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The red‐crowned crane (Grus japonensis), a Class I protected animal in China, inhabits Northeast Asia, including China, Russia, and Japan. As sex‐monomorphic birds, red‐crowned cranes cannot be directly distinguished between females and males through observation. Molecular methods are accurate and stable for sex identification in birds and are widely used in zoos and farms. With the development of isothermal techniques, recombinase‐aided amplification (RAA) has provided novel insights into bird sexing owing to its low equipment dependence and rapid amplification. Advancements in the Pyrococcus furiosus Argonaute (PfAgo) biosensor have facilitated clinical detection. In this study, an innovative sex identification system was developed by integrating RAA and PfAgo in red‐crowned cranes. The RAA‐PfAgo system identified both females and males with remarkable accuracy. Via proper design of primers set, gDNA and probe, the RAA‐PfAgo system can complete visual detection, with detection limits between 0.35 ng/μL and 0.035 ng/μL under optimal conditions. The test samples exhibited strong green fluorescence in females, whereas no fluorescence was observed in males under blue light. The results of RAA‐PfAgo in the field were consistent with those obtained using conventional PCR. This study provides a high degree of rapidity, accuracy, and sensitivity for the sex identification of red‐crowned cranes. This study presents an innovative approach for sex identification in the red‐crowned crane (Grus japonensis) by combining recombinase‐aided amplification (RAA) with the Pyrococcus furiosus Argonaute (PfAgo) biosensor. The RAA‐PfAgo system enables rapid, accurate, and highly sensitive molecular sexing of these sex‐monomorphic birds, with a key advantage being the visual detection of fluorescence. The system achieves a detection limit as low as 0.35 ng/μL and demonstrates consistent performance with traditional PCR methods.

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, induces severe vomiting and acute watery diarrhea in unweaned piglets. The pig industry has suffered tremendous financial losses due to the high mortality rate of piglets caused by PEDV. Consequently, a simple and rapid on-site diagnostic technology is crucial for preventing and controlling PEDV. This study established a detection method for PEDV using recombinase-aided amplification (RAA) and Pyrococcus furiosus Argonaute (PfAgo), which can detect 100 copies of PEDV without cross-reactivity with other pathogens. The entire reaction of RAA and PfAgo to detect PEDV does not require sophisticated instruments, and the reaction results can be observed with the naked eye. Overall, this integrated RAA-PfAgo cleavage assay is a practical tool for accurately and quickly detecting PEDV. Key points • PfAgo has the potential to serve as a viable molecular diagnostic tool for the detection and diagnosis of viral genomes • The RAA-PfAgo detection technique has a remarkable level of sensitivity and specificity • The RAA-PfAgo detection system can identify PEDV without needing advanced equipment

Abstract Escherichia coli O157:H7 (E. coli O157:H7) is a significant foodborne pathogen that causes severe illnesses worldwide. In this study, a novel detection system combining recombinase polymerase amplification and Pyrococcus furiosus Argonaute (PfAgo) was developed for the rapid and sensitive identification of the conserved rfbE gene. The system integrates dual-fluorescence probes labelled with 6-carboxyfluorescein (FAM) and X-rhodamine (ROX) for parallel signal verification, which enhances detection reliability and minimises the risk of false positives. The optimised system achieved a limit of detection of 6.14 × 101 copies/μl, 100% specificity and repeatability, with a coefficient of variation below 10%. Validation using artificially contaminated milk and bottled water samples demonstrated complete agreement with the results of polymerase chain reaction, yielding 100% accuracy. With high sensitivity and specificity, this system enables rapid, accurate point-of-care screening for food safety and clinical diagnostics, offering broad potential in public health surveillance and foodborne pathogen control. Graphical Abstract Graphical Abstract

Monkeypox virus (MPXV), the pathogen responsible for the infectious disease monkeypox, causes lesions on the skin, lymphadenopathy, and fever. It has posed a global public health threat since May 2022. Highly sensitive and specific detection of MPXV is crucial for preventing the spread of the disease. Pyrococcus furiosus Argonaute (PfAgo) is an artificial DNA-guided restriction cleavage enzyme programmable with 5′-phosphorylated ssDNA sequences, which can be developed to specifically detect nucleic acids of pathogens. Here, a PfAgo-based system was established for the detection of MPXV-specific DNA targeting the F3L gene. A short amplicon of 79 bp could be obtained through a fast PCR procedure, which was completed within 45 min. Two 5′-phosphorylation guide DNAs were designed to guide PfAgo to cleave the amplicon to obtain an 18 bp 5′-phosphorylation sequence specific to MPXV, not to other orthopoxviruses (cowpox, variola, and vaccinia viruses). The 18 bp sequence guided PfAgo to cleave a designed probe specific to MPXV to emit fluorescence. With optimized conditions for the PfAgo-MPXV system, it could be completed in 60 min for the detection of the extracted MPXV DNA with the limit of detection (LOD) of 1.1 copies/reaction and did not depend on expensive instruments. Successful application of the PfAgo-MPXV system in sensitively detecting MPXV in simulated throat swabs, skin swabs, sera, and wastewater demonstrated the system’s good performance. The PfAgo platform, with high sensitivity and specificity established here, has the potential to prevent the spread of MPXV.

A  Pyrococcus furiosus Argonaute ( Pf Ago)-based biosensor is presented for alkaline phosphatase (ALP) activity detection in which the ALP-catalyzed hydrolysis of 3′-phosphate-modified functional DNA activates the strand displacement amplification, and the amplicon mediates the fluorescent reporter cleavage as a guide sequence of Pf Ago. Under the dual amplification mode of Pf Ago-catalyzed multiple-turnover cleavage activity and pre-amplification technology, the developed method was successfully applied to ALP activity determination with a detection limit (LOD) of 0.0013 U L −1 (3σ) and a detection range of 0.0025 to 1 U L −1 within 90 min. The Pf Ago-based method exhibits satisfactory analytic performance in the presence of potential interferents and in complex human serum samples. The proposed method shows several advantages, such as rapid analysis, high sensitivity, low-cost, and easy operation, and has great potential in disease evolution fundamental studies and clinical diagnosis applications. Graphical abstract

PfAgo, a thermophilic Argonaute nuclease from Pyrococcus furiosus, is widely used in various fields due to its high DNA‐guided DNA cleavage activity. However, its high‐temperature‐dependent cleavage activity largely restricts its applications in moderate‐temperature scenarios. In this study, PfAgo is engineered for cold adaptation based on its ternary complex structure and the attributes of cold‐adapted enzymes, yielding a series of variants with better performance at moderate temperatures. Among those, mPfAgo (K617G, L618G) exhibits significantly promoted cleavage activity at 37 °C and a wider catalytic temperature range of 30–95 °C. Its high‐temperature cleavage activity is also greatly improved, enabling its application in DNA detection with attomolar sensitivity in the presence of Mg2+. Additionally, mPfAgo shows versatile cleavage activities, including DNA cleavage guided by 5′OH‐gDNA, 5′P‐gDNA, or 5′COOH‐gDNA, as well as RNA cleavage with 5′OH‐gDNA, 5′P‐gDNA, 5′P‐gRNA, or 5′COOH‐gDNA as guides. Further analysis through far‐UV CD spectra and DSF indicates that mPfAgo has a more flexible structure than wild‐type PfAgo. Furthermore, this established strategy is applied to engineer TtdAgo, likewise obtaining its variants with enhanced moderate‐temperature activity and expanded substrate spectrum. In summary, this work provides a novel method for the rational design of thermophilic Agos, thereby greatly expanding their application scopes. This work presents a strategy for engineering thermophilic Ago proteins to obtain wide catalytic temperature range and broad substrate spectrum. This strategy is applied to engineer PfAgo and TtdAgo, and the biochemical properties of the engineered PfAgo are systematically characterized. The study further elucidates the structural differences involved. Overall, this work provides an alternate approach to obtaining desired Ago proteins.

Conventional Escherichia coli (E. coli) detection methods are often time-consuming, while molecular diagnostics typically rely on expensive thermocycling equipment. To address these limitations, this study developed a rapid nucleic acid detection method for E. coli by integrating multienzyme isothermal rapid amplification (MIRA) with Pyrococcus furiosus Argonaute (PfAgo)-mediated targeted cleavage. The conserved housekeeping gene phoA was selected as the target, and specific MIRA primers and 5′-phosphorylated guide DNAs (gDNAs) were designed accordingly. After exponential amplification at 39 °C, the amplicons were specifically recognized by PfAgo at 95 °C, leading to molecular beacon cleavage and generation of a detectable FAM fluorescence signal. Among the tested guides, gDNA6 showed the highest cleavage efficiency. Optimal performance was achieved with 1 μM PfAgo, 0.5 μM gDNA, and 5 mM MnCl2. The optimized MIRA-PfAgo assay demonstrated a limit of detection of 100 copies/μL, comparable to qPCR, and exhibited high specificity with no cross-reactivity against common enteric pathogens. In 28 clinical and environmental samples, the assay results were fully consistent with those of qPCR. Overall, the MIRA-PfAgo platform provides a rapid, sensitive, and specific approach for E. coli detection, demonstrating strong potential to reduce reliance on precision thermal cyclers for resource-limited applications.

The advancement in CRISPR-Cas biosensors has transmuted the detection of plant viruses owing to their rapid and higher sensitivity. However, false positives and restricted multiplexing capabilities are still the challenges faced by this technology, demanding the exploration of novel methodologies. In this study, a novel detection system was developed by integrating reverse transcriptome (RT) techniques with recombinase polymerase isothermal amplification (RPA) and Pyrococcus furiosus Argonaute (PfAgo). The RT-RPA-PfAgo system enabled the simultaneous detection of rice ragged stunt virus (RRSV), rice grassy stunt virus (RGSV), and rice black streaked dwarf virus (RBSDV). Identifying targets via guide DNA without being hindered by protospacer adjacent motif sequences is the inherent merit of PfAgo, with the additional advantage of it being simple, cost-effective, and exceptionally sensitive, with detection limits between 3.13 and 5.13 copies/µL, in addition to it effectively differentiating between the three distinct viruses. The field evaluations were also in accordance with RT-PCR methods. The RT-RPA-PfAgo system proved to be a robust, versatile, highly specific, and sensitive method with great potential for practicality in future plant virus diagnostics.

Background Malaria remains a serious public health problem worldwide, particularly in Africa. Resistance to antimalarial drugs is an essential issue for malaria control and elimination. Currently, polymerase chain reaction (PCR) combined with Sanger sequencing is regarded as the gold standard for mutation detection. However, this method fails to meet the requirements of point-of-care testing (POCT) because of its time-consuming, expensive instruments and professional dependence. To support this strategy, we developed a novel diagnostic platform that combines recombinase polymerase amplification (RPA) with the Pyrococcus furiosus argonaute ( Pf Ago) protein and was designed to detect gene mutations related to antimalarial drug resistance. The Pfcrt haplotypes CVMNK and CVIET of chloroquine resistance (CQR) were used as examples and were assessed in this study. Methods By meticulously designing strategies, RPA primers, guide DNAs, and probes were screened, the reaction was optimized, and the resulting parameters were employed to ascertain the genotype of Pfcrt . The recombinant plasmids pUC57/Pfcrt -CVIET and pUC57/Pfcrt -CVMNK were constructed and diluted for sensitivity detection. The pUC57/Pfcrt -CVIET plasmid mixture was added to the pUC57/Pfcrt -CVMNK plasmid mixture in different additions to configure several specific proportions of mixed plasmid mixtures. The RPA- Pf Ago platform was used, and the mixed plasmid was detected simultaneously via nest-PCR (nPCR) and Sanger sequencing. The platform was then evaluated on 85 clinical samples and compared with Sanger sequencing. Results The entire process achieves the key mutation Pfcrt -CVMNK/CVIET genotype identification of CQR within 90 min. The platform achieved 1.8 × 10 4 copies/μL sensitivity and could detect as little as 3% CVIET in mixed plasmids, which is a higher sensitivity than that of Sanger sequencing (5%). Notably, the platform shows 100% concordance with the gold standard method when 85 clinical samples are tested. The sensitivity and specificity were 100% for the 85 clinical samples. Conclusions This study established an RPA- Pf Ago platform for genotyping the key mutation Pfcrt -CVMNK/CVIET of CQR. This method can rapidly produce reliable results and avoid the disadvantages of nPCR with sequencing. This approach has the characteristics of a short operation time, low device dependence, and a good match to the POCT strategy, suggesting that the platform can be easily applied locally or on site. Graphical abstract

To develop and validate dual detection platforms integrating recombinase polymerase amplification (RPA) with Pyrococcus furiosus Argonaute (PfAgo) for the rapid and specific identification of serotype 2. The conserved gene was selected as the molecular target. Key RPA parameters and PfAgo reaction conditions were systematically optimized, including temperature, reaction time, MnCl concentration, gDNA design and probe concentration. Specificity and sensitivity were evaluated using plasmid dilutions and multiple serotypes together with other common swine pathogens. A total of 41 clinical samples were also tested and compared with the national standard PCR assay (GB/T 19915.3-2005). Two assay formats were established: real-time fluorescence system (RPA-PfAgo-RTF) and lateral flow dipstick system (RPA-PfAgo-LFD). The RPA-PfAgo-RTF assay achieved a detection limit of 10 copies/μL, while the RPA-PfAgo-LFD assay detected 10 copies/μL. Both formats showed high specificity without cross-reactivity. Among 41 field samples, six were SS2-positive, and results showed 100% agreement with the reference PCR method. Total detection time for either assay was < 1 h. Both assay formats provide rapid, sensitive, and accurate tools for SS2 detection suitable for laboratory use and on-farm point-of-care testing.