Bei Wang, Liwei Liu, Xiaoqing Wang, Kai Yang, Daojun Chen, Dongdong Yin
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引用次数: 0
Abstract
In this study, a novel nucleic acid detection method based on multienzyme isothermal rapid amplification (MIRA) combined with the Pyrococcus furiosus Argonaute (PfAgo) cleavage system (MIRA-PfAgo) was developed for the detection of Tembusu virus (TMUV), an important pathogen threatening the waterfowl industry. The method targets the highly conserved NS5 gene of TMUV. Target nucleic acids are rapidly enriched by MIRA under isothermal conditions at 39°C, followed by PfAgo-mediated cleavage guided by sequence-specific guide DNA (gDNA), enabling visual detection through fluorescence signal output. Systematic optimization identified 0.5 μM gDNA, 1 μM PfAgo, and 5 mM Mn²⁺ as the optimal reaction conditions. The assay exhibited excellent specificity for TMUV, with no cross-reactivity observed with common waterfowl pathogens, including H9 subtype avian influenza virus (AIV-H9), duck enteritis virus (DEV), goose parvovirus (GPV), Muscovy duck reovirus (MDRV), Newcastle disease virus (NDV), and duck circovirus (DuCV). The sensitivity was 10⁰ copies/μl. In the analysis of 21 clinical samples, the results of the MIRA-PfAgo assay were fully consistent with those obtained by RT-PCR. This method does not require complex thermal cycling equipment and combines high sensitivity, strong specificity, and operational simplicity, providing a reliable and practical technical tool for rapid on-site monitoring of TMUV.RESEARCH HIGHLIGHTSThis is the first study to utilize the MIRA-PfAgo detection assay for the detection of TMUV.The results are observable by the unaided eye.The developed assay potentially offers an alternative means for detection of TMUV, requiring minimal equipment.
期刊介绍：
Avian Pathology is the official journal of the World Veterinary Poultry Association and, since its first publication in 1972, has been a leading international journal for poultry disease scientists. It publishes material relevant to the entire field of infectious and non-infectious diseases of poultry and other birds. Accepted manuscripts will contribute novel data of interest to an international readership and will add significantly to knowledge and understanding of diseases, old or new. Subject areas include pathology, diagnosis, detection and characterisation of pathogens, infections of possible zoonotic importance, epidemiology, innate and immune responses, vaccines, gene sequences, genetics in relation to disease and physiological and biochemical changes in response to disease. First and subsequent reports of well-recognized diseases within a country are not acceptable unless they also include substantial new information about the disease or pathogen. Manuscripts on wild or pet birds should describe disease or pathogens in a significant number of birds, recognizing/suggesting serious potential impact on that species or that the disease or pathogen is of demonstrable relevance to poultry. Manuscripts on food-borne microorganisms acquired during or after processing, and those that catalogue the occurrence or properties of microorganisms, are unlikely to be considered for publication in the absence of data linking them to avian disease.
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