A sensitive one-step real-time RT-PCR method for detection of deformed wing virus and black queen cell virus in honeybee Apis mellifera.
A one-step real-time RT-PCR based on SYBR Green (SG) chemistry was developed for the detection, differentiation and quantification of two of the most common viruses on the honeybee Apis mellifera L., deformed wing virus and black queen cell virus. Two sets of primers specific for each virus, were designed in conserved regions of the viral genome for their use in the one-step real-time RT-PCR. Both reactions were optimized for highest sensitivity and specificity and SG-based real-time was used to achieve quantitative detection. All samples evaluated in this study were from Spanish honeybee colonies. Viral detection and identification was confirmed by sequencing of the PCR products. The described one-step real-time SG RT-PCR proved to be a fast, accurate and useful technique to detect and even quantify these honeybee viruses that cause unapparent infections, and might contribute with other factors to the increasing honeybee colonies depopulation.
Kukielka
Deborah
D
Animal Health Department, Faculty of Veterinary Science, Universidad Complutense de Madrid, Avda/Puerta de Hierro s/n, 28040 Madrid, Spain.
Esperón
Fernando
F
Higes
Mariano
M
Sánchez-Vizcaíno
Jose Manuel
JM
D000818:Animals; D001516:Bees / Q000821:virology*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D012367:RNA, Viral