引用本文:冯微宏,肖勇,钱燕华,等.两种水源性诺如病毒富集方法的比较[J].环境科学研究,2020,33(6):1549-1554.
FENG Weihong,XIAO Yong,QIAN Yanhua,et al.Comparison of Two Concentration Methods for Norovirus in Water[J].Research of Environmental Sciences,2020,33(6):1549-1554.]
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两种水源性诺如病毒富集方法的比较
冯微宏1,2, 肖勇2, 钱燕华2, 许秋瑾1,3
1. 南京医科大学公共卫生学院全球健康中心, 江苏 南京 211166;2. 无锡市疾病预防控制中心, 江苏 无锡 214023;3. 中国环境科学研究院, 环境基准与风险评估国家重点实验室, 北京 100012
摘要:
评估阳离子膜吸附-洗脱法和阴离子膜吸附-洗脱法在富集水源性诺如病毒(NV)过程中的病毒回收效果、消耗时间及使用成本,可以获得更适用于水源性诺如病毒实际监测工作的较优富集方法.以无锡市疾病预防控制中心保存的诺如病毒阳性标准质粒为模板,采用荧光定量RT-PCR(反转录酶-聚合酶链反应)方法绘制病毒定量标准曲线,并应用于病毒的绝对定量.将含诺如病毒的粪便悬液先稀释成3个不同浓度,并加入灭菌水中,分别经过阴离子膜(硝酸纤维素滤膜)或阳离子膜(Nanoceram膜)吸附洗脱,最终相同体积洗脱液提取核酸后用荧光定量RT-PCR方法进行绝对定量,并进一步计算最终病毒回收率.结果表明:①样本病毒浓度为8×101~8×1010 copies/μL时,病毒拷贝数的lg对数值与Ct(荧光定量PCR中达到设定阈值所经过的循环数)呈线性关系.②随着样本病毒浓度的增加,阴离子膜法和阳离子膜法的病毒回收率都相应增加.③当检测样本中病毒浓度分别为8×101、4×102 copies/μL时,阴离子膜法回收率分别为22.2%±4.1%和36.8%±6.2%,阳离子膜法回收率分别为17.4%±1.5%和28.8%±6.1%,说明阴离子膜吸附-洗脱法对病毒的富集回收率显著高于阳离子膜吸附-洗脱法(P<0.05).研究显示,综合富集效果、耗费时间和成本等因素,阴离子膜吸附-洗脱法比阳离子膜吸附-洗脱法更适用于日常水源性诺如病毒的富集工作.
关键词:  诺如病毒  阴离子膜  阳离子膜  病毒富集
DOI:10.13198/j.issn.1001-6929.2020.05.04
分类号:X52;R123
基金项目:国家自然科学基金项目(No.41673122)
Comparison of Two Concentration Methods for Norovirus in Water
FENG Weihong1,2, XIAO Yong2, QIAN Yanhua2, XU Qiujin1,3
1. Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing 211166, China;2. Wuxi Center for Disease Control and Prevention, Wuxi 214023, China;3. State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China
Abstract:
In order to obtain a more suitable enrichment method for practical work, the recovery rate, consumption time and use-cost of norovirus concentration methods using electropositive membrane and electronegative membrane were compared. Using the norovirus-positive standard plasmid in Wuxi City Center for Disease Control and Prevention as a template, a standard curve of virus quantification was drawn by RT-PCR. Feces suspensions with three different diluted concentrations were added into sterilized water, and then filtered through a electronegative membrane (nitrocellulose membrane) or a electropositive membrane (Nanoceram membrane), After enrichment, RNA was extracted from the concentrates with the same volume and subjected to fluorescent quantitative RT-PCR. The standard curve of NV was generated by RT-PCR, and the virus recovery rates were calculated according to the standard curve and compared statistically. The results showed that when the sample virus concentration was 8×101-8×1010 copies/μL, the log value of the virus copy number was linearly related to the Ct(the Ct is defined as the number of cycles required for the fluorescent signal to cross the threshold). With the increase of sample virus concentration, the virus recovery rate of the concentration methods using electronegative membrane or electropositive membrane increased correspondingly. When the virus concentration was 8×101 and 4×102 copies/μL, the recovery rates of concentration methods using electronegative membrane were 22.2%±4.1% and 36.8%±6.2%, respectively, which were statistically higher than the recovery rates when electropositive membrane was applied (17.4%±1.5% and 28.8%±6.1%) (P<0.05). In this study, considering the recovery rate, consumption time and use-cost, the electronegative membrane adsorption elution method is more suitable for daily enrichment of water-borne norovirus than the electropositive membrane adsorption elution method.
Key words:  norovirus  electropositive membrane  electronegative membrane  virus concentration