Pyrococcus furiosus argonaute combined with loop-mediated isothermal amplification for rapid, ultrasensitive, and visual detection of fowl adenovirus serotype 4

Zhaorong Yu; Daoming Shi; Yanli Dong; Ying Shao; Zhe Chen; Fanyu Cheng; Yu Zhang; Zhenyu Wang; Jian Tu; Xiangjun Song

doi:10.1016/j.psj.2024.103729

Pyrococcus furiosus argonaute combined with loop-mediated isothermal amplification for rapid, ultrasensitive, and visual detection of fowl adenovirus serotype 4

Poult Sci. 2024 Apr 3;103(7):103729. doi: 10.1016/j.psj.2024.103729. Online ahead of print.

Authors

Zhaorong Yu¹, Daoming Shi¹, Yanli Dong¹, Ying Shao¹, Zhe Chen¹, Fanyu Cheng¹, Yu Zhang¹, Zhenyu Wang¹, Jian Tu¹, Xiangjun Song²

Affiliations

¹ Anhui Province Engineering Laboratory for Animal Food Quality and Bio-safety, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, PR China; Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, PR China.
² Anhui Province Engineering Laboratory for Animal Food Quality and Bio-safety, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, PR China; Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, PR China. Electronic address: sxj@ahau.edu.cn.

Abstract

Since 2015, an outbreak of an infectious disease in broilers caused by fowl adenovirus serotype 4 (FAdV-4) has occurred in China, resulting in substantial economic losses. Rapid, accurate, and specific detection are significant in the prevention and control of FAdV-4. In this study, an FAdV-4 detection method combining loop-mediated isothermal amplification (LAMP) and Pyrococcus furiosus Argonaute (PfAgo) was established. Specific primers, guide DNAs (gDNAs), and molecular beacons were designed to target a conserved region of the FAdV-4 hexon gene. After optimizing the reaction conditions, the minimum detection of this assay could reach 5 copies. It only amplified FAdV-4, and there was no cross-reactivity with other pathogens. The assay took about only 50 min, and the results could be visualized with the naked eye under ultraviolet or blue light, getting rid of specialized instruments. This novel LAMP-PfAgo assay was validated by using 20 clinical samples and the results were identical to gold-standard real-time polymerase chain reaction method. In summary, the LAMP-PfAgo assay established in the paper provides a rapid, reliable, convenient, ultra-sensitive and highly specific tool for the on-site detection and clinical diagnosis of FAdV-4.

Keywords: detection; fowl adenovirus serotype 4; loop-mediated isothermal amplification; pyrococcus furiosus argonaute.