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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

Jagadish, Anupama and Khajje, Diksha and Tony, Merinrose and Nilsson, Anna and Rodrigues De Miranda, Joachim and Terenius, Olle and Dubey, Himanshu and Mishra, Rakesh K. and Ponnuvel, Kangayam M. (2021). 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. Journal of Microbiological Methods. 186 , 106238
[Research article]

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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.

Authors/Creators:Jagadish, Anupama and Khajje, Diksha and Tony, Merinrose and Nilsson, Anna and Rodrigues De Miranda, Joachim and Terenius, Olle and Dubey, Himanshu and Mishra, Rakesh K. and Ponnuvel, Kangayam M.
Title: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
Series Name/Journal:Journal of Microbiological Methods
Year of publishing :2021
Volume:186
Article number:106238
Number of Pages:7
Publisher:ELSEVIER
ISSN:0167-7012
Language:English
Publication Type:Research article
Article category:Scientific peer reviewed
Version:Published version
Copyright:Creative Commons: Attribution 4.0
Full Text Status:Public
Subjects:(A) Swedish standard research categories 2011 > 1 Natural sciences > 106 Biological Sciences (Medical to be 3 and Agricultural to be 4) > Microbiology (Microbiology in the medical area to be 30109)
Keywords:Silkworm, Bombyx mori, Pebrine, Nosema bombycis, TaqMan, beta-Tubulin
URN:NBN:urn:nbn:se:slu:epsilon-p-112680
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-p-112680
Additional ID:
Type of IDID
DOI10.1016/j.mimet.2021.106238
Web of Science (WoS)000659532600005
ID Code:24814
Faculty:NJ - Fakulteten för naturresurser och jordbruksvetenskap
Department:(NL, NJ) > Dept. of Ecology
(S) > Dept. of Ecology
Deposited By: SLUpub Connector
Deposited On:07 Jul 2021 11:26
Metadata Last Modified:03 Aug 2021 14:02

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