Development and application of a novel recombinase polymerase amplification-Pyrococcus furiosus argonaute system for rapid detection of goose parvovirus.

Rapid and accurate detection of goose parvovirus (GPV) is crucial for controlling outbreaks and mitigating their economic impact on the poultry industry. This study introduces recombinase polymerase amplification combined with the Pyrococcus furiosus argonaute (RPA-PfAgo) system, a novel diagnostic platform designed to address the limitations of traditional GPV detection methods. Capitalizing on the rapid DNA amplification of RPA and stringent nucleic acid cleavage by the PfAgo protein, the RPA-PfAgo system offers high specificity and sensitivity in detecting GPV. Our optimization efforts included primer and probe configurations, reaction parameters, and guided DNA selection, culminating in a detection threshold of 102 GPV DNA copies per microlitre. The specificity of the proposed method was rigorously validated against a spectrum of avian pathogens. Clinical application to lung tissues from GPV-infected geese yielded a detection concordance of 100%, surpassing that of qPCR and PCR in both rapidity and operational simplicity. The RPA-PfAgo system has emerged as a revolutionary diagnostic modality for managing this disease, as it is a promising rapid, economical, and onsite GPV detection method amenable to integration into broad-scale disease surveillance frameworks. Future explorations will extend the applicability of this method to diverse avian diseases and assess its field utility across various epidemiological landscapes.

Development of an optimized RPA-PfAgo detection system for MTHFR C677T polymorphism genotyping.

This study introduces an efficient RPA-PfAgo detection system for the MTHFR C677T polymorphism, proposing a potential strategy to simplify the genotyping process. By optimizing recombinase polymerase amplification (RPA) with Pyrococcus furiosus Argonaute (PfAgo) nucleases, we achieved DNA amplification at a constant temperature. The assay was fine-tuned through meticulous primer and guide DNA selection, with optimal conditions established at 2.0 µL of MgAc, a reaction temperature of 42 °C, and a 10-minute reaction time for RPA. Further optimization of the PfAgo cleavage assay revealed the ideal concentrations of MnCl2, guide DNA, molecular beacon probes, the PfAgo enzyme, and the RPA product to maximize sensitivity and specificity. Clinical validation of 20 samples showed 100% concordance with Sanger sequencing, confirming the method's precision. The RPA-PfAgo system is a promising tool for on-site genotyping, with broad applications in personalized medicine and disease prevention.

Establishment of a simple, sensitive, and specific ASFV detection method based on Pyrococcus furiosus argonaute.

African swine fever (ASF), which is casued by African swine fever virus (ASFV), is a fatal infectious disease of pigs that results in significant losses to the breeding industry. Therefore, screening and detection are crucial for the control and prevention of the ASFV. Argonaute is a new detection tool that is being extensively used due to its high specificity and programmability. This study reports on a new nucleic acid assay method, termed REPD, which uses recombinase-aided amplification and restriction endonuclease-assisted Pyrococcus furiosus argonaute (PfAgo) detection. One-pot REPD was developed for the detection of ASFV. The one-pot REPD could detect a single copy of ASFV nucleic acid and showed no cross-reactivity with other pathogens. Detection in clinical samples was 100% consistent with the results of real-time PCR analysis. The results showed that the one-pot REPD assay is convenient, sensitive, specific, and potentially adaptable to the detection of ASFV. In summary, this study highlights a novel method that can be employed for the detection of pathogens.