2.2.2.3.2. Multiplex PCR for detection of Nosema and differentiation between N. apis, N. bombi and N. ceranae

For multiplex PCR amplification of partial 16S rRNA (=SSU rRNA) gene fragments, we recommend the following primer combination, though others from Table 1 (standard or qPCR primers) may be more suitable for different purposes and in different laboratories:

Primers were designed based on alignment of all available sequence data in GenBank of the 16S rRNA gene from N. apis, N. bombi and N. ceranae.

Mnapis-F               forward primer: 5’-GCATGTCTTTGACGTACTATG-3’

Mnbombi-F            forward primer: 5’-TTTATTTTATGTRYACMGCAG-3’

Mnceranae-F         forward primer: 5’- CGTTAAAGTGTAGATAAGATGTT-3’

Muniv-R:               reverse primer: 5’- GACTTAGTAGCCGTCTCTC-3’

Note that the Mnbombi-F primer contains variable sites to account for the sequence diversity observed for this species.

PCR product size:

  • for N. ceranae: 143 bp
  • for N. bombi: 171 bp
  • for N. apis: 224 bp

 

2.2.2.3.2.3. Controls