西生菜中低污染量GⅡ型诺如病毒的富集与定量检测研究

Enrichment and quantitative detection of low artificially contaminated GⅡ type norovirus in fresh romaine lettuce

中文关键词:  西生菜  GⅡ型诺如病毒  富集  实时荧光RT-PCR  微滴式数字RT-PCR

英文关键词:romaine lettuce  GⅡ type norovirus  enrichment  real-time fluorescent RT-PCR  droplet digital RT-PCR

中文摘要:

      目的 优化新鲜西生菜中低污染量的GⅡ型诺如病毒富集方法, 比较实时荧光RT-PCR与微滴式数字RT-PCR检测方法, 研究适用于新鲜西生菜中低污染量的GⅡ型诺如病毒的定量检测方法。方法 选取洗脱液种类、沉淀剂种类、沉淀剂作用温度及沉淀剂作用时间4个因素进行研究, 以确定较优的GⅡ型诺如病毒富集条件。结合较优的富集条件, 分别采用实时荧光RT-PCR方法和微滴式数字RT-PCR方法进行定量检测。结果 病毒富集方法研究表明: 洗脱液为牛肉膏-甘氨酸洗脱液、沉淀剂为PEG6000+PEG8000、沉淀剂作用温度为4 ℃, 沉淀剂作用时间为4 h为较优条件, 此时富集后的平均病毒浓度最高, 平均回收率可达53.43%。实时荧光RT-PCR的检测限为1.44×104 copies/g; 而微滴式数字RT-PCR的检测限为7.86×102 copies/g。结论 通过优化西生菜中低污染量的GⅡ诺如病毒富集方法, 比较实时荧光RT-PCR方法与微滴式数字RT-PCR方法定量检测病毒的效果, 建立了适用于西生菜样品中低污染量GⅡ诺如病毒的定量检测方法。

英文摘要:

      Objective To optimize the enrichment method of low artificially contamininated GⅡ type norovirusin in fresh romaine lettuce, compare the detection of the real-time fluorescent RT-PCR and the droplet digital RT-PCR, and develop a method for the quantitative detection of low artificially contaminated GⅡ type norovirus in fresh romaine lettuce. Methods The optimum conditions were determined by the orthogonal experiment using 4 factors of eluent type, precipitant type, precipitation temperature and time. Under the optimum conditions, the norovirus concentrations were detected by the real-time fluorescent RT-PCR and the droplet digital RT-PCR, respectively. Results The optimum conditions were as follows: eluent type was beef-glycine eluent, precipitant type was PEG6000+PEG8000, precipitation temperature was 4 ℃ and time was 4 h. Under these conditions, the average recovery rate could reach 53.43%. The limit of detection of the real-time fluorescent RT-PCR was 1.44×104 copies/g while the limit of detection of the droplet digital RT-PCR was 7.86×102 copies/g. Conclusion By optimizing the enrichment and comparing the detection effects of the real-time fluorescent RT-PCR and the droplet digital RT-PCR, a method for the quantitative detection of low artificially contaminated GⅡ type norovirus in fresh romaine lettuce was developed