Polymerase chain reaction (PCR), which was invented by Mullis(Mullis and Erlich, 1988), has a profound impact on the development of life science since its inception(Higuchi, 1989). Traditional PCR usually runs in 30–40 cycles, and even if the initial sample contains only a minimal amount of DNA, a million-fold amplification can be achieved within a few hours, reaching a level of quantity sufficient for analysis (Chen et al., 2022a). With its significant advantages of ease of operation, high specificity and sensitivity, PCR has rapidly permeated every corner of life sciences, including medical diagnosis, forensic science, agricultural science, et.al (Kadri, 2019). Meanwhile, the continuous advancement of PCR technology has further facilitated the development of POCT. This innovative approach integrates nucleic acid amplification into portable and automated platforms, enabling rapid on-site diagnosis outside central laboratories. Such progress has significantly reduced detection time and operational complexity(Nguyen et al., 2017). As a result, molecular diagnostics has become particularly valuable in resource-limited settings, emergency scenarios, and field applications, thereby supporting timely clinical and public health decision-making(Zhu et al., 2020).

In the early days, the reaction required manual addition of heat-labile DNA polymerases (such as Klenow fragment) and adjustment of temperature in each cycle. Later, the introduction of Taq DNA polymerase greatly simplified the PCR process, as this enzyme remains active at high temperatures, allowing for automated thermal cycling(Saiki, 1990). Then, it has undergone multiple improvements, such as nested PCR(He et al., 2024a), quantitative real-time PCR(qRT-PCR)(Spiess et al., 2023; Wen et al., 2023), multiplex PCR (mPCR)(Ewart et al., 2018), and digital PCR (dPCR)(Cao et al., 2017; Huggett et al., 2015; Wainman et al., 2024). Due to the development of microfluidic technology (e.g., continuous-flow PCR(Li et al., 2019), oscillating-flow PCR(Zhou et al., 2024) and natural convective PCR (Guo et al., 2025)) and the emergence of photonic PCR(You et al., 2020) have further promoted technological progress and application in this field (Table 1).

Nested PCR involves using two pairs of primers for two rounds of PCR, making it a highly specific and sensitive nucleic acid amplification technique(Green and Sambrook, 2019), but the process is complicated and less precise in quantitative analysis. Quantitative real-time PCR incorporates fluorescent substances into the reaction system and monitors the fluorescence changes in real time, thereby achieving quantitative analysis of the amplicons(Xu et al., 2024). Multiplex PCR can simultaneously amplify different target genes with multiple pairs of specific primers(Kim and Oh, 2021; Zhang et al., 2019). However, due to the competition among different primer pairs, it may lead to false positive PCR products. Digital PCR does not require the standard curves for quantification. It can realize precise absolute quantification of nucleic acid molecules by partitioning the sample into droplet emulsions (digital droplet PCR) (Hou et al., 2023) or physically isolated chambers (microchamber PCR) (Li et al., 2023a), which can realize single DNA molecule detection(Lei et al., 2021; Quan et al., 2018; Salipante and Jerome, 2020). Another emerging approach, photonic PCR, utilizes photothermal effects to accelerate thermal cycling, significantly reducing thermal inertia and enabling ultrafast amplification with lower energy consumption. This technology has achieved advantages such as ultrafast heating rates, high sensitivity, high specificity, low cost, and miniaturization, demonstrating the potential of next-generation ultrafast PCR technologies(Zhang et al., 2026). In recent years, PCR technology has given rise to many branches. However, most reports only focus on a certain branch technology, and there is still lack of comprehensive reports on the overall development of PCR. Therefore, we have summarized the latest progress of various PCR technologies, as well as their future development directions and prospects.