Development of isothermal amplification assay for detection of Nosema bombycis infection in silkworm Bombyx mori targeting polar tube protein 1 gene

Abstract

Microsporidiosis of the silkworm Bombyx mori is caused by the highly virulent parasite Nosema bombycis (Nageli). The infection can be deleterious due to horizontal and vertical transmission, causing heavy damage to the sericulture industry. In recent years, molecular diagnostics has revolutionized the possibility to detect diseases in terms of rapidity and simplicity, however, most of them are time consuming, require sophisticated instruments and skilled personnel. In this study, the polar tube protein 1 gene of N. bombycis (Indian isolate) was cloned, characterized and utilized for the development of rapid and simple loop mediated isothermal amplification assay (LAMP) for detection of microsporidiosis in silkworm B. mori. The LAMP reaction conditions were optimized to 65 °C for 60 min. The developed method demonstrated a higher sensitivity and the detection limit was found to be 2-fold higher than conventional PCR. This is the first report on loop mediated isothermal amplification assay that could be used to diagnose microsporidiosis at various developmental stages of the silkworm. This method serves as a robust alternative technique to conventional PCR and aids in the rapid diagnosis of N. bombycis infecting silkworm B. mori.

Keywords

• microsporidiosis
• Nosema bombycis
• loop mediated isothermal amplification
• polar tube protein 1