Kukielka Deborah, Sánchez-Vizcaíno José Manuel
Departamento de Sanidad Animal Facultad de Veterinaria, UCM, Avda Puerta de Hierro s/n, Madrid, Spain.
J Virol Methods. 2009 Nov;161(2):240-6. doi: 10.1016/j.jviromet.2009.06.014. Epub 2009 Jun 25.
Two one-step real-time RT-PCR assays, based on SYBR Green (SG) chemistry, were developed or adapted respectively, for the detection, differentiation, and quantitation of two important honeybee viruses: Sacbrood virus (SBV) and Acute bee paralysis virus (ABPV). Both reactions were optimized to yield the highest sensitivity and specificity. The genome equivalent copies (GEC) detection limit per reaction was 389.3 for the ABPV RT-PCR. The GEC detection limit per reaction was 298.9 for the SBV RT-PCR. Viral detection and identification were confirmed by melting curve analysis and sequencing of the PCR products. Both techniques were used to evaluate Spanish field samples and establish the distribution of these viruses. Acute bee paralysis virus was not detected, and Sacbrood virus was present at low frequencies. The one-step real-time SG RT-PCR methods are fast, accurate, and useful for detecting and quantifying these honeybee viruses, which cause inapparent infections and contribute to the increasing depopulation of honeybee colonies.
分别开发或改编了两种基于SYBR Green（SG）化学的一步法实时逆转录聚合酶链反应（RT-PCR）检测方法，用于检测、区分和定量两种重要的蜜蜂病毒：囊状幼虫病毒（SBV）和急性蜜蜂麻痹病毒（ABPV）。对这两种反应均进行了优化，以实现最高的灵敏度和特异性。ABPV RT-PCR每个反应的基因组等效拷贝数（GEC）检测限为389.3。SBV RT-PCR每个反应的GEC检测限为298.9。通过熔解曲线分析和PCR产物测序确认病毒检测和鉴定。这两种技术均用于评估西班牙野外样本并确定这些病毒的分布情况。未检测到急性蜜蜂麻痹病毒，囊状幼虫病毒以低频率存在。一步法实时SG RT-PCR方法快速、准确，对于检测和定量这些导致隐性感染并促使蜜蜂蜂群数量不断减少的蜜蜂病毒非常有用。
