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KMID : 0893420210220030040
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Journal of Veterinary Science
2021 Volume.22 No. 3 p.40 ~ p.40
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Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR
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Truong A-Tai
Sevin Sedat
Kim Seon-Mi
Yoo Mi-Sun
Cho Yun-Sang
Yoon Byoung-Su
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Abstract
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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.
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KEYWORD
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Nosema ceranae, microscopic enumeration, ultra-rapid real-time PCR, nosemosis prevention
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