裂谷热病毒实时荧光定量PCR检测方法的建立

目的建立一种快速、敏感、特异的RealtimePCR（实时荧光定量PCR）方法，用于裂谷热病毒（RVFV）的检测。方法根据裂符热病毒N蛋白基因的保守序列设计并合成一对引物及特异性TaqMan探针。通过条件优化，以10倍系列稀释重组质粒为标准品，进行Real-timePCR扩增，绘制标准曲线，并进行重复性、准确性、特异性及敏感性检测。结果建立的Real-timePCR方法检测裂谷热病毒所绘制标准曲线的相关系数大于0．99，灵敏度为1．0×10^1拷贝，高于常规PCR方法（1．0×10^3拷贝）；除裂谷热病毒外的其他7种对照烈性病病原体基因检测均呈阴性；批内重复和批问重复的变异系数均小于1％。结论建立的裂谷热病毒Real-timePCR检测方法敏感性和特异性较高，可用于裂谷执病毒感染懊涑诊眯斤殛流行病学谰杏．

Development of a real-time PCR technique to detect Rift Valley fever virus

Objective To establish a rapid and sensitive technique to detect Rift Valley fever virus （RVFV） using real- time fluorescence quantitative PCR. Methods A pair of primers and a TaqMan probe were designed in accordance with the conserved sequence of the RVFV nucleoprotein gene. Real-time PCR was done using optimized parameters and stand ard samples prepared by 10-fold serial dilution of recombinant plasmid and plotting a standard curve. In addition, the re- peatability, accuracy, specificity, and sensitivity of the devised technique were tested. Results Real time PCR results and the RVFV standard curve had a correlation coefficient greater than 0.99; the developed technique was sensitive e- nough to detect as few as 1.0 ×10^1 copies, which was more sensitive than conventional PCR （1.0× 10^1 copies）. The de- veloped technique yielded negative results when testing for 7 control pathogens that cause fulminant disease. The intra- batch and inter-batch coefficients of variation were both less than 1 ~. Conclusion The real-time PCR technique devel- oped here has laid the foundation for rapid confirmation and epidemiological study of outbreaks of Rift Valley fever.