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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 Kunjin virus (KUNV) is a flavivirus and is a subtype of West Nile virus (WNV). Due to the existence of its popular ecological conditions in our country, there is a great risk of transmission. Therefore, it is urgent to establish a rapid, sensitive and efficient TaqMan fluorescent RT-PCR method for the monitoring of KUNV at the border crossings to prevent its introduction. Methods KUNV whole gene sequence was retrieved from GenBank. The sequences were analyzed by MEGA 4 software and Blast. The E gene fragment was selected as a template. The primers and probes for the template were designed by Beacon Designer 7.0 software. TaqMan fluorescence RT-PCR was used to detect and optimize the reaction conditions and primer concentration. The annealing temperature was set at 50, 55 and 60 ℃, the final concentration of primers was set at 200 and 400, 800nmol / L, the final concentration of the probe was set to 100, 200, 400nmol / L, the optimal annealing temperature, the best primer and the final concentration of the probe were determined by the analysis of the amplification curve and the Ct value, and the sensitivity and specificity of the method were verified Sex. Results The optimal annealing temperature of KUNV fluorescence RT-PCR was 55 ℃, the final concentration of primer was 400nmol / L, and the final concentration of probe was 200nmol / L. The sensitivity of the method to detect KUNV nucleic acid was 1.83 × 102copies / μl, and the positive rate of KUNV nucleic acid was the same as that of La Crosse virus (LACV), Snowshoe hare virus (SSHV) Barmah Forest virus (BFV), Mayaro virus (MAYV), Japanese encephalitis virus (JEV), Yellow fever virus (YFV), Southeast Asia There was no cross reaction between the two RNA viruses, Banna virus (BAV). Conclusion The established TaqMan fluorescence RT-PCR method has high sensitivity and specificity and is suitable for the rapid detection of KUNV, which has application value.