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Development of a loop-mediated isothermal amplification (LAMP) and a direct LAMP for the specific detection of Nosema ceranae, a parasite of honey bees

Abstract

Nosema ceranae is a ubiquitous microsporidian pathogen infecting the midgut of honey bees. The infection causes bee nosemosis, a disease associated with malnutrition, dysentery, and lethargic behavior, and results in considerable economic losses in apiculture. The use of a rapid, sensitive, and inexpensive DNA-based molecular detection method assists in the surveillance and eventual control of this pathogen. To this end, a loop-mediated isothermal amplification (LAMP) assay targeting the single-copy gene encoding the polar tube protein 3 (PTP3) has been developed. Genomic DNA of N. ceranae–infected forager bees sampled from distant geographic regions could be reliably amplified using the established LAMP assay. The N. ceranae-LAMP showed higher sensitivity than a classical reference PCR (98.6 vs 95.7%), when both approaches were applied to the detection of N. ceranae. LAMP detected a ten-fold lower infection rate than the reference PCR (1 pg vs 10 pg genomic DNA, respectively). In addition, we show highly specific and sensitive detection of N. ceranae from spore preparations in a direct LAMP format. No cross-reactions with genomic DNA and/or spores from N. apis, often co-infecting A. mellifera, or from N. bombi, infecting bumble bees, were observed. This low-cost and time-saving molecular detection method can be easily applied in simple laboratory settings, facilitating a rapid detection of N. ceranae in honey bees in epidemiological studies, surveillance and control, as well as evaluation of therapeutic measures against nosemosis.

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Funding

This research has been financially supported by grants from the University of Moron, Argentina (PI3/18-09-LS-07, Code: 800201702000023UM), the Argentine Ministry of Science, Technology and Productive Innovation (PICT-2014-1585), and the National Institute of Agricultural Technology, Argentina (2019-PE-E1-I017-001).

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Conceptualization: Leonhard Schnittger. Formal analysis: Lucas Lannutti and Leonhard Schnittger. Investigation: Lucas Lannutti, Anabela Mira, and Victoria Silva. Resources: Marina Basualdo, Graciela Rodriguez, Silvio Erler, Sebastian Gisder, and Elke Genersch. Writing–original draft: Lucas Lannutti, Monica Florin-Christensen, and Leonhard Schnittger. Writing–review and editing: Silvio Erler, Elke Genersch, and Leonhard Schnittger. Visualization: Lucas Lannutti. Supervision: Leonhard Schnittger. Funding acquisition: Leonhard Schnittger, Marina Basualdo, and Monica Florin-Christensen.

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Correspondence to Leonhard Schnittger.

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Lannutti, L., Mira, A., Basualdo, M. et al. Development of a loop-mediated isothermal amplification (LAMP) and a direct LAMP for the specific detection of Nosema ceranae, a parasite of honey bees. Parasitol Res 119, 3947–3956 (2020). https://doi.org/10.1007/s00436-020-06915-w

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  • DOI: https://doi.org/10.1007/s00436-020-06915-w

Keywords

  • Loop-mediated isothermal amplification
  • LAMP
  • Microsporidia
  • Nosema ceranae
  • Nosema apis
  • Nosemosis
  • Apis mellifera
  • Apis cerana