Detection of Fowl Adenovirus Serotype 4 by Recombinase Aided Amplification Combined with CRISPR/Cas13a

Purpose To establish an efficient, sensitive and specific detection method for fowl adenovirus serotype 4 (FAdV-4).

Methods Recombinase aided amplification (RAA) was used in combination with the clustered regularly interspaced short palindromic repeats associated protein 13a (CRISPR-Cas13a) to design RAA primers and CRISPR RNA (crRNA) targeting the conserved region of the FAdV-4 Hexon gene. The RAA technology was employed to amplify the sample nucleic acid, followed by CRISPR-Cas13a fluorescence detection. The established method was evaluated for sensitivity, specificity, and consistency compared to the qPCR control method.

Results The method established in this study was performed at 37 ℃ with a minimum amplification copy number of 10¹ copies/μL, it was sensitive and had no cross-reactivity with avian-derived pathogens such as FAdV-1, FAdV-7, FAdV-8b, FAdV-9, FAdV-10, avian infectious laryngotracheitis virus, infectious bursal disease virus, infectious bronchitis virus, avian influenza subtype H9, newcastle disease virus. The detection rate of 30 clinical samples by this method was consistent with that of qPCR.

Conclusion The RAA-Cas13a method is simple, sensitive and specific for the detection of FAdV-4, and can be used for rapid detection and epidemiological surveillance of FAdV-4.