Direct and sensitive detection of a microsporidian parasite of bumblebees using loop-mediated isothermal amplification (LAMP)

Yuto Kato,Maki N Inoue,Madoka Nakai,Takahiro Yanagisawa,Ken Komatsu

doi:10.1038/s41598-020-57909-8

Yuto Kato, Maki N Inoue + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-57909-8

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Abstract

The reduction of bumblebee populations has been reported in the last decades, and the microsporidian parasite Nosema bombi is considered as one of the factors contributing to such reduction. Although the decline of bee populations affects both wild plants and human food supply, the effects of Nosema spp. infections are not known because it is difficult to obtain infective spores from wild bees due to their low prevalence. Microscopical observation of fecal samples or midgut homogenates and/or PCR are generally used for N. bombi detection. However, the germination rate of microsporidian spore declines if they are kept at 4 °C for a long time or frozen. It is therefore crucial to minimize the diagnosis and isolation time of infective spores from field-collected samples. Therefore, we performed a loop-mediated isothermal amplification (LAMP) assay for the direct detection of N. bombi in bumblebee midgut homogenates. Using this method, we could detect N. bombi from individuals from which it was visible under the microscope and directly from wild individuals.

Highlights

The reduction of bumblebee populations has been reported in the last decades, and the microsporidian parasite Nosema bombi is considered as one of the factors contributing to such reduction
N. ceranae was found to infect A. mellifera leading to the reduction of the survival rate of workers and collapse of colonies, suggesting that the host switch between A. mellifera and A. cerana contributed to the reduction of A. mellifera populations[18,19]
To optimize the reaction temperatures of the loopmediated isothermal amplification (LAMP) assay for detecting N. bombi, we conducted a LAMP assay from 61 °C to 64 °C using the DNA extracted from the spore suspension of N. bombi (c. 1 ng/μl)

Summary

Introduction

The reduction of bumblebee populations has been reported in the last decades, and the microsporidian parasite Nosema bombi is considered as one of the factors contributing to such reduction. We performed a loopmediated isothermal amplification (LAMP) assay for the direct detection of N. bombi in bumblebee midgut homogenates. Using this method, we could detect N. bombi from individuals from which it was visible under the microscope and directly from wild individuals. Two additional “loop-primers”, LF and LB, contribute to increase the specificity of the LAMP reaction and to accelerate it[31] In this method, the amplification can be detected without electrophoresis using magnesium pyrophosphate as a by-product[32,33,34]. We tested LAMP as a method for detecting N. bombi from midgut homogenates without DNA extraction, which improves the versatility of LAMP

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Conclusion