Research Note: Rapid detection of avian infectious laryngotracheitis virus with real-time fluorescence-based recombinase-aided amplification.

Wang, Wenjing; Wang, Chunguang; Zhang, Zichuang; Zhang, Peng; Yao, Shanshan; Liu, Jingru; Zhai, Xianghe; Zhang, Tie

In this study, specific primers and fluorescent probes were designed to target the thymidine kinase (TK) gene sequence of avian infectious laryngotracheitis virus (ILTV). Through specificity and sensitivity tests, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method for detecting ILTV was established. The results showed that the method was specific and could be used to accurately detect ILTV, and there was no cross-reaction with Newcastle disease virus (NDV), avian influenza virus (AIV), or infectious bronchitis virus (IBV). Real-time fluorescence-based recombinase-aided amplification had high sensitivity, and the lowest detectable limit (LDL) for ILTV could reach 10 copies/μL, 1,000 times more sensitive than conventional PCR (104 copies/μL), to rival that of real-time fluorescence-based quantitative PCR (RFQ-PCR) (10 copies/μL). This method and RFQ-PCR were used to detect 96 samples of chicken throat swabs with ILT initially diagnosed in clinic from the north of China, and the coincidence rate of the 2 methods was 100%. The RF-RAA reaction required only 20-30 minutes to completing, and its sensitivity was much higher than that of conventional PCR. Real-time fluorescence-based recombinase-aided amplification is similar to RFQ-PCR and has the advantages of specificity, sensitivity, and high efficiency, so it is suitable for early clinical detection and epidemiological investigation of ILTV.