副流感病毒5型荧光定量RT-PCR检测方法的建立

Development of fluorescence quantitative RT-PCR for detection of Parainfluenza virus 5(PIV5)

关键词:  副流感病毒5型  荧光定量  方法

Key words:  Parainfluenza virus 5  fluorescence quantitative  method

摘要:

为建立检测副流感病毒5型(PIV5)的荧光定量RT-PCR方法,针对PIV5 L基因设计筛选出一对特异性引物和探针,对退火温度、引物浓度、探针浓度进行了优化,并进行了敏感性、特异性和重复性试验。结果显示：最适退火温度为54.7℃,最适引物浓度为0.5μ mol/L,最适探针浓度为0.1μ mol/L。检测灵敏度为10 copies、0.8-1TCID50,特异性结果显示与BPIV3等22种常见病毒均无交叉,重复性试验显示不同模板浓度重复检测变异系数均小于1.5%,重复性良好。

Abstract:

To establish a fluorescence quantitative RT-PCR method for detection of parainfluenza virus 5 ( PIV5 ), a pair of specific primers and probes were designed and screened for PIV5 L gene. The annealing temperature, primer concentration and probe concentration were optimized, and the sensitivity, specificity and repeatability tests were revaluated respectively. The results showed that the optimal annealing temperature was 54.7 °C, the optimal primer concentration was 0.5 μ mol/L, and the optimal probe concentration was 0.1 μ mol / L. The detection sensitivity was 10 copies and 0.8-1 TCID_50,the specificity results showed that there was no cross-over with 22 common viruses such as BPIV3, and the repeatability test showed that the coefficient of variation of different template concentrations was less than 1.5 %, and the repeatability was good.