基于建立的SYBR Green Ⅰ实时荧光定量PCR检测方法,检测新疆部分地区家畜血清样品中流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)的流行情况.利用PCR扩增JEV毒株的E基因片段,将其克隆到pEASY-Blunt载体,构建重组质粒,建立SYBR Green Ⅰ实时荧光定量PCR检测方法,并验证其灵敏度、特异性和重复性.收集2021-2022年新疆哈密、阿勒泰、伊犁、阿克苏、喀什地区家畜血清样品,应用建立的检测方法进行检测并分析阳性率,以监测新疆地区JEV在家畜中的流行情况.结果显示,建立的检测方法呈良好的线性关系,检测区间在4.03 × 102～4.03 ×109拷贝/μL,相关系数为0.995,斜率为-3.431,灵敏度下限极值为4.03 × 102拷贝/μL.该方法用于检测寨卡病毒(Zika virus,ZIKV)、登革病毒(Dengue virus,DENV)未出现特异性扩增.重复性的组内变异系数为0.53％～1.27％,组间变异系数为0.48％～1.43％.使用本方法检测2021-2022年新疆地区家畜血清样品,总阳性率为3.11％,其中马、牛和羊中的阳性检出率分别为2.35％、5.26％和3.74％,鉴定为基因Ⅰ型JEV.结果表明,建立了基因Ⅰ型JEV的SYBR GreenⅠ实时荧光定量PCR检测方法,新疆地区马、牛、羊血清中均检测到JEV,总阳性率为3.11％.
Abstract
The SYBR Green Ⅰ real-time fluorescence quantitative PCR detection method was used to determine the prevalence of Japanese encephalitis virus(JEV)in mosquito vectors and cattle se-rum samples in Xinjiang.The E gene fragment of the JEV strain was amplified by PCR,cloned into a pEASY-Blunt vector,produced as a recombinant plasmid,and its sensitivity,specificity and re-producibility were verified.Between 2021 and 2022,serum samples were taken in the regions of Hami,Altay,Ili,Aksu,and Kashi in order to monitor the prevalence of JEV in livestock in Xin-jiang.The positive rate was discovered and evaluated using the established detection method.The established detection method showed a good linear relationship,and the detection interval was 4.03X102-4.03×109 copies/pL.The correlation coefficient was 0.995,the slope was-3.431,and the extreme value of the lower limit of sensitivity was 4.03 × 102 copies/pL.This method has no specific amplification for Zika virus(ZIKV)and Dengue virus(DENV).The intra group coefficient of variation of reproducibility was 0.53％-1.27％,and the inter group coefficient of variation was 0.48％-1.43％.Using this method to detect serum samples from livestock in Xinjiang from 2021 to 2022,the total positive rate was 3.28％,with positive detection rates in horses,cows,and sheep being 2.35％,6.77％,and 3.74％respectively,the virus was identified as Type Ⅰ JEV.A SYBR Green Ⅰ real-time PCR method for the detection of genotype 1 JEV was established.JEV was de-tected in the serum of horses,cattle and sheep in Xinjiang,with a total positive rate of 3.11％.
关键词
流行性乙型脑炎病毒/流行病学/E基因/检测方法
Key words
Japanese encephalitis virus/epidemiology/E gene/test method
维普
万方数据