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Development of Portable Fluorescence Microplate Reader Equipped with Indium Tin Oxide Glass Heater for Loop-mediated Isothermal Amplification

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Development of Portable Fluorescence Microplate Reader Equipped with Indium Tin Oxide Glass Heater for Loop-mediated Isothermal Amplification

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Foodborne bacterial infections and diseases have been considered to be a major threat for public health in the worldwide. Increased incidence of human diseases caused by foodborne pathogens have been correlated with growing world population and mobility. Loop-mediated isothermal amplification (LAMP) has been regarded as an innovative gene amplification technology and emerged as an alternative to PCR-based methodologies in both clinical laboratory and food safety testing. Nowadays, LAMP has been applied to detection and identification on pathogens from microbial diseases, as it showed significant advantage in high sensitivity, specificity and rapidity. The high sensitivity of LAMP enables detection of the pathogens in sample materials even without time consuming sample preparation. An overview of LAMP mainly containing the development history, reaction principle and its application to four kind of foodborne pathogens detection are presented in this paper. As concluded, with the advantages of rapidity, simplicity, sensitivity, specificity and robustness, LAMP is capable of applications for clinical diagnosis as well as surveillance of infection diseases. Moreover, the main purpose of this paper is to provide theoretical basis for the clinical application of LAMP technology.
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Point-of-care (POC) molecular diagnostics plays a pivotal role for the prevention and treatment of infectious diseases. In spite of recent advancement in microfluidic based POC devices, there are still rooms for development to realize rapid, automatic and cost-effective sample-to-result genetic analysis. In this study, we propose an integrated rotary microfluidic system that is capable of performing glass microbead based DNA extraction, loop mediated isothermal amplification (LAMP), and colorimetric lateral flow strip based detection in a sequential manner with an optimized microfluidic design and a rotational speed control. Rotation direction-dependent coriolis force and siphon valving structures enable us to perform the fluidic control and metering, and the use of the lateral flow strip as a detection method renders all the analytical processes for nucleic acid test simplified and integrated without the need of expensive instruments or human intervention. As a proof of concept for point-of-care DNA diagnostics, we identified the food-borne bacterial pathogen which was contaminated in water or milk. Not only monoplex Salmonella Typhimurium but also multiplex Salmonella Typhimurium and Vibrio parahaemolyticus were analysed on the integrated rotary genetic analysis microsystem with a limit of detection of 50 CFU in 80min. In addition, three multiple samples were simultaneously analysed on a single device. The sample-to-result capability of the proposed microdevice provides great usefulness in the fields of clinical diagnostics, food safety and environment monitoring.
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In this study, we fabricate a high efficiency heater consisting of the indium tin oxide (ITO) nanoparticle (NP)-paste and polydimethylsiloxane (PDMS) and investigate the effect of PDMS on temperature maintenance of the heater through the comparison with the PDMS-free ITO film heater. Compared to the ITO film heater, the temperature of the PDMS/ITO film heater lasts 1.5 times longer. And the power consumption of the PDMS/ITO film heater is reduced by 35%, owing to the low thermal conductivity of the PDMS layer.
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Rapid, sensitive, and selective pathogen detection is of paramount importance in infectious disease diagnosis and monitoring of treatment. Currently available diagnostic assays based on polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) are timeconsuming, complex, and relatively expensive, thus limiting their utility in resource-limited settings. Loop-mediated isothermal amplification (LAMP) techniques have been used extensively in the development of rapid and sensitive diagnostic assays for pathogen detection and nucleic acid analysis and hold great promise for revolutionizing point-of-care molecular diagnostics. Here, we review novel LAMP-based lab-on-a-chip (LOC) diagnostic assays developed for pathogen detection over the past several years. We review various LOC platforms based on their design strategies for pathogen detection and discuss LAMP-based platforms still in development and already in the commercial pipeline. This review is intended as a guide to the use of LAMP techniques in LOC platforms for molecular diagnostics and genomic amplifications.
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Here we report one of the smallest real-time polymerase chain reaction (PCR) systems to date with an approximate size of 100 mm × 60 mm × 33 mm. The system is an autonomous unit requiring an external 12 V power supply. Four simultaneous reactions are performed in the form of virtual reaction chambers (VRCs) where a ≈200 nL sample is covered with mineral oil and placed on a glass cover slip. Fast, 40 cycle amplification of an amplicon from the H7N9 gene was used to demonstrate the PCR performance. The standard curve slope was -3.02 ± 0.16 cycles at threshold per decade (mean ± standard deviation) corresponding to an amplification efficiency of 0.91 ± 0.05 per cycle (mean ± standard deviation). The PCR device was capable of detecting a single deoxyribonucleic acid (DNA) copy. These results further suggest that our handheld PCR device may have broad, technologically-relevant applications extending to rapid detection of infectious diseases in small clinics.
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Point-of-care (POC) genetic diagnostics critically depends on miniaturization and integration of sample processing, nucleic acid amplification, and detection systems. Polymerase chain reaction (PCR) assays have extensively applied for the diagnosis of genetic markers of disease. Microfluidic chips for microPCR with different materials and designs have been reported. Temperature cycling systems with varying thermal masses and conductivities, thermal cycling times, flow-rates, and cross-sectional areas, have also been developed to reduce the nucleic acid amplification time. Similarly, isothermal amplification techniques (e.g., loop-mediated isothermal amplification or LAMP), which are still are emerging, have a better potential as an alternative to PCR for POC diagnostics. Isothermal amplification techniques have: (i) moderate incubation temperature leading to simplified heating and low power consumption, (ii) yield high amount of amplification products, which can be detected either visually or by simple detectors, (iii) allow direct genetic amplification from bacterial cells due to the superior tolerance to substances that typically inhibit PCR, (iv) have high specificity, and sensitivity, and (v) result in rapid detection often within 10-20 min. The aim of this review is to provide a better understanding of the advantages and limitations of microPCR and microLAMP systems for rapid and POC diagnostics.
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A microchannel chip for continuous-flow polymerase chain reaction (PCR) was developed using transparent materials. The microchannel was fabricated on a quartz glass substrate using standard photolithography and wet-etching techniques and was sealed by another quartz glass substrate. Two indium-tin-oxide (ITO) films were deposited on the etched substrate as a thermal source. To confirm the temperature distribution in the microchannel, we measured the fluorescence spectra of an aqueous solution of 1-pyrenesulfonic acid sodium salt (PS-Na), which is a temperature-indicator dye, in the microchannel under a continuous solution flow. The results confirm that the temperature distribution on the microchannel’s ITO films was almost uniform (within ±2 °C) under two flow rates (56 and 152 nl/min). The slightness of this deviation indicates that the ITO films integrated into the microchannel chip can be very useful as a thermal source for PCR. An amplification of a 450 bp segment of Escherichia coli HB101 was successfully performed by two-stage (94 and 67 °C) thermal cycling on the chip device.
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In this work, we developed a portable integrated microchip of loop-mediated isothermal nucleic acid amplification (LAMP). This chip, with sample-to-answer capability, could perform rapid DNA release, exponential signal amplification and naked-eye result read-out in single or multiplex format. We call it iμLAMP, namely integrated micro-LAMP, which was successfully used for point-of-care identification of bacteria.
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An integrated continuous-flow microfluidic chip was fabricated on glass substrate with polydimethylsiloxane (PDMS)-based microchannels, cell lysis and Polymerase chain reaction (PCR) modules on the same chip. While gold-microelec- trode was used for electrochemical cell lysis, indium-tin-oxide (ITO) microheater was used for thermal cycling during PCR reaction. The fabricated device was used for PCR amplification of pancreatic cancer DNA marker (SMAD4) from non-tumorigenic MCF10a human cell lines. The PCR product (193 bp) was verified for MCF10a cells by agarose gel electrophoresis after 20 cycles of reaction on the microchip, whereas no product was detected in case of tumorigenic MCF7 cells. The total time required for the entire reaction was less than 45 min. Therefore, the proposed mi- crochip can be helpful in predicting the risk of metastatic cancer by analysis of genetic tumor markers from human samples and can also be used for other genetic analysis involving PCR reaction. Index Terms—Electrochemical cell lysis, microfluidics, micro- heater, pancreatic cancer, polymerase chain reaction (PCR) microchip, polydimethylsiloxane (PDMS), SMAD4.
Article
We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem–loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem–loop DNA and a new stem–loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 109 copies of target in less than an hour. The final products are stem–loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.
Article
A thermostat chip of indium-tin oxide glass substrate for static chip polymerase chain reaction (PCR) is, for the first time, introduced in this paper. The transparent conductive layer was used as an electro-heating element. Pulse width modulation and fuzzy proportional integration-differentiation algorithm were adopted in the temperature programming of the chip. The temperature distribution was investigated, and a dynamic control precision within +/-2 degrees C was achieved. The highest ramping rates were 37 degrees Cs(-1) for heating and 8 degrees Cs(-1) for cooling with an electric fan. The PCR reaction vials were constructed with polyethylene tubes or poly(dimethylsiloxane) directly on the thermostat chip; the chip had a typical size of 25 mm x 25 mm and a thickness of 1.1mm. Static chip PCR was successfully demonstrated either in a single vial or in an up to 8-parallel array vials. In situ real time fluorescence monitoring during PCR of a lambda DNA fragments (236bp) with SYBR Green I was demonstrated using a blue light emission diode as a light source and a photomultiplier as a detector. The method proposed here is characterized by open access, easy fabrication and low cost. This work could be the basis for developing a portable real time PCR system with disposable chips for point of care tests.
  • J Fischbach
  • N C Xander
  • M Frohme
  • J F Glökler
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