Microfluidic Polymerase Chain Reaction Research Articles

(Invited) COVID-19 As a Technology Accelerator for High-Speed Polymerase Chain Reaction (PCR)

Limit of detection (LOD), speed and cost for some of the most important diagnostics tools for COVID-19 i.e., lateral flow assays (LFA), enzyme-linked immunosorbent assay (ELISA) and Polymerase Chain Reaction (PCR) all benefitted from both the financial and regulatory support brought about by the pandemic. Before COVID-19, LFA was the fastest and least expensive but came with the poorest limit of detection (LOD). The slowest, most expensive but with the best (lowest) LOD was PCR. In between those two extremes, in terms of cost, LOD and speed, was an ELISA . Today, through the “COVID-19 accelerator effect” there are point of care (POC) reverse transcriptase quantitative PCR (ie., RT-qPCR) COVID-19 tests on the market that provide sample to answer in under 20 minutes, while retaining the same very low LOD but at a lower cost [1]. There are even POC PCR tests under development that come in at less than 10 minutes: beating both LFA and ELISAs in terms of time and LOD [2]. Although RT-qPCR has gained the most in overall performance, signal-amplified POC LFAs are possible now with an LOD that rivals that of an ELISA.However, implementing the next generation PCR equipment in point-of-care settings, remains very challenging because of difficulties in reaching a 10 minute or below sample to answer time , instrument complexity and cost. In this work, we discuss the engineering challenges involved in the development of high-speed microfluidic PCR equipment. To guide this review/comparison of PCR advances we introduce three parameters. The first one is the overall sample to answer time (t), the second is called lambda (λ) and is a measure of the degree to which stochastic effects interfere with obtaining analytical results and thereby setting the minimal number of copies required per volume. The third parameter gamma (γ), introduced first by Dong et al [3], is the ratio of thermal cycling time (in min) to the heat cycled sample volume (in mL). It describes the trade-off between the speed of the PCR system and the desired level of sensitivity. It can be interpreted as a system efficiency: the most efficient system heat cycles the largest amount of sample in the smallest amount of time.With these three criteria in mind we set out to suggest designs for an optimized POC reverse transcriptase (RT) quantitative qPCR or RT-qPCR system. Applications of RT-qPCR include quantifying gene expression levels, validating RNA interference (RNAi), and detecting pathogens such as viruses. The aim of this specific POC RT-qPCR system is to provide sample to answer in ten minutes or less for a respiratory pathogens panel, with an LOD of 500 copies/mL and the capability to multiplex (8 chambers) while remaining portable and low cost.Finally we introduce two of the most daunting, remaining, microfluidics challenges that both need to be tackled to implement an ideal microfluidic platform like the POC RT-qPCR system described above. One is liquid storage and the other is proportional valves on a disposable microfluidic platform. Liquid storage is essential when the assay requires repeated washes, if these washes can come from one and the same reservoir via a proportional valve, real estate is gained and system complexity is reduced. The storage issue is further complicated when the design also calls for storing lyophilized or dried reagents on the same disposable. We compare some of the most recent approaches to liquid storage and proportional valving that we believe will enable the fast POC diagnostic platforms of the future. 1. Tsang, Hin Fung, Wai Ming Stanley Leung, Lawrence Wing Chi Chan, William Chi Shing Cho, and Sze Chuen Cesar Wong. “Performance Comparison of the Cobas® Liat® and Cepheid® GeneXpert® Systems on SARS-CoV-2 Detection in Nasopharyngeal Swab and Posterior Oropharyngeal Saliva.” Expert Review of Molecular Diagnostics 21, no. 5 (May 4, 2021): 515–18. 2. BUCKLAND, Justin, Tom JELLICOE, Alex STOKOE, and Amaru ARAYA-WILLIAMS. “VARIABLE TEMPERATURE REACTOR, HEATER AND CONTROL CIRCUIT FOR THE SAME.” Patent Application, January 30, 2020. 3. Dong, Xiaobin, Luyao Liu, Yunping Tu, Jing Zhang, Guijun Miao, Lulu Zhang, Shengxiang Ge, Ningshao Xia, Duli Yu, and Xianbo Qiu. “Rapid PCR Powered by Microfluidics: A Quick Review under the Background of COVID-19 Pandemic.” Trends in Analytical Chemistry 143 (October 2021): 116377.

Fast Thermocycling in Custom Microfluidic Cartridge for Rapid Single-Molecule Droplet PCR.

The microfluidic droplet polymerase chain reaction (PCR), which enables simultaneous DNA amplification in numerous droplets, has led to the discovery of various applications that were previously deemed unattainable. Decades ago, it was demonstrated that the temperature holding periods at the denaturation and annealing stages in thermal cycles for PCR amplification could be essentially eliminated if a rapid change of temperature for an entire PCR mixture was achieved. Microfluidic devices facilitating the application of such fast thermocycling protocols have significantly reduced the time required for PCR. However, in microfluidic droplet PCR, ensuring successful amplification from single molecules within droplets has limited studies on accelerating assays through fast thermocycling. Our developed microfluidic cartridge, distinguished for its convenience in executing single-molecule droplet PCR with common laboratory equipment, features droplets positioned on a thin glass slide. We hypothesized that applying fast thermocycling to this cartridge would achieve single-molecule droplet PCR amplification. Indeed, the application of this fast protocol demonstrated successful amplification in just 22 min for 30 cycles (40 s/cycle). This breakthrough is noteworthy for its potential to expedite microfluidic droplet PCR assays, ensuring efficient single-molecule amplification within a remarkably short timeframe.

Designing Microfluidic PCR Chip Device Using CFD Software for the Detection of Malaria

Polymerase chain reaction (PCR) technique is one of the molecular methods in amplifying DNA for the detection of malaria. However, the collection and transportation of samples and the processing and dissemination of results via conventional PCR, especially when used for routine clinical practice, can hamper the technique’s sensitivity and specificity. The rampancy of such disease in the Philippines is aggravated by the limited supply of medical machinery and the poor economic state of the country; thus, the need to innovate a device for the early detection of malaria is necessary. With that, this study focuses on designing a microfluidic device that will mimic the function of a conventional genus-specific PCR based on the 18S rRNA gene to detect malaria parasites (Plasmodium falciparum) at low-grade parasitemia. The design was intended to be portable, accessible, and economical, which none from past literature has dealt with specifically for malaria detection. This in silico design is a first in the country specially crafted for such reasons. The proposed device was developed and simulated using ANSYS software for Computational Fluid Dynamics (CFD) analyses. The simulation shows that adding loops to the design increases its relative deviation but minimally compared to having only a straight path design. This indicates that looping is acceptable in designing a microfluidic device to minimize chip length. It was also found that increasing the cross-sectional area of the fluid path decreases the efficiency of the design. Lastly, among the three materials utilized, the chip made of polypropylene is the most efficient, with a relative deviation of 0.94 compared to polycarbonate and polydimethylsiloxane, which have relative deviations of 2.78 and 1.92, respectively. Future researchers may mesh the 44-cycle microfluidic chip due to the limitations of the software used in this study, and other materials, such as biocomposites, may be assessed to broaden the application of the design.

Rapid detection and diagnosis of herpetic keratitis using quantitative microfluidic polymerase chain reaction system for herpes simplex and varicella-zoster virus DNA: a case series

BackgroundA microfluidic real-time polymerase chain reaction (PCR) system can rapidly detect the viral DNA in specimens. Detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA in tears is a useful diagnostic tool for herpes simplex virus keratitis (HSK) and herpes zoster ophthalmicus (HZO).MethodsIn total, 20 patients were included in this cross-sectional study. Among them, 8 patients with infectious epithelial HSK and 12 patients with HZO were included in HSK and HZO groups, respectively. In addition, 8 patients with non-herpetic keratitis and 4 healthy individuals without keratitis were included in the control group. Numbers of HSV and VZV DNA copies in tears of all patients and individuals were evaluated using a microfluidic real-time PCR system. Regarding HSV/VZV DNA test, tear specimens were collected by filter paper method using Schirmer’s test paper, and subsequently, DNA was extracted from the filter paper using an automated nucleic acid extractor. Afterward, quantitative PCR was performed using a microfluidic real-time PCR system.ResultsFrom tear collection to real-time PCR result determination, the HSV/VZV DNA test took approximately 40 min. In the HSK group, the sensitivity and specificity of the HSV DNA tests were 100% each. The median value (range) of number of HSV DNA copies for affected eyes was 3.4 × 105 copies/μL (under a lower detection limit of 7.6). In the HZO group, the sensitivity and specificity of the VZV DNA tests were 100% each. The median value (range) of number of VZV DNA copies for affected eyes was 5.3 × 105 copies/μL (under a lower detection limit of 5.6 × 10–2).ConclusionIn conclusion, quantitative PCR for HSV and VZV DNA in tears using a microfluidic real-time PCR system is useful for diagnosing and monitoring HSK and HZO.

(Digital Presentation) An Electrochemical Sensor in a Disc for Real-Time PCR Assisted with Thermal Convective Flow

Nowadays, the polymerase chain reaction (PCR) has extensively adapted to microfluidic devices for several features such as the low consumption of reagents and sample, portability, high-throughput, and low fabrication costs [1]. The reduction of the amplification time has been a relevant parameter since the invention of the PCR, where rapid microfluidic PCR is especially preferred for the detection of infectious diseases [1,2]. The continuous-flow-based PCR is a type of microfluidic PCR that proposes the mobilization of the PCR mixture through a microchannel with different fixed temperature zones to achieve the required thermal-cycling [3]. This approach has smaller thermal inertia because only the PCR mixture needs to be heated and cooled instead of the entire microfluidic device. The continuous-flow approach allows rapid-thermal cycling with lower power consumption and, therefore, reduces the overall amplification time. The Lab-on-a-disc, or centrifugal microfluidic platform, has adapted the continuous-flow PCR by utilizing the flow generated by thermal convection in a ring-structured microchannel [4]. During spinning, embedded heaters in the bottom of the CD heat specific section of the microchannel (see Figure 1) at a fixed temperature and, subsequently, create a natural convection flow in the PCR mixture and move through the microchannel. The centrifugal-assisted thermal convection (CATC) technique not only enables the recirculation pathway for the continuous-flow PCR but overcomes the unidirectional nature of centrifugation force on the CD. Despite the advantage of rapid PCR on Lab-on-a-Disc, the CATC technique lacks the implementation of a detection method with high sensitivity and resolution, aiming for a fully integrated microfluidic platform. Electrochemical detection can provide fast responses with minimum power consumption for miniaturized devices. This work proposes the integration of an electrochemical sensor to the CATC technique in a circular microchannel for the real-time monitoring of DNA amplification. The electrochemical sensor consists of interdigitated gold electrodes located above the ring microchannel to quantify the amplicon concentration after each cycle mediated by the intercalation of Methylene Blue between the newly-generated DNA (see figure 1A). The characterization of the sensor is performed through voltammetric techniques such as cyclic voltammetry (CV) and linear sweep voltammetry (LSV), where the latter is utilized for amplicon quantification. To optimize the CATC technique combined with the electrochemical sensor, a COMSOL simulation is performed to improve the amplification ratio and amplification time in the CD by analyzing the microchannel geometry and flow rate (see figure 1B). Both the thermal control for the thermal-assisted microchannel and the electrochemical sensor are powered and controlled via the “electrified Lab-on-a-Disc” (eLoaD) board [5] (see figure 1C).[1] Y. Zhang, P. Ozdemir, Anal. Chim. Acta 638 (2009) 115–125.[2] X. Dong, L. Liu, Y. Tu, J. Zhang, G. Miao, L. Zhang, S. Ge, N. Xia, D. Yu, X. Qiu, TrAC - Trends Anal. Chem. 143 (2021) 116377.[3] M.B. Kulkarni, S. Goel, Eng. Res. Express 2 (2020).[4] M. Saito, K. Takahashi, Y. Kiriyama, W.V. Espulgar, H. Aso, T. Sekiya, Y. Tanaka, T. Sawazumi, S. Furui, E. Tamiya, Anal. Chem. 89 (2017) 12797–12804.[5] S.M. Torres Delgado, J.G. Korvink, D. Mager, Biosens. Bioelectron. 117 (2018) 464–473. Figure 1

A Portable Continuous-Flow Polymerase Chain Reaction Chip Device Integrated with Arduino Boards for Detecting Colla corii asini.

Food security is a significant issue in modern society. Because morphological characters are not reliable enough to distinguish authentic traditional Chinese medicines, it is essential to establish an effective and applicable method to identify them to protect people’s health. Due to the expensive cost of the manufacturing process and the large volume of the analytical system, the need to build a portable and cheap device is urgent. This work describes the development of a portable nucleic acid amplification device integrated with thermal control and liquid pumping connecting to Arduino boards. We present a novel microfluidic polymerase chain reaction (PCR) chip with symmetric isothermal zones. The total chip volume is small, and only one Arduino board is needed for thermal control. We assemble a miniaturized liquid pump and program an Arduino file to push the sample mixture into the chip to implement the PCR process. In the proposed operation, the Nusselt number of the sample flow is less than one, and the heat transfer is conduction only. Then we can ensure temperature uniformity in specific reaction regions. A Colla corii asini DNA segment of 200 bp is amplified to evaluate the PCR performance under the various operational parameters. The initial concentration for accomplishing the PCR process is at least 20 ng/μL at the flow rate of 0.4 μL/min in the portable continuous flow PCR (CFPCR) device. To our knowledge, our group is the first to introduce Arduino boards into the heat control and sample pumping modules for a CFPCR device.

Microfluidic Digital Quantitative PCR to Measure Internal Cargo of Individual Liposomes.

Lipid nanoparticles serve as drug delivery vehicles for biopharmaceutical products. The lipid membrane shields internal nucleic-acid drug cargo from enzymatic degradation and facilitates cellular uptake of the drug. However, existing methods to assess drug loading within liposomes are limited to averaged bulk measurements, which obscures heterogeneity of the biopharmaceutical formulation. This report describes the development of a single-liposome analysis method to measure copy numbers of DNA within liposomes and assess population heterogeneity. This novel measurement was achieved by integrating two orthogonal polymerase chain reaction (PCR) techniques─digital PCR (dPCR) and quantitative PCR (qPCR)─within a single microfluidic assay. The dPCR dimension quantified liposomes to validate their capture in the single-liposome analysis regime. The qPCR dimension quantified DNA copy numbers packaged within each liposome. The ability of digital quantitative PCR (dqPCR) to analyze large numbers of individual liposomes in parallel revealed significant population heterogeneity, which could not be obtained from standard bulk analysis methods. Our innovative measurement of internal DNA cargo from single liposomes has the potential to inform liposome synthesis procedures and create more uniform liposomal biopharmaceutical formulations to enhance drug safety and efficacy.

Miniaturized and IoT Enabled Continuous-Flow-Based Microfluidic PCR Device for DNA Amplification.

Herein, a continuous-flow driven microfluidic device has been designed and fabricated using the CO2 laser ablation method for polymerase chain reaction (PCR). The device consists of a polymethyl methacrylate (PMMA) microfluidic channel with 30 serpentine thermal cycles, an arduino board, two custom-made cartridge heaters, and thermocouple sensors. The portable thermal management system, with aluminium blocks placed on a wooden substrate, working on the PID controller principle, is low-cost, battery-powered, automated, integrated, and IoT-enabled. The device with dimensions 80×72×36 mm3 (L × W × H) has a temperature accuracy of ±0.2 °C. The IoT module enables accessing and storage of real-time temperature values directly onto the smartphone through ThingSpeak analytics. It was developed to achieve desirable accurate temperature at two thermal zones, denaturation and annealing (95 °C and 60 °C) on the microfluidic thermal management platform. A PCR mixture of [Formula: see text] was infused into the serpentine-based microchannel using a syringe pump. Amplification of DNA template with 594-base pair (bp) fragment of the rat GAPDH gene was successfully performed on the miniaturized thermal management system. The total time required for a complete PCR reaction was 32 min at an optimum flow rate of [Formula: see text]/min. The amplified sample of the target DNA obtained from the PCR microchannel was then separated by agarose gel electrophoresis and was further analyzed using a gel-doc system. Finally, the obtained results were compared to the conventional PCR instrument showing excellent performance.

Continuous polymerase chain reaction microfluidics integrated with a gold-capped nanoslit sensing chip for Epstein-Barr virus detection

We present the first combination of a microfluidic polymerase chain reaction (PCR) with a gold nanoslit-based surface plasmon resonance (SPR) sensor for detecting the DNA sequence of latent membrane protein 1 (LMP1). The PCR microchannel was produced through a laser scribing technique, and the SPR nanoslit chip was manufactured via hot-embossing nanoimprinting lithography. Afterward, the LMP1 DNA probe was adsorbed onto the SPR chip of the integrated device through electrostatic interactions for further detection. The device can complete the analytical procedure in around 36 min, while the traditional machine requires 105 min to achieve similar signals under the same PCR thermal cycles. The calibration curve with serially diluted LMP1 DNA exhibited the accuracy (R2 > 0.99) and sensitivity (limit of detection: ∼10−11 g/mL) of the device. Moreover, extracted DNA from Epstein-Barr virus (EBV)-positive cells were directly detected through the integrated chip. In brief, this all-in-one chip can amplify gene fragments at the front-end and detect them at the back-end, decreasing the time required for the analysis without compromising accuracy or sensitivity. We believe this label-free, real-time, low-cost device has enormous potential for rapid detection of various viruses, such as EBV and COVID-19.

Ultrafast and Real-Time Nanoplasmonic On-Chip Polymerase Chain Reaction for Rapid and Quantitative Molecular Diagnostics.

Advent and fast spread of pandemic diseases draw worldwide attention to rapid, prompt, and accurate molecular diagnostics with technical development of ultrafast polymerase chain reaction (PCR). Microfluidic on-chip PCR platforms provide highly efficient and small-volume bioassay for point-of-care diagnostic applications. Here we report ultrafast, real-time, and on-chip nanoplasmonic PCR for rapid and quantitative molecular diagnostics at point-of-care level. The plasmofluidic PCR chip comprises glass nanopillar arrays with Au nanoislands and gas-permeable microfluidic channels, which contain reaction microchamber arrays, a precharged vacuum cell, and a vapor barrier. The on-chip configuration allows both spontaneous sample loading and microbubble-free PCR reaction during which the plasmonic nanopillar arrays result in ultrafast photothermal cycling. After rapid sample loading less than 3 min, two-step PCR results for 40 cycles show rapid amplification in 264 s for lambda-DNA, and 306 s for plasmids expressing SARS-CoV-2 envelope protein. In addition, the in situ cyclic real-time quantification of amplicons clearly demonstrates the amplification efficiencies of more than 91%. This PCR platform can provide rapid point-of-care molecular diagnostics in helping slow the fast-spreading pandemic.

On-Chip PCR Based Plasmonic Microfluidic Platform: Ultrafast Point-of-Care Diagnostics of SARS-CoV-2

It is critically important to have rapid screening and identification of contagious viral diseases such as the current COVID-19 pandemic that is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Rapid and accurate diagnostic is essential for preventing worldwide spread of virus and ensuring in-time care for patients during the fast spread of pandemic diseases. Nanobiotechnology enabled tools have allowed to develop advanced polymerase chain reaction (PCR) based diagnostics of contagious viral diseases. To this end, microfluidic on-chip PCR platforms have shown huge promise for highly efficient, rapid and small-volume bioassay for point-of-care (POC) diagnostic applications in mitigating the challenges of SARS-CoV-2. Here, we discuss latest advances in ultrafast, real-time, and on-chip nanoplasmonic PCR for rapid and quantitative molecular diagnostics at POC level.

Miniaturized Thermal Monitoring Module With CO₂ Laser Ablated Microfluidic Device for Electrochemically Validated DNA Amplification

Polymerase chain reaction (PCR) is the gold standard method to amplify deoxyribonucleic acid (DNA) fragments. Several methods have been used to miniaturize and integrate PCR to execute in a microfluidic platform. A continuous-flow-based microfluidic PCR device permits the required rapid thermal cycling and multiplexing, which are hard to realize in a benchtop PCR or microchamber PCR. This work aims to develop a microfluidic PCR platform using a custom-made Internet of Things (IoT)-enabled portable thermal monitoring module. The microfluidic device was fabricated on polymethyl methacrylate (PMMA) using a CO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> laser. Apart from 30 serpentine cycles, the PCR microchannel also had initial denaturation and final elongation regions to enhance the overall amplification. As a proof-of-application, rat GAPDH gene, with a fragment size of 594 base pair, has been amplified on the miniaturized platform. Furthermore, the agarose gel electrophoresis technique was used to separate the DNA molecules validated using a gel-doc system. Subsequently, the acquired results were quantitatively compared with the conventional thermocycler using ImageJ software. Furthermore, this was also validated with electrochemical DNA detection using differential pulse voltammetry (DPV). The results from the smart low-power device showed promising performance for the nucleic acid amplification process while reducing the overall amplification time and achieving benchmarked efficiency.

Design and storage stability of reference materials for microfluidic quantitative PCR-based equine gene doping tests.

One method of gene doping in horseracing is administering of exogenous genetic materials, known as transgenes. Several polymerase chain reaction (PCR)-based methods have been developed for detecting transgenes with high sensitivity and specificity. However, novel designs for reference materials (RMs) and/or positive template controls (PTCs) are necessary for simultaneous analysis of multiple transgene targets. In this study, we designed and developed a novel RM for simultaneously detecting multiple targets via microfluidic quantitative PCR (MFQPCR). Twelve equine genes were selected as targets in this study. A sequence region including primers and probes for quantitative PCR was designed, and a 10 bp sequence was inserted to allow the RM to be distinguished from the original transgene sequences. The sequences of individual detection sites were then connected for 12 genes and cloned into a single plasmid vector. We performed fragment size analysis to distinguish between the PCR products of the original transgene sequence and those of the RM, enabling identification of RM contamination. PTCs diluted to 10,000, 1,000, 100, and 10 copies/µl with horse genomic DNA from RM were stably stored at 4°C for 1 year. As digital PCR enabled absolute quantification, the designed substances can serve as an RM. These findings indicate that the RM design and storage conditions were suitable for gene doping tests using MFQPCR.

Plasmonic and label-free real-time quantitative PCR for point-of-care diagnostics.

In response to the world's medical community's need for accurate and immediate infectious pathogen detection, many researchers have focused on adapting the standard molecular diagnostic method of polymerase chain reaction (PCR) for point-of-care (POC) applications. PCR technology is not without its shortcomings; current platforms can be bulky, slow, and power-intensive. Although there have been some advances in microfluidic PCR devices, a simple-to-operate and fabricate PCR device is still lacking. In the first part of this paper, we introduce a compact plasmonic PCR thermocycler in which fast DNA amplification is derived from efficient photothermal heating of a colloidal reaction mixture containing gold nanorods (AuNRs) using a small-scale vertical-cavity surface-emitting laser (VCSEL). Using this method, we demonstrate 30 cycle-assay time of sub-ten minutes for successful Chlamydia trachomatis DNA amplification in 20 μL total PCR sample volume. In the second part, we report an ultrasensitive real-time amplicon detection strategy which is based on cycle-by-cycle monitoring of 260 nm absorption of the PCR sample. This was accomplished by irradiating the PCR sample using a UV LED and collecting the transmitted optical power with a photodetector. The UV absorption dependency on the nucleotides' structural degree of freedom gives rise to distinctive features in the shape of UV amplification curves for the determination of PCR results, thus circumventing the need for the complicated design of target-specific probes or intercalating fluorophores. This amplicon quantification method has a high detection sensitivity of one DNA copy. This is the first demonstration of a compact plasmonic thermocycler combined with a real-time fluorophore-free quantitative amplicon detection system. The small footprint of our PCR device stems from hardware miniaturization, while abundant sample volume facilitates highly sensitive detection and fluid handling required for in-field sample analysis, thereby making it an excellent candidate for POC molecular diagnostics.

Simulation and Optimization of a Single Heater Convective PCR Chip and Its Controller for Fast Salmonella Enteritidis Detection

The design and optimization of a Rayleigh- Bénard convection based polymerase chain reaction (PCR) microchip is presented. This PCR microchip simplifies the fabrication, implementation and control of microfluidic PCR while enhancing the detection of the bacterial pathogen Salmonella Enteritidis. The device's dimensions were optimized using multiphysics simulation then it was fabricated using high precision milling of Poly(methyl methacrylate) (PMMA) substrate. The device incorporated a single heater at the bottom end of its cylindrical chamber and two sensors at both ends. The heater was controlled using a fuzzy proportional, integral, derivative (PID) controller to maintain denaturation and annealing temperatures necessary for efficient PCR to take place. Salmonella Enteritidis DNA was successfully detected using the device while reducing the required assay time by 66%.

A Miniaturized Device for Performing PCR, Integrated with an Electrochemical DNA Biosensor for Detection of Corynebacterium Diphtheriae

Introduction Corynebacterium diphtheriae by secreting harmful toxin causes lethal disease called diphtheria. It occurs mainly in underdeveloped areas of the world with low vaccination rate [1]. Rapid detection of this pathogen is crucial to prevent serious health complications of the patient and spreading of the epidemic. As an alternative to time-consuming phenotypic analysis, a device has been designed, which allows the fast detection of the pathogen based on biological interactions at the molecular level, which reduces the risk of error. This device consists of a microfluidic system for performing a fast polymerase chain reaction (PCR), which is integrated with a DNA biosensor. It enables fast and unambiguous detection of bacteria in the sample, therefore it can be potentially used as a point-of-care device. Microfluidic PCR device The concept of rapid bacterial analysis is based on the amplification of a DNA fragment specific for a pathogen followed by its fast detection with e.g. biosensor. A miniaturized system for carrying out PCR was developed. The geometry of the microchannels was designed using CAD software and then micromilled in poly(methyl methacrylate). The microsystem was connected with miniaturized peristaltic pump and with a valve to enable automatic introduction, circulation and removal of the solution from the system. Moreover, a set of six heaters was used to divide the microsystem into three temperature zones. One cycle of the solution in a microchip corresponded with one reaction cycle. Temperatures of three zones were measured and controlled by resistance temperature sensor and set to the suitable values for following reaction steps: denaturation, annealing and elongation. Using the microsystem, any number of reaction cycles could be carried out and peristaltic pump enabled the medium flow in wide range (4 - 150µL/min). A number of both standard and asymmetric PCR reactions were carried out in the microchip using various polymerases, of which the Phire polymerase gave the most promising results. The products were analyzed by electrophoretic techniques and compared with the results of reactions carried out in thermocyclers. The ultimate goal is direct detection of the reaction product using a DNA biosensor. DNA biosensor An electrochemical DNA biosensor has been developed as detection part of a device. Its receptor layer was based on a hairpin like DNA probe labeled with methylene blue (MB). After the asymmetric PCR reaction the hybridization process took place which results in formation of double stranded DNA. The changes in hairpin like structure influenced the registered current and the same the biosensor response, proportional to PCR product concentration [2]. Results and Conclusions The miniaturized device consisting of a microchip with designed channel geometry, connected to the pump and valve was constructed. It enabled to automatically control the flow of a solution in a microchip and to carry out any number of reaction cycles. The reaction mixture changed its temperature rapidly when it flowed between different zones of the microchip, which contributed to a significant reduction in the reaction time. Asymmetric PCR was successfully carried out using the designed device in just 15 minutes. The electrochemical genosensor enabled detection of ssDNA, and its electrochemical signal was directly proportional to the concentration of the analyte in 0.0125-0.25µmol/L range. Studies on the effects of different factors, such as probe sequence, temperature, magnesium ion concentration and addition of dimethyl sulfoxide on electrochemical signal of a biosensor have also been performed.The constructed microchip allowed to significantly shorten the PCR reaction time, when compared with the thermocycler. Integration of microPCR with the genosensor might results in construction of fully automatized portable device enabling rapid and reliable pathogen detection. This type of instrument could be used in medical diagnostics as the point-of-care device contributing to the rapid detection of the disease, hence preventing the spread of the epidemic [3].

Evaluation of the performance of Acuitas® Resistome Test and the Acuitas Lighthouse® software for the detection of β-lactamase-producing microorganisms

ObjectivesGiven the international spread of multidrug-resistant Gram-negative bacteria the need for prompt and precise characterization of underlying resistance traits has evolved into the cornerstone of infection control strategies. Novel commercial molecular tests enable rapid simultaneous testing for multiple resistance genes. We aimed to evaluate the performance of OpGen’s Acuitas® Resistome Test and the Acuitas Lighthouse® software. MethodsThe test is tailored towards detecting 46 β-lactamase genes (SHV and TEM variants associated with wild-type penicillin resistance, extended-spectrum β-lactamases [ESBLs], acquired AmpCs and carbapenemases) via a microfluidic polymerase chain reaction (PCR) array. In total 118 isolates part of the collection of the Bacteriology Laboratory of the Hellenic Pasteur Institute, specifically 96 enterobacterial isolates and 21 Acinetobacter baumannii, of divergent origins, with previously characterized β-lactamase content, were tested. ResultsIn the enterobacterial group all 69 carbapenemase genes of the KPC, VIM, NDM and OXA-48 types were correctly identified (sensitivity, specificity, positive predictive value [PPV] and negative predictive value [NPV] of 100%). Non-ESBL SHV enzymes, ESBLs (CTX-M, GES, VEB types) and acquired AmpC enzymes were also correctly characterized. Of the 35 SHV-ESBLs harboured, correct identification was possible in 32/35 isolates, with overall sensitivity, specificity, PPV and NPV for the Klebsiella pneumoniae group of 89.29%, 100%, 100% and 91.18%, respectively. For the A. baumannii group the test exhibited an overall sensitivity for carbapenemase detection of 96.55% and 100% PPV. ConclusionsThe OpGen Acuitas Resistome Test is an efficient molecular tool that can identify resistance threats in health care institutions with high diagnostic accuracy and be integrated into targeted surveillance protocols.

A lateral flow strip in combination with microfluidic device and polymerase chain amplification for rapid and visual detection of lymphatic filarial infection

Background: Lymphatic filariasis (LF) is a neglected tropical disease caused by the filarial nematode parasites which is transmitted by the bites of infective mosquitoes. The diagnosis of LF traditionally relies on the detection of circulating microfilariae (mf) using Giemsa-stained thick blood smears which has several limitations. Materials and methods: In the present study, we developed a lateral flow (LF) strip to be used in combination with a novel microfluidic device and polymerase chain reaction (PCR) for a rapid and visual detection of lymphatic filariae, Brugia malayi/ Wuchereria bancrofti infection in human blood samples. Results: The assay targets B. malayi HhaI gene and W. bancrofti Ssp gene. Prior to perform the assay, no DNA product clean up step required, thus, it can shorten time and reduce cost. The LF strip can detect as low as 10 pg of DNA product and no cross-reactivity with DNA of other parasites such as Gnathostoma spinigerum, cysticercisis soliumnor with DNA of other filariae i.e. Brugia pahangi, Dirofilaria immitis andD. repens. Conclusions: The developed LF strip shows high sensitivity and specificity, in combination with a novel microfluidic device and PCR, can be used as an alternative tool for the diagnosis of lymphatic filariasis.

Shunting microfluidic PCR device for rapid bacterial detection

Polymerase chain reaction (PCR) is commonly used for the analysis of nucleic acids in a variety of applications including clinical. There is, however, a need for a low cost portable PCR device that allows rapid identification of pathogenic bacteria. We report a shunting PCR microfluidic device comprising: polycarbonate microfluidic PCR chip; shunting thermal cycler and fluorescence detector. The microfluidic PCR chip – fabricated using micro-milling and thermal fusion bonding for sealing of the cover – was shunted between three double side temperature zones for thermal cycling. Rapid amplification was observed with heating and cooling rates of 1.8 °C/s and 2 °C/s respectively. Lock-in photodetector for fluorescence detection of the microfluidic PCR chip achieved at 95% confidence an LOD of 75pM FITC and 0.7 ng μl−1 of dsDNA using a QuantiFluor assay kit. The device was validated using universal primers - based on chromosomal DNA extracted from non-pathogenic K-12 subtype of Escherichia coli (E. coli) – for amplification of fragments of 250, 552 and 1500 bp. PCR amplification was demonstrated, with annealing temperatures ranging between 54 °C and 68 °C, and confirmed using gel electrophoresis. The developed shunting PCR microfluidic device will allow for low cost and portable nucleic acid amplification for the detection of infectious diseases.

Peanut Detection Using Droplet Microfluidic Polymerase Chain Reaction Device

In this study, we integrated genetic detection for polymerase chain reaction (PCR) with microfluidics technology for the detection of peanut DNA. A cross-junction microchannel was used to induce emulsion droplets of water in oil for PCR on a chip. Compared with the single-phase flow, the emulsion droplet flow exhibited a 7.24% lower evaporation amount and prevented air bubble generation. PCR results of the droplet microfluidic PCR chip for peanut DNA fragment detection was verified by comparison with a commercial PCR thermal cycler and increased fluorescence intensity in SYBR Green reagent-based PCR. Moreover, PCR on the microfluidic PCR chip was successful for sesame, Salmonella spp., and Staphylococcus aureus. The droplet microfluidic PCR device developed in this study can be applied for peanut detection in the context of food allergy.