Taqman-MGB探针RT-PCR方法检测食品中脊髓灰质炎病毒

Detection of Polioviruses in Foodstuffs by Real-time RT-PCR Based on Taqman-MGB Probe

目的:建立食品中脊髓灰质炎病毒Taqman-MGB探针实时荧光RT-PCR检测方法。方法:在脊髓灰质炎病毒基因组2A区设计引物和探针,对3种血清型脊髓灰质炎病毒进行单独和同时检测。通过参照分子建立标准曲线,对人工接种病毒的牡蛎、蓝莓、生菜和水食品样品进行检测。结果:建立的检测体系分别高度特异于相应血清型病毒检测,对3种血清型脊髓灰质炎病毒进行单独和同时检测的下限均为2拷贝参照分子DNA。基于参照分子建立的4条标准曲线具有很好线性关系(R2=0.999)和较高反应效率(1.01～1.04)。对4种食品样品的分析表明,建立的实时荧光RT-PCR方法可稳定检测到各添加浓度脊髓灰质炎病毒。结论:建立的Taqman-MGB探针实时荧光RT-PCR检测体系适于食品中脊髓灰质炎病毒的检测。

This study was aimed to develop three sero-specific real-time RT-PCR detection systems based on Taqman-MGB probes for the assay of polioviruses in foodstuffs. Primer and Taqman-MGB probe sets based on the 2A region of poliovirus genome were designed to detect three serotypes of polioviruses separately and simultaneously. Four artificially inoculated food samples, including oyster, blueberry, lettuce and water were analyzed based on the established standard curves using three reference molecules as calibrators. Sensitivity tests suggested that at least 2 copies of the reference molecule DNA could be stably detected both in the separate and simultaneous detection systems. Good linearity with the correlation coefficient above 0.999 and high reaction efficiency （1.01-1.04） were observed in four standard curves employing serially diluted reference molecule DNA as the calibrator. Three serotypes of polioviruses in four inoculated samples were successfully detected using the established real-time RT-PCR detection systems. The developed real-time RT-PCR detection systems based on Taqman-MGB probes are most suitable for polioviruses detection in foodstuffs.