5.1.1.3. Primer design and secondary structures

  • Design primers (80 – 150 bp) with Primer3Plus (Untergasser et al., 2007).
  • Evaluate secondary structures of amplicon with MFold (Zuker, 2003) for 60°C, 50 mM Na+ and 3 mM Mg2+. Selected candidate reference genes for P. larvae are listed in Table 5.

 

Table 5. Reference genes for P. larvae.

Name

Sequence

Predicted gene product

AFB_rpoD_fw

AFB_rpoD_rv

 

5'-AACTTGCCAAACGGATTGAG-3'

5'-AAGCCCCATGTTACCTTCCT-3'

RNA polymerase sigma factor RpoD

 

AFB_gyrA_fw

AFB_gyrA_rv

 

5'-ATGCGGTCATCCCTATTGAG-3'

5'-GGTCATCTTCCCGCAAATTA-3'

DNA gyrase subunit A

AFB_cmk_fw

AFB_cmk_rv

 

5'-GTACAGGGCGATTACCTGGA-3'

5'-GCCATCAACGAATACCTGCT-3'

Cytidylate kinase

AFB_sucB_fw

AFB_sucB_rv

 

5'-ATTGCCAAGGGTGTTGTAGC-3'

5'-TTCAGCCCGGATTCATTTAG-3'

Succinyl-CoA synthetase subunit β

AFB_eftu_fw

AFB_eftu_rv

 

5'-TAACATCGGTGCCCTTCTTC-3'

5'-CCACCCTCTTCGCTAGTCAG-3'

Elongation factor Tu

AFB_fum _fw

AFB_fum _rv

 

5'-CCAAAATATGCGGAGCTGAT-3'

5'-GTGAACCGCAATTTCCCTTA-3'

Fumarate hydratase

AFB_purH_fw

AFB_purH_rv

5'-TTCTCTCGGGGCTTTTGATA-3'

5'-CTACTGTTGGCTCACGGTCA-3'

Bifunctional phosphoribosyl aminoimidazole carboxamide formyltransferase / IMP cyclohydrolase

 

AFB_adk_fw

AFB_adk_rv

 

5'-TCAACAGGTGATGCTTTTCG-3'

5'-TGTGATTTCGTCAGGAACCA-3'

Adenylate kinase

AFB_gapdh_fw

AFB_gapdh_rv

 

5'-TGTTGAAGCTGGTGAAGGTG-3'

5'-TCCGCTTTTTCTTTTGCAGT-3'

Glyceraldehyde-3-phosphate dehydrogenase