三种分子生物学方法检测牡蛎中诺如病毒的比较研究

Comparative Study of Detection of Norovirus in Oysters by RT-PCR,RT-Seminested PCR and RT-LAMP

采用RT-PCR、RT-半巢式PCR(seminested PCR)和RT-环介导等温扩增(LAMP)三种分子生物学方法分别检测了牡蛎中经粪便污染的诺如病毒。三种方法所采用的特异性引物均针对诺如病毒高度保守的N/S结构域。结果显示:一步法RT-PCR较两步法结果理想但仍不能有效去除食品中的PCR反应抑制物。RT-半巢式PCR和RT-LAMP的特异性和敏感性都远远优于RT-PCR,但是RT-半巢式PCR操作繁琐费时。RT-LAMP扩增程序简单、反应时间短,且不需要精密的温度循环装置,在产物中加入SYBR Green Ⅰ染料后可用肉眼直接判断反应结果。因此,RT-LAMP有望发展成为快速检测牡蛎中诺如病毒的有效手段。

Reverse transcription polymerase chain reaction （RT-PCR）, RT-seminested PCR and reverse transcription loop- mediated isothermal amplification （RT-LAMP） assays were compared to detect norovirus in oysters. All the three assays targeted the highly conserved capsid N-terminal/shell （N/S） domain of norovirus with specific primers. Although one-step RT-PCR presented more apparent results than two-step approach, it still had the limitation in eliminating inhibitors contained in food matrix, and both RT-seminested PCR and RT-LAMP showed significantly higher specificity and sensitivity than RT-PCR. However, the operation of RT-seminested PCR is tedious and time-consuming. In comparison to other two approaches, the amplification program of RT-LAMP is simple, and the reaction time is short. Without special apparatus needed, RT-LAMP can be performed under isothermal condition and the results may be visually ascertained by a simple color reaction using SYBR Green I dye. Therefore, RT-LAMP is of great potential to become a valuable method for rapid detection of norovirus in oysters.