Duplex PCR for Nosema spp. Identification

A duplex PCR protocol by [29 (link)] was used for the preparation of the reaction mixture, which was modified as follows: 2.5 µL buffer, 2 µL MgCl₂, 0.5 µL dNTPs, 0.3 µL DNA polymerase (FIREPol DNA Polymerase produced by Solis BioDyne, Tarfu, Estonia), and was used for a single sample. Then, we proceeded as instructed in their protocol. In order to diagnose species of Nosema spp., two 30 picomole primers were added to the reaction mixture, 0.6 µL to each (Nosema apis for the amplification of 321 bp) APIS FOR-5′-GGGGCCATGTCTTTGACGTACTATGTA-3′ and 321 APIS REV 5′-GGGGGGCGTTTAAAATGTGAAACAACTATG-3′ and (Nosema ceranae for the amplification of 218 bp) MITOC FOR-5′CGGCGACGATGTGATATGAAAATATTAA-3′ and 218 MITOC REV 5′-CCCGGTCATTCTCAAACAAAAAAACCG-3. 2.5 µL of DNA template was added to the mixture, and PCR water was filled to final volume of 25 µL.
PCR was performed using a VWR RISTRETTO Personal Thermocycler according to the protocol of Ostroverkhová et al., 2020. ELFO was performed using 1.5% agarose gel after PCR reaction had been completed. After assessing the results using a UV Transilluminator, the DNA concentration was measured using the NanoDrop, and the PCR products were sent for sequencing. The obtained sequences were compared with the sequences stored in the gene bank using BLAST program. https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 20 November 2022).