Recombinase-aided amplification-lateral flow dipstick assay-a specific and sensitive method for visual detection of avian infectious laryngotracheitis virus

Abstract

The purpose of this study was to explore a specific, simple, and sensitive method for diagnosis of avian infectious laryngotracheitis virus. Recombinase-aided amplification (RAA) and lateral flow dipstick (LFD) were combined for labeling the optimized RAA probe with 6-carboxyfluorescein (FAM) and the 5'-end of the downstream primer with biotin, respectively. By optimizing the reaction time, temperature, and primer concentration of RAA, a RAA-LFD assay, which could be used for detection of infectious laryngotracheitis, was established. After the specificity and sensitivity test, the target gene fragments could be amplified by RAA-LFD assay in 20 min under isothermal conditions (37°C), and the amplification products could be visually observed and determined by LFD within 3 min. There was no cross-reaction with nucleic acids of other avian pathogens, the lowest detectable limit of RAA-LFD was 10² copies/μL, and the sensitivity of this method was 100 times higher than that of conventional PCR with the lowest detectable limit of 10⁴ copies/μL. The results showed that RAA-LFD assay was highly sensitive, easy to use, and more suitable for clinical detection.

Keywords: avian infectious laryngotracheitis virus; lateral flow dipstick; recombinase-aided amplification.

Figure 1

Screening and determination of the best reaction conditions for the detection of ILTV by RAA–LFD. (A) Optimization of RAA–LFD reaction temperature for ILTV detection. The amplified reaction works effectively within a wide range of reaction temperatures from 35°C to 39°C. A light colored band appeared in the detection area at 35°C. With the increase of temperature, the amplification product increased, and the band color darkened. The amplification product decreased, and the band color became lighter at 41°C. The optimum reaction temperature was determined to be 37°C. (B) Optimization of RAA–LFD reaction time for ILTV detection. After 20 min, the amplified reaction works effectively. After 14 min, the band appeared in the detection area, and the band of the detection line became clearer after 20 min. Considering the experimental time and the reliability of the results, the best reaction time was determined to be 20 min. (C) Optimization of primer and probe concentrations of RAA–LFD assay for ILTV detection. The amplified reaction works effectively within a wide range of primer concentration of 1,250 nmol/L–10,000 nmol/L. When the concentration of primer is 1,250 nmol/L, the detection line was clear and the required concentration was the lowest, so the best concentration of the primer and probe was determined to be 1,250 nmol/L. Abbreviations: ILTV, infectious laryngotracheitis virus; LFD, lateral flow dipstick; RAA, recombinase-aided amplification.

Figure 2

Specificity test by RAA–LFD assay for ILTV detection. It was found that the test line and quality control line appeared on dipstick at the same time for the RAA products of ILTV, whereas only the quality control line appeared for the products of other viruses (IB virus, ND virus, and AIV) and the negative control (N). Abbreviations: IB, ILTV, infectious laryngotracheitis virus; LFD, lateral flow dipstick; ND, Newcastle disease; RAA, recombinase-aided amplification.

Figure 3

Compare the sensitivities of the 3 methods for ILTV detection. (A) Sensitivity of RAA–LFD assay for ILTV detection. N, negative control; 1–6, template concentration of 10⁰–10⁵ copies/μL. The results showed that the lowest detectable limit was 10² copies/μL for RAA–LFD assay. (B) Sensitivity of PCR assay for ILTV detection. M, marker; 1–8, concentration of 10⁷–10⁰ copies/μL; 9, negative control. The results showed that the lowest detectable limit was 10⁴ copies/μL for PCR. (C) Sensitivity of RFQ-PCR assay for ILTV detection. 0, negative control; 1–7, 10⁰–10⁶ copies/μL. The results showed that the lowest detectable limit was 10 copies/μL for RFQ-PCR. Abbreviations: ILTV, infectious laryngotracheitis virus; LFD, lateral flow dipstick; RAA, recombinase-aided amplification; RFQ-PCR, real-time fluorescence-based quantitative-PCR.