关键词: 罗湖病毒（TiLV）, 特异性引物, 探针, 实时荧光定量RT-PCR（RT-qPCR）

Abstract: To establish a fluorescent RT-PCR method for the detection of tilapia lake virus (TiLV), according to the segment 1 codified for the RNA polymerase enzyme, a pair of specific primers and probe was designed and a reverse transcription-quantitative real-time PCR (RT-qPCR) method was established. The reaction system and conditions were optimized. The results showed that the optimized 20 μL reaction system contained 10 μL 2×Probe RT-PCR Master Mix, 0.2 μL QN Probe RT-Mix, 5 μmol/L of all 1 μL primer TiLV-qF, TiLV-qR and probe TiLV-qP of working concentration, 1 μL template, and RNase-free water was added to 20 μL. The optimized reaction conditions were as follows: reverse transcription 45℃ for 20 min; Pre-denaturation at 95℃ for 5 min; 40 cycles of amplification were performed at 95℃ for 15 s and 60℃ for 1 min. Fluorescence collection was set at 60℃ for annealing extension. The RT-qPCR method of TiLV was superior in specificity, repeatability and sensitivity. The sensitivity value was 2.5×10^-8 ng/μL. It provided technical support for monitoring and prevention of TiLV.

Key words: tilapia lake virus(TiLV), specific primer, probe, reverse transcription-fluorogenic quantitative PCR