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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 and Guillot, Sylvain and Antùnez, Karina and Köglberger, Hemma and Kryger, Per and Rodrigues De Miranda, Joachim and Franco, Stéphanie and Chauzat, Marie-Pierre and Thiéry, Richard and Ribière, Magali (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. Journal of Virological Methods. 197, 7-13
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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).

Authors/Creators:Blanchard, Philippe and Guillot, Sylvain and Antùnez, Karina and Köglberger, Hemma and Kryger, Per and Rodrigues De Miranda, Joachim and Franco, Stéphanie and Chauzat, Marie-Pierre and Thiéry, Richard and Ribière, Magali
Title: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
Series/Journal:Journal of Virological Methods (0166-0934)
Year of publishing :2014
Volume:197
Page range:7-13
Number of Pages:7
Publisher:Elsevier
ISSN:0166-0934
Language:English
Publication Type:Journal article
Refereed:Yes
Article category:Scientific peer reviewed
Version:Accepted version
Full Text Status:Public
Agris subject categories.:L Animal production > L73 Animal diseases
Subjects:(A) Swedish standard research categories 2011 > 1 Natural sciences > 106 Biological Sciences (Medical to be 3 and Agricultural to be 4) > Biochemistry and Molecular Biology
Agrovoc terms:viruses, quantitative analysis, honey bees, apis mellifera
Keywords:sacbrood virus (SBV), real-time RT-PCR, validation, field survey, apis mellifera
URN:NBN:urn:nbn:se:slu:epsilon-e-2815
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-e-2815
Additional ID:
Type of IDID
Web of Science (WoS)000331484400002
DOI10.1016/j.jviromet.2013.09.012
ID Code:11859
Faculty:NJ - Fakulteten för naturresurser och jordbruksvetenskap
Department:(NL, NJ) > Dept. of Ecology
(S) > Dept. of Ecology
Deposited By: SLUpub Connector
Deposited On:30 Jul 2015 06:53
Metadata Last Modified:22 Feb 2016 12:06

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