文章摘要
蓝莓中诺如病毒检测方法的优化及其在果蔬中的应用
Optimization of methods for detecting norovirus on blueberries and application in fruits and vegetables
投稿时间:2016-08-03  修订日期:2016-08-03
DOI:
中文关键词: 诺如病毒  蓝莓  方法优化  病毒富集  实时荧光RT-PCR
英文关键词: Norovirus  Blueberry  OptimizingSmethod  SVirus enrichment  Quantitative real-time PCR
基金项目:浙江省公益性项目(NO. 2015C32017);浙江省医药卫生科技计划项目(骨干人才)(NO. 2015RCA003)
作者单位E-mail
施晓峰 浙江中医药大学生命科学学院 浙江 杭州浙江省疾病预防控制中心 浙江 杭州 494549698@qq.com 
程东庆 浙江中医药大学生命科学学院 浙江 杭州  
章荣华 浙江省疾病预防控制中心 浙江 杭州  
廖宁波 浙江省疾病预防控制中心 浙江 杭州 nbliao@cdc.zj.cn 
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中文摘要:
      目的: 建立蓝莓等浆果类和蔬菜类食品中GII型诺如病毒 ( NoV GII) 的有效检测方法。方法:利用已知NoV GII病毒样本对阴性的蓝莓进行人工染毒,分别比较了7种洗脱液和2种浓缩方法的回收效果,优化了RNA提取方法,最后采用荧光定量PCR方法进行检测;并运用新建立的诺如病毒浓缩检测方法,对7种市场上购买的浆果和蔬菜样品进行检测并研究其最低检出限。结果:实时荧光 RT-PCR 检测结果表明,人工染毒后的蓝莓等浆果样品(表皮和基质)采用TP ALK Elution洗脱,再经酶处理后用超滤法浓缩,最后采用QIAamp Viral RNA Mini Kit提取浓缩病毒的RNA步骤后,诺如病毒的回收效率达到较高的水平,且其灵敏度可达3.5-350 PCRU/10g,同时将该方法运用于草莓、杨梅、葡萄、小番茄、生菜、胡萝卜、黄瓜等七种水果蔬菜中诺如病毒的检测,其中杨梅和小番茄的检测灵敏度能达到3.5-3500PCRU/10g,草莓、生菜和胡萝卜次之,为35-35000PCRU/10g,黄瓜和葡萄的灵敏度较低。结论:所建立的果蔬中诺如病毒富集方法和核酸提取方法操作简便,灵敏度较高,适合于蓝莓等浆果和蔬菜类样品中NoV GII的检测。
英文摘要:
      Objective: Establish an effective method for detecting norovirus (NoV GII) in fruits and vegetables such as blueberry. Method: First, a known amount of NoV GII was spiked onto the surface and matrix of blueberries and seven different buffers previously described for eluting NoV GII from fruits and vegetables were evaluated. Second, to optimize a method for concentrating the recovered NoV GII, Two concentration methods (PEG precipitation and ultrafiltration) were investigated. Finally, two different methods for nucleic acid extraction were evaluated. Results: NoV GIIs were extracted from the food surface and matrix by a direct elution method in the ALK (pH 9.5) buffer, concentrated by ultrafiltration and then followed by QIAamp viral RNA mini kit for RNA extraction. Occasionally, PCR inhibitors were present in the processed berry samples, which gave relatively poor detection limits. However, this problem was overcome by adding a pectinase treatment in the protocol, which markedly improved the sensitivity of the method. After optimization, this concentration method was applied in combination with real-time reverse transcription-PCR (RT-PCR) using specific primers in various types of berries and vegetables. Most of the tested fresh fruits and vegetables except for the cucumber and grape (>3500), the average detection limits for NoV GII were 35-350 RT-PCR units per 10 g of food. Conclusion: Based on our results, it is concluded that this procedure is suitable to detect and quantify noro- viruses within a short time (about 6-8 h) and can be applied for surveillance of noroviruses in fresh fruits and vegetables.
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