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Research article - Peer-reviewed, 2014

Development and validation of a real-time two-step RT-qPCR TaqMan (R) assay for quantitation of Sacbrood virus (SBV) and its application to a field survey of symptomatic honey bee colonies

Blanchard, Philippe; Guillot, Sylvain; Antùnez, Karina; Köglberger, Hemma; Kryger, Per; Rodrigues De Miranda, Joachim; Franco, Stéphanie; Chauzat, Marie-Pierre; Thiéry, Richard; Ribiere, Magali

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 subcuticular 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 (R) 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 0.11 log(10). The detection and quantitation limits were established respectively at 50 copies and 100 copies of SBV genome, for a template volume of 5 mu l 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). (C) 2013 Elsevier B.V. All rights reserved.

Keywords

Sacbrood virus (SBV); Real-time RT-PCR; Validation; Field survey; Apis mellifera

Published in

Journal of Virological Methods
2014, volume: 197, pages: 7-13
Publisher: ELSEVIER SCIENCE BV

Authors' information

Blanchard, Philippe
French Agency for Food, Environmental and Occupational Health & Safety (ANSES)
Guillot, Sylvain
French Agency for Food, Environmental and Occupational Health & Safety (ANSES)
Antùnez, Karina
Instituto de Investigaciones Biologicas Clemente Estable
Köglberger, Hemma
AGES, Institut für Saat- und Pflanzgut, Pflanzenschutzdienst und Bienen
Kryger, Per
Swedish University of Agricultural Sciences, Department of Ecology
Franco, Stéphanie
French Agency for Food, Environmental and Occupational Health & Safety (ANSES)
Chauzat, Marie-Pierre
French Agency for Food, Environmental and Occupational Health & Safety (ANSES)
Thiéry, Richard
French Agency for Food, Environmental and Occupational Health & Safety (ANSES)
Ribiere, Magali
French Agency for Food, Environmental and Occupational Health & Safety (ANSES)

UKÄ Subject classification

Biochemistry and Molecular Biology

Publication Identifiers

DOI: https://doi.org/10.1016/j.jviromet.2013.09.012

URI (permanent link to this page)

https://res.slu.se/id/publ/63765