Simultaneous detection of bee viruses by multiplex PCR

Sguazza, Guillermo Hernán; Reynaldi, Francisco José; Galosi, Cecília Mônica; Pecoraro, Marcelo Ricardo Ítalo

doi:10.1016/j.jviromet.2013.08.003

Cited by 49 publications

(40 citation statements)

References 32 publications

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“…The multiplex PCR method offers a significant cost-saving advantage, especially when large numbers of samples are analyzed (Sguazza et al 2013). Furthermore, multiplex PCR is able to detect multiple target DNA sequences in a single reaction, with the simultaneous identification of two or more kinds of pathogens, simplifying the workflow and processing time, which are significantly reduced (Bilgic et al 2013), among other advantages (Edwards and Gibbs 1994).…”

Section: Discussionmentioning

confidence: 99%

Simultaneous detection of Nosema spp., Ascosphaera apis and Paenibacillus larvae in honey bee products

Guimarães-Cestaro¹,

Serrão²,

Message³

et al. 2016

JHR

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Honey bees are responsible for pollinating many native and cultivated plant species. These insects can be affected by many pathogens, including fungi and bacteria, both of which can form spores that are easily dispersed within the colony by means of the stored products, among other routes. The objective of this study was to develop a method to detect spores of the honey bee pathogens Nosema apis, Nosema ceranae, Ascosphaera apis and Paenibacillus larvae in samples of honey, bee pollen and royal jelly. The method was standardized for each product individually, and then analyzed by monoplex and multiplex PCR, which showed the same detection thresholds: 1.25 spores/mL of honey for N. ceranae; 7.5 spores/mL of honey for A. apis; and 0.4 spore/mL of honey for P. larvae, respectively. The standardized technique was effective and rapid for the detection of these pathogens in bee products and can be used for the establishment of official methods of sanitary control of bee products, considering the growing national and international trade of these products and the movement or migration of colonies between regions.

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Section: Discussionmentioning

confidence: 99%

Simultaneous detection of Nosema spp., Ascosphaera apis and Paenibacillus larvae in honey bee products

Guimarães-Cestaro¹,

Serrão²,

Message³

et al. 2016

JHR

View full text Add to dashboard Cite

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“…They rely on specific primers/probes and require known sequences in the target nucleic acid (Santos et al, 2004;Arya et al, 2005). Microarrays and multiplex PCR come in handy when one needs to analyze different viruses in one experiment (Wang et al, 2002;Chen et al, 2004;Liu et al, 2005;Topley et al, 2005;Glover et al, 2011;Sguazza et al, 2013).…”

Section: Viral Infections -Detection Nucleic Acid-based Assaysmentioning

confidence: 99%

“…C sophisticated method C expensive equipment needed C indirect viral genome analysis C siRNA coverage of virus genome is unevenly distributed EM (electron microscopy) (Sguazza et al, 2013;Aubert et al, 2007) C no specific reagents are needed (method independent of virus type) C provides structural information C expensive and time consuming C low specificity due to similarity in size and shape of viruses Serological methods (ELISA, Western Blot, immunodiffusion) (Chen et al, 2004;Sguazza et al, 2013;Aubert et al, 2007) C useful in detection of similar, unknown viruses C when modified into LFT, the methods are very quick C require specific antibodies (production may be expensive) C lower specificity and sensitivity than molecular methods C require laboratory equipment (except ready-to-use diagnostic tests) C time consuming in standard format Using ELISA one can estimate virus concentration in a sample, if a reference material is available (Sguazza et al, 2013). Although serological methods do not require knowledge of antigen structure or sequence, there are still drawbacks associated with their use.…”

Section: Protein-based Assaysmentioning

confidence: 99%

Viral infections of Apis mellifera : its importance, dynamics, diagnostics and treatment opportunities

Matusiewicz¹,

Margas²,

Mazurek³

et al. 2016

bta

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Honey bees (Apis mellifera ) play a crucial function in terrestrial ecosystems by maintaining floral biodiversity and providing sustainable agriculture through pollination. Bees affect 35% of the world's crop production, hence, they are essential for economic sustainability, agriculture and food security. The economic value of pollination by bees goes beyond agricultural production. An important role of bees is pollination of the plants that provide food to the wild animals, and this is part of the proper functioning of the ecosystem. On the one hand Apis mellifera has great adaptive potential and it may be found in highly diverse climates almost everywhere in the world, but on the other hand bees are under constant threat by numerous pathogens including bacteria, viruses, fungi, Microsporidia, mites and insects. Other stress factors include environmental change-related stresses, pesticides, malnutrition and migratory beekeeping. The interactions among these, along with possibly other unknown factors, are causing widespread decline in honey bee populations -Colony Collapse Disorder (CCD) syndrome. Viruses are the most recent and still poorly investigated pathogens of the honey bees. Simultaneously, Varroa destructor mites and viruses are currently the high-profile suspects in collapsing bee colonies. Varroa acts as a vector for a range of RNA viruses of honey bees which are transferred when the mite feeds on hemolymph from the developing pupa. This publication aims to present an overview of the most important honey bee viruses, their diagnostics methods and treatment opportunities with emphasis on the most common and devastating viruses from Dicistroviridae and Iflaviridae families.

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“…The virus specific primers were based on the previous reports for DWV, BQCV, IAPV, SBV, ABPV, and CBPV (Sguazza et al, 2013), and KBV (Shen et al, 2005) (Tab. 1).…”

Section: Reverse Transcription-polymerase Chain Reaction (Rt-pcr) Ampmentioning

confidence: 99%

“…1). The reaction mixture and thermal cycling profiles of cDNA synthesis (Reverse Transcription System, Promega, Madison, WI) and the amplification, were performed as previously described (Sguazza et al, 2013). A negative and a positive DNA control were performed for each PCR reaction.…”

Section: Reverse Transcription-polymerase Chain Reaction (Rt-pcr) Ampmentioning

confidence: 99%

Multiple Virus Infections and the Characteristics of Chronic Bee Paralysis Virus in Diseased Honey Bees (Apis Mellifera L.) in China

Jia

Wang

et al. 2015

Journal of Apicultural Science

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A b s t r a c t China has the largest number of managed honey bee colonies globally, but there is currently no data on viral infection in diseased A. mellifera L. colonies in China. In particular, there is a lack of data on chronic bee paralysis virus (CBPV) in Chinese honey bee colonies. Consequently, the present study investigated the occurrence and frequency of several widespread honey bee viruses in diseased Chinese apiaries, and we used the reverse transcription-polymerase chain reaction (RT-PCR) assay. Described was the relationship between the presence of CBPV and diseased colonies (with at least one of the following symptoms: depopulation, paralysis, dark body colorings and hairless, or a mass of dead bees on the ground surrounding the beehives). Phylogenetic analyses of CBPV were employed. The prevalence of multiple infections of honey bee viruses in diseased Chinese apiaries was 100%, and the prevalence of infections with even five and six viruses were higher than expected. The incidence of CBPV in diseased colonies was significantly higher than that in apparently healthy colonies in Chinese A. mellifera aparies, and CBPV isolates from China can be separated into Chinese-Japanese clade 1 and 2. The results indicate that beekeeping in China may be threatened by colony decline due to the high prevalence of multiple viruses with CBPV.Keywords: Apis mellifera L, China, chronic bee paralysis virus (CBPV), diseased honey bees, multiple viruses, phylogeny. INTRODUCTIONHoney bees (Apis mellifera L.) are one of the most important and economically valuable pollinators of crops worldwide and are the most commonly managed pollinator species. Colonies of A. mellifera serve as biological indicators and provide invaluable data on the environmental and ecological impacts of particular factors on nature in general (Celli and Maccagnani, 2003). However, the abundance of honey bees is currently declining (vanEngelsdorp et al., 2011). The possible reasons for this decline are pathogens and pesticides (Vejsnaes et al., 2010). To date, at least 18 viruses have been reported in honey bees colonies (de Miranda et al., 2013) with the following viruses being the most common: deformed wing virus (DWV), black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), sacbrood virus (SBV), acute bee paralysis virus (ABPV), Kashmir bee virus (KBV), and chronic bee paralysis virus (CBPV). Multiple viral infections in honey bees have been reported in several countries (Berényi et al., 2006;Todd et al., 2007;Chen et al., 2011;Morimoto et al., 2012). Multiple viral infections are considered to (Allen and Ball, 1996;Chantawannakul et al., 2006). Thus, monitoring the occurrence and frequency of these viruses is vital for maintaining successful apiaries. It has been reported that the following viruses can be present in apparently healthy honey bee colonies: DWV, BQCV, IAPV, SBV, ABPV, and KBV (Berényi et al., 2006;Ai et al., 2012;Morimoto et al., 2012;Yang et al., 2013;Jia et al., 2014). The majority of DWV infected bees were asympt...

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Simultaneous detection of bee viruses by multiplex PCR

Cited by 49 publications

References 32 publications

Simultaneous detection of Nosema spp., Ascosphaera apis and Paenibacillus larvae in honey bee products

Simultaneous detection of Nosema spp., Ascosphaera apis and Paenibacillus larvae in honey bee products

Viral infections of Apis mellifera : its importance, dynamics, diagnostics and treatment opportunities

Multiple Virus Infections and the Characteristics of Chronic Bee Paralysis Virus in Diseased Honey Bees (Apis Mellifera L.) in China

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