A Duplex polymerase chain reaction (PCR) was developed for the simultaneous detection and differentiation among Nosema spp. and Vairimorpha spp. from Lepidoptera insects. Two sets of primers were selected from different genomic sequences to specifically amplify an 831 bp amplicon within the SSU rRNA gene, specific for both Nosema spp. and Vairimorpha spp. (MSSR primer); a 542 bp amplicon within the SSU rRNA gene, specific for Vairimorpha spp. (VSSU primer). Using the primers in conjunction (duplex PCR) it was possible to detect Nosema spp. and Vairimorpha spp. to differentiate between them. The sensitivity of this PCR assay was approximately 10 spores per milliliter. It is proposed that the duplex PCR is a sensitive, specific and rapid tool that can serve as a useful differential diagnostic tool for detecting Nosema spp. and Vairimorpha spp. in Lepidoptera insect.

Microsporidia are obligate fungal intracellular parasites of all animal taxa. Among them the genus Nosema (Nosematidae) is known as the most common entomopathogen. Of these parasites, the ribosomal organization is one of the most pronounced molecular characteristics. One type is the normalarrangement of small subunit (SSU)-internal transcribed spacer (ITS)-large subunit (LSU) in the DNA sequence order. The other is the reverse arrangement of LSU-ITS-SSU. The latter is assigned to be the ‘true’ Nosema in the Nosema/Vairimorpha clade. However, we found that the SSU sequence of a strain of Nosema species having the normal arrangement of its rRNA sequence seemed to be more closely related to the ‘true’ Nosemagroup. Consequently we have further analyzed the complete sequence of rRNA. The results imply that there might be arecombination event in its rRNA evolution and/or the strain may form a novel group near the ‘true’ Nosema group. Interestingly both SSU and LSU of the ‘true’ Nosema and others may be under different selection pressure. We have also found that the size of ITS is distinct between the ‘true’ Nosema and other microsporidian species within the Nosema/Vairimorpha clade. This feature should be a useful diagnostic tool to distinguish the ‘true’ Nosema from others in the clade.