Development and optimization of a TaqMan assay for Nosema bombycis, causative agent of pébrine disease in Bombyx mori silkworm, based on the β-tubulin gene
A. Jagadish, Diksha Khajje, Merinrose Tony, Anna Nilsson, Joachim R. de Miranda, Olle Terenius, Himanshu Dubey, Rakesh K. Mishra, Kangayam M. Ponnuvel
Abstract
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.