蜜蜂残翅病毒两步法荧光定量PCR检测方法的建立  

Establishment of Two-step Fluorescence Quantitative PCR Method for Detection of Deformed Wing Virus in Honeybee

作　　者：王红坤 王艺桦 周丹银 董坤 张炫 WANG Hongkun;WANG Yihua;ZHOU Danyin;DONG Kun;ZHANG Xuan(Faculty of Animal Science and Technology,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南农业大学动物科学技术学院,云南昆明650201

出　　处：《河南农业科学》2022年第3期154-161,共8页Journal of Henan Agricultural Sciences

基　　金：国家自然科学基金项目(31560667);国家现代蜂产业技术体系项目(CARS-44-kxj13)。

摘　　要：为了建立一种灵敏度更高的蜜蜂残翅病毒两步法荧光定量PCR检测方法,根据残翅病毒保守基因片段设计引物,采取反转录过程与荧光定量PCR过程分步进行的方式,建立基于SYBR染料法检测残翅病毒的两步法实时荧光定量PCR检测方法,并对其特异性、灵敏性、稳定性和可行性进行验证。结果显示,建立的两步法荧光定量PCR检测方法采用的引物特异性良好,在6.58×10^(1)~6.58×10^(8)拷贝/μL质粒标准品间具有良好线性关系,R^(2)为0.9997,扩增效率为100.8%。该方法的灵敏度为6.58拷贝/μL,与蜜蜂黑蜂王台病毒和以色列急性麻痹病毒不发生交叉反应,组内变异系数为0.20%~0.66%,组间变异系数为0.08%~0.98%,对蜜蜂样品残翅病毒的阳性检出率明显高于普通PCR,具有良好的特异性、重复性和实用性。综上,成功建立了一种灵敏度高、适用性好的残翅病毒两步法实时荧光定量PCR检测方法,为我国蜜蜂残翅病的流行病学调查、疫情监测和预警机制的构建提供新的技术支持。In order to establish a more sensitive two-step fluorescence quantitative PCR detection method for deformed wing virus in honeybee,primers were designed according to the conservative gene fragment of deformed wing virus,and a two-step real-time fluorescence quantitative PCR detection method based on SYBR dye was established by conducting reverse transcription process and fluorescence quantitative PCR step by step,and its specificity,sensitivity,stability and feasibility were verified.The results indicated that the primer specificity of the two-step real-time fluorescence quantitative PCR method established in this study was good,and there was a good linear relationship in 6.58×10^(1)—6.58×10^(8) copies/μL of plasmid standard.R^(2) was 0.9997 and the amplification efficiency was 100.8%.The sensitivity of the method was 6.58 copies/μL,and there were no cross-reactions between deformed wing virus and black queen cell virus,Israeli acute paralysis virus.The coefficient of variation was 0.20%—0.66%within the group and 0.08%—0.98%between the groups.The deformed wing virus infection rate of honeybee samples detected by this method was higher than that by ordinary PCR.The method showed good specificity,repeatability and practicability.In conclusion,a two-step real-time quantitative PCR method with higher sensitivity and better applicability was successfully established,which provides new technical support for epidemiological investigation,epidemic surveillance and early warning mechanism construction of bee deformed wing virus disease in China.

关 键 词：西方蜜蜂 东方蜜蜂 残翅病毒 两步法荧光定量PCR SYBR染料法 灵敏度 

分 类 号：S895.9[农业科学—特种经济动物饲养]