Detection by PCR of the silkworm infected with Nosema bombycis

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Primers for polymerase chain reaction (PCR) were designed, which were based on the sequence of DNA fragment from Nosema bombycis. One pair of primers successfully amplified DNA extracted from either N.bombycis spores or silkworms (pupae or moths) harboring N.bombycis. The extent of specificity of those primers was tested, using reference stocks of N.bombycis, N.bombycis-like-isolates i.e.one from Spodoptera depravata and the other from S.exigus, Nosema necatrix, and Vairimorpha sp. isolates M_<11> and M_<12>. Of DNA from those microsporidia, DNA from reference stocks of N.bombycis only was amplified, while DNA from all microsporidia used were amplified by the primers which are known to amplify consensus part of small subunit rDNA (SSUrDNA). The sequences of SSUrDNA of the reference stock of N.bombycis and that of S.depravata isolate were determined and compared each other. The length of both rDNA was the same, 1226 bp.Of them, nine bps near c-terminal, ranging from 1201th to 1225th, were different. They appeared to be closely related, but the primers were sensitive enough to discrimiate between them.Investigations are now underway to apply this detection method to the silkworm female moth examination for pebrine.