Polymerase Chain Reaction Device Research Articles

SPEED: an integrated, smartphone-operated, handheld digital PCR Device for point-of-care testing

This study elaborates on the design, fabrication, and data analysis details of SPEED, a recently proposed smartphone-based digital polymerase chain reaction (dPCR) device. The dPCR chips incorporate partition diameters ranging from 50 μm to 5 μm, and these partitions are organized into six distinct blocks to facilitate image processing. Due to the superior thermal conductivity of Si and its potential for mass production, the dPCR chips were fabricated on a Si substrate. A temperature control system based on a high-power density Peltier element and a preheating/cooling PCR protocol user interface shortening the thermal cycle time. The optical design employs four 470 nm light-emitting diodes as light sources, with filters and mirrors effectively managing the light emitted during PCR. An algorithm is utilized for image processing and illumination nonuniformity correction including conversion to a monochromatic format, partition identification, skew correction, and the generation of an image correction mask. We validated the device using a range of deoxyribonucleic acid targets, demonstrating its potential applicability across multiple fields. Therefore, we provide guidance and verification of the design and testing of the recently proposed SPEED device.

Design of a Novel PID Controller Based on Machine Learning Algorithm for a Micro-Thermoelectric Cooler of the Polymerase Chain Reaction Device

Design of a Novel PID Controller Based on Machine Learning Algorithm for a Micro-Thermoelectric Cooler of the Polymerase Chain Reaction Device

Enhanced enrichment of collected airborne coronavirus and influenza virus samples via a ConA-coated microfluidic chip for PCR detection

The severe acute respiratory syndrome (SARS-CoV-2) outbreak triggered global concern and emphasized the importance of virus monitoring. During a seasonal influenza A outbreak, relatively low concentrations of 103–104 viral genome copies are available per 1 m3 of air, which makes detection and monitoring very challenging because the limit of detection of most polymerase chain reaction (PCR) devices is approximately 103 viral genome copies/mL. In response to the urgent need for the rapid detection of airborne coronaviruses and influenza viruses, an electrostatic aerosol-to-hydrosol (ATH) sampler was combined with a concanavalin A (ConA)-coated high-throughput microfluidic chip. The samples were then used for PCR detection. The results revealed that the enrichment capacity of the ATH sampler was 30,000-fold for both HCoV-229E and H1N1 influenza virus, whereas the enrichment capacities provided by the ConA-coated microfluidic chip were 8-fold and 16-fold for HCoV-229E and H1N1 virus, respectively. Thus, the total enrichment capacities of our combined ATH sampler and ConA-coated microfluidic chip were 2.4 × 105-fold and 4.8 × 105-fold for HCoV-229E and H1N1 virus, respectively. This methodology significantly improves PCR detection by providing a higher concentration of viable samples.

A 3D-printed oscillatory polymerase chain reaction system using a single heater

Nucleic acid amplification is a process that arises in all organisms. Polymerase chain reaction (PCR) is a standard laboratory nucleic acids amplification technology in vitro widely used in medical detection and biometric-related research. This device represents the first demonstration of 3D-printed oscillatory thermocycling within a portable PCR device. This study uses a single Peltier element to create the three temperature regions required for PCR. The overall control system uses an Arduino microcontroller with the advantages of easy development, abundant resources, and easy access to device software design. The sample-driving system integrated with the 3D printed object and the laser-cut acrylic component to achieve the linear reciprocating motion of the sample is conducive to the realisation of the portable design of the device. After system optimisation, the reaction tube moves from the denaturation zone to the aluminum block for cooling, and it moves to the groove of the denaturation zone from the annealing zone to accelerate the sample temperature rise. The time duration reduces from 180 s to 85 s to complete one thermal cycle. Combining the 5 mm instead of 3 mm thickness of the acrylic material and the configuration of the acrylic end exposed in the air creates a more significant temperature difference from 25 °C to 40 °C. Using this oscillatory PCR device, we successfully amplified a 219 bp PCR product of turmeric DNA after 30 thermal cycles within 50 min. The developed PCR device will cost less than $300 USD in our work. Compared with previous works, the cost of this research device is relatively low, and the total volume of the device is quite small. This work should be interesting to persons involved in diagnosing the infectious diseases of viruses and bacteria and identifying the fake healthy food in the low-cost bioreaction chambers in fields and remote areas.

Rational PCR Reactor Design in Microfluidics.

Limit of detection (LOD), speed, and cost for some of the most important diagnostic tools, i.e., lateral flow assays (LFA), enzyme-linked immunosorbent assays (ELISA), and polymerase chain reaction (PCR), all benefited from both the financial and regulatory support brought about by the pandemic. From those three, PCR has gained the most in overall performance. However, implementing PCR in point of care (POC) settings remains challenging because of its stringent requirements for a low LOD, multiplexing, accuracy, selectivity, robustness, and cost. Moreover, from a clinical point of view, it has become very desirable to attain an overall sample-to-answer time (t) of 10 min or less. Based on those POC requirements, we introduce three parameters to guide the design towards the next generation of PCR reactors: the overall sample-to-answer time (t); lambda (λ), a measure that sets the minimum number of copies required per reactor volume; and gamma (γ), the system's thermal efficiency. These three parameters control the necessary sample volume, the number of reactors that are feasible (for multiplexing), the type of fluidics, the PCR reactor shape, the thermal conductivity, the diffusivity of the materials used, and the type of heating and cooling systems employed. Then, as an illustration, we carry out a numerical simulation of temperature changes in a PCR device, discuss the leading commercial and RT-qPCR contenders under development, and suggest approaches to achieve the PCR reactor for RT-qPCR of the future.

Smartphone integrated handheld (SPEED) digital polymerase chain reaction device

We demonstrate a smartphone integrated handheld (SPEED) digital polymerase chain reaction (dPCR) device for point-of-care application. The device has dimensions of ≈100 × 200 × 35 mm3 and a weight of ≈400 g. It can perform 45 PCR cycles in ≈49 min. The device also features integrated, miniaturized modules for thermal cycling, image taking, and wireless data communication. These functions are controlled by self-developed Android-based applications. The only consumable is the developed silicon-based dPCR chip, which has the potential to be recycled. The device's precision and accuracy are comparable with commercial dPCR machines. We have verified the SPEED dPCR prototype's utility in the testing of severe acute respiratory syndrome coronavirus 2, the detection of cancer-associated gene sequences, and the confirmations of Down syndrome diagnoses. Due to its low upfront capital investment, as well as its nominal running cost, we envision that the SPEED dPCR device will help to perform cancer screenings and non-invasive prenatal tests for the general population. It will also aid in the timely identification and monitoring of infectious disease testing, thereby expediting alerts with respect to potential emerging pandemics.

A low-cost and hand-hold PCR microdevice based on water-cooling technology

Polymerase chain reaction (PCR) has become a powerful tool for detecting various diseases due to its high sensitivity and specificity. However, the long thermocycling time and the bulky system have limited the application of PCR devices in Point-of-care testing. Herein, we have proposed an efficient, low-cost, and hand-hold PCR microdevice, mainly including a control module based on water-cooling technology and an amplification module fabricated by 3D printing. The whole device is tiny and can be easily hand-held with a size of about 110mm × 100mm × 40mm and a weight of about 300g at a low cost of about $170.83. Based on the water-cooling technology, the device can efficiently perform 30 thermal cycles within 46min at a heating/cooling rate of 4.0/8.1 ℃/s. To test our instrument, plasmid DNA dilutions were amplified with this device; the results demonstrate successful nucleic acid amplification of the plasmid DNA and exhibit the promise of this device for Point-of-care testing.

Ultrafast Microfluidic PCR Thermocycler for Nucleic Acid Amplification.

The polymerase chain reaction (PCR) is essential in nucleic acid amplification tests and is widely used in many applications such as infectious disease detection, tumor screening, and food safety testing; however, most PCR devices have inefficient heating and cooling ramp rates for the solution, which significantly limit their application in special scenarios such as hospital emergencies, airports, and customs. Here, we propose a temperature control strategy to significantly increase the ramp rates for the solution temperature by switching microfluidic chips between multiple temperature zones and excessively increasing the temperature difference between temperature zones and the solution; accordingly, we have designed an ultrafast thermocycler. The results showed that the ramp rates of the solution temperature are a linear function of temperature differences within a range, and a larger temperature difference would result in faster ramp rates. The maximum heating and cooling ramp rates of the 25 μL solution reached 24.12 °C/s and 25.28 °C/s, respectively, and the average ramp rate was 13.33 °C/s, 6-8 times higher than that of conventional commercial PCR devices. The thermocycler achieved 9 min (1 min pre-denaturation + 45 PCR cycles) ultrafast nucleic acid amplification, shortening the time by 92% compared to the conventional 120 min nucleic acid amplification, and has the potential to be used for rapid nucleic acid detection.

Ultra-fast, sensitive and low-cost real-time PCR system for nucleic acid detection.

Nucleic acid detection directly identifies the presence of pathogenic microorganisms and has various advantages, such as high sensitivity, commendable specificity and a short window period, and has been widely used in many fields, such as early tumor screening, prenatal diagnosis and infectious disease detection. Real-time PCR (polymerase chain reaction) is the most commonly used method for nucleic acid detection in clinical practice, but it always takes about 1-3 hours, severely limiting its application in particular scenarios such as emergency testing, large-scale testing and on-site testing. To solve the time-consuming problem, a real-time PCR system based on multiple temperature zones was proposed, which realized the speed of temperature change of biological reagents from 2-4 °C s-1 to 13.33 °C s-1. The system integrates the advantages of fixed microchamber-type and microchannel-type amplification systems, including a microfluidic chip capable of fast heat transfer and a real-time PCR device with a temperature control strategy based on the temperature difference. The detection of HCMV biological samples using the real-time PCR system in this research took only 15 min, which was 75% shorter compared to the commercial qPCR instrument such as BIO-RAD, and the detection sensitivity remained essentially the same. The system could complete nucleic acid detection within 9 min under extreme conditions, characterized by fast detection speed and high sensitivity, providing a promising solution for ultra-fast nucleic acid detection.

Smartphone Integrated Handheld (Speed) Digital Polymerase Chain Reaction Device

Smartphone Integrated Handheld (Speed) Digital Polymerase Chain Reaction Device

Simulation of Rapid Thermal Cycle for Ultra-Fast PCR.

The polymerase chain reaction (PCR) technology is a mainstream detection method used in medical diagnoses, environmental monitoring, food hygiene, and safety. However, the systematic analysis of a compact structure with fast temperature changes for an ultra-fast PCR device that is convenient for on-site detection still lacks investigation. To overcome the problems of low heating efficiency and non-portability of PCR devices currently used, a miniaturized PCR system based on a microfluidic chip, i.e., lab-on-chip technology, has been proposed. The main objective of this paper is to explore the feasibility of using a heat resistor that can reach a fast heating rate and temperature uniformity combined with air cooling technology for rapid cooling and to investigate the influences of various pattern designs and thicknesses of the resistor on heating rates and temperature uniformity. Additionally, a PCR chip made of various materials with different thermal properties, such as surface emissivity, thermal conductivity, mass density, and heat capacity at constant pressure is analyzed. In addition to the heat loss caused by the natural convection of air, the radiation loss of the simulation object is also considered, which makes the model much closer to the practical situation. Our research results provide a considerable reference for the design of the heating and cooling modules used in the ultra-fast PCR protocol, which has great potential in In Vitro Diagnosis (IVD) and the PCR detection of foodborne pathogens and bacteria.

Hundreds-Dollar-Level Multiplex Integrated RT-qPCR Quantitative System for Field Detection.

The COVID-19 pandemic poses a threat to global health. Due to its high sensitivity, specificity, and stability, real-time fluorescence quantitative (real-time PCR) detection has become the most extensively used approach for diagnosing SARS-CoV-2 pneumonia. According to a report from the World Health Organization, emerging and underdeveloped nations lack nucleic acid detection kits and polymerase chain reaction (PCR) instruments for molecular biological detection. In addition, sending samples to a laboratory for testing may result in considerable delays between sampling and diagnosis, which is not favorable to the timely prevention and control of new crown outbreaks. Concurrently, there is an urgent demand for accurate PCR devices that do not require a laboratory setting, are more portable, and are capable of completing testing on-site. Hence, we report on HDLRT-qPCR, a new, low-cost, multiplexed real-time fluorescence detection apparatus that we have developed for on-site testing investigations of diverse diseases in developing nations. This apparatus can complete on-site testing rapidly and sensitively. The entire cost of this instrument does not exceed USD 760. In order to demonstrate the applicability of our PCR instrument, we conducted testing that revealed that we achieved gradient amplification and melting curves comparable to those of commercially available equipment. Good consistency characterized the testing outcomes. The successful detection of target genes demonstrates the reliability of our inexpensive PCR diagnostic technique. With this apparatus, there is no need to transport samples to a central laboratory; instead, we conduct testing at the sampling site. This saves time on transportation, substantially accelerates overall testing speed, and provides results within 40 min.

Effect of calcium fluoride nanoparticles in prevention of demineralization during orthodontic fixed appliance treatment: a randomized clinical trial.

White spot lesions (WSLs) are the most common complications of fixed appliance orthodontic treatment. To evaluate the effectiveness of calcium fluoride nanoparticles-containing orthodontic primer (nCaF2-primer) in preventing the incidence of WSLs during orthodontic treatment. Single-centre, double-blinded, split-mouth, randomized clinical trial. The sample involved 31 orthodontic patients (≥12 years). Participants were recruited using a simple nonstratified randomization. Data collection, measurements, and analysis were performed blindly. Outcome measures included comparing the effect of nCaF2-primer with control primer (Transbond) regarding the degree of demineralization (DIAGNOdent pen), Streptococcus mutans (S. mutans) bacterial counting [real-time polymerase chain reaction device (PCR)], and WSLs incidence (pre- and post-operative photographs). The measurements were performed before bonding, 1, 3, and 6 months after bonding and after appliance removal. A two-way repeated measure analysis of variance test (for DIAGNOdent pen scores), and Wilcoxon signed-rank test (for the difference between bacterial counting and WSLs incidence) were used (P < 0.05). Thirty-one patients were recruited and randomized (mean age 17.9 ± 2.45 years). For the primary outcome (DIAGNOdent pen scores) and secondary outcome of S. mutans counting: 31 patients (310 teeth for each group) were included in scoring at T1 and T3, and 30 patients (300 teeth) were included at T6. While for the photographic scores, 26 patients were included after bracket bonding. The demineralization scores showed significant differences at all-time intervals within the 6 months after bracket bonding which was more noticeable after the first month. There was a significant difference in bacterial count between the two primer groups at the T1 only. Regarding photographic scores, there were no significant differences in the WSLs incidence between the two primers groups after brackets removal. No harm was detected during treatment, except the usual pain/gingival irritation. nCaF2-primer effectively decreased demineralization scores within the 6 months after bracket bonding. Moreover, it significantly reduced S. mutans colonization after the first month. However, the tested primer did not have an extra advantage in preventing WSLs development at the clinical level after appliance removal. The trial was registered with ClinicalTrials.gov on 8 May 2021 (registration number: NCT04994314).

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.

Portable Heating System Based on a Liquid Metal Bath for Rapid PCR

With the outbreak of COVID-19 around the world, rapid and accurate detection of new coronaviruses is the key to stop the transmission of the disease and prevent and control the novel coronavirus, among which polymerase chain reaction (PCR) is the mainstream nucleic acid detection method. A temperature cycling device is the core of the PCR amplification micro-device. The precision of the temperature control and temperature change rate directly affect the efficiency of PCR amplification. This study proposes a new PCR method based on rapid PCR chip optimization of a liquid metal bath, which realizes precise and rapid temperature rise and fall control. We systematically explored the feasibility of using liquid metals with different melting points in the system and proposed a 47 °C bismuth-based liquid metal bath as the heat conduction medium of the system to optimize the system. The heat conduction properties of the thermally conductive silicone oil bath were compared. Compared with the thermally conductive silicone oil bath, thermal cycle efficiency is improved nearly 3 times. The average heating rate of the liquid metal bath is fast, and the temperature control stability is good, which can significantly reduce the hysteresis, and the temperature change curve is more gentle, which can greatly improve the efficiency of PCR amplification. The results of gene amplification using rat DNA as the template and SEC61A as the target also indicate that the system can be successfully used in PCR devices, and the types of PCR containers can be not limited to PCR tubes. Based on the experiment, we proved that the PCR method optimized by the liquid metal bath multi-gene rapid PCR chip can further improve the temperature response speed. It has the advantages of accurate data, fast response speed, low price, safety, and environmental protection and can effectively reduce the time of PCR and improve the application efficiency. As far as we know, this is the first international report on using a liquid metal bath to do rapid-cooling PCR.

Performance Evaluation of a Novel Ultrafast Molecular Diagnostic Device Integrated With Microfluidic Chips and Dual Temperature Modules.

Ultrafast, portable, and inexpensive molecular diagnostic platforms are critical for clinical diagnosis and on-site detection. There are currently no available real-time polymerase chain reaction (PCR) devices able to meet the demands of point-of-care testing, as the heating and cooling processes cannot be avoided. In this study, the dual temperature modules were first designed to process microfluidic chips automatically circulating between them. Thus, a novel ultrafast molecular diagnostic real-time PCR device (approximately 18 and 23 min for DNA and RNA detection, respectively) with two channels (FAM and Cy5) for the detection of 12 targets was developed. The device contained three core functional components, including temperature control, optics, and motion, which were integrated into a portable compact box. The temperature modules accurately control temperature in rapid thermal cycles with less than ±0.1 °C, ±1 °C and ±0.5 °C for the temperature fluctuation, uniformity, and error of indication, respectively. The average coefficient of variation (CV) of the fluorescence intensity (FI) for all 12 wells was 2.3% for FAM and 2.7% for Cy5. There was a good linear relationship between the concentrations of fluorescent dye and the FIs of FAM and Cy5(R 2 = 0.9990 and 0.9937), and the average CVs of the Ct values calculated by the embedded software were 1.4% for FAM and Cy5, respectively. The 100 double-blind mocked sputum and 249 clinical stool samples were analyzed by the ultrafast real-time PCR device in comparison with the DAAN Gene SARS-CoV-2 kit run on the ABI 7500 instrument and Xpert C. difficile/Epi, respectively. Among the 249 stool samples, the ultrafast real-time PCR device detected toxigenic C. difficile in 54 samples (54/249, 21.7%) with a specificity and positive predictive values of 99.0 and 96.3%, which were higher than the Xpert C. difficile/Epi values of 94.4 and 88.1% (p > 0.05). The ultrafast real-time PCR device detected 15 SARS-CoV-2 positive samples, which has a 100% concordance with that obtained by the DAAN Gene SARS-CoV-2 kit. This study demonstrated that the ultrafast real-time PCR device integrated with microfluidic chips and dual temperature modules is an ultrafast, reliable, easy-to-use, and cost-effective molecular diagnostic platform for clinical diagnosis and on-site testing, especially in resource-limited settings.

Ultrafast DNA Amplification Using Microchannel Flow-Through PCR Device.

Polymerase chain reaction (PCR) is limited by the long reaction time for point-of-care. Currently, commercial benchtop rapid PCR requires 30–40 min, and this time is limited by the absence of rapid and stable heating and cooling platforms rather than the biochemical reaction kinetics. This study develops an ultrafast PCR (<3 min) platform using flow-through microchannel chips. An actin gene amplicon with a length of 151 base-pairs in the whole genome was used to verify the ultrafast PCR microfluidic chip. The results demonstrated that the channel of 56 μm height can provide fast heat conduction and the channel length should not be short. Under certain denaturation and annealing/extension times, a short channel design will cause the sample to drive slowly in the microchannel with insufficient pressure in the channel, causing the fluid to generate bubbles in the high-temperature zone and subsequently destabilizing the flow. The chips used in the experiment can complete 40 thermal cycles within 160 s through a design with the 56 µm channel height and with each thermal circle measuring 4 cm long. The calculation shows that the DNA extension speed is ~60 base-pairs/s, which is consistent with the theoretical speed of the Klen Taq extension used, and the detection limit can reach 67 copies. The heat transfer time of the reagent on this platform is very short. The simple chip design and fabrication are suitable for the development of commercial ultrafast PCR chips.

Effects of Different Centrifugation Protocols on the Detection of EGFR Mutations in Plasma Cell-Free DNA.

ObjectivesVarious preanalytical factors, including the collection tube, storage conditions, and centrifugation, affect the detection results of plasma cell-free DNA (cfDNA). We compared the effect of different centrifugation protocols on the detection of EGFR mutations in cfDNA.MethodsWe analyzed 117 plasma specimens from 110 patients with non–small cell lung cancer using the cobas EGFR Mutation Test v2 (Roche Diagnostics). We compared the identified EGFR mutations and semiquantitative index values from the 1- and 2-step centrifugation groups and confirmed the clinical impact of differences in the results after further high-speed centrifugation.ResultsWe detected EGFR mutations in 44 (37.6%) and 47 (40.2%) samples that were centrifuged once and twice, respectively; the 2 groups showed an 89.7% (105/117) concordance and a strong correlation in their semiquantitative index values (r = 0.929). Among the 12 inconsistent result pairs, 9 samples of 2-step centrifugation (75%) were consistent with the results of a recent tissue biopsy.ConclusionsAdditional high-speed centrifugation has been shown to increase the sensitivity of EGFR mutation detection in a commercial in vitro diagnostic real-time polymerase chain reaction device and is an optimal preanalytical factor for detecting low-allele frequency gene mutations using low concentrations of cfDNA.

A Thermocycler Using a Chip Resistor Heater and a Glass Microchip for a Portable and Rapid Microchip-Based PCR Device.

This study proposes a rapid and inexpensive thermocycler that enables rapid heating of samples using a thin glass chip and a cheap chip resistor to overcome the on-site diagnostic limitations of polymerase chain reaction (PCR). Microchip PCR devices have emerged to miniaturize conventional PCR systems and reduce operation time and cost. In general, PCR microchips require a thin-film heater fabricated through a semiconductor process, which is a complicated process, resulting in high costs. Therefore, this investigation substituted a general chip resistor for a thin-film heater. The proposed thermocycler consists of a compact glass microchip of 12.5 mm × 12.5 mm × 2 mm that could hold a 2 μL PCR sample and a surface-mounted chip resistor of 6432 size (6.4 mm × 3.2 mm). Improving heat transfer from the chip resistor heater to the PCR reaction chamber in the microchip was accomplished via the design and fabrication of a three-dimensional chip structure using selective laser-induced etching, a rapid prototyping technique that allowed to be embedded. The fabricated PCR microchip was combined with a thermistor temperature sensor, a blower fan, and a microcontroller. The assembled thermocycler could heat the sample at a maximum rate of 28.8 °C/s per second. When compared with a commercially available PCR apparatus running the same PCR protocol, the total PCR operating time with a DNA sample was reduced by about 20%.

Rapid membrane-based photothermal PCR for disease detection

The worldwide incidence of deadly infectious diseases has increased in recent years. Therefore, a rapid and accurate nucleic acid diagnostic system is in high demand. Polymerase chain reaction (PCR) is the standard method for amplifying and quantifying nucleic acid biomarkers for disease detection. Although several rapid PCR systems have been developed to overcome the limitations of conventional PCR, such as post-processing for analysis, complicated handling, and highly specialized instruments, a new PCR testing approach is still required. In this study, we developed a rapid membrane-based photothermal PCR that enables quantitative fluorescence detection. It requires no post-PCR procedures and provides user-friendly micro-scale solution control and sample loading. Au nanostructure was deposited on the reaction pad as a light-to-heat converter for PCR thermocycling. The automatically controlled 785 nm light source heated a solution in the reaction pad with photothermal effect. The temperature of the reaction solution, which was absorbed in a membrane, was cycled 25 times between 63 °C and 95 °C within 6 min with a photothermal cycler. The amplified samples on the membrane were then quantitatively analyzed by measuring the fluorescence signal of intercalating SYBR dye within 12 min. DNA samples extracted from Staphylococcus aureus and unpurified cells were amplified and analyzed. This membrane-based PCR approach can provide easy connectivity with various membrane-based sample pre-treatment processes, and therefore the proposed procedure could be used as the basis for an all-in-one molecular diagnosis platform. This simple, rapid, and sensitive PCR device could also serve as the basis for advanced molecular diagnostics.