实时荧光PCR检测Nam Dinh病毒方法的建立及初步应用  

The establishment and initial application of real-time fluorescent PCR detection of Nam Dinh virus

作　　者：梁克峰[1] 刘渠[2] 王德全[1] 周健明[2] 金玉娟[2] 陈应坚[2] 李静媚[2] 甘莉萍[2] 杨慧[2] 

机构地区：[1]广东药学院,广州510310 [2]深圳市龙岗区疾病预防控制中心

出　　处：《中国媒介生物学及控制杂志》2015年第5期447-450,共4页Chinese Journal of Vector Biology and Control

基　　金：广东省自然科学基金(2014A030313583)~~

摘　　要：目的建立一种快速、特异检测Nam Dinh病毒（NDi V）的实时荧光PCR方法。方法根据Gen Bank和本实验室分离的NDi V进行序列比对,找出保守序列（Rd Rp）并设计特异引物和Taq Man-MGB探针,通过调整引物、探针浓度,优化其反应条件,对方法做灵敏度、特异性、稳定性实验,以评价反应体系。结果 NDi V的Taq Man-MGB实时荧光PCR检测方法用时短,灵敏度高,最低检测下限为0.1 PFU。与登革热1-4血清型病毒、流行性乙型脑炎、人呼吸道合胞病毒、轮状病毒、星状病毒、腺病毒无交叉反应,特异性良好;将4份核酸含量不同的标准样品重复检测5次,平均Ct值变异系数范围为1.67%-3.68%,稳定性较高。通过监测,龙岗区蚊虫携带NDi V概率为18.00%。结论NDi V的Taq Man-MGB实时荧光PCR方法是一种快速、特异、灵敏、稳定性好的方法,可应用于流行病学环境监测,提高病毒的快速检测能力。Objective To establish a rapid and specific real-time fluorescent PCR method for Nam Dinh virus （NdiV）. Methods According to the sequence alignment of GenBank and NDiV isolated by our laboratory, we found out the conservative sequence （RdRp） and design specific primers and TaqMan- MGB probe. In order to evaluate the reaction system, the concentration of primers and probe were adjusted to optimize the reaction conditions, and the sensitivity, specificity, stability tests for our method were also conducted. Results TaqMan- MGB real- time fluorescence PCR detection method of NDiV were less time-consuming and highly sensitive, and the low detection limit was 0.1 PFU. It had a good specificity characteristic for having no cross reaction with dengue serotype 1-4, epidemic encephalitis b virus, respiratory syncytial virus, rotavirus, stellate virus, and adenovirus; The five times repeated testing of four nucleic acid content in different standard samples revealed that the average coefficient of variation range of Ct was 1.67%-3.68%, and thus it had high stability. Through monitoring, the positive probability of mosquitoes collected from Longgang district for NDiV was 18.00%. Conclusion NDiV TaqMan-MGB real-time fluorescent PCR method is a rapid, specific, sensitive and stable method, it can be applied to epidemiological monitoring in order to improve the ability of rapid detection of viruses.

关 键 词：NAM Dinh病毒 实时荧光定量PCR TAQ Man-MGB探针 应用 

分 类 号：R373[医药卫生—病原生物学]