摘要
目的库京病毒(Kunjin virus,KUNV)为黄病毒属病毒,是西尼罗病毒(West Nile virus,WNV)的亚型。由于我国存在其流行的生态学条件,传入的风险很大。因此,亟待建立快速、灵敏、高效的TaqMan荧光RT-PCR方法用于国境口岸KUNV的监测,防止其传入。方法从GenBank中检索KUNV全基因序列,通过MEGA 4软件进行序列比对和Blast进行保守序列搜索分析,选取E基因片段为模板。利用Beacon Designer 7.0软件针对模板设计引物和探针,进行TaqMan荧光RT-PCR检测并优化反应条件和引物浓度,退火温度设定为50、55、60℃,引物终浓度设定为200、400、800nmol/L,探针终浓度设定为100、200、400nmol/L,通过扩增曲线与Ct值分析确定最佳退火温度、最佳引物及探针终浓度并验证该方法的敏感性和特异性。结果通过分析比较,确定KUNV荧光RT-PCR检测方法最佳退火温度为55℃,引物终浓度为400nmol/L,探针终浓度为200nmol/L。该方法检测KUNV核酸的敏感度达1.83×102copies/μl,且与正布尼亚病毒属拉克罗斯病毒(La Crosse virus,LACV)、雪靴野兔病毒(Snowshoe hare virus,SSHV),甲病毒属巴马哈森林病毒(Barmah Forest virus,BFV)、马雅罗病毒(Mayaro virus,MAYV),黄病毒属乙脑病毒(Japanese encephalitis virus,JEV)、黄热病毒(Yellow fever virus,YFV),东南亚十二节段RNA病毒属版纳病毒(Banna virus,BAV)均无交叉反应。结论建立的TaqMan荧光RT-PCR方法灵敏度高、特异性好,适合于KUNV的快速检测,具有应用价值。
Objective The Kunjin virus（KUNV）is a member of the Flavivirus genus and is a clade of the WNV group.A rapid,sensitive,and efficient method of detecting KUNV needs to be established because of the increasing risk of imported KUNV. Methods GenBank was searched for the complete genomic sequence of the KUNV.The conserved sequences of viral strains were aligned and compared using MEGA 4software.Beacon Designer 7.0software was used to design specific primers and a TaqMan probe based on the E gene of the KUNV.Reaction conditions and the concentrations of primers and probes were optimized. Results The optimized annealing temperature for real-time RT-PCR was55℃,and the concentrations of primers and the probe were 400nmol/L and 200nmol/L,respectively.The established method had a detection limit of 1.83×102 copies/μl,and there was no cross-reaction with the La Crosse virus,Snowshoe hare virus,Barmah Forest virus,Mayaro virus,Japanese encephalitis virus,Yellow fever virus,or Banna virus under the same reaction conditions. Conclusion A TaqMan real-time RT-PCR assay for detection of KUNV has been developed and this method proved to be highly reproducible,sensitive,and specific.This method could be used to screen for KUNV in mosquitoes.
出处
《中国病原生物学杂志》
CSCD
北大核心
2015年第7期594-597,共4页
Journal of Pathogen Biology
基金
质检公益专项(No.201410014)
