检测蜜蜂急性麻痹病毒TaqMan实时荧光RT-PCR方法的建立和初步应用

Establishment and preliminary application of a real-time RT-PCR assay with TaqMan probe for detecting acute bee paralysis virus

【背景】蜜蜂急性麻痹病毒(Acute Bee Paralysis Virus,ABPV)是一种高毒力的蜜蜂病毒,可以引起蜜蜂的大批死亡和蜂群衰竭。【目的】建立一种快速、灵敏的ABPV实时荧光RT-PCR检测方法。【方法】根据ABPV衣壳蛋白基因保守序列设计引物和探针,通过对引物、探针浓度和退火温度等反应条件进行优化,建立基于TaqMan探针检测ABPV的实时荧光RT-PCR方法,并对方法的灵敏性、特异性和稳定性进行验证。【结果】ABPV实时荧光RT-PCR检测方法在9.8×101-9.8×108 copies/μL之间呈现良好的线性关系,线性相关系数R2为0.998,扩增效率为103.8%。该方法的检测灵敏度为9.8copies/μL;对其他蜜蜂病毒不发生交叉反应,具有良好的特异性;重复性试验结果显示组内和组间的变异系数分别为0.19%-0.80%和0.57%-1.07%,重复性良好。对2018年-2019年在福建地区采集的70份蜜蜂样品进行ABPV检测,阳性率为2.86%。【结论】建立的ABPV实时荧光RT-PCR检测方法能用于该病的实验室检测、流行病学调查和疫情监测。

[Background]Acute bee paralysis virus(ABPV) is a highly virulent bee virus that causes the death of honeybee and colony exhaustion.[Objective]This experiment was conducted to develop a rapid and sensitive real-time RT-PCR method for the detection of ABPV.[Methods]The primers and probe were designed according to the conservative sequence of capsid protein gene of ABPV registered in GenBank.By optimizing the reaction conditions such as primers,probe concentration,and alternating temperature,a detection method for ABPV based on TaqMan probes was successfully established,and the sensitivity,specificity and stability of the method were tested.[Results]The real-time RT-PCR assay showed a good linear relationship between 9.8×101-9.8×108 copies/μL,linear correlation coefficient R2 was 0.998,and amplification efficiency was 103.8%.The sensitivity limit of the method was 9.8 copies/μL,and there was no cross-reaction with other honey bee viruses,which represents showing high sensitivity and specificity.The coefficient of variation(CV) for intra-assay and inter-assay repeatability were 0.19%-0.80% and 0.57%-1.07% respectively.In 70 samples of honey bee collected from Fujian regions in 2018 and 2019,the ABPV detection rate was 2.86%.[Conclusion]The ABPV real-time RT-PCR detection method can be used for laboratory testing,epidemiological investigation and epidemic monitoring.