蜜蜂残翼病毒TaqMan-MGB探针荧光定量RT-PCR检测方法的建立  

Development of a Quantitative PCR Method Using a TaqMan?-MGB Fluorescent Probe for Detection of the Deformed Wing Virus

作　　者：王琛 马跃宇 黄思超 孙莉 李明 韩喜彬 高志峰 费东亮 马鸣潇 WANG Chen;MA Yueyu;HUANG Sichao;SUN Li;LI Ming;HAN Xibin;GAO Zhifeng;FEI Dongliang;MA Mingxiao(Laboratory Animal Center,Jinzhou Medical Unitversity,Jinchou 121000,China;Liaomng Province Agricultural Development Service Center,Shenyang 110015,China)

机构地区：[1]锦州医科大学实验动物中心,锦州121000 [2]辽宁省农业发展服务中心,沈阳110015

出　　处：《病毒学报》2021年第3期686-694,共9页Chinese Journal of Virology

基　　金：国家自然基金(项目号:31972626),题目:蜜蜂残翅病毒(DWV)结构蛋白与宿主互作蛋白筛选及其在感染中的作用研究;辽宁省省自然科学基金指导计划(项目号:20170540346),题目:Rab9基因修饰的巨噬细胞抗肿瘤效应研究。

摘　　要：蜜蜂残翅病毒(Deform wing virus,DWV)已成为世界上最著名、分布最广、研究最为深入的昆虫病原。虽然DWV以前存在于蜜蜂种群中,但一种新的媒介—外寄生螨Varroa破坏体的到来和全球传播,极大地促进了DWV的流行病学。为了建立快速、准确且能定量分析的蜜蜂残翅病毒,DWV检测方法,本研究参照GenBank中已登录的DWV高度保守的3C-RdRp基因区域,设计了1对特异性引物和带有羧基荧光素(Fast Auxiliary Memory,FAM)与淬灭基团(Eclipse)标记的TaqMan探针,并对反应条件进行优化,建立了检测DWV的TaqMan-MGB探针荧光定量RT-PCR检测方法(TaqMan qRT-PCR),同时对该检测方法的特异性、敏感性、重复性进行了试验,并对临床样品进行检测。结果显示:该方法最佳上下游引物和探针浓度分别为12.5μmol/L和10μmol/L,最佳退火温度为59℃;敏感性试验中,对DWV质粒标准品检测下限为2.58×10^(1)拷贝/μL;本方法特异性较好,对蜜蜂常见病毒—蜜蜂慢性麻痹病毒(Chronic bee paralysis virus,CBPV)、蜜蜂急性麻痹病毒(Acute paralysis virus,ABPV)、黑蜂王台病毒(Black queen cell virus,BQCV)、囊状幼虫病毒(Sacbrood virus,SBV)、以色列急性麻痹病毒(Israeli acute paralysis virus,IAPV)和中蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)无交叉反应;且批内变异系数和批间变异系数均小于2%。利用该方法对49份临床疑似样本进行检测,其中DWV阳性样本为35份,检出率高于常规RTPCR方法,且病毒载量大于1×10^(7)拷贝/只样本数共24份,表明辽宁和河北部分地区感染DWV蜂群中病毒载量较高,流行情况比较严重。因此,本研究建立的TaqMan探针荧光定量RT-PCR方法具有灵敏度高、特异性强、重复性好等特点,能够用于DWV病原监测、流行病学调查和病毒定量分析等相关研究。Deformed wing virus(DWV)has become the most well-known,widespread,and intensively studied insect pathogen in the world.Although DWV was previously present in honeybee populations,the arrival and global spread of a new vector,the ectoparasitic mite Varroa destructor,has dramaticallyaltered DWV epidemiology.We wished to develop an assay for rapid detection and quantification of the DWV.We designed a pair of specific primers and a TaqMan?probe labeled with carboxyl fluorescein and a quenching group(eclipse)according to the highly conserved 3C-RdRp gene region of the DWV.A TaqMan?minor groove binder(MGB)probe-based,fluorescence real-time reverse transcription-quantitative polymerase chain reaction(RTqPCR)was established.Then,the specificity,sensitivity and stability of the assay were assessed.We found that the optimal concentration of the primers and probe was 12.5μmol/L and 10μmol/L,respectively,the optimal annealing temperature was 59°C,and the limit of detection for the DWV plasmid standard was as low as 2.58×10^(1)copies/μL.The specificity of the assay was high and there were no cross-reactivity with the bee chronic paralysis virus,bee acute paralysis virus,black bee queen station virus,sacbrood virus,Israeli acute paralysis virus or Chinese sacbrood virus.The intra-assay and inter-assay coefficient of variation was<2%.The assay was also used to detect the DWV in 35 clinical samples,and its efficiency compared with that of conventional RT-PCR.In 24 samples,the viral load was>1×10^(7)copies.A high viral load was documented in colonies infected with the DWV in some areas of Hebei Province,China and Liaoning Province,China.Our TaqMan MGB-based fluorescent probe RT-qPCR method for the DWV was more sensitive than that for the other methods tested.Our assay was reliable,fast and sensitive,and could be used to detect DWV in clinical samples.This technology could be a useful tool for rapid detection of the DWV.This method could be used in monitoring,epidemiological investigation and quantitative analyses of the DWV

关 键 词：蜜蜂残翼病毒(DWV) TAQMAN-MGB探针 qRT-PCR方法 临床检测 

分 类 号：S855.3[农业科学—临床兽医学] S895.1[农业科学—兽医学]