title: Development and validation of a real-time two-step RT-qPCR TaqMan(®) assay for quantitation of Sacbrood virus (SBV) and its application to a field survey of symptomatic honey bee colonies.
abstract: Sacbrood virus (SBV) is the causal agent of a disease of honey bee larvae, resulting in failure to pupate and causing death. The typical clinical symptom of SBV is an accumulation of SBV-rich fluid in swollen sub-cuticular pouches, forming the characteristic fluid-filled sac that gives its name to the disease. Outbreaks of the disease have been reported in different countries, affecting the development of the brood and causing losses in honey bee colonies. Today, few data are available on the SBV viral load in the case of overt disease in larvae, or for the behavioural changes of SBV-infected adult bees. A two-step real-time RT-PCR assay, based on TaqMan(®) technology using a fluorescent probe (FAM-TAMRA) was therefore developed to quantify Sacbrood virus in larvae, pupae and adult bees from symptomatic apiaries. This assay was first validated according to the recent XP-U47-600 standard issued by the French Standards Institute, where the reliability and the repeatability of the results and the performance of the assay were confirmed. The performance of the qPCR assay was validated over the 6 log range of the standard curve (i.e. from 10(2) to 10(8) copies per well) with a measurement uncertainty evaluated at 0.11log10. The detection and quantitation limits were established respectively at 50 copies and 100 copies of SBV genome, for a template volume of 5μl of cDNA. The RT-qPCR assay was applied during a French SBV outbreak in 2012 where larvae with typical SBV signs were collected, along with individuals without clinical signs. The SBV quantitation revealed that, in symptomatic larvae, the virus load was significantly higher than in samples without clinical signs. Combining quantitation with clinical data, a threshold of SBV viral load related to an overt disease was proposed (10(10) SBV genome copies per individual).
authors.affiliation: Anses, Sophia-Antipolis Laboratory, Bee Diseases Unit, BP 111, 06902 Sophia Antipolis, France. Electronic address: philippe.blanchard@anses.fr.
authors.forename: Philippe
authors.initials: P
authors.lastname: Blanchard
authors.affiliation: Anses, Sophia-Antipolis Laboratory, Bee Diseases Unit, BP 111, 06902 Sophia Antipolis, France.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Guillot
authors.affiliation: Laboratorio de Microbiologia, Instituto de Investigaciones Biologicas Clemente Estable, Avenida Italia 3318, CP 11600 Montevideo, Uruguay.
authors.forename: Karina
authors.initials: K
authors.lastname: Antùnez
authors.affiliation: AGES, Institut für Saat- und Pflanzgut, Pflanzenschutzdienst und Bienen, Abteilung für Bienenkunde und Bienenschutz, Spargelfeldstrasse 191, 1220 Wien, Austria.
authors.forename: Hemma
authors.initials: H
authors.lastname: Köglberger
authors.affiliation: University of Aarhus, Faculty of Agricultural Sciences, Department of Integrated Pest Management, Forsogsvej 1, DK-4200 Slagelse, Denmark.
authors.forename: Per
authors.initials: P
authors.lastname: Kryger
authors.affiliation: Department of Ecology, Swedish University of Agricultural Sciences, 750-07 Uppsala, Sweden.
authors.forename: Joachim R
authors.initials: JR
authors.lastname: de Miranda
authors.affiliation: Anses, Sophia-Antipolis Laboratory, Bee Diseases Unit, BP 111, 06902 Sophia Antipolis, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Franco
authors.affiliation: Anses, Sophia-Antipolis Laboratory, Bee Diseases Unit, BP 111, 06902 Sophia Antipolis, France.
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Chauzat
authors.affiliation: Anses, Sophia-Antipolis Laboratory, Bee Diseases Unit, BP 111, 06902 Sophia Antipolis, France. Electronic address: richard.thiery@anses.fr.
authors.forename: Richard
authors.initials: R
authors.lastname: Thiéry
authors.affiliation: Anses, Sophia-Antipolis Laboratory, Bee Diseases Unit, BP 111, 06902 Sophia Antipolis, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Ribière
mesh_terms: D000818:Animals; D001516:Bees / Q000821:virology*; D005602:France; D010849:Picornaviridae / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019562:Viral Load / Q000379:methods*
keywords: Apis mellifera; Field survey; Real-time RT-PCR; Sacbrood virus (SBV); Validation