Introduction

Before developing nucleic acid (NA) amplification based diagnostic method in 19831, humanity experienced significant losses due to pandemics of infectious diseases caused by viruses, bacteria, and fungi2. In fact, pandemics, such as the Spanish Flu3, Asian Flu4, Seventh Cholera5, and Hong Kong Flu6 reached the global death toll of more than 1 million because slow and inaccurate serological diagnostics7 hindered effective containment. However, post-1983, the casualties of severe acute respiratory syndrome (SARS)8, influenza9, and Middle East respiratory syndrome (MERS)10 pandemics in 2002, 2009, and 2012, respectively, were under 200,000 as shown in Fig. 1, because of developing a polymerase chain reaction (PCR) which amplifies NAs by repeating a temperature cycle and is a standard method for molecular diagnosis that can accurately detect small amounts of NAs1. However, even under PCR era, COVID-19 causing 777 million cases, 7 million deaths, and major social and economic impact worldwide11 during the last six years because of their high transmissibility in modern society where the prompt containment of infection is highly demanded based on quick and accurate diagnosis by individuals. Consequently, faster, cheaper, more sensitive, and more accurate NA amplification-based point-of-care testing (PoCT) platforms have been demanded globally12,13,14,15 to control viral spread in the early stages of emerging infectious diseases.

Fig. 1

Pandemic death toll. Graph showing the number of deaths from infectious diseases that have been prevalent worldwide since the 20th century. Looking at before and after the time of PCR development, the number of deaths from infectious diseases has decreased significantly since 1983 when PCR was developed. These statistics show that the spread of infectious diseases can be effectively controlled through rapid and accurate diagnosis of infectious diseases

While passing through the COVID-19 pandemic, worldwide research for developing NA amplification-based rapid and accurate PoCT methods has been intensively carried out16. Especially, isothermal amplification methods, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), have been intensively investigated for developing NA amplification-based PoCT because they can amplify NA without bulky and complex thermal cyclers of PCR, but only requires a simple thermal control system to keep isothermal temperatures of 65 °C17 and 37 °C18, respectively. However, the isothermal amplification methods generally result in higher non-specific amplification19,20 than PCR, resulting in false positives. In addition, LAMP requires six primers, which poses significant challenges for multiplexed detection because of the potential primer-dimer formation and increased analytical complexity. Furthermore, both LAMP and RPA have limited quantification because of unclear correlation between color change and NA concentration because of colorimetric reading method which is highly dependent on individual’s interpretation of the color intensity. Therefore, the PCR will be a good candidate for developing NA amplification-based PoCT with 6S: simplicity, speed, small, sustainability, sensitivity, and specificity.

To diagnose the infectious diseases by PCR-based PoCT with 6S, sample preparation and injection should be simple to use by untrained personnels, speedy thermal cyclers should be equipped for the rapid NA amplification, and the size of PCR-based PoCT should be small enough to carry, while maintaining sensitivity and specificity. In addition, the sustainability in manufacturing disposable parts of the PCR-based PoCT and landfills of them should be considered as well. However, up to present, the PCR-based PoCT which can meet 6S has not been reported yet. Thus, this review presents a guide for the practical development of the PCR-based PoCT with 6S, called QUICK-PCR: quick, ubiquitous, integrated, and cost-efficient molecular diagnostic kit based on PCR system.

To develop the prospective QUICK-PCR, this review first analyzes main fundamental elements in a PCR diagnostic system: the sample preparation process, thermal cycler system, and result readout system. Based on the analyzed main elements, innovative technologies in the main elements to meet 6S were briefly introduced and comparatively summarized key technologies to implement them in developing the QUICK-PCR and analyzed the disposable parts with the view of the sustainable manufacturing system including their landfills. In addition, the review considered the clinical validity of any innovative technologies and parts in the PCR-based PoCT which will be shrunk the size and fabricate with a high-throughput additive manufacturing method to meet simplicity, speed, small, and sustainability while compromising the sensitivity and specificity to meet the regulations21 in FDA and CE-IVD. As such, the prospective QUICK-PCR by addressing a way of resolving 6S and issues of clinical validation can practically guide researchers to develop rapid, cost-effective, and universally accessible diagnostics for serving as an efficient tool for future pandemic response and other public health crises.

Polymerase chain reaction: gold standard molecular diagnosis method

Although during the COVID-19 pandemic, a substantial number of publications emerged on molecular diagnostics-based PoCT using methods such as PCR, LAMP, and RPA, only PCR has been clinically proven accurate through many technological advances and numerous clinical experiments22,23,24, and international standards and regulatory systems are well established25,26 for global use. Therefore, it is used as the gold standard of molecular diagnosis. Thus, we provide a comprehensive overview of RT-PCR technology in this section.

The standard diagnostic process for PCR consists of four steps[27](https://www.nature.com/articles/s41378-025-01057-4#ref-CR27 “Lorenz, T. C. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J. Vis. Exp. e3998 https://doi.org/10.3791/3998

. (2012).“) respectively, by sample collection, sample preparation, thermal cycling, and fluorescence detection. In the case of COVID-19, a nasopharyngeal swab is used to collect target NAs from a patient (Fig. 2a). The extraction of target NAs from the collected swab involves several key steps as shown in Fig. 2b. In general, the cell membranes are first lysed, and then, contaminants are removed via chemicals such as phenol or chloroform, or enzymatic treatment by using silica-based columns. After the pre-treatment, the purified target NAs are retrieved by centrifuging the treated solution from the previous stage. After injecting purified target NAs, the thermal cycling of the PCR consists of three main temperature steps (Fig. 2c): the first step is denaturation, which occurs in the temperature range of 93–95 °C and is the process of separating the double strand DNA to a single strands. The second step is annealing, which depends on the melting temperature of the primer, and usually occurs in the temperature range of 55–65 °C. This is the process of attaching forward and reverse primers, which have a nucleotide sequence specific to the target amplicon to the 5’-end of each single strand DNA. As the third step, elongation step begins at 72 °C, where the polymerase and dNTP react to replicating the target amplicon sequence, starting from the primer-attached position. For RNA amplification, complementary DNA (cDNA) is synthesized from RNA before starting the thermal cycles using an isothermal reverse transcription step with the help of reverse transcriptase. Through this process, a small amount of the initial target NA is amplified by a factor of 2n (where n is the number of PCR cycles), facilitating the more accurate detection and analysis of the target NAs. As a readout step, the amplified DNA can be analyzed using three different methods (Fig. 2d): 1. Gel electrophoresis28, 2. real-time amplification curves29 and 3. end-point detection30. Based-on these fundamental three steps in PCR, it can be categorized into three generations according to the result analysis methods, as follows:

Fig. 2

Overview of the PCR-based molecular diagnostic process. a Sample collection: Biological samples were collected from patients using nasopharyngeal swabs. b Sample preparation: Collected samples release NA through lysis, and RNA is then extracted and purify RNA using column-based methods. c RT-PCR amplification: Extracted RNA was converted to cDNA through reverse transcription and then subjected to thermal cycling for target gene amplification. d Detection: Amplified DNA is analyzed using gel electrophoresis, real-time fluorescence detection, or digital PCR

The first-generation PCR, conventional PCR method is used gel electrophoresis28 to detect DNA after thermal cycling. Gel electrophoresis is effective in separating DNA fragments by size, but it is difficult to distinguish minute variations due to the low resolution between fragments of similar size, and errors in experimental results may occur because they are sensitive to gel concentration and applied voltage. In addition, the experimental process is prolonged, which provides information about presence or absence of DNA but not accurate quantitative analysis. Moreover, gel electrophoresis is performed on separate equipment rather than on a thermal cycler requiring transfer of the amplified solution which can result in cross-contamination.

Real-time PCR or qPCR alleviates the problem of cross-contamination by eliminating the additional process for gel electrophoresis and replacing it with a real-time analysis method29, thereby simplifying the detection process. qPCR uses fluorescence signals to quantify DNA in real-time during thermal cycles using dye-based31 (e.g., SYBR Green) and probe-based32 (e.g., TaqMan) approaches. Furthermore, multiplex detection is achievable for diagnosing various infectious diseases using a single PCR by attaching different types of fluorophores to the end of the probe33. Despite increasing the feasibility of PoCT by replacing bulky optical reading systems with smartphone LED and camera34,35, qPCR still relies on standard curves and is sensitive to inhibitors and contamination.

Digital PCR (dPCR) enhances the limit of detection (LoD) and more reliable sensitivity for the low-abundance targets by partitioning samples into numerous individual PCR reactions (microwell plates, oil emulsions, and microwell chips), allowing the absolute quantification of NA at very low concentrations36. Individual partitions dPCR effectively increases the local concentration of the target within each reaction volume and reduces inhibition and contamination. After thermal cycling, Poisson statistics were used to analyze the number of positive partitions to accurately estimate the number of NA copies37. Droplet-based dPCR (ddPCR)38 and chip-based dPCR (cdPCR)39 are two dPCR methods that utilize microfluidic chips. However, the PoCT application of dPCR faces challenges, such as thermal conductivity fluctuations due to droplet formation40, which require adequate ramping rates to minimize temperature differences. It also requires a droplet generator to generate uniform droplets, increases the complexity and time requirements. Moreover, cdPCR mitigates the issues of ramp rate and eliminates droplet generation, thereby shortening partitioning time and facilitating multiplexing.

Limitations in recent PCR technology

Advances in PCR technology have improved accuracy, thermal cycling speed, and diagnostic efficiency, but it is still difficult to implement practical QUICK-PCR because of the complex operation process, long sample-to-answer turnaround time, and the limitation that bulky equipment is required for result analysis. Typical sample preparation to obtain purified NAs takes about 30 min through a complex multi-step process by skilled personnel. These conditions limit the applicability of PCR in field diagnosis, where simple, rapid, cost-effective, and easy use are required.

To realize QUICK-PCR, current PCR methods should address three major limitations while maintaining its diagnostic accuracy and sensitivity. First, sample preparation should be simplified using microfluidic chip-based implementations which can be used in the field by a general population. Second, diagnostic speed should be improved by improving thermal cycling methods while keeping them portable and low powered. Finally, readout system should be simplified by implementing smartphone-based fluorescence, colorimetric reader or implementing electrochemical based methods eliminating bulky optical systems.

Strategy for realizing QUICK-PCR

Sample preparation methods for ubiquitous access to diagnostics

To diagnose the infectious diseases by individuals, PCR-based PoCT should be simple to use and rapid to check out the results, while maintaining accuracy. Recently, methods for easy and quick extraction of NAs from bodily fluids such as saliva41,42, blood43,44,45, and urine46,47 with simple workflow by incorporating microfluidic chips which exploits mechanical48 and chemical innovations49, have been developed. These bodily fluids contain a variety of impurities which can interfere the activation of PCR enzymes and inhibit amplification50, thus high-purity NA extraction is required for PCR. As a typical clinical example, sample of whole blood consists of blood cells (~45%) and plasma (~55%), with target NA. Most of the remaining impurities in the blood cells are hemoglobin, heme group, and cell debris which can chelate Mg2+ ions, essential for PCR amplification, inhibiting polymerase activity51, and cause non-specific binding, thereby significantly reducing the amplification efficiency. Therefore, high-purity plasma separation should be performed with easy-of-use format for a high-efficiency and accurate PCR reaction. This section discusses the development of a user-friendly sample preparation system with high-efficiency and implementation strategies of a field-type system, and a comparative analysis is presented in Table 1.

Microfluidic chip-based sample preparation methods

Microfluidic chips utilize micro-sized channels to direct the fluid flow enabling various methods such as mixing, filtration, transport in compact and automated device[52](https://www.nature.com/articles/s41378-025-01057-4#ref-CR52 “Kumar, A., Parihar, A., Panda, U. & Parihar, D. S. Microfluidics-based point-of-care testing (POCT) devices in dealing with waves of COVID-19 pandemic: the emerging solution. ACS Appl. Bio. Mater. https://doi.org/10.1021/acsabm.1c01320

(2021).“). A typical application involves cell lysis, NA extraction, purification from whole blood and saliva using microfluidic chips minimizing human error and sample processing time in the field. Using an integrated microfluidic device, the sample preparation process can be simplified to enable rapid quantitative diagnosis by extracting NAs from whole blood samples. A demonstration of self-powered integrated microfluidic PoC low-cost enabling (SIMPLE) chip applies the principle of separating plasma from whole blood using a trench structure (Fig. 3a) and incorporates a vacuum battery to maintain fluid flow without an external pump or power source53. Plasma separation was completed with an extraction efficiency of 95% in 12 min from 100 μL of whole blood. Likewise, plasma separation was completed with 100% extraction efficiency within 10 min from 5 μL of whole blood using degas-driven flow and trench filter structures54. Another group succeeded in plasma separation in 4 min from whole blood at a 10 μL capacity using gravity and dielectrophoresis techniques55, but the plasma separation efficiency was reduced to 44% while shortening the time. The vacuum battery method used in these devices can effectively remove air in the microfluidic chip, but over time, air is replenished through porous PDMS block, which can reduce the temperature uniformity and accuracy of fluorescence signals. In addition, microfluidic chips have difficulty controlling the fluid precisely, and a complex design is required to implement precise chip functions, which increases production costs because of the complex manufacturing process.

Fig. 3

Various sample preparation methods enable ubiquitous access to diagnostics. The techniques include a µ-fluidic automation adapted from ref. 53, b paper-based extraction adapted from ref. 57, c centrifugal µ-fluidic, reprinted with permission from ref. 45, Elsevier, d ultrasonic lysis, reprinted with permission from ref. 44, Elsevier, e magnetic bead separation, adapted from ref. 65 under a Creative Commons license CC BY 4.0. and f direct PCR, adapted from ref. 66, under a Creative Commons license CC BY 4.0. Each method is optimized for PoC applications. These methods aim to simplify and expedite sample processing, making diagnostic tests accessible in diverse settings

By sequentially processing cell lysis, NA extraction, and purification using the capillary force of paper-based microfluidic systems, we can implement a cost-effective, disposable, and portable sample pretreatment system by moving samples to a series of areas containing reagents56. An easy application to a variety of biological samples at a small capacity without the need for an external pump or power source, the paper-based platform provides an ideal solution for on-site diagnosis in environments with limited human and material resources. Tang, R. et al. developed a device for extracting DNA by automatically inducing reagents and samples using sponge-based buffer reservoirs, paper-based valves, and channels of various lengths were developed (Fig. 3b)57. The device can extract DNA from 30 µL of whole blood, serum, saliva, sputum, or bacterial suspension within 2 min. Kim et al. completed plasma separation, lysis, and NA purification in 30 min by injecting 3 mL of whole blood into an acrylic-based microfluidic device derived by finger-actuator58. Upon implementation of cartridge body using biodegradable paper and polymers, an environmentally friendly field diagnosis system can be developed, thereby it can be realizing a sustainable QUICK-PCR platform.

The disc-type microfluidic system (Lab-on-a-disc, LoaD) can implement plasma separation, cell lysis, NA extraction, and reagent mixing in a single chip by automating liquid movement along microchannels using centrifugal force. Centrifugation is a widely used method59 for separating plasma from whole blood by separating components in a mixture according to differences in density. When whole blood is placed in a centrifuge and rotated quickly, a centrifugal force acts and heavier components, such as red blood cells, white blood cells, and platelets, are pushed down to the bottom of the test tube, and the low-density plasma remains on top, thereby obtaining high-purity plasma. These systems provide consistency and accuracy in diagnosis by reducing the likelihood of human error and minimizing human intervention. Recently, a cost-effective LoaD-based sample preparation and lysis system has been developed by incorporating roll-to-roll (R2R) printing technology (Fig. 3c)45. This device does not require an external pump or mechanical valve, but a miniature motorized centrifuge. Instead, after injecting 150 µL of whole blood using a photosensitive wax valve, plasma separation, cell lysis, and reagent mixing were continuously performed on one chip to complete NA extraction within 30 min. When using the centrifugation method, reducing the amount of input whole blood can shorten the plasma separation time60,61. Despite these advantages, the dependence on dedicated centrifugation equipment compatible with the chip limits its application in field diagnostic environments.

Other sample preparation methods

Nucleic acids can be extracted by lysing the cell membrane using ultrasonic waves and then separating the NAs using magnetic force. Ultrasound creates cavitation bubbles, and the mechanical force generated when this bubble collapses acts on the cell membrane, lysing the cell membrane and releasing NAs from the cell substrate62. The efficiency of cell lysis depends on the frequency of ultrasonic waves and the solvent composition63, and target NAs are captured from the hemolyzed blood or tissue homogenate using magnetic force and specific binding molecules64. Magnetically bound NAs can be separated using an external magnetic field and eluted to obtain high-purity NAs. This magnetic bead-based method is widely used in NA extraction systems because of its high efficiency and ease of automation, as NAs bound to beads can be effectively separated from unnecessary pollutants. As shown in Fig. 3d44, a NA extraction system was developed using a method in which magnetic beads were dispersed within a supersonic wavelength to generate acoustic pressure. The induced particles concentrated at the background vibration point of a standing wave, and genomic DNA was efficiently extracted within 8 min from 1 µL of whole blood. The oil-immersed lossless total analysis system (OIL-TAS) was combined with a magnetic-based separation method to complete RNA extraction and detection from a 30 μL dose of nasopharyngeal swab specimen in a single system in 30 min65 (Fig. 3e). The OIL-TAS system minimizes the risk of cross-contamination and sample loss by using magnetism to move separated droplets to the next chamber while covered with oil. The system proposes a method to ensure rapid and high reliability for the examination of infectious diseases such as SARS-CoV-2 by efficiently purifying the analyte through magnetic extraction and then performing isothermal amplification to quickly detect NAs.

Taking advantage of the relatively fragile nature of the membrane of a viral pathogen and engineered polymerase, a “direct PCR” method has been developed to perform direct amplification of NAs without a NAs extraction step. Direct PCR, which skips the complicated sample preparation process, has the advantage to simply operate the PCR. In the COVID-19 test, the NA extraction process was omitted, and a direct RT-PCR method (Fig. 3f) using an inert or dissolved sample with heat was introduced66. In this method, after lysis using a surfactant, Triton X-100, mixed with saliva or throat/spinal swab, SARS-CoV-2 was directly detected by RT-PCR. When a high concentration of Triton X-100 (5%) was used, the C**t value slightly increased, and the qPCR fluorescence was slightly reduced, which did not affect the RT-PCR result. This method was clinically verified using COVID-19, resulting in 96% of sensitivity, 99.8% of specificity, and 98.8% of accuracy. Therefore, the method of inactivation using a surfactant may be effectively used for large-scale rapid SARS-CoV-2 screening tests. On the other hand, direct PCR with blood samples67 for the diagnosis of malaria showed 93% clinical sensitivity and 100% clinical specificity using a special enzyme called OmniKlentaq polymerase, which is resistant to PCR inhibitors, and a PCR enhancement cocktail. This result showed high agreement with PCR performed on purified DNA and demonstrated that sufficiently sensitive diagnosis was possible when PCR was performed directly from blood using special enzymes and reagents. In contrast, when the clinical performance of the PCR system was directly evaluated for various respiratory tract infections using nasal swab samples68, it achieved 97.5% sensitivity and 98.6% specificity due to fewer inhibitors compared to blood samples, resulting in 98.6% diagnostic accuracy. This clinical validation supports a practical way in developing the QUICK-PCR, utilizing the direct PCR, since it can eliminate two important barriers, simplicity and disposability in the PCR to transform into the QUICK-PCR. Thus, impurities in running PCR, such as hemoglobin, cell residue, and protein, which inhibit polymerase activity, should be nullified by selecting an engineered polymerase and adding designed molecules or ions. In fact, the inhibitory effect has been reduced by using special reagents, such as an inhibitor resistant polymerase, and optimizing the reaction buffer in clinical tests69,70.

Thermal cycler for quick diagnosis

Commercially available PCR device can accurately and stably control the temperature using a block thermal cycler, but its speed and volume are slow and bulky, respectively, limiting the overall diagnosis speed and miniaturization of the device. To implement a QUICK-PCR system, it is required to design a small thermal cycler and to control the temperature quickly and efficiently. PCR repeats the denaturation, annealing, and extension steps within a specific temperature range. The temperature instability in the annealing/extension step, non-specific amplification can occur71, and the instability of temperature in the denaturation step can cause damage to the reagent72, resulting in reduce the amplification efficiency. This section introduces the latest technologies for realizing fast temperature control using Joule-, thermoelectric-, and plasmonic-based thermal cycling methods, and a summary and comparative analysis are presented in Table 2.

Joule heating, also known as resistive or ohmic heating, is a phenomenon in which electrical energy is converted into heat when electrons in a conductor collide with atoms when current passes through it73 (Fig. 4a). The amount of heat generated is directly proportional to the square of the current and the resistance of the conductor. Because energy conversion is performed without an intermediate step or other forms of energy intervention, the theoretical efficiency of Joule heating is approximately 100%74. However, the cooling depends on the surrounding environment temperature and passive cooling mechanisms, such as conduction, convection, and radiation. The local heating effect of Joule heating minimizes thermal inertia and directly heats the microfluidic channel or reaction chamber, resulting in a thermal circulation that is much faster than that of the conventional thermal block. Kim et al. developed a rapid PCR kit that significantly shortened NA amplification time and improved accessibility and efficiency using a Joule heater75. The kit was prepared by combining a nichrome-based thin-film heater with a lateral flow paper strip (Fig. 4b). By achieving a heating rate of up to 16.3 °C/s and a cooling rate of 3.4 °C/s through fast and precise temperature control of Joule heating, SARS-CoV-2 RNA with a volume of 3 µL could be detected within 30 min (Fig. 4c) with 3.36 W of electrical power. In addition, Jeon et al. developed a Ag/carbon fiber film-based resistive heater76, and human coronavirus was detected within 10 min at a heating rate of ~4.5 °C/s with 5 W of electrical power. Although the heat conversion efficiency of the Joule heater is excellent, its use in the PoCT field is limited by technical limitations, such as slow cooling rates due to natural heat dissipation and physical/chemical side effects, such as electrolytic reactions or bubble generation77.

Fig. 4: Various thermal cycling technologies based rapid PCR.

a Joule heating, when electrical current passing through a resistive metal generates heat owing to electron collisions (M: metal atoms, e-: free electron). b Joule heater-integrated lateral flow PCR kit, and c 30 cycles of a NiCr thin film-based Joule heater adapted from ref. 75. d Thermoelectric heating uses Peltier-elements, in which, when a voltage is applied, heat is absorbed at the n-type material and dissipated at the p-type material, enabling precise temperature control. e Peltier-element-based thermocycler, and f temperature profiles of Peltier-element-based thermal cycler, reprinted with permission from ref. 79, Elsevier, g Plasmonic heating, when light strikes a metallic nanoparticle, the oscillating electric field of the light induces a collective oscillation of the conduction electrons within the nanoparticle. During the damping process of these oscillations, hot electrons are generated as the energy from the plasmons transfers to the electrons. These hot electrons subsequently lose their energy through electron-phonon interactions, converting it into localized heat, which results in significant temperature increases near the surface of the nanoparticle. h Plasmonic optical wells (POWs) based photothermal conversion effect, and i temperature profiles, reprinted with permission from ref. [84](https://www.nature.com/articles/s41378-025-01057-4#ref-CR84 “