Search Results (89)

Chicken infectious anemia virus (CIAV) is a major immunosuppressive pathogen that causes significant economic losses to the global poultry industry. Conventional detection methods for CIAV are limited by poor timeliness, high equipment requirements, and insufficient sensitivity. To address these challenges, this study developed a novel one-tube integrated RPA-CRISPR/Cas12a assay targeting the highly conserved VP3 gene of CIAV for rapid and accurate detection. The performance of the assay was comprehensively evaluated in terms of sensitivity, specificity, and repeatability. Its clinical utility was assessed by testing 80 clinical suspected samples, with quantitative real-time PCR (qPCR) serving as the reference method. The results showed that the limit of detection (LoD) of the developed method was 10 copies/reaction, comparable to that of qPCR. No cross-reactivity with common avian pathogens was observed. The intra- and inter-assay coefficients of variation (CV%) for the time to threshold (Tt) were both below 10%. In clinical sample detection, the assay achieved a total coincidence rate of 97.5%, with a sensitivity of 100% and specificity of 96% relative to qPCR. In conclusion, the RPA-CRISPR/Cas12a assay developed in this study offers rapid detection, high sensitivity and specificity, operational simplicity, low equipment dependency, and excellent repeatability. It provides a practical tool for early and rapid diagnosis of CIAV, clinical sample screening in grassroots veterinary laboratories, and on-site epidemiological surveillance in poultry farms, holding significant potential for the precise prevention, control, and eradication of CIAV in the poultry industry. Full article

Rapid detection of Pseudomonas aeruginosa and its virulence factor ExoU is essential for improving patient outcomes. In this study, a CRISPR–Cas13a-based diagnostic assay combined with recombinase polymerase amplification (RPA) was developed to detect P. aeruginosa and the exoU gene in blood samples. The assay demonstrated robust amplification, with detection limits of 6 log₁₀ and 8 log₁₀ CFU/mL in Luria–Bertani medium and blood, respectively, and a 100% specificity, without cross-reactivity against four Gram-negative bacilli and Staphylococcus aureus reference strains. The utilisation of a fluorescence-based readout facilitated unambiguous discrimination between P. aeruginosa and P. aeruginosa/exoU⁺ isolates vs. negative controls. In conclusion, these results support the potential of RPA/CRISPR-Cas13a diagnostics for the rapid identification of P. aeruginosa and its ExoU virulence factor. Further optimisation and clinical validation are required to confirm its utility as a bedside diagnostic test, where its application would speed up clinical decisions in the treatment of these infections. Full article

Anthracnose, caused by Colletotrichum gloeosporioides, is a globally distributed phytopathogenic disease with a broad host range, posing a serious threat to the healthy growth of forest trees, including Cunninghamia lanceolata. To enable rapid and accurate on-site detection of this pathogen, this study developed a comprehensive field-deployable detection method. The approach integrates the EZ-D method (EASY DNA extraction) for rapid nucleic acid extraction with recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system. A specific target gene, designated Cglo6922, was identified for the detection of C. gloeosporioides. The entire detection process can be completed within approximately 25 min, comprising a 10-min isothermal RPA at 39 °C followed by a 15-min Cas12a cleavage reaction. Specificity evaluation showed that the method successfully detected two C. gloeosporioides isolates derived from different hosts, while no cross-reactivity was observed against a panel of 32 other isolates, including ten Colletotrichum species, eight Phytophthora species, six Pythium species, seven Fusarium species, and one Botryosphaeria dothidea isolate, demonstrating robust species-level specificity. Sensitivity testing revealed that the method achieved a limit of detection (LOD) of 10 pg/μL of genomic DNA for C. gloeosporioides. Furthermore, by incorporating the EZ-D rapid extraction method (requiring only one minute for DNA extraction at a cost of approximately $0.03 USD per sample), target nucleic acid was successfully extracted from artificially inoculated Cunninghamia lanceolata branch samples and proved compatible with the RPA-CRISPR/Cas12a detection system. In conclusion, this study establishes a novel field-deployable detection method for C. gloeosporioides that is rapid, cost-effective, highly specific, and highly sensitive, providing a powerful tool for point-of-care testing (POCT) of this disease. Full article

Background/Objectives: Rapid identification of foodborne pathogens is of high practical significance because it enables prompt epidemiological response, timely patient management, and effective sanitary control of food products. In this study, we developed an integrated molecular platform combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology for rapid, sensitive, and specific detection of Salmonella enterica. Methods: Four virulence genes (sirA, stn, siiD, and pagN) were selected as targets to ensure reliable pathogen identification. Reaction conditions were optimized using the Moraxella bovoculi Cas12a (MbCas12a) nuclease. The study focused on integrating isothermal amplification with a custom-engineered hardware solution for visual fluorescence detection. Results: The developed method demonstrated sensitive and specific detection, with no cross-reactivity to non-target microorganisms. Optimization allowed for a substantially reduced assay time of approximately 30 min. As a result, a portable fluorescence visualization approach was developed, featuring a 3D-printed housing and an integrated ultraviolet light source for direct visual fluorescence detection. This allows rapid differentiation of samples without specialized laboratory equipment, making it suitable for field applications. Conclusions: The combination of isothermal amplification, MbCas12a-based detection, and the portable fluorescence visualization approach provides a versatile platform for rapid diagnostics and food safety monitoring. This approach has strong potential to improve public health outcomes and enhance the resilience of food supply chains by enabling accessible, field-deployable pathogen detection. Full article

Microbial contamination in aquatic environments poses severe threats to aquaculture sustainability, ecological balance and public health. Traditional culture-based detection methods, while standardized, are time-consuming and labor-intensive, often failing to meet the urgent need for rapid on-site monitoring required to prevent disease outbreaks and manage water quality effectively. By integrating latest research advances (2020–2025), this study reviews advances in rapid detection technologies for aquatic microorganisms, including the evolution of nucleic acid amplification strategies, with a focused comparison of the analytical sensitivity and field deployability of quantitative polymerase chain reaction (qPCR) and mainstream isothermal amplification techniques (loop-mediated isothermal amplification, LAMP; recombinase polymerase amplification, RPA). Furthermore, this study reports on the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) systems as next-generation diagnostic tools, highlighting their integration with microfluidic Lab-on-a-Chip (LOC) platforms to achieve attomolar sensitivity. We also consider the application of portable nanopore sequencing for real-time pathogen identification and the growing role of Artificial Intelligence (AI) in analyzing complex diagnostic datasets. Advanced molecular methods have achieved significant reductions in time consumption—from days to less than one hour—while challenges regarding sample preparation and environmental matrix inhibition remain. The future of aquatic monitoring lies in integrated, automated systems that combine the specificity of CRISPR-Cas diagnostics with the connectivity of IoT-enabled biosensors. Comparative analysis indicates that isothermal amplification methods (LAMP, RPA) coupled with CRISPR-Cas systems offer the optimal balance of sensitivity, speed, and field deployability for point-of-care aquaculture diagnostics, while qPCR/dPCR remain indispensable for quantitative regulatory applications. We propose a structured technology selection framework to guide researchers and practitioners in choosing appropriate detection modalities based on specific sensitivity, cost, throughput, and deployment requirements. Full article

Bacillus and Paenibacillus species are common and widely distributed microorganisms in food systems, often implicated in food spoilage and quality issues. Bacillus subtilis, in particular, has been associated with gas production and package bulging in seasoned foods. In this study, we developed a rapid and visual detection method for Bacillus subtilis by integrating (Recombinase Polymerase Amplification) RPA with (Clustered Regularly Interspaced Short Palindromic Repeats) CRISPR/Cas12a technology (designated as RPA-CRISPR/Cas12a). Specific RPA primers and probes were designed based on the conserved gyrB gene of Bacillus subtilis. Two sets of crRNA were designed according to the number of T-rich PAM sites on the RPA-amplified target sequence, and the reaction conditions were optimized in combination with the CRISPR/Cas12a trans-cleavage detection technology. Under optimized conditions, the crRNA3 guide (with a TT-rich PAM site) demonstrated superior cleavage efficiency compared to crRNA2 (TTT-rich PAM), while crRNA1 (TTTT-rich PAM) showed no activity. The assay achieved a detection limit of 150 pg/μL for genomic DNA and 5.5 CFU/mL for bacterial suspensions within 10 min at 37 °C. The method exhibited high specificity and sensitivity, providing a robust tool for early and on-site detection of Bacillus subtilis in food products. Full article

Bacilus cereus and Pseudomonas fluorescens are major foodborne psychrotrophic bacteria posing global health and economic risks. B. cereus has a 23.8% food prevalence worldwide. P. fluorescens is a leading cause of spoilage in refrigerated products. Their rapid detection is crucial for food safety. However, existing detection methods often rely on open-tube operations, risking aerosol contamination. In this study, we developed two independent one-tube RPA-CRISPR/Cas12a visual detection assays for B. cereus and P. fluorescens. Using a physical separation design, the recombinase polymerase amplification (RPA) and CRISPR/Cas12a detection were pre-assembled in a single reaction tube. After incubation, a brief centrifugation combined the components for enclosed detection. This step is compatible with portable mini-centrifuges. The assays can be completed within 40 min at 37 °C, with results visualized directly under blue light. Both assays demonstrated good specificity against six common non-target pathogens. The visual detection limits were 5.1 × 10¹ copies/μL for B. cereus and 2.1 × 10¹ copies/μL for P. fluorescens. Each assay was applied to 14 types of real-world food samples (naturally contaminated and uncontaminated, confirmed by PCR), achieving 100% concordance with conventional PCR. The one-tube assays are tailored for psychrotrophic bacteria in refrigerated foods. They minimize aerosol contamination risk and provide a reliable solution for on-site cold-chain food safety monitoring. Full article

Viral hemorrhagic fevers (VHFs) are highly lethal diseases that often present non-specific, influenza-like symptoms in their early stages, making clinical recognition and differentiation from other febrile illnesses difficult. This overlap underscores the critical need for diagnostic tests that are both sensitive and specific. Point-of-care (POC) diagnostic tests are an invaluable tool for detecting and controlling the spread of pathogens that threaten public health, such as VHFs, as these require fast, accurate diagnostics to ensure biosafety and appropriate mobilization of resources during outbreaks. Current molecular and serological diagnostic tests, while efficient and effective, lack the characteristics required of a POC test (POCT) to quickly and easily respond to a VHF outbreak while maintaining a low cost. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tests have gained popularity as POCTs due to their inherent attractive qualities, including high sensitivity and specificity, adaptability, low cost, quick turnaround time, and ease of use. However, studies on the development of CRISPR-based POC diagnostic tests for VHFs are limited. This review summarizes the current CRISPR-based POCTs for VHFs, including Ebola virus (EBOV), Lassa virus (LASV), Dengue virus (DENV), and Crimean–Congo hemorrhagic fever virus (CCHF). The isothermal pre-amplification methods commonly paired with CRISPR-based tests, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), are also discussed. Full article

Duck plague virus (DPV), a highly contagious α-herpesvirus in the livestock and poultry environment, poses a significant threat to the healthy growth of ducks, potentially causing substantial economic losses. Effective control of DPV requires the development of specific diagnostic tools. A new fluorescent biosensor (R-C-CHA) was developed to detect virulent strains of DPV. It combined recombinase polymerase amplification (RPA), a CRISPR/Cas12a system, and catalytic hairpin assembly (CHA) for signal enhancement. The RPA primers were specifically designed to target the conserved DPV-CHv UL2 gene region, allowing for the rapid, efficient amplification of the target nucleic acids in isothermal conditions. The CRISPR/Cas12a system was used for sequence-specific recognition, activating its lateral cleavage activity. Furthermore, the CHA cascade reaction was utilized for enzyme-free fluorescent signal amplification. The results showed that the R-C-CHA biosensor completed the detection process in 40 min with a detection limit of 0.02 fg/μL, which was an approximate five-fold improvement compared to traditional RPA-CRISPR/Cas12a biosensors. The R-C-CHA biosensor also demonstrated perfect consistency with clinical detection and polymerase chain reaction (PCR) diagnosis, highlighting its strong potential for rapid detection in livestock and poultry farming settings. Full article

As an emerging threat to global food security, wheat blast necessitates the development of a rapid and field-deployable detection system to facilitate early diagnosis, enable effective management, and prevent its further spread to new regions. In this study, we aimed to validate and improve a Recombinase Polymerase Amplification coupled with PCRD lateral flow detection (RPA-PCRD strip assay) kit for the rapid and specific identification of Magnaporthe oryzae pathotype Triticum (MoT) in field samples. The assay demonstrated exceptional sensitivity, detecting as low as 10 pg/µL of target DNA, and exhibited no cross-reactivity with M. oryzae Oryzae (MoO) isolates and other major fungal phytopathogens under the genera of Fusarium, Bipolaris, Colletotrichum, and Botrydiplodia. The method successfully detected MoT in wheat leaves as early as 4 days post-infection (DPI), and in infected spikes, seeds, and alternate hosts. Furthermore, by combining a simplified polyethylene glycol-NaOH method for extracting DNA from plant samples, the entire RPA-PCRD strip assay enabled the detection of MoT within 30 min with no specialized equipment and high technical skills at ambient temperature (37–39 °C). When applied to field samples, it successfully detected MoT in naturally infected diseased wheat plants from seven different fields in a wheat blast hotspot district, Meherpur, Bangladesh. Training 52 diverse stakeholders validated the kit’s field readiness, with 88% of trainees endorsing its user-friendly design. This method offers a practical, low-cost, and portable point-of-care diagnostic tool suitable for on-site genomic surveillance, integrated management, seed health testing, and quarantine screening of wheat blast in resource-limited settings. Furthermore, the RPA-PCRD platform serves as an early warning modular diagnostic template that can be readily adapted to detect a wide array of phytopathogens by integrating target-specific genomic primers. Full article

Hepatitis B virus (HBV) persists in infected hepatocytes through covalently closed circular DNA (cccDNA), a stable episomal form that serves as the transcriptional template for viral replication. Accurate and sensitive quantification of intrahepatic cccDNA is crucial for evaluating antiviral therapies, particularly those targeting a functional cure. Here, we report the development of a novel, cccDNA-specific detection system combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a-based fluorescence detection. We designed and validated CRISPR RNAs (crRNAs) targeting HBV cccDNA-specific regions conserved across genotypes A–D. Reaction conditions for both RPA and Cas12a detection were optimized to enhance sensitivity, specificity, and accuracy. The system reliably detected as few as 10 copies of cccDNA-containing plasmid per reaction and showed no cross-reactivity with non-cccDNA forms in serum or plasma, indicating assay specificity. When applied to liver tissue samples from 10 HBV-infected and 6 non-HBV patients, the RPA-CRISPR/Cas12a assay exhibited a high sensitivity (90%) and a strong correlation with qPCR results (R² = 0.9155), confirming its accuracy. In the conclusion, the RPA-CRISPR/Cas12a system provides a robust, cost-effective, and scalable platform for sensitive and specific quantification of intrahepatic HBV cccDNA. This method holds promises for research and high-throughput therapeutic screening applications targeting cccDNA clearance. Full article

Early detection of high-risk human papillomavirus (HPV), particularly HPV16 E7, is critical for cervical cancer prevention. Here, we report a novel, portable, and instrument-free biosensing platform that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated detection on a microfluidic paper-based analytical device (μPAD) for colorimetric, visual readout of double-stranded DNA (dsDNA). The μPAD features seven functional zones, including lyophilized RPA and CRISPR reagents, and immobilized streptavidin and anti-FAM antibodies for signal generation. Upon target recognition, Cas12a’s trans-cleavage activity releases biotinylated-FAM-labeled reporters that form a sandwich complex with gold nanoparticle (AuNP)-conjugated anti-FAM antibodies, producing a visible red signal at the test zone. The gray value of the colorimetric signal correlates linearly with target concentration, enabling the quantitative detection of HPV16 E7 dsDNA down to 100 pM within 60 min. The assay demonstrated high accuracy and reproducibility in spiked samples. By combining isothermal amplification, CRISPR specificity, and paper-based microfluidics, this platform offers a rapid, low-cost, and user-friendly solution for point-of-care HPV screening in resource-limited settings. This work advances the integration of CRISPR diagnostics with μPAD, paving the way for scalable point-of-care molecular diagnostics beyond HPV. Full article

The widespread circulation of Eurasian avian-like H1N1 (EA H1N1) swine influenza virus poses significant zoonotic and pandemic risks worldwide. However, current diagnostic methods are difficult to deploy in the field, as they generally require specialized laboratory infrastructure and trained personnel. Here, we present a novel dual-signal detection platform that combines reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR/Cas12a technology for rapid, on-site EA H1N1 detection. We established an integrated one-tube assay by designing and optimizing RT-RPA primers targeting a conserved region of the hemagglutinin (HA) gene, together with engineered CRISPR/Cas12a guide RNAs exhibiting high specificity. The platform incorporates two complementary readout modes: real-time fluorescence monitoring and visual colorimetric detection using a smartphone. The assay shows excellent analytical specificity, with no cross-reactivity observed against other swine influenza virus subtypes or common swine pathogens, (including CSFV, PRRSV, PEDV, PCV, TGEV, and RV). The detection limit is 2 copies/μL, and the entire procedure can be completed within 30 mins using simple portable equipment. When evaluated on 86 clinical samples, the assay demonstrated 94.18% concordance with RT-qPCR. Compared with conventional diagnostic methods, this RT-RPA–CRISPR/Cas12a assay offers greater convenience and cost-effectiveness. Its strong potential for field-based rapid testing underscores promising application prospects in swine influenza surveillance and control programs. Full article

Paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic intestinal disease in ruminants. PTB is difficult to diagnose, control, and eradicate, leading to substantial economic losses. Thus, sensitive and specific detection methods are urgently required. crRNA and primers targeting the MAP ATPase FtsK gene were designed for recombinase polymerase amplification (RPA) and nested PCR. Fecal DNA was amplified using RPA or nested PCR, purified with Tris-saturated phenol-chloroform-isoamyl alcohol, and detected via CRISPR-Cas12a. Moreover, signals were read using a qPCR instrument, fluorescence reader, or lateral flow strips. RPA–CRISPR-Cas12a and nested PCR–CRISPR-Cas12a assays were optimized and validated on 50 clinical samples and 7 MAP cultures. The limits of detection were 1 × 10⁻¹⁰ μg/μL for RPA–CRISPR-Cas12a and 1 × 10⁻¹⁴ μg/μL for nested PCR–CRISPR-Cas12a. Efficient cleavage of the ssDNA reporter occurred at DNA concentrations of ≥1 × 10⁻⁴ μg/μL, producing a strong fluorescent signal. All three detection methods showed perfect agreement with reference assays across both sample sets. This study presents the first integration of RPA or nested PCR with CRISPR-Cas12a for MAP detection, enabling rapid, specific, and highly sensitive diagnosis. Flexible detection options allow adaptation to available resources and bacterial loads, supporting practical use in PTB control. Full article

Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor’s response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application. Full article