Development and optimization of a TaqMan assay for Nosema bombycis, causative agent of pebrine disease in Bombyx mori silkworm, based on the beta-tubulin gene

Abstract

"Pe ' brine" is a devastating disease of Bombyx mori silkworms that is highly contagious and can completely destroy an entire crop of silkworms and is thus a serious threat for the viability and profitability of sericulture. The disease is most commonly attributed to microsporidians of the genus Nosema, which are obligate intracellular parasites that are transmitted through spores. Nosema infections in silkworms are diagnosed primarily through light microscopy, which is labour intensive and less reliable, sensitive, and specific than PCR-based techniques. Here, we present the development and optimization of a new TaqMan based assay targeting the beta-tubulin gene in the pe ' brine disease causing agent Nosema bombycis in silkworms. The assay displayed excellent quantification linearity over multiple orders of magnitude of target amounts and a limit of detection (LOD) of 6.9 x 102 copies of target per reaction. The method is highly specific to N. bombycis with no cross-reactivity to other Nosema species commonly infecting wild silkworms. This specificity was due to three nucleotides in the probe-binding region unique to N. bombycis. The assay demonstrated a high reliability with a Coefficient of variation (CV) <5% for both intra-assay and inter-assay variability. The assay was used to trace experimental N. bombycis infection of silkworm larvae, in the fat body, midgut and ovary tissues, through pupation and metamorphosis to the emerging female moth, and her larval off-spring, confirming the vertical transmission of N. bombycis in silkworms. The TaqMan assay revealed a gradual increase in infection levels in the post-infection samples. The assay is reliable and simple to implement and can be a suitable complement to microscopy for routine diagnostics and surveillance in silkworm egg production centres with appropriate infrastructure.

Keywords

Silkworm; Bombyx mori; Pebrine; Nosema bombycis; TaqMan; beta-Tubulin

Published in

Journal of Microbiological Methods
2021, Volume: 186, article number: 106238
Publisher: ELSEVIER

SLU Authors

• Nilsson, Anna

  • Department of Ecology, Swedish University of Agricultural Sciences
    

• Rodrigues de Miranda, Joachim

  • Department of Ecology, Swedish University of Agricultural Sciences
    

• Terenius, Olle

  • Department of Ecology, Swedish University of Agricultural Sciences
    

UKÄ Subject classification

Microbiology


Publication identifier

DOI: https://doi.org/10.1016/j.mimet.2021.106238

Permanent link to this page (URI)

https://res.slu.se/id/publ/112680