罗湖病毒(TiLV)RT-qPCR方法的建立
Establishment of reverse transcription-quantitative real-time PCR(RT-qPCR)for detection of tilapia lake virus
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摘要: 研究根据罗湖病毒(Tilapia lake virus,TiLV)编码RNA聚合酶的基因片段1设计1对特异性引物和探针,建立一种检测TiLV的实时荧光定量RT-PCR (RT-qPCR)方法,并对该检测方法进行了反应体系和条件优化。结果表明,优化的20 μL反应体系:2×Probe RT-PCR Master Mix 10 μL,QN Probe RT-Mix 0.2 μL,5 μmol/L的引物TiLV-qF、TiLV-qR、探针TiLV-qP各1 μL,模板1 μL,补充RNase-free water至20 μL。优化的反应条件:反转录45℃ 20 min;预变性95℃ 5 min;扩增95℃ 15 s,60℃ 1 min,40个循环。荧光收集设置在60℃退火延伸时进行。本文建立的RT-qPCR方法检测TiLV的特异性好,重复性好,灵敏度高,灵敏度为2.5×10-8 ng/μL,可为TiLV的监测和预防提供技术支撑。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.
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