Abstract
Microsporidiosis (Pebrine) caused by the microsporidian parasite is one of the important devastating disease which affect the silk production leading to an unprofitable harvest. Till date ribosomal RNA (rRNA) gene was used as a target for detection of microsporidian species. In this study, we describe conventional and SYBR green based real-time PCR techniques alternatively targeting β-tubulin gene for quantitative detection of microsporidia infecting both the mulberry and non-mulberry silkworms. The modified DNA extraction method followed in our study was found to be easy, economical and could be used for both conventional and real time PCR as template. The real time qPCR revealed the expression of β-tubulin gene in different infected tissues of the silkworm Bombyx mori. The sensitivity of the SYBR green based real time PCR was found to be 100 times more than the conventional PCR and PCR was found more sensitive than the microscopic examination. The developed method did not produce any false positive results with the other silkworm pathogens and healthy silkworm. The data suggest that both the developed PCR methods targeting β-tubulin gene could be used effectively in quarantine process at seed centres for early detection of microsporidian infection in silkworms.
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Acknowledgements
The authors would like to acknowledge Dr. G Subramaniyam, Dr. Rajesh Kumar CMERTI, Lahdoigarh, Assam, Dr. K. Velayudhan, ESSPC, Hosur and Dr. Wazid Hassan SBRL Bangalore for providing the infected silk moth and larval samples.
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Funding was provided by Department of Biotechnology, Ministry of Science and Technology (BT/PR15405/TDS/121/14/2015 dt 09.02.2017).
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K.M. Ponnuvel designed the experiment, Ms. Vijaya Gowri, Ms. M. Aarthi and Ms. M. Shruthi performed the laboratory experiments. Ms. Vijaya Gowri, Dr. K.M. Ponnuvel and Ms. Tania Gupta wrote the manuscript.
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Esvaran, V.G., Mohanasundaram, A., Mahadeva, S. et al. Development and comparison of real-time and conventional PCR tools targeting β-tubulin gene for detection of Nosema infection in silkworms. J Parasit Dis 43, 31–38 (2019). https://doi.org/10.1007/s12639-018-1053-4
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DOI: https://doi.org/10.1007/s12639-018-1053-4