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目的:以青葱与葡萄为材料,建立以猪胃黏蛋白偶联磁珠(PGM-MB)和聚乙二醇8000(PEG8000)富集检测水果、蔬菜中诺如病毒的方法。方法:确定病毒原液中的相对病毒量,梯度稀释病毒原液并进行实时荧光-聚合酶链式反应检测,以每个反应管内的荧光信号达到设定的域值时所经历的循环数值与病毒量(实时荧光-聚合酶链式反应单位数)的常用对数值绘制标准曲线和线性方程;人工接种诺如病毒于青葱与葡萄表面,洗脱后,分别用PEG8000和PGM-MB富集诺如病毒,实时荧光-聚合酶链式反应扩增,用标准曲线对回收的病毒进行相对定量。结果:基质为青葱时,高接种量条件下,两种富集方法的病毒回收效果相当,低接种量下,PGM-MB法的富集回收率高于PEG8000法,且PGM-MB的检测下限更低;基质为葡萄时,PGM-MB法的富集回收率均高于PEG8000法,且检测下限更低。结论:PGM-MB富集效果良好,快速方便,适合应用于水果和蔬菜中的诺如病毒的富集检测。
OBJECTIVE: To establish a method for the enrichment detection of norovirus in fruits and vegetables by using porcine gastric mucoadhesion magnetic beads (PGM-MB) and polyethylene glycol 8000 (PEG8000). Methods: To determine the relative amount of virus in the virus stock solution, to dilute the virus stock solution in a gradient manner and to perform real-time fluorescence-polymerase chain reaction to measure the number of cycles of the fluorescent signal in each reaction tube when the fluorescence signal reaches the set value. (Real-time fluorescence - polymerase chain reaction units) common logarithm plot standard curve and linear equation; artificial inoculation of norovirus on the surface of green onion and grape, after elution, respectively PEG8000 and PGM-MB enrichment Norovirus , Real-time fluorescence-polymerase chain reaction amplification, using a standard curve of the recovered virus relative quantification. Results: When the substrate was green, the recoveries of the two enrichment methods were comparable under high inoculum size. The enrichment recovery of PGM-MB method was higher than that of PEG8000 under low inoculum size, and the detection limit of PGM-MB Lower. When the matrix was grapes, the recoveries of PGM-MB method were higher than that of PEG8000 method, and the detection limit was lower. Conclusion: The PGM-MB enrichment effect is rapid, convenient and suitable for the enrichment detection of Norovirus in fruits and vegetables.