One-step real-time quantitative PCR assays for the detection and field study of Sacbrood honeybee and Acute bee paralysis viruses

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-PCF. 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. (C) 2009 Elsevier B.V. All rights reserved.