Continuous-flow Polymerase Chain Reaction Research Articles

Spatial continuous-flow polymerase chain reaction structure controlled by single-temperature driver

A spatial continuous-flow polymerase chain reaction microfluidic chip was designed with the traditional plane channel replaced by a vertical-cavity channel by a single-temperature driver. To optimize the design, the temperature distribution in the microchannel of this chip was simulated by the finite-element method with various structural parameters and fluid velocities. The results show that the fluid produces three temperature zones of 94–95, 54–56, and 71–73 °C when the chip size is 54 mm (x) × 78 mm (y) × 47 mm (z), the wall thickness is 2 mm (x) × 20 mm (y) × 2 mm (z), and the pipe diameter is 1 mm. To increase the polymerase chain reaction amplification efficiency, we considered fluid velocities in the range of 0.0005–0.002 m/s. The optimal temperature for polymerase chain reaction amplification occurs at a fluid velocity of 0.0005 m/s.

Detection of Periodontal Pathogens Based on an Integrated Continuous Flow PCR and Capillary Electrophoresis Microfluidic Chip

Continuous-flow PCR (CF-PCR) can realize rapid DNA amplification because of the high temperature variation rate. However, off-line detection methods for PCR may induce cross contamination. To overcome this problem, we herein fabricated an integrated CF-PCR and electrophoresis microfluidic chip. The optimal voltage applied in the electrophoresis part of the microfluidic chip was achieved by simulation in COMSOL. Coating the inside wall of the microchannel can inhibit electroosmotic flow and improve the resolution for DNA fragments. The temperature distribution of the serpentine part can meet the PCR and has no obvious suppressive effect on sample separation. Finally, we have performed the amplification of target genes for Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola and detected the corresponding PCR products in the microfluidic chip within 11 min. Such work provides a new method for the rapid detection of bacteria.

Performance Analysis of a Phase Changing Material Based Thermocycler for Nucleic Acid Amplification

Abstract Modeling, simulation, and thermal performance analysis of a thermocycler for the continuous-flow polymerase chain reaction (CF-PCR), with a phase changing material (PCM)-laden annealing flow path, is presented. The incessant threat of microorganisms such as viruses, bacteria, and fungi has fostered effective, quick, and miniature detection devices in order to curtail the wide-spreading of infections. Microfluidics-based CF-PCR systems are compact and ideal for faster response. The thermal cycling process involves a sequential exposure of a given liquid sample to various temperature conditions when it is taken through the continuous-flow path. As a result, a prescribed periodic change of temperature suitable for deoxyribonucleic acid (DNA) amplification is achieved. A rapid temperature reduction and maintenance of isothermal conditions to facilitate the annealing phase of CF-PCR process by a PCM-assisted cooling is envisaged in the present study. Unsteady, two-dimensional, incompressible fluid flow, and internal convection heat transfer in a microchannel annealing path with melting of tetracosane (C24H50) boundary has been simulated using semi-implicit method for pressure linked equations-consistent (SIMPLEC) algorithm based finite volume solver. Solver validation is carried out against the experimental data on internal convection heat transfer in a rectangular microchannel. A detailed numerical study has been performed to assess the spatiotemporal heat transfer characteristics of internal convection in the microfluidic path when the flow triggers the melting of encapsulated PCM. A minimum sample flowrate with PCM encapsulation of less than 600 μm is found to be ideal for achieving desired thermal performance. The present study evidences the swift temperature reduction and management of isothermal conditions congenial for the annealing process in the CF-PCR system for various sample flowrates and PCM masses. The study offers valuable design input for the development of a microfluidic thermocycler for CF-PCR applications.

Lower fluidic resistance of double-layer droplet continuous flow PCR microfluidic chip for rapid detection of bacteria

BackgroundRapid diagnosis of harmful microorganisms demonstrated its great importance for social health. Continuous flow PCR (CF-PCR) can realize rapid amplification of target genes by placing the microfluidic chip on heaters with different temperature. However, bubbles and evaporation always arise from heating, which makes the amplification not stable. Water-in-oil droplets running in CF-PCR microfluidic chip with uniform height takes long time because of the high resistance induced by long meandering microchannel. To overcome those drawbacks, we proposed a double-layer droplet CF-PCR microfluidic chip to reduce the fluidic resistance, and meanwhile nanoliter droplets were generated to minimize the bubbles and evaporation. ResultsExperiments showed that (1) fluidic resistance could be reduced with the increase of the height of the serpentine microchannel if the height of the T-junction part was certain. (2) Running speed, the size and the number of generated droplets were positively correlated with the cross-sectional area of the T-junction and water pressure. (3) Droplet fusion happened at higher water pressure if other experimental conditions were the same. (4) 0.032 nL droplet was created if the cross-sectional area of T-junction and water pressure were 1600 μm2 (40 × 40 μm) and 7 kPa, respectively. Finally, we successfully amplified the target genes of Porphyromonas gingivalis within 11′16″ and observed the fluorescence from droplets. Significance and noveltySuch a microfluidic chip can effectively reduce the high resistance induced by long meandering microchannel, and greatly save time required for droplets CF-PCR. It offers a new way for the rapid detection of bacterial.

Simultaneous amplification of DNA in a multiplex circular array shaped continuous flow PCR microfluidic chip for on-site detection of bacterial.

Based on time to place conversion, continuous flow polymerase chain reaction (CF-PCR) can realize a rapid amplification of DNA by running the PCR reagent in a serpentine microchannel but a larger space is required for each sample, which greatly reduces the efficiency of the CF-PCR. Herein, we propose a multiplex circular array shaped CF-PCR microfluidic chip for on-site detection of bacteria. There were 12 serpentine microchannels which were distributed on the disc in an annular form, and each microchannel consisted of an inlet for sample injection, and an outlet for the detection of the PCR products based on fluorescence. Samples could be simultaneously driven into each inlet by a one-to-twelve diverter through a syringe. Moreover, the method of adding fluorescent dyes at the end of the microchannel can solve the inhibition effect of excessive fluorescent dyes on the PCR reaction. The process finished with simultaneous amplification of 12 different target genes from Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Escherichia coli, and on-site detection of their corresponding positives within 23 min. The fastest detectable PCR reaction time was 5.38 ± 0.2 min at a flow rate of 1 mL h-1. For E. coli, the minimum detectable concentration was 2.5 × 10-3 ng μL-1 in this microfluidic system. Such a system can increase the throughput of CF-PCR for point-of-care testing of pathogens.

(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.

Multi-objective optimisation of polymerase chain reaction continuous flow systems

A surrogate-enabled multi-objective optimisation methodology for a continuous flow Polymerase Chain Reaction (CFPCR) systems is presented, which enables the effect of the applied PCR protocol and the channel width in the extension zone on four practical objectives of interest, to be explored. High fidelity, conjugate heat transfer (CHT) simulations are combined with Machine Learning to create accurate surrogate models of DNA amplification efficiency, total residence time, total substrate volume and pressure drop throughout the design space for a practical CFPCR device with sigmoid-shape microfluidic channels. A series of single objective optimisations are carried out which demonstrate that DNA concentration, pressure drop, total residence time and total substrate volume within a single unitcell can be improved by up to sim5.7%, sim80.5%, sim17.8% and sim43.2% respectively, for the practical cases considered. The methodology is then extended to a multi-objective problem, where a scientifically-rigorous procedure is needed to allow designers to strike appropriate compromises between the competing objectives. A series of multi-objective optimisation results are presented in the form of a Pareto surface, which show for example how manufacturing and operating cost reductions from device miniaturisation and reduced power consumption can be achieved with minimal impact on DNA amplification efficiency. DNA amplification has been found to be strongly related to the residence time in the extension zone, but not related to the residence times in denaturation and annealing zones.

A continuous flow PCR array microfluidic chip applied for simultaneous amplification of target genes of periodontal pathogens.

The concept of time to place conversion makes using a continuous flow polymerase chain reaction (CF-PCR) microfluidic chip an ideal way to reduce the time required for amplification of target genes; however, it also brings about low throughput amplicons. Although multiplex PCR can simultaneously amplify more than one target gene in the chip, it may easily induce false positives because of cross-reactions. To circumvent this problem, we herein fabricated a microfluidic system based on a CF-PCR array microfluidic chip. By dividing the chip into three parts, we successfully amplified target genes of Porphyromonas gingivalis (P.g), Tannerella forsythia (T.f) and Treponema denticola (T.d). The results demonstrated that the minimum amplification time required for P.g, T.d and T.f was 2'07'', 2'51'' and 5'32'', respectively. The target genes of P.g, T.d and T.f can be simultaneously amplified in less than 8'05''. Such a work may provide a clue to the development of a high throughput CF-PCR microfluidic system, which is crucial for point of care testing for simultaneous detection of various pathogens.

A sample-to-answer DNA detection microfluidic system integrating sample pretreatment and smartphone-readable gradient plasmonic photothermal continuous-flow PCR.

As the gold standard for nucleic acid detection, full-process polymerase chain reaction (PCR) analysis often falls into the dilemma of complex workflow, time-consuming, and high equipment costs. Therefore, we designed and optimized a DNA quantification microfluidic system by strategically integrating sample pretreatment and a smartphone-readable gradient plasmonic photothermal (GPPT) continuous-flow PCR (CF-PCR). Through preloading and sequential injection of immiscible extraction reagents, combined with magnetic bead (MB) manipulation, the microfluidic chip successfully purified and concentrated 100 μL of HBV-DNA spiked plasma into a 20-μL purified sample within 14 minutes. With a digital PCR platform, the optimized experiments showed that the DNA extraction efficiency can reach 69% at an immiscible reagent configuration ratio of 10 : 10 : 1 : 12 : 2 (sample : lysis/binding buffer : MB : silicone oil : eluent) and a flow rate of 25 μL min-1. For the first time, we used gold nanorod (AuNR)-doped PDMS to prepare a CF-PCR submodule for the amplification of a 40 μL PCR mixture. Due to the plasmonic photothermal effect of AuNRs and the gradient intensity of an expanded laser spot, the PCR thermal gradient was formed on a coin-sized area. The compact annular thermal-microfluidic layout, optimized DNA dye concentration, and chip transmittance synergistically enable a rarely reported smartphone-based fluorescence CF-PCR, greatly simplifying thermal control and detection setup. Prototype construction and validation experiments show that the microsystem can complete the sample-to-answer quantification of HBV-DNA with a dynamic linear range from 1.2 × 101 to 1.2 × 106 copies per μL in ∼37 minutes. This novel microfluidic solution effectively bridges the technical gap between the CF-PCR, sample pretreatment and result characterization, making the workflow standardized and rapid and requiring <15% of the commercial instrument cost. The simplicity, rapidity and low cost of this work make it promising for applications in decentralized laboratories and low-resource settings.

Miniaturized DNA amplification platform with soft-lithographically fabricated continuous-flow PCR microfluidic device on a portable temperature controller

Miniaturized DNA amplification platform with soft-lithographically fabricated continuous-flow PCR microfluidic device on a portable temperature controller

Multiplex amplification of target genes of periodontal pathogens in continuous flow PCR microfluidic chip.

Porphyromonas gingivalis (P.g), Treponema denticola (T.d), and Tannerella forsythia (T.f) are believed to be the major periodontal pathogens that cause gingivitis, which affects 50-90% of adults worldwide. Microfluidic chips based on continuous flow PCR (CF-PCR) are an ideal alternative to a traditional thermal cycler, because it can effectively reduce the time needed for temperature transformation. Herein, we explored multi-PCR of P.g, T.d and T.f using a CF-PCR microfluidic chip for the first time. Through a series of experiments, we obtained two optimal combinations of primers that are suitable for performing multi-PCR on these three periodontal pathogens, with amplicon sizes of (197 bp, 316 bp, 226 bp) and (197 bp, 316 bp, 641 bp), respectively. The results also demonstrated that by using multi-PCR, the amplification time can be reduced to as short as 3'48'' for the short-sized amplicons, while for T.f (641 bp), the minimum time required was 8'25''. This work provides an effective way to simultaneously amplify the target genes of P.g, T.d and T.f within a short time, and may promote CF-PCR as a practical tool for point-of-care testing of gingivitis.

Optimisation of microfluidic polymerase chain reaction devices

The invention and development of Polymerase Chain Reaction (PCR) technology have revolutionised molecular biology and molecular diagnostics. There is an urgent need to optimise the performance of these devices while reducing the total construction and operation costs. This study proposes a CFD-enabled optimisation methodology for continuous flow (CF) PCR devices with serpentine-channel structure, which enables the optimisation of DNA amplification efficiency and pressure drop to be explored while varying the width (W) and height (H) of the microfluidic (μ) channel. This is achieved by using a surrogate-enabled optimisation approach accounting for the geometrical features of a μCFPCR device by performing a series of simulations using COMSOL Multiphysics 5.4®. The values of the objectives are extracted from the CFD solutions, and the response surfaces are created using polyharmonic splines. Genetic algorithms are then used to locate the optimum design parameters. The results indicate that there is the possibility of improving the DNA concentration and the pressure drop in a PCR cycle by ~2.1 % ([W, H] = [400 μm, 50 μm]) and ~95.2 % ([W, H] = [400 μm, 80 μm]) respectively, by modifying its geometry.

Compressed Air-Driven Continuous-Flow Thermocycled Digital PCR for HBV Diagnosis in Clinical-Level Serum Sample Based on Single Hot Plate.

We report a novel compressed air-driven continuous-flow digital PCR (dPCR) system based on a 3D microfluidic chip and self-developed software system to realize real-time monitoring. The system can ensure the steady transmission of droplets in long tubing without an external power source and generate stable droplets of suitable size for dPCR by two needles and a narrowed Teflon tube. The stable thermal cycle required by dPCR can be achieved by using only one constant temperature heater. In addition, our system has realized the real-time detection of droplet fluorescence in each thermal cycle, which makes up for the drawbacks of the end-point detection method used in traditional continuous-flow dPCR. This continuous-flow digital PCR by the compressed air-driven method can meet the requirements of droplet thermal cycle and diagnosis in a clinical-level serum sample. Comparing the detection results of clinical samples (hepatitis B virus serum) with commercial instruments (CFX Connect; Bio Rad, Hercules, CA, USA), the linear correlation reached 0.9995. Because the system greatly simplified the traditional dPCR process, this system is stable and user-friendly.

Efficient Bond of PDMS and Printed Circuit Board with An Application on Continuous-flow Polymerase Chain Reaction

A simple and efficient bond technique for integrating polydimethylsiloxane (PDMS) and printed circuit board (PCB) is presented in this paper by using half-cured PDMS technique. We take advantage of the mature PCB technology for fabrication of microchannel mold and heating electrodes leading to a cost-effective and mass manufacturing method. PDMS is applied for fabricating microfluidic chip by replica molding while a seamless and reliable bond is formed between cured PDMS chip and half-cured PDMS film on PCB substrate. To demonstrate the reliability of the system, the bond strengths of the PDMS film to PCB substrate as well as the PDMS chip to PDMS film are investigated. An application of a continuous-flow PCR (CF-PCR) chip is also fabricated based on the bond technique. Successful DNA amplification is obtained and compared to the conventional PCR reaction. The proposed bond method is a promising attempt for the integration between polymer and PCB which can be critical for the microfluidic applications requiring low cost, high efficiency and portability.

A digital PCR system based on the thermal cycled chip with multi helix winding capillary

This paper presents a digital PCR system based on a novel thermal cycled chip, which wraps microchannels on a trapezoidal structure made of polydimethylsiloxane (PDMS) in a multi-helix manner for the first time. It is found that compared to the single helix chip commonly used in previous reports, this kind of novel multi-helix chip can make the surface temperature in the renaturation zone more uniform, and even in the case of rapid fluid flow, it can improve the efficiency of the polymerase chain reaction. What’s more, the winding method of multi helix (such as double helix, six helix and eight helix) can obtain better temperature uniformity than the winding of odd helix (such as single helix and three helix). As a proof of concept, the temperature-optimized double-helical chip structure is applied to continuous-flow digital PCR and there is no need to add any surfactant to both the oil phase and reagent. In addition, we successfully analyzed the fluorescence signal of continuous-flow digital PCR by using CMOS camera. Finally, this method is applied for the absolute quantification of the clinical serum sample infected by HBV. The accuracy of the test results has been confirmed by commercial instruments.

Fully automatic integrated continuous-flow digital PCR device for absolute DNA quantification

Existing digital polymerase chain reaction (PCR) devices consist of multiple independent devices, such as a droplet generator, a PCR thermocycler, and a droplet reader, which have the disadvantages of low integration, complex equipment structure, and high operation difficulty. This paper proposes a fully automatic integrated digital PCR device based on continuous-flow digital PCR theory. By simply adding the sample to the entrance of the integrated instrument, a series of procedures required for digital PCR detection can be fully automated, including sample injection, droplet generation, PCR thermal cycling, fluorescence acquisition, and signal analysis. In contrast to traditional techniques, sample testing requires only one integrated device rather than three separate instruments. For full automation, we design complete control and data processing software, which can complete a test by one-step operation. Therefore, the disadvantages of traditional instruments, such as multi-step operation, and hence, potential environmental pollution, are avoided. Moreover, the system can be powered by solar cells and does not require an external power supply.As a proof of concept, the proposed device is used for absolute quantitative detection of the hepatitis B virus in serum samples. The capacity of the system is validated by absolute quantification of three orders of magnitude from 103 to 105 IU/mL. The results have a good linear correlation (0.9986) with those of the traditional quantitative (qPCR), thus confirming the reliability of the instrument. In summary, we believe that our work can promote the development of integrated digital PCR systems.

Numerical method for optimization of thermal cycling in 3D-printed polymerase chain reaction device

A continuous-flow polymerase chain reaction (PCR) device with integrated heaters for DNA amplification was studied. We developed a numerical model to predict the temperature variations of a 3D-printed PCR device for different heater temperatures. The target temperatures at thermal zones for a successful PCR are 95 °C, 55 °C, and 72 °C for denaturation, annealing and extension, respectively. A numerical simulation was conducted to determine the heater temperatures required to compensate for heat loss. Our numerical model and optimization method were confirmed by comparison with the experimental results. Consequently, the compensated heater temperatures required to achieve the target temperatures are 97.9 °C, 56.6 °C and 74.1 °C. The proposed optimization method and numerical simulations will be useful in achieving highly effective thermal cycling in the 3D-printed PCR device.

Heat and pressure-resistant room temperature irreversible sealing of hybrid PDMS-thermoplastic microfluidic devices via carbon-nitrogen covalent bonding and its application in a continuous-flow polymerase chain reaction.

In this study, we have introduced a facile room-temperature strategy for irreversibly sealing polydimethylsiloxane (PDMS) elastomers to various thermoplastics using (3-aminopropyl)triethoxysilane (APTES) and [2-(3,4-epoxycyclohexyl)ethyl]trimethoxysilane (ECTMS), which can resist heat and pressure after sealing due to the high chemical reactivity of the used chemicals. An irreversible chemical bond was realized at RT within 30 min through the initial activation of PDMS and thermoplastics using oxygen plasma, followed by surface modification using amino- and epoxy-based silane coupling reagents on either side of the substrates and then conformally contacting each other. Surface characterizations were performed using contact angle measurements, fluorescence measurements, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) to verify the successful surface modification of PDMS and thermoplastics. The tensile strengths of the bonded devices were 274.5 ± 27 (PDMS–PMMA), 591.7 ± 44 (PDMS–PS), 594.7 ± 25 (PDMS–PC), and 510 ± 47 kPa (PDMS–PET), suggesting the high stability of interfacial bonding. In addition, the results of the leakage test revealed that there was no leakage in the indigenously fabricated hybrid devices, even at high pressures, which is indicative of the robust bond strength between PDMS and thermoplastics obtained through the use of the chemical bonding method. Moreover, for the first time, the heat and pressure-resistant nature of the bonded PDMS–PC microfluidic device was assessed by performing a continuous-flow polymerase chain reaction (CF-PCR), which requires a high temperature and typically generates a high pressure inside the microchannel. The results demonstrated that the microfluidic device endured high heat and pressure during CF-PCR and successfully amplified the 210 bp gene fragment from the Shiga-toxin gene region of Escherichia coli (E. coli) O157:H7 within 30 min.

Design and fabrication of portable continuous flow PCR microfluidic chip for DNA replication.

The reasons for restricting continuous flow polymerase chain reaction (CF-PCR) microfluidic chip from lab to application are that it is not portable and requires costly external precision pumps for sample injection. Herein, we employed water as the substitute for PCR solution, and investigated the effect of the cross-section, width-to-depth ratio, and the length ratio for three temperature zones of the micro channel on the thermal and flow distribution of fluid in micro tube by finite element analysis. Results show that the central velocity is uniform and stable velocity occupies the most if the cross-section is rectangular. The deviation between predefined temperature and theoretical temperature is slight and the fluid flux is the most if width-to-depth ratio is 1:1. It is suitable for the short DNA replication if the high temperature zone Wh is larger than the low temperature zone Wl, and vice versa. Then a portable CF-PCR microfluidic chip was fabricated and an automatic sample injection system was developed. As an application, we have successfully amplified the DNA of Treponema denticola in the chip within 8min. Such a study may offer new insight into the design of CF-PCR microfluidic chip and promote it from lab-scale research to full-scale application.