罗非鱼罗湖病毒RT-PCR检测方法的建立及初步应用

Development and Application of a RT-PCR Assay of Tilapia Lake Virus

【目的】建立快速检测罗非鱼罗湖病毒(Tilapia Lake Virus,Ti LV)的RT-PCR方法。【方法】参照GenBank中罗非鱼罗湖病毒全基因序列,依据其假定蛋白基因片段3设计合成1对特异性扩增引物,提取罗湖病毒RNA,逆转录成cDNA,进行PCR扩增,通过优化扩增条件和扩增体系,建立快速检测罗非鱼罗湖病毒的RT-PCR方法。用该方法对自然感染罗湖病毒发病的罗非鱼样品进行RT-PCR扩增。【结果】RT-PCR扩增得到与试验设计相符的499 bp的特异性目的条带,而对健康罗非鱼、野生罗非鱼及其他病毒对照组的扩增结果均为阴性。测序比对结果表明,该方法检测结果准确,最低可检测出约10fg/mL的质粒DNA,具有较好的特异性和较高的敏感性。用该方法对332份临床样品进行RT-PCR检测,其中36份样品扩增出特异性的目的条带,经测序比对,检测结果正确。【结论】建立的检测方法可用于TiLV的检测。

【Objective】To establish a method for rapidly detecting tilapia lake virus(TiLV) by RT-PCR.【Method】A pair of specific primers were designed according to the complete genome hypothetical protein segment 3 of a novel RNA virus, called TiLV, and RNA of TiLV was extracted and reversely transcribed to cDNA as template for PCR. A rapid RT-PCR method was established for detection of TiLV by optimization of the reaction parameters. And the Tilapia which naturally infected with TiLV was detected by the RT-PCR method.【Result】The specific band of 499 bp was amplified from the positive sample, but no specific band was found from healthy tilapia, wild tilapia and other control.Sequencing analysis indicated that this method was accurate, and 10 fg/mL DNA plasmid of the TiLV could be detected. 332 clinical samples were collected and 36 of those were positive, and the detection result was verified by sequencing and sequence alignment.【Conclusion】 The findings indicated that the RT-PCR assay could be used for the detection of TiLV. The established RT-PCR assay could lay the foundation for the detection and epidemiological investigation of TiLV infection.