Currently, the mainstream technology for nucleic acid detection involves using polymerases to specifically synthesize target DNA before performing detection. Polymerase chain reaction (PCR) technology was proposed by Mullis^[25]in 1985. Its core principle is to use an in vitro enzymatic synthesis reaction catalyzed by DNA polymerase, combined with temperature cycling controlled by a thermocycler, to achieve exponential amplification of the target DNA sequence. This molecular-level thermodynamic control mechanism enables target DNA fragments to be amplified by 10⁶to 10⁹-fold within 2 hours. With its high sensitivity, high specificity, and high efficiency, PCR technology has been widely applied in fields such as basic life science research, molecular medical diagnostics, agriculture, forensic science, and environmental monitoring, greatly advancing technological progress in these areas^[26]. Building on PCR technology, researchers have subsequently developed a variety of derivative techniques. For example, by combining reverse transcriptase (RT) with PCR technology, reverse transcription PCR (RT-PCR) was created, thereby expanding the application scope of PCR in RNA virus detection, biomedical research, and clinical diagnostics. In addition, in the 1990s, Vogelstein and Kinzler^[27]proposed the concept of digital PCR (dPCR). dPCR achieves absolute quantification of nucleic acids by partitioning the reaction system into micro-units and combining this with fluorescence signal detection. Compared with traditional PCR, dPCR offers higher sensitivity, specificity, and precision, and it demonstrates significant advantages, particularly in the detection of low-abundance targets and in copy number variation analysis^[28-29].

The implementation of PCR technology relies on Taq DNA polymerase, which is heat-resistant and enables efficient DNA amplification under high-temperature conditions. In contrast, isothermal amplification technologies have gradually gained attention due to their simplicity, speed, and efficiency. This method does not require complex temperature-cycling equipment and can achieve rapid nucleic acid amplification at a constant temperature, making it well-suited for use in resource-constrained settings. Nucleic acid sequence-based amplification (NASBA) primarily relies on the synergistic action of AMV reverse transcriptase, T7 RNA polymerase, and ribonuclease H (RNase H). A pair of specific primers is used to amplify RNA under constant 42°C conditions^[30]. Rolling circle amplification (RCA) is an isothermal DNA amplification technique that uses short primers and a DNA polymerase with strand-displacement activity (such as φ29 DNA polymerase) to generate long single-stranded DNA molecules from a circular DNA template at a constant temperature^[31]. RCA is characterized by its high efficiency and isothermal nature, enabling rapid amplification of target DNA^[32]; however, its efficiency is lower when amplifying larger circular DNA templates, and it depends on single-primer binding and extension. Multiple-primer rolling circle amplification (MP-RCA) achieves efficient amplification through the use of random primers and φ29 DNA polymerase, enhancing both amplification efficiency and selectivity^[33]. Building on the principles of MP-RCA, the field of single-cell sequencing has further developed multiple displacement amplification (MDA) technology, which uses random primers and φ29 DNA polymerase under mild conditions to perform whole-genome amplification of single-cell genomes, thereby further improving amplification uniformity and coverage^[34]. This technology has driven the advancement of single-cell sequencing and provides strong technical support for research in areas such as cellular heterogeneity, developmental biology, and disease mechanisms^[35]. In addition, loop-mediated isothermal amplification (LAMP)^[36-39]and recombinase polymerase amplification (RPA) are widely used in pathogen detection, genotyping, and other fields, and are particularly well suited for POCT applications^[40-43].

In addition, various other innovative detection methods have achieved precise analysis of pathogen nucleic acids through unique technologies. Nucleic acid mass spectrometry is an advanced nucleic acid detection method that combines multiplex PCR, high-throughput chips, and time-of-flight mass spectrometry (MALDI-TOF-MS). By precisely measuring mass differences among nucleic acid molecules, this technique can rapidly and efficiently detect genetic variants, polymorphisms, methylation patterns, and other characteristics^[44-45]. The technology offers advantages such as high throughput, high sensitivity, ease of operation, and cost-effectiveness^[46-47]. In situ hybridization (ISH) or fluorescence in situ hybridization (FISH) is a technique used to detect specific nucleic acid sequences in cell or tissue sections. It achieves localization and detection of target genes or transcripts by enabling specific hybridization between labeled nucleic acid probes and the target nucleic acid sequences^[48-49]. These technological advances provide multidimensional complementarity with isothermal amplification systems: isothermal amplification focuses on rapid nucleic acid enrichment, mass spectrometry excels in fine variant analysis, and in situ hybridization enables spatially resolved detection.

In recent years, real-time nucleic acid detection technologies based on the CRISPR-Cas12 system have garnered widespread attention. CRISPR-Cas12a (formerly known as Cpf1), as a unique member of the Type II CRISPR-Cas system, has demonstrated significant advantages in the field of nucleic acid detection^[56](Fig. 2). Compared with other Cas proteins, Cas12a not only accurately recognizes and cleaves double-stranded DNA target sequences under the guidance of crRNA, but also activates its non-specific single-stranded DNA cleavage activity upon target binding. This unique trans-cleavage property provides an ideal molecular tool for developing highly sensitive nucleic acid detection methods^[57].

DETECTR (DNA endonuclease-targeted CRISPR trans reporter) technology was developed in 2018 by the Doudna team^[58]. The underlying principle is that, after the Cas12a protein, guided by gRNA, recognizes and cleaves the target double-stranded DNA, it activates its non-specific single-stranded DNA cleavage activity, thereby cleaving a fluorescently labeled single-stranded DNA reporter molecule and generating a detectable signal^[59]. This method offers rapid detection with high sensitivity and specificity, and the results are visually intuitive, but it is limited to detecting DNA samples only and requires the design of specific guide sequences. HOLMES (One-hour low-cost multipurpose highly efficient system) technology was proposed in 2018 by Li et al.^[60]. It similarly exploits the dual cleavage activity of Cas12a under gRNA guidance, featuring rapid detection with high sensitivity and specificity, but is also limited to the detection of DNA samples^[61]. HOLMESv2 represents a further upgrade, employing a heat-stable Cas12b protein that can specifically distinguish single nucleotide polymorphisms (SNPs) and accurately detect viral RNA, human cell mRNA, and circular RNA. By combining LAMP amplification technology, HOLMESv2 enables one-step quantitative nucleic acid detection under isothermal conditions, effectively preventing cross-contamination between samples. In addition, it can be combined with bisulfite treatment to precisely quantify the degree of DNA methylation, demonstrating extremely broad application prospects in molecular diagnostics and epigenetic research^[62]. The sPAMC (Suboptimal PAM of Cas12a-based test with enhanced flexibility, speed, sensitivity and reproducibility) technology developed by Lu et al.^[63] utilizes a suboptimal PAM sequence and combines isothermal amplification (RPA) with Cas12a-mediated detection to achieve rapid, highly sensitive, and specific detection of SARS-CoV-2 RNA. Furthermore, Shi et al.^[64] developed a DNA diagnostic technology called CONAN (CRISPR-Cas-only amplification network). This method integrates Cas12a’s stringent target recognition, helicase activity, and trans-cleavage activity into a positive feedback loop to achieve isothermal amplification-based detection of genomic DNA. The method boasts advantages such as single-enzyme operation, one-step procedure, real-time detection, high sensitivity, and single-base specificity. In optimizing traditional Cas12a-based detection systems, Yue et al.^[65], by optimizing reaction conditions and employing droplet-based Cas12a detection with dual crRNA targeting, significantly improved detection sensitivity, quantitative analysis capability, and specificity compared to the single-crRNA approach, providing a more precise tool for molecular diagnostics. Meanwhile, Ding et al.^[66] developed an all-integrated dual CRISPR-Cas12a (AIOD-CRISPR) detection technology that uses a dual crRNA design to overcome the limitations imposed by PAM sequences in traditional Cas12a-based detection. Coupled with a "one-pot" strategy, this technology significantly enhances detection sensitivity, enabling the detection of extremely low copy numbers of SARS-CoV-2 nucleic acid.

Although the Cas12a enzyme has traditionally been used for DNA detection, the SAHARA (Split activator for highly accessible RNA analysis) technology ingeniously leverages Cas12a to enable RNA detection. The core principle is that when a short DNA strand binds near the PAM sequence, the distal end of the PAM sequence can bind to RNA, thereby activating Cas12a’s trans-cleavage activity and enabling efficient RNA detection. This technology not only allows the use of the Cas12a enzyme for detecting RNA substrates but also enhances sensitivity to mutations at specific sites^[67]..

Following the Cas12a technology, CRISPR-Cas13a, as a representative member of the Type VI CRISPR-Cas system, has demonstrated great potential in the field of point-of-care nucleic acid testing due to its precise RNA recognition and cleavage capabilities^[68-70]. Guided by crRNA, Cas13a can specifically recognize target RNA and, upon activation, exhibits non-specific cleavage of surrounding single-stranded RNA, providing an ideal mechanism for signal amplification. In 2017, the Zhang lab^[71]first proposed the SHERLOCK technology, which leverages this "collateral cleavage" property. By designing fluorescently labeled RNA reporter molecules, SHERLOCK enables highly sensitive detection of target RNA, thereby significantly advancing the development of POCT applications. Subsequently, Liu et al.^[72]developed the FIND-IT technology, which uses Cas13a and the Csm6 nuclease in tandem. Under amplification-free conditions and in combination with chemically stable activators, this technology enables rapid detection of SARS-CoV-2viral RNA, with a sensitivity of approximately 30 molecules per microliter and a detection time of within 20 minutes. In addition, Fozouni et al.^[4]proposed an amplification-free CRISPR-Cas13a detection technology that, when combined with a mobile phone microscope, enables high-sensitivity, low-cost on-site detection.

In addition to detection technologies based on Cas12a and Cas13a, other CRISPR systems also demonstrate unique advantages in nucleic acid detection. The CRISDA (CRISPR-Cas9-triggered nicking endonuclease-mediated strand displacement amplification) technology was invented in 2018 by Zhou et al.^[73]It leverages the precise recognition and cleavage capabilities of the Cas9 nickase (Cas9n) to generate a single-strand nick on the non-target strand of the target DNA, and through DNA polymerase–mediated strand displacement amplification, achieves highly sensitive and specific detection of the target DNA. The LEOPARD (Leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection) technology was proposed in 2021 by Jiao et al.^[74]This technology reprograms tracrRNA (Rptr) to specifically hybridize with the target RNA, forming an atypical crRNA that guides the Cas9 nuclease to recognize and cleave the complementary DNA sequence. Another technique that uses Rptr’s specific binding to the target RNA to activate the Cas9 protein is AGATHA (Atypical gRNA-activated transcription halting alarm). After activation, Cas9 binds to a specific AGATHA DNA, blocking transcription by disrupting the secondary structure at its 3’ end, while continuously generating Broccoli RNA aptamers that bind to fluorescent substrates to produce a signal. By designing multiple Rptr variants, the AGATHA technology achieves highly specific, strongly amplified multiplex RNA detection, demonstrating rapid and precise detection capabilities^[75].

Among the numerous detection technologies described above, most are limited to detecting a single target, making it difficult to meet the demand for simultaneous multi-target analysis in complex biological systems. Multi-target detection technologies aim to overcome the limitation of traditional methods, which can only target a single analyte, thereby addressing the need for concurrent analysis of multiple indicators in complex biological systems. Microfluidic chip-based technologies enable the combination of multiple targets and stepwise reactions by precisely controlling ultra-small reaction volumes, providing an ideal platform for high-throughput, multiplexed detection.

CARMEN (Combinatorial arrayed reactions for multiplexed evaluation of nucleic acids) technology was developed by the Blainey team in 2020. It achieves self-organized pairing of nanoliter-volume reaction systems in the form of droplets within a micro-well array, and uses fluorescence microscopy to read the color codes of the droplets, thereby identifying sample–assay pairs (Fig. 4a). By merging droplets under an electric field to initiate the reaction, CARMEN can simultaneously detect thousands of sample–crRNA target pairs, ensuring both high sensitivity and high specificity while effectively reducing detection costs^[87]. Building on CARMEN technology, mCARMEN represents a further upgrade: by implementing multiplexed nucleic acid testing for multiple samples and pathogens on commercial microfluidic chips, it significantly streamlines clinical workflows and enhances its potential for broader applications in the POCT field^[88](Fig. 4b). Xu et al.^[89]developed a multiplex nucleic acid detection technology called MiCaR (Microfluidic space coding for multiplexed nucleic acid detection via CRISPR-Cas12a and recombinase polymerase amplification) (Fig. 4c). This technology integrates microfluidic spatial coding and, using a single fluorescent probe, can simultaneously detect 30 nucleic acid targets, completing highly sensitive and specific rapid detection within 40 minutes. In addition, MiCaR technology demonstrates broad application prospects in rapid pathogen diagnostics. With its simple operation, low cost, and suitability for POCT, it provides an efficient solution for multiplex nucleic acid testing in resource-limited settings. Another technology, the Microfluidic dual-droplet device (M-D3), combines CRISPR-Cas12a with RPA technology, using pressure/vacuum-driven generation of microdroplets to separately encapsulate Cas12a/crRNA, fluorescent reporter molecules, and samples, thereby enabling rapid and sensitive simultaneous detection of HPV16 and HPV18 (Fig. 4d). With its high sensitivity, rapid detection capability, and extremely low sample consumption, this technology exhibits great application potential in the field of multiplex nucleic acid POCT, particularly suitable for clinical management and large-scale population screening^[90].

In clinical sample testing, pathogen concentrations are often extremely low, and traditional nucleic acid detection methods typically require amplification prior to detection. However, amplification-free nucleic acid detection technology based on microfluidic chips, through innovative design, enables direct detection of low-concentration samples, offering a new solution for clinical diagnosis.

Tian et al.^[91]developed the "ultra-local Cas13a assay" technology, which confines the RNA-triggered Cas13a catalytic system within cell-sized reactors. By leveraging microdroplet technology to simultaneously enhance the local concentrations of both the target RNA and the reporter molecule, this approach enables absolute digital single-molecule quantification of multiple RNA molecules. With its high sensitivity, specificity, and the advantage of not requiring nucleic acid amplification, this technology shows great promise for broad applications in clinical diagnostics, pathogen detection, and liquid biopsies. In particular, its simplicity and low cost make it an ideal tool for viral load detection. Similarly, Yue et al.^[65]developed the "microdroplet Cas12a assay" technology, which optimizes Cas12a reaction parameters and confines the reaction within microdroplets, thereby successfully achieving absolute single-molecule-level quantification of DNA while avoiding the risk of cross-contamination associated with traditional amplification methods (Figure 5a).

The STAMP (Self-digitization through automated membrane-based partitioning) technology employs a different strategy^[92].This technology uses automated membrane partitioning to separate the reaction system into tiny, independent reaction units by mixing the sample with the detection reagent and then loading the mixture onto a polycarbonate membrane, thereby enabling amplification-free, highly sensitive, and specific absolute quantification of HIV-1 viral RNA (Figure 5b). Meanwhile, the “digital microfluidic hybridization detection technology” developed by Mou et al.^[93]integrates picoliter-scale droplet technology with magnetic microbeads to achieve highly sensitive HPV nucleic acid detection without amplification. This technology is easy to operate, low in cost, and fast in detection, making it suitable for POCT and providing an efficient solution for viral load testing in resource-limited settings (Figure 5c). Notably, the amplification-free digital CRISPR/Cas13a detection technology developed by Wang et al.^[94]innovatively combines magnetic bead capture and enrichment with micro-well dispersion detection, ensuring that each micro-well contains only one magnetic bead, thereby significantly enhancing detection sensitivity. This method can complete detection in just 50 minutes, avoiding the complex procedures and potential biases associated with traditional amplification methods, and provides an efficient and reliable solution for detecting low-concentration samples (Figure 5d). These amplification-free detection technologies not only simplify the operational process and reduce costs but also open up new possibilities for POCT.

Among the detection methods mentioned above, most rely on external equipment to complete the testing process. In the field of microfluidic POCT, reducing dependence on specialized equipment is a crucial direction for enhancing the portability and practicality of testing.

The MAV chip technology innovatively designs a magnetic bead-single-stranded DNA-platinum nanoparticle (BDNP) complex as a reporting molecule. This system leverages the strong binding affinity between biotin and streptavidin, as well as the stable coordination bond between thiol and platinum nanoparticles, to achieve visual quantitative detection without the need for fluorescence (Figure 6). Upon activation of the Cas12a enzyme, its trans-cleavage activity releases platinum nanoparticles, which then catalyze the decomposition of hydrogen peroxide to produce oxygen, driving the movement of red ink in the chip’s channels. The test result can be intuitively determined by observing the distance the ink has moved^[95]. This technology not only enables visual quantitative detection of target nucleic acids but also eliminates the reliance on fluorescent labeling in traditional Cas12a assays, significantly enhancing the convenience and practicality of the detection process.