convergence of nanotechnology and molecular biology and postgenomics medicine, play a major role in the field of public health. This overview summarizes the potentiality of piezoelectric sensors, and in particular, of quartz-crystal microbalance (QCM), a physical nanogram-sensitive device. QCM enables the rapid, real time, on-site detection of pathogens with an enormous burden in public health, such as influenza and other respiratory viruses, hepatitis B virus (HBV), and drug-resistant bacteria, among others. Further, it allows to detect food allergens, food-borne pathogens, such as Escherichia coli and Salmonella typhimurium, and food chemical contaminants, as well as water-borne microorganisms and environmental contaminants. Moreover, QCM holds promises in early cancer detection and screening of new antiblastic drugs. Applications for monitoring biohazards, for assuring homeland security, and preventing bioterrorism are also discussed.

Combination of multiplex reverse-transcription loop-mediated isothermal amplification with an immunochromatographic strip for subtyping influenza A virus

Considering the fatal human victims and economic loss by the annual epidemic influenza virus, the development of a rapid and convenient genetic analysis methodology is demanding for timely on-site pathogen detection. In this study, we utilized reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for multiplex target gene amplification, and the resultant amplicons were analyzed on the immunochromatographic strip (ICS) for subtyping influenza A virus. Through the optimized primer design, reaction temperature and time, and concentration of enzymes (Bst DNA polymerase and AMV reverse transcriptase) and dNTP, the HA (H1, H3, and H5 gene) and conserved M gene were amplified. The ICS contains two test lines in addition to a control line in order to detect the presence of the HA and M gene, thereby informing us of influenza virus A type as well as its subtype (H1N1, H3N2, and H5N1). The combination of the multiplex RT-LAMP with the ICS could be complete in 40min and the pathotyping and subtyping of influenza A virus were performed even with 10 copies of viral RNA templates. Moreover, the subtyping of clinical samples, which were obtained from patients infected by influenza A virus was successfully confirmed using the multiplex RT-LAMP and ICS techniques, showing great feasibility of our methodology for real sample analysis with high speed, simplicity and sensitivity.

Self-propelled continuous-flow PCR in capillary-driven microfluidic device: Microfluidic behavior and DNA amplification

Continuous-flow polymerase chain reaction (CF-PCR) in microfluidic devices has a great potential for on-site detection of various pathogens and food species because of their high speed deoxyribo nucleic acid (DNA) amplification. However, flow-controlled pumps such as syringe pumps are absolutely necessary, which cause complex and difficult operations. Here, we present a self-propelled CF-PCR (SP-CF-PCR) in a microfluidic device which requires no external pumps to control the flow. The PCR solution is simply dropped onto the inlet and is autonomously transported by capillary forces. One of the difficulties in addressing the capillary flow in PCR microfluidic device is that the temperature of the PCR solution is periodically switched. Differing from previous theoretical approaches which deal with single steady-state temperature zone, for the first time, the displacement of capillary flow during temperature switching was mathematically formulated and was simulated by using experimental values of viscosities and capillary pressures at each temperature as parameters for simulation. Basing on the excellent matching between simulated and experimental data of the capillary flow, we were able to reveal the optimized design of 150-μm-wide and 150-μm-deep microchannel that successfully transported over 1600mm for carrying out PCR within less than 14min solely by capillary forces. We also demonstrated the SP-CF-PCR for verifying our concept. The specific amplifications of 295bp of β-actin from human genome, 232bp of AH1pdm influenza virus and 95bp of 16S rDNA of Escherichia coli genomic DNA were successfully achieved, proving the application potentials of our device.

A microfluidic system integrated with buried optical fibers for detection of Phalaenopsis orchid pathogens

Orchids of the genus Phalaenopsis are some of the most economically important plants in Taiwan. Fast, accurate, and on-site detection of pathogens in these orchids is therefore of critical importance in order to prevent or suppress costly disease outbreaks. Traditional pathogen detection methods are time-consuming, require well-equipped laboratories with highly trained personnel, and cannot be conducted in situ. In this study, a microfluidic system integrated with buried optical fibers was developed to detect viral pathogens of Phalaenopsis spp. Briefly, virus-specific ribonucleic acid (RNA) purification was achieved by a pre-treatment incubation with magnetic beads, and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was used subsequently to amplify the viral RNA. Positive RT-LAMP reactions resulted in the precipitation of magnesium pyrophosphate, which caused a change in turbidity that could be seen by the naked eye. A buried optical fiber-based detection module and a micro-stirring device were then integrated into the microfluidic chip to detect the RT-LAMP reaction product directly on the chip itself by measuring the change in the optical signals caused by the turbidity change associated with a positive amplification. The limit of detection for this system was found to be 25fg, which is of similar sensitivity to existing, more laborious methods. Therefore, by using the integrated microfluidic system, a sensitive, rapid, accurate, and automatic diagnosis of viral pathogens in Phalaenopsis spp. orchids could be achieved within only 65min.

Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP) for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals

Rapid, low-cost, and user-friendly strategies are urgently needed for early disease diagnosis and timely treatment, particularly for on-site screening of pathogens in aquaculture. In this study, we successfully developed a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP), which was capable of simultaneously detecting 10 pathogenic bacteria in aquatic animals, i.e., Nocardia seriolae, Pseudomonas putida, Streptococcus iniae, Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio rotiferianus, and Vibrio vulnificus. The assay provided a nearly-automated approach, with only a single pipetting step per chip for sample dispensing. This technique could achieve limits of detection (LOD) ranging from 0.40 to 6.42pg per 1.414μL reaction in less than 30min. The robust reproducibility was demonstrated by a little variation among duplications for each bacterium with the coefficient of variation (CV) for time to positive (Tp) value less than 0.10. The clinical sensitivity and specificity of this on-chip LAMP assay in detecting field samples were 96.2% and 93.8% by comparison with conventional microbiological methods. Compared with other well-known techniques, on-chip LAMP assay provides low sample and reagent consumption, ease-of-use, accelerated analysis, multiple bacteria and on-site detection, and high reproducibility, indicating that such a technique would be applicable for on-site detection and routine monitoring of multiple pathogens in aquaculture.