猪胃黏蛋白偶联磁珠和聚乙二醇富集检测青葱和葡萄中诺如病毒的比较研究

摘要

Objective： To establish and compare two detection methods for human norovirus （HuNoV） in fruit （grape） and vegetable （green onion） samples using porcine gastric mucin-conjugated magnetic beads （PGM-MB） and polyethylene glycol 8000 （PEG8000）, respectively. Methods： 1 RTU was defined from a consistently （triplicate） positive qRT-PCR signal of viral RNA extract with 4 × 10-6 dilution. A standard curve （1 to 10000 RTU） was established to convert Ct values to corresponding RTUs with an equation of y （Ct） = -3.6967 lg （RTU） ＋39.12 with R2 of 0.999. Green onion and grape samples were inoculated with various amounts of HuNoV, eluted and concentrated using PGM-MB or PEG8000 method. Viral RNA was extracted and quantified by qRT-PCR. Student＇s t-test was used for statistical analysis. Results： The virus recovery rate by PGM-MB method （（31.61 ± 15.04）%） was significantly higher than that obtained by PEG precipitation method （（6.09 ± 1.56）%） at all doses of HuNoV inoculated onto grapes （P 〈0.001）. For virus-inoculated green onions, the virus recovery rates from higher inoculation doses （625 RTU and 125 RTU） were comparable between both methods. However, the virus recovery rate by PGM-MB method from a lower dose of HuNoV （25 RTU） inoculated onto green onion samples was significant higher （（4.49 ± 0.79）%） than that from PEG precipitation method （（1.93 ± 0.18）%）. In addition, PGM-MB method showed a lower detection limit than PEG precipitation method. Conclusion： PGM-MB method requires less time than PEG precipitation method, produces higher yield of HuNoV from various food samples, and hence exhibits higher sensitivity.%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。