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

Author:
Wang, Wenjing, Wang, Chunguang, Zhang, Zichuang, Zhang, Peng, Yao, Shanshan, Liu, Jingru, Zhai, Xianghe, Zhang, Tie
Source:
Poultry science 2020 v.99 no.10 pp. 4809-4813
ISSN:
0032-5791
Subject:
Avian orthoavulavirus 1, Gallid alphaherpesvirus 1, Infectious bronchitis virus, Influenza A virus, chickens, cross reaction, disease diagnosis, epidemiological studies, fluorescent dyes, low density lipoprotein, nucleotide sequences, quantitative polymerase chain reaction, rapid methods, throat, thymidine kinase, China
Abstract:
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.
Agid:
7007531