罗湖病毒实时荧光定量RT-PCR检测方法的建立

Development of Real-time Fluorescence Quantitative RT-PCR Detection Method for Tilapia Lake Virus

【目的】建立一种快速、灵敏、特异的罗非鱼(Oreochromis spp)罗湖病毒定量检测方法。【方法】根据罗湖病毒节段8保守区设计一对特异性引物,建立检测罗湖病毒的SYBR GreenⅠ实时荧光定量RT-PCR方法,并对该方法的特异性、灵敏度和重复性进行了评价。【结果】标准曲线在2.5×10^8~2.5×10^0拷贝数之间有良好的线性关系(相关系数R2=0.999),检测限为2.5×100个拷贝。试验内及试验间变异系数分别为0.11%~0.43%与0.54%~1.13%,重复性强;对水生动物其他病毒和细菌均无扩增反应,有很好的特异性。【结论】新建立的罗湖病毒实时荧光定量PCR检测方法特异性好、灵敏度高、重复性强,可用于TiLV的早期的快速诊断、流行病学调查和防控。

【Objective】To establish a rapid, sensitive and specific quantitative detection method for Tilapia Lake Virus(TiLV).【Method】To design a pair of specific primers based on the conserved region of Lo Wu virus segment 8 and to establish SYBR Green I real-time quantitative RT-PCR for detection of Ti LV.The specificity, sensitivity, and repeatability of the method were evaluated.【Results】The standard curve had a good linear relationship between 2.5×10~8 and 2.5×10~0 copy number(correlation coefficient R^2 = 0.999), and the detection limit was 2.5×10~0 copy numbers.The intra-and inter-assay coefficients of variation were 0.11%-0.43% and 0.54%-1.13%, respectively.The repeatability was strong.There was no amplification reaction for other viruses and bacteria in aquatic animals, and it had good specificity.【Conclusion】The newly established real-time fluorescent quantitative PCR detection method of TiLV has good specificity, high sensitivity and high reproducibility, and can be used for early rapid diagnosis, epidemiological investigation, prevention and control of TiLV.