Development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians, nucleopolyhedrovirus, and densovirus infecting silkworms

Single and multiplex PCR amplifications of Nosema bombycis (794 bp), BmNPV (471 bp) and BmDNV (391 bp). M: Molecular weight marker; 1–3: Individual PCR products; 4: Multiplex PCR products; DNA from uninfected Bombyx mori (lane 5), and mulberry (lane 6) were used as controls for the reaction. 2.5 μl of PCR product was loaded on to a 1.30% agarose gel. [Display omitted] ► A multiplex PCR assay was developed for the detection of three major pathogens of the silkworm, Bombyx mori. ► First report describing the multiplex PCR for the simultaneous detection of three different pathogens in the silkworm. ► The assay can be extended to the same or closely related pathogens of other silkworms and insects. We have developed a novel PCR-based assay for individual and simultaneous detection of three major pathogens (microsporidians, nucleopolyhedrovirus (NPV) and densovirus (DNV)) infecting the silkworm, Bombyx mori. Multiplex PCR, using three primer pairs, two of which were designed from the conserved regions of 16S small subunit ribosomal RNA gene of microsporidians, and polyhedrin gene of NPVs respectively, and a third primer pair designed from the internal sequences of B. mori DNVs ( BmDNV), showed discrete and pathogen specific PCR products. The assay showed high specificity and sensitivity for the pathogenic DNA. Under optimized PCR conditions, the assay yielded a 794 bp DNA fragment from Nosema bombycis, 471 bp fragment from B. mori NPV ( BmNPV) and 391 bp fragment from BmDNV. Further, this detection method was successfully applied to other silkworm species such as Antheraea mylitta and Samia cynthia ricini, in detecting same or similar pathogens infecting them. This method is a valuable supplement to the conventional microscopic diagnostic methods and can be used for the early detection of pathogens infecting silkworms. Furthermore it can assist research and extension centers for the safe supply of disease-free silkworms to farmers.