PCR-based detection of microsporidia in silkworms using non-conventional RNA polymerase primers

Abstract

Microsporidia are obligate intracellular, spore-forming parasites that infect both invertebrates and vertebrates. They infect silkworms causing the deadly pebrine disease leading to heavy crop loss in sericulture. Because of the horizontal and vertical transmittance, outbreaks should be detected at an early stage and persistent infections should also be identifi ed to prevent further transmittance. So far, microscopic examination method remains the conventional detection method for screening of microsporidia in sericulture. Molecular diagnosis tools have an advantage over microscopic detection as they are more specifi c, sensitive and aid in early detection. Microsporidia detection by PCR method using primers designed from SSU-rRNA is widely used. In this study, we developed a PCR assay for the detection of microsporidia using primers designed from the conserved regions of RNA polymerase gene. Under optimized PCR conditions, the assay yielded a ~650 bp DNA fragment from microsporidia infected silkworms, Bombyx mori and Antheraea mylitta. Sequence analysis of the amplifi ed products has shown homology to various microsporidia including Nosema bombycis and N. antheraea. No non-specifi c products were observed. This method could help in early detection of microsporidia infection at any developmental stage of the silkworm and thereby reducing the crop loss.