Optimization of reaction conditions for NDV detection by RT-RAA-LFD. (A) Screening of reaction temperature. The band appeared in the test area at 37°C and 39°C but not at 33°C, 35°C, and 41°C. As per the criteria for the best reaction conditions (obvious test line and lowest reaction temperature), 37°C was determined as the best reaction temperature. (B) Screening of reaction time. There was band in the test area at 23 and 25 min but not at 13, 15, 17, 19, and 21 min. As per the criteria for the best reaction conditions (obvious test line and shortest reaction time), 23 min was determined as the best reaction time (C) Screening of primer and probe concentration. 10,000, 5,000, 2,500, and 1,250 nmol/L test area showed bands, and no bands were found in 625 nmol/L test area. As per the criteria for the best reaction conditions (obvious test line and lowest primer or probe concentration), 1,250 nmol/L was determined as the best primer and probe concentration. Abbreviations: NDV, Newcastle disease virus; RT-RAA-LFD, reverse transcription recombinase–aided amplification-lateral flow dipstick.