Nucleic Acid Detection Research Articles

Cis- and trans-cleavage activity of CRISPR-Cas9 and its new developments in diagnostics

CRISPR-Cas system is an adaptive immune system of prokaryotes against foreign invading viruses. Type II CRISPR-Cas9 system, as the earliest of the Class II systems to be discovered and applied to gene editing, had its new discovery of sharing trans-cleavage activity with other Class II systems not long ago. In this review, we summarize the molecular mechanism of cis- and trans-cleavage of target nucleic acids by the Class II type II CRISPR-Cas system, introduce the nucleic acid detection platforms developed based on its trans-cleavage activity, and compare them with those developed based on the type V, type VI, and type I CRISPR-Cas systems.

A rapid and naked-eye methicillin resistant Staphylococcus aureus screening method based on CRISPR/Cas12a and hybridization chain reaction

Methicillin-resistant Staphylococcus aureus (MRSA), a widely drug-resistant bacterium, poses a significant threat to global health. Current culturing and nucleic acid detection methods are time-consuming and require complex instruments, which do not meet the detection needs. Herein, we developed a rapid and visual MRSA detection method (MCFHCR) using ssDNA-functionalized magnetic beads as a trigger chain combined with the trans-cleavage activity of the Cas12a protein and fluorescence signal amplification of the hybridization chain reaction (HCR). MCFHCR is a signal-off platform for the detection of MRSA. In the absence of DNA targets, the trans-cleavage activity of Cas12a is inactivated, allowing HCR to proceed and form long double-stranded DNA, resulting in an increased fluorescent signal. In the presence of the DNA targets, the trans-cleavage activity of CRISPR/Cas12a is activated to cleave the trigger strand, failing HCR and leading to a decrease in the fluorescence signal. Combined with RPA, MCFHCR was completed within 35 min, achieving a limit of detection (LOD) of five copies/μL for mecA DNA and 8 CFU/mL for MRSA. In detecting clinical strains, MCFHCR demonstrated comparable performance to qPCR and drug sensitivity testing. Therefore, with its simple, rapid operation and convenient signal acquisition, MCFHCR shows significant practical applicability in detecting MRSA.

Inverted Molecular Beacons as Reaction-Based Hybridization Probes for Small-Molecule Activation by Nucleic Acid Inputs.

Nucleic acid-based hybridization probes that produce a fluorescent signal in the presence of DNA or RNA target molecules are essential components of nucleic acid computation and detection strategies. Commonly, the fluorescence activation of reporter gates is triggered by separation of a fluorophore-quencher pair upon target hybridization or strand displacement. In order to expand the utility of DNA computing by providing a chemical reaction as the ultimate output, reporter systems have been designed that carry reactive groups, which undergo a proximity-induced reaction upon oligonucleotide hybridization. The downside of published reporter gate designs is that they are composed of two separate, chemically modified oligonucleotides, which need to be taken into consideration when designing upstream circuits. Here, we report a novel hairpin-forming nucleic acid reporter probe that utilizes template-induced proximal reactivity to activate a small molecule in the presence of an unmodified nucleic acid input molecule. This DNA hairpin reporter gate consists of a duplex between a blocking strand and a hairpin-forming reporter strand. In the presence of input, the blocking strand is displaced, triggering hairpin formation allowing the proximity-driven templated activation of a vinyl ether-caged fluorophore by a tetrazine via an inverse electron demand Diels-Alder reaction. This new approach demonstrates robust small-molecule activation in vitro and in cells through logic operations in the presence of input DNA molecules.

Colorimetric Biosensor for Nucleic Acid Detection Using Hybridization Chain Reaction and Gold Nanoparticles: Effect of DNA Hairpin Sequence Modifications on Biosensor Performance.

In this work, a pair of DNA hairpins was specifically designed for detecting and amplifying a target DNA via hybridization chain reaction (HCR). The biosensor exploits the interactions between gold nanoparticles (AuNPs) and DNA hairpins as well as HCR products, resulting in salt-induced aggregation (SIA) of the AuNPs, observable through visible color changes and UV-vis spectral shifts. This study builds on previous findings regarding the optimal length and sequence of hairpin toeholds and stems, which provide AuNPs with greater protection against SIA. The HCR-AuNP biosensor was developed by applying design guidelines for HCR hairpins and incorporating features that enhance their adsorption onto AuNPs. Intriguingly, hairpins with a 12-14 bp stem and a mixed-base toehold (6-8 nt) containing only a single adenine base provided adequate AuNP stabilization and enabled efficient HCR without leakage. The limit of detection (LOD) for the final HCR-AuNP biosensor was 10.7 nM, with the AuNP color changing from red to blue in the presence of the target DNA. This biosensor is advantageous due to its ease of operation, isothermal nature, and rapid results, all achieved without the need for enzymes or expensive equipment.

Rapid and Ultrasensitive Detection of Staphylococcus aureus by a One-Pot System Integrating Pyrococcus furiosus Argonaute with Loop-Mediated Isothermal Amplification.

Staphylococcus aureus (S. aureus) is one of the most common pathogens associated with oral and maxillofacial space infections (OMSI), significantly impairing patients' quality of life and posing substantial public health risks. Traditional detection methods are usually time-consuming and operationally complex, which limits their applicability for rapid on-site detection. This study introduces a novel, ultrasensitive nucleic acid detection system combining loop-mediated isothermal amplification (LAMP) with Pyrococcus furiosus Argonaute (PfAgo) in a one-pot strategy. The combination of LAMP and PfAgo not only improves specificity and sensitivity but also effectively reduces the risk of aerosol contamination and false positives. The system detects the nuc gene of S. aureus, enabling rapid detection within 50 min with a limit of detection (LOD) of 100 Colony Forming Units (CFU)/ml for bacterial solutions and 10-5 ng/μl for plasmids. This system exhibited exceptional specificity by effectively differentiating S. aureus from other common OSMI bacteria, and was successfully applied to samples from animal OMSI infection models. This innovative one-pot system offers a rapid, reliable, and effective solution for S. aureus detection, with potential applications in human health and public safety.

Dual-Check CRISPR-SERS strategy for sensitively detecting Monkeypox DNA and its single-base mutated DNA.

This study presents a convenient and efficient dual-check strategy for detecting Monkeypox DNA utilizing the SERS (Surface-enhanced Raman Spectroscopy)-CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system.The Monkeypox plasmid DNA (mpDNA) is recognized by the CRISPR RNA (crRNA)-Cas12a protein complex, where crRNA encompasses a targeting complementary sequence. Upon recognition, the trans-cleavage activity of Cas12a is activated and trans-cleaves the probe DNA (Cy3-ssDNA) which is modified on Au nanoparticles (AuNPs). As the ssDNA strand is cleaved, Cy3 molecules are released in the solution, while the amount of Cy3 modified on the AuNPs decreases. The free Cy3 molecules are collected from the supernatant, and their SERS intensities are measured using the silver nanopillar (AgNRs) substrate. The mpDNA with varying concentration from 5nM to 0.5 fM can be quantitatively determined based on the SERS signals of free Cy3 and the collected nanotag. This strategy allows the detection of mpDNA with a concentration of 50 fM within 60min. Moreover, the strategy can successfully detect single-base mutated mpDNA. Owing to the non-specific trans-cleavage activity of the protein, this strategy can be adapted to various nucleic acid detection scenarios by designing complementary RNA and DNA sequences.

Expanding horizons of CRISPR applications beyond genome editing.

Expanding horizons of CRISPR applications beyond genome editing.

Molecular Encoded Beads with DNA Duplex Programming Fluorophore-Quencher Distance for Multiplexed Detection.

Currently, the leading-edge bead encoding technique adopts the dye-doped strategy, which requires the construction of an encoded bead library in advance and is limited by strict material and technical barriers. Here, we report a simple, versatile, and plug-and-play bead encoding strategy, which uses bead surface DNA encoding instead of a dye-doped strategy. This is a fluorophore-quencher (F-Q) distance encoded strategy through the different Q-labeled positions at the DNA duplex, thereby changing the FRET efficiency. The strategy can simultaneously realize target capture and encoding on a bead surface, which greatly simplifies the process and reduces detection costs. In this work, we encoded 9-plex barcodes through the strategy and decoded them by 2-channel fluorescence flow cytometry. It is a modular technique that enables seamless integration with additional signal amplifier units. Herein, we constructed three kinds of multiplexed nucleic acid detection models, including "direct capture", "capture and then amplify", and "amplify and then capture", that enrich the encoding toolbox for bead-based multiplexed detection.

CRISPR/Cas-Based Biosensing Strategies for Non-Nucleic Acid Contaminants in Food Safety: Status, Challenges, and Perspectives.

Non-nucleic acid targets (non-NATs), such as heavy metals, toxins, and pesticide residues, pose critical threats to food safety. Although CRISPR/Cas systems were initially developed for nucleic acid detection, recent advances have enabled their adaptation to non-NATs analysis by transducing target recognition into nucleic acid signals. Unlike previous reviews categorized by target type, this work establishes a mechanism-centric framework, systematically classifying non-NAT-to-nucleic acid signal conversion methodologies into three paradigms: (1) aptamer-based systems, (2) catalytic nucleic acid-based methods (e.g., DNAzymes), and (3) protein-mediated strategies (e.g., antibodies, transcription factors). When integrated with CRISPR/Cas, these systems achieve rapid, sensitive detection at picomolar (pM) levels without relying on chromatographic or spectroscopic instruments. Furthermore, we critically discuss challenges, including the limited diversity of recognition elements, inefficient signal conversion, and inflexible signal outputs, proposing solutions including synthetic-biology-driven bioreceptor design and artificial-intelligence-based data analysis. By bridging mechanistic principles with applications in complex food matrices, this review provides actionable insights to advance CRISPR-based tools for rapid, on-site, food safety monitoring.

SPARC: An Orthogonal Cas12a/Cas13a Dual-Channel CRISPR Platform for Reliable SNV Identification and Mutation Confirmation.

Rapid and reliable detection of single nucleotide variants (SNVs) is essential for accurate pathogen diagnostics, genetic mutation screening, and personalized medicine. However, existing CRISPR-based nucleic acid detection platforms frequently suffer from ambiguous signal interpretation, specificity limitations, and complex assay workflows. Herein, we introduce SPARC (specific and precise mutation recognition with Cas12a/Cas13a), a novel orthogonal dual-channel CRISPR assay that significantly enhances the SNV detection reliability. SPARC integrates Acidaminococcus sp. Cas12a (AsCas12a), which specifically detects a conserved region as an internal reference, with our recently identified DNA-activated Leptotrichia buccalis Cas13a (LbuCas13a), which exhibits exceptionally high intrinsic SNV specificity without requiring engineered crRNA mismatches. The orthogonal design uniquely resolves the common diagnostic ambiguity between genuine SNVs and target absence. Combined with recombinase polymerase amplification (RPA) and T7 exonuclease digestion, the SPARC platform achieved a sensitivity as low as 1 aM. We demonstrated the platform's robust clinical applicability through successful detection and accurate differentiation of hepatitis B virus (HBV) and clinically significant YMDD resistance mutations. This work presents an innovative and versatile CRISPR-based solution, highlighting substantial potential for advancing clinical diagnostics and precision medicine.

Analysis on the epidemic and genetic characteristics of varicella in Fengtai District, Beijing City, 2024-2025

This study analyzed the epidemiological characteristics and strain genotypic distribution of varicella in Fengtai District, Beijing, utilizing population-based surveillance data from March 2024 to February 2025 combined with laboratory nucleic acid detection and genotyping.We reported 522 varicella cases with male predominance (282 cases, 54.41%) and a majority aged >15 years (358 cases, 68.58%).A bimodal incidence pattern peaked in May and November, while 161 breakthrough infections (30.8%) occurred predominantly among students aged 6-20 years.The multivariate logistic regression model analysis showed that, compared to the 0-5 years age group, individuals aged 6-14 years (OR: 2.729, 95%CI: 1.083-6.88), 15-20 years (OR: 2.495, 95%CI: 1.158-5.378), and>20 years (OR: 5.382, 95%CI: 2.478-11.689) exhibited progressively higher odds of oderate-to-severe rash; females demonstrated a lower risk compared to males (OR: 0.485, 95%CI: 0.286-0.822); regarding vaccination status, recipients of one vaccine dose (OR: 0.301, 95%CI: 0.161-0.564) and two doses (OR: 0.203, 95%CI: 0.086-0.48) showed significantly reduced risks relative to unvaccinated individuals. Genotyping of 174 specimens identified 161 VZV-positive samples, with successful ORF22/38/62 sequencing in 142 samples confirming Clade 2 predominance (141 strains, 99.3%) and one Clade 5 strain; local isolates exhibited high vaccine-strain homology (ORF22 nucleotide:99.5%-100%, amino acid:99.3%-100%) with mutation sites partially overlapping other Chinese regions.

LUPP-LAMP: one-pot multiplatform universal LAMP assay for simultaneous detection and genotyping of DENV and ZIKV

Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne viruses that cause severe health problems upon infection, necessitating timely and accurate diagnostic testing for effective prevention and control of their transmission. In this study, a novel multiplex nucleic acids detection method was introduced based on ligation-based universal primer and probe loop-mediated isothermal amplification (LUPP-LAMP) for the simultaneous detection and genotyping of DENV and ZIKV. By employing ligation of universal primers and probes using splint DNA ligase, LUPP-LAMP simplifies primer design, eliminates primer dimer formation, and achieves high sensitivity and specificity at low temperatures, with a detection limit of 10 copies/reaction. The robustness of this strategy allows for precise differentiation even at low concentrations and in cases of co-infection, which is crucial for addressing early diagnostic challenges due to similar symptoms. LUPP-LAMP is further adapted for point-of-care testing (POCT) by integrating lateral flow test strips and fluorescence imaging, offering rapid and user-friendly results in resource-limited settings. This method is also applied to DENV genotyping (serotypes 1–4) using melting curve analysis (MCA) in cases of co-infection, aiding in epidemiological surveillance and intervention strategies. In summary, LUPP-LAMP represents a significant advancement in the detection and genotyping of DENV and ZIKV, offering a versatile tool for laboratory and POCT applications, thereby enhancing public health outcomes in the control of mosquito-borne diseases.Graphical

Progress in the application of isothermal amplification technology in the diagnosis of infectious diseases

Rapid detection of infectious diseases is critical for global public health prevention and control. However, the use of traditional molecular diagnostic methods, including PCR, has been limited because of their cumbersome procedures, complex equipment requirements, operation at different temperatures, and the level of expertise required for operation. Isothermal amplification technology (IAT) provides a rapid, sensitive, specific, simple and less costly method for diagnosing infectious diseases, which has led to revolutionary breakthroughs in molecular diagnostics. This paper summarizes recent progress in IAT technology, which focuses on the principles and applications of core technologies such as NASBA, LAMP, RPA, and RAA. In addition, the combination of IATs with the CRISPR/Cas system, which further revolutionizes nucleic acid detection technology, is explored in this review.

Wide Dynamic Range Multiplex Digital Clustered Regularly Interspaced Short Palindromic Repeat Chip for Rapid Detection and Absolute Quantification of Nucleic Acids.

Rapid detection and accurate quantification of multiplex nucleic acids are crucial for infectious diagnosis and pathogen identification, yet they still represent a significant challenge that deserves further attention and investigation. Herein, this research developed a wide dynamic range multiplex digital clustered regularly interspaced short palindromic repeat (CRISPR) (WDRM-dCRISPR) chip and established a rapid, sensitive, multiplexed nucleic acid detection (RSMND) platform that can achieve absolute quantification of multiplex nucleic acids with a limit of detection of 90 copies/mL within 30 min. The chip integrates the rapid amplification capability of RPA, the specific cleavage activity of CRISPR/Cas12a, and the portability of microfluidics, enabling precise and reliable digital absolute quantification of multiplex nucleic acids. As a proof of concept, this chip allows for multiplex absolute quantification of foodborne pathogens with a linear dynamic range of 9 × 101 to 1.8 × 106 copies/mL (R2 > 0.9999), high sensitivity, high specificity, and strong tolerance to background interference. The platform was successfully used for the analysis of foodborne pathogens in milk. Compared to qPCR, the RSMND platform exhibited superior accuracy and sensitivity in nucleic acid quantification. Overall, this study demonstrates that the platform provides a powerful tool for multiplex nucleic acid detection and holds significant potential for applications in point-of-care testing.

A novel assay incorporating CRISPR with RPA in a single pot for visual and accurate detection of Aphelenchoides besseyi in soybean

Aphelenchoides besseyi is a highly prevalent plant parasitic nematode which has a substantial impact and poses an economic risk to soybean cultivation, with a reported 2017 outbreak resulting in significant yield losses of up to 60%. Therefore, more effective control of this nematode depends on early and accurate nucleic acid detection. One of the promising detection approaches is to combine the CRISPR technology with the isothermal RPA. However, incorporating the RPA amplicon with the CRISPR ingredients in a single pot remains a significant challenge due to their incompatibility. In the current research, we propose a visual nucleic acid detection technique that takes less than thirty minutes and is highly sensitive for detecting A. besseyi. First, we conduct the RPA amplification, then we perform the CRISPR reaction using either a portable thermal cup or our body heat temperature. We tested this new assay on forty-four soybean samples exhibiting GSFR syndrome symptoms, and it effectively detected samples containing the A. besseyi. We designed three different ways for data collection and visualization to suit the requirements of various environments. Our findings confirm that the suggested new low-instrumentation portable single-pot RPA-CRISPR assay is durable, specific, and has strong nucleic acid sensitivity in the open field.

A rapid and ultrasensitive RPA-assisted CRISPR-Cas12a/Cas13a nucleic acid diagnostic platform with a smartphone-based portable device.

A rapid and ultrasensitive RPA-assisted CRISPR-Cas12a/Cas13a nucleic acid diagnostic platform with a smartphone-based portable device.

Fluorescence damping as primary interference mechanism of humic acids on qPCR quantification of SARS-CoV-2 in wastewater surveillance.

Fluorescence damping as primary interference mechanism of humic acids on qPCR quantification of SARS-CoV-2 in wastewater surveillance.

Sensitive and Visualized Detection of Hantavirus Using CRISPR/Cas12a Based on AutoCORDSv2 Design.

In recent years, detection technologies based on the CRISPR/Cas12a method have been extensively utilized in the fields of nucleic acid, enzyme, and macromolecule detection, thereby reinforcing their significant role in the detection landscape. Enhancing the simplicity of design, efficiency, and automation of the CRISPR/Cas12a detection system is essential for advancing its application in diagnostics. Recently, we developed an automated CRISPR/Cas12a design system named AutoCORDSv2. This system can process published genomic sequences of pathogenic bacteria in a high-throughput manner and automatically generate conserved and highly specific crRNA sequences, along with primer sequences for target amplification. This capability facilitates the specific and precise design of the CRISPR/Cas12a detection system. In this study, crRNAs targeting the Hantaan virus (HTNV) and Seoul virus (SEOV), as well as RT-PCR primers and RT-RPA primers, were designed using AutoCORDSv2. The experimental results demonstrated that the CRISPR/Cas12a system, automatically designed by AutoCORDSv2, was specific for the detection of both the HTNV and SEOV, with no cross-reactivity observed with other pathogens. The detection sensitivity reached 6 copies/μL (equivalent to 111 copies per amplification reaction), whether measured by a microplate reader or directly observed with the naked eye. The detection results for 50 samples were consistent with those obtained from commercial RT-qPCR kits, indicating high precision. Furthermore, the CRISPR/Cas12a system designed by AutoCORDSv2 can also be utilized for the development of a single-tube detection system with a sensitivity of 42 copies per reaction. This system combined with a 5-min extraction step and RT-RPA, further underscoring its potential for application.

An Aptamer-Conjugated AIEgen for Targeted Bioimaging and Photodynamic Therapy of Cancer Cells.

Photodynamic therapy (PDT) has emerged as a promising noninvasive treatment for cancer and other diseases. Aggregation-induced emission (AIE) photosensitizers are excellent candidates for enhancing the efficacy of image-guided PDT due to their unique properties. DNA-modified AIE luminogens (AIEgens) offer an effective approach for nucleic acid detection and biospecies sensing, but their applications in tumor cell imaging and image-guided PDT have been rarely reported. Herein, we designed a light-up probe, named ApApt, for targeted imaging and image-guided PDT of cancer cells. The probe was prepared by conjugating AIEgen with aptamer and displayed specific targeting ability to EpCAM (epithelial cell adhesion molecule)-positive cancer cells. ApApt exhibits good water solubility and shows very low emission in aqueous solution. However, it emits strong fluorescence as a result of the restricted intramolecular motion (RIM) activation of AIEgens, which is triggered by the specific binding between the aptamer SYL3C and EpCAM protein. Upon delivery to cancer cells, ApApt efficiently generates ROS for targeted PDT under light irradiation.

From lab to field: Innovative RPA‒CRISPR/Cas12a platform for early short-beak and dwarfism syndrome virus nucleic acids detection.

From lab to field: Innovative RPA‒CRISPR/Cas12a platform for early short-beak and dwarfism syndrome virus nucleic acids detection.