Journal of Veterinary Science

The Korean Society of Veterinary Science (대한수의학회)
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Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR

  • Truong, A-Tai (Department of Life Science, College of Fusion Science, Kyonggi University) ;
  • Sevin, Sedat (Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Ankara University) ;
  • Kim, Seonmi (Department of Life Science, College of Fusion Science, Kyonggi University) ;
  • Yoo, Mi-Sun (Parasitic and Honeybee Disease Laboratory, Bacterial Disease Division, Department of Animal & Plant Health Research, Animal and Plant Quarantine Agency) ;
  • Cho, Yun Sang (Parasitic and Honeybee Disease Laboratory, Bacterial Disease Division, Department of Animal & Plant Health Research, Animal and Plant Quarantine Agency) ;
  • Yoon, Byoungsu (Department of Life Science, College of Fusion Science, Kyonggi University)
  • Received : 2020.06.06
  • Accepted : 2021.04.12
  • Published : 2021.05.31
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Abstract

Background: The microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen. Objectives: The present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees. Methods: A procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration. Results: UR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 104 spores/bee, and the stable detection level was ≥ 2.40 × 105 spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 104 copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min. Conclusions: UR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.

Keywords

Acknowledgement

This work was supported by the "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01408002)" Rural Development Administration of the Republic of Korea, as well as by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry, through the Agri-Bio Industry Technology Development Program (318093-03), partially funded by the Animal and Plant Quarantine Agency (Grant No. B-1543081-2019-21-03).

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