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Optimization of duplex real-time PCR with melting-curve analysis for detecting the microsporidian parasites Nosema apis and Nosema ceranae in Apis mellifera1

  • Karen L. Burgher-MacLellan (a1), Geoffrey R. Williams (a2), Dave Shutler (a3), Kenna MacKenzie (a1) and Richard E.L. Rogers (a4)
  • DOI: http://dx.doi.org/10.4039/n10-010
  • Published online: 01 April 2012
Abstract
Abstract

Honey bees, Apis mellifera (L.) (Hymenoptera: Apidae), are parasitized by the microsporidians Nosema apis (Zander) and Nosema ceranae (Fries). Molecular techniques are commonly used to differentiate between these parasites because light microscopy is inadequate. Our objectives were to (i) adapt the previously published duplex polymerase chain reaction (PCR) targeting the 16S rRNA gene of N. apis (321APIS-FOR, 321APIS-REV) and N. ceranae (218MITOC-FOR, 218MITOC-REV) using qualitative real-time PCR assay with SYBR® Green I dye (R-T PCR) and DNA melting-curve analysis, and (ii) determine whether the two Nosema species can be detected simultaneously in honey bees. Total spore counts and purified total genomic DNA were obtained from 37 bee samples (19 individual workers and 18 pooled samples of 15 workers) collected in Nova Scotia, Prince Edward Island, and Newfoundland, Canada. Overall, the prevalence of Nosema species was 86.5% (32/37 samples of bee DNA), based on conventional PCR and the optimized R-T PCR assay. The melting-curve analysis showed three groups of curve profiles that could determine the prevalence of N. apis, N. ceranae, and co-infection (21.9%, 56.2%, and 21.9%, respectively). The duplex R-T PCR assay was efficient, specific, and more sensitive than duplex conventional PCR because co-infection was identified in 5.4% (n = 2) more samples. Sequencing of R-T PCR products confirmed the results of the melting-curve analysis. Duplex R-T PCR with melting-curve analysis is a sensitive and rapid method of detecting N. apis, N. ceranae, and co-infection in honey bees.

Résumé

Les abeilles domestiques, Apis mellifera (L.) (Hymenoptera: Apidae) sont parasitées par les microsporidies Nosema apis (Zander) et N. ceranae (Fries). Parce que la microscopie optique est inadéquate, on utilise couramment des méthodes moléculaires pour distinguer ces parasites. Nos objectifs sont 1) d'adapter la méthode déjà publiée de la réaction de PCR (amplification en chaîne par polymérase) duplex qui cible le gène 16S de l'ARNr de N. apis (321APIS-FOR et 321APIS-REV) et de N. ceranae (218MITOC-FOR et 218MITOC-REV) à l'aide d'un test qualitatif au vert de SYBR I en temps réel avec une analyse de la courbe de fusion de l'ADN (R-T PCR) et 2) de voir s’il est possible de détecter simultanément les deux espèces de Nosema chez les abeilles. Nous avons obtenu les dénombrements de spores et l'ADN génomique total purifié dans 37 échantillons d'abeilles (19 ouvrières individuelles et 18 échantillons collectifs de 15 ouvrières) récoltés en Nouvelle-Écosse, à l'Île-du-Prince-Édouard et à Terre-Neuve, Canada. La prévalence globale de Nosema est de 86,5 % (32/37 échantillons d'ADN d'abeilles) d'après les analyses de PCR conventionnelle et de R-T PCR optimisée. L’analyse de la courbe de fusion révèle l'existence de trois groupes de profils de courbes qui permettent d'identifier les prévalences de N. apis, de N. ceranae et de co-infections (respectivement 21,9 %, 56,2 % et 21,9 %). Le test de la R-T PCR duplex est efficace, spécifique et plus sensible que la PCR duplex ordinaire parce que la co-infection a pu être décelée dans 5,4 % (n=2) plus d'échantillons. Le séquençage des produits de la R-T PCR confirme les résultats de l'analyse de la courbe de fusion. La PCR duplex au vert SYBR I en temps réel avec une analyse de la courbe de fusion est une méthode sensible et rapide de détection de N. apis, de N. ceranae et des co-infections chez les abeilles.

[Traduit par la Rédaction]

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2 Corresponding author (e-mail: burgherk@agr.gc.ca).
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Contribution No. 2365 from the Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, Kentville, Nova Scotia.

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