Table 2. Melt-MAMA primers targeting canSNPs for new phylogenetic branches in Francisella tularensis subsp. tularensis A.I in United States*.
| SchuS4† position | SNP state, der/anc‡ | Primers, 5′ → 3′§ | Con¶ | Temp, °C# | ||
|---|---|---|---|---|---|---|
| Subgroup | ||||||
| Major | Minor | |||||
| NA | A.I.7 | 1005448** | C/T | A: TATTTCAATTTTTGCGATGGTAgGT | 0.80 | 55 |
| D: ggggcggggcggggcTATTTCAATTTTTGCGATGGTAcTC | 0.20 | |||||
| C: AAGTATGTTGGCAAGTAAAGTGAGAAGA | 0.20 | |||||
| A.I.12 | NA | 142781†† | C/G | A: GCTTATCGCCGACATTCATCtAC | 0.20 | 60 |
| D: ggggcggggcggggcgggCTTATCGCCGACATTCATCcAG | 0.20 | |||||
| C: GGTATGGCAAAAAATACTTATGGTACG | 0.20 | |||||
| A.I.12 | A.I.13 | 1833651‡‡ | T/C | A: CTTTCAATCATGTAACCATCATTATTTAaGC | 0.80 | 60 |
| D: cggggcggggcggggcggggCTTTCAATCATGTAACCATCATTATTTAgGT | 0.20 | |||||
| C: CTTAATGAACTTGGTGTAATGGGTAGATA | 0.20 | |||||
| A.I.12 | A.I.16 | 273622 | T/C | A: AAACTTAAAAAAGAGCAAGAACTTAATGATcTC | 0.60 | 60 |
| D: ggggcggggcggggcgAAACTTAAAAAAGAGCAAGAACTTAATGATaTT | 0.15 | |||||
| C: CATCTTCATTAAAAGTCTTATTGTTTAAACGC | 0.15 | |||||
| A.I.12 | A.I.15 | 1210286 | A/G | A: TCTTAAAACATCGACACTCTCAACcTG | 0.80 | 60 |
| D: ggggcggggcggggcGATCTTAAAACATCGACACTCTCAACtTA | 0.20 | |||||
| C: gtatcattcagatcataatgaagcaactatc | 0.20 | |||||
| A.I.12 | A.I.14 | 1296147 | T/C | A: ATCATACTGGTTATATTGGCGGTcTC | 0.80 | 60 |
| D: cggggcggggcggggcggggATCATACTGGTTATATTGGCGGTgTT | 0.20 | |||||
| C: GATGAGTCGCTATTAGCTTCTCGAAAG | 0.20 | |||||
| A.I.8 | NA | 1150298 | G/A | A: TAGTCAATCTTGGAACTCCAGAtAA | 0.75 | 60 |
| D: ggggcggggcggggcTAGTCAATCTTGGAACTCCAGAaAG | 0.15 | |||||
| C: TCTATTACTCTAGGGTCAGATAGAAATTC | 0.15 | |||||
| A.I.8 | A.I.9 | 1453599 | C/T | A: GCTGCTGCTAGATTAGCTATgCT | 0.15 | 60 |
| D: ggggcggggcggggcGCTGCTGCTAGATTAGCTATcCC | 0.15 | |||||
| C: TCAAGCAATCAACAATAATTTTACTAT | 0.15 | |||||
| A.I.8 | A.I.10 | 797599 | T/G | A: GATCAATTGGTGGTGTTcCG | 0.80 | 60 |
| D: ggggcggggcggggcGTGATCAATTGGTGGTGTTtCT | 0.20 | |||||
| C: AACGTTTTATCCTCTTGAATATCAACTAT | 0.20 | |||||
| A.I.8 | A.I.11 | 1278606 | G/A | A: AAGGAACAAAAAACATCATCATTgCT | 0.20 | 60 |
| D: ggggcggggcggggcAAAAGGAACAAAAAACATCATCATTaCC | 0.20 | |||||
| C: TCATACTAACAACGGCTATTCAGGGA | 0.20 | |||||
| A.I.3 | NA | 1233898 | T/G | A: GCTTGACAATATTAGCTTATAAAACTATAgTG | 0.15 | 60 |
| D: ggggcggggcggggcGCTTGACAATATTAGCTTATAAAACTATAaTT | 0.15 | |||||
| C: TTTTTTCCATATTTCTGTAAAAAATATACTATTATG | 0.15 | |||||
| A.I.3 | A.I.4 | 830715§§ | T/C | A: GTTAAGTCGGTAAGTATCGACAAaTC | 0.60 | 60 |
| D: ggggcggggcggggcGTTAAGTCGGTAAGTATCGACAAgTT | 0.20 | |||||
| C: CAAATCTTCTAGTATCTCTTTATCTTCAG | 0.20 | |||||
| A.I.3 | A.I.5 | 113671 | G/A | A: cgggcgggcgggcgggGCTTGAGTTTATTTTTTGTTTAATGTgTA | 0.20 | 60 |
| D: GCTTGAGTTTATTTTTTGTTTAATGTaTG | 0.20 | |||||
| C: GGACAAAACTGTGGACGTTAAGAA | 0.20 | |||||
| A.I.3 | A.I.6 | 580153 | G/A | A: cgggcgggcgggcgggTATAATGGTAACTCATGATCAAGAAcAA | 0.20 | 60 |
| D: TTATAATGGTAACTCATGATCAAGAAaAG | 0.20 | |||||
| C: ATCTGTCATGATACCAATTCTTGTCG | 0.20 | |||||
*Melt-MAMA, melt–mismatch amplification mutation assay; SNP, single nucleotide polymporphism; canSNP, canonical SNP; con, concentration, μmol/L; temp, annealing temperature, °C; NA, not applicable; der, derived SNP state; anc, ancestral SNP state; D, derived allele primer; A, ancestral allele primer; C, common primer. †Genomic position in reference A.I SchuS4 strain (GenBank accession no. NC_006570). ‡SNP states are listed according to their orientation in the SCHU S4 reference genome (GenBank accession no. AJ749949.2). §Melt-MAMA primer sequences; primer tails and antepenultimate mismatch bases are in lower case. ¶Final concentration of each primer in Melt-MAMA genotyping assays. #Assay annealing temperature. **Assay designed on the reverse complement. ††SNP from (6). ‡‡Assay supplemented with 0.025 U of Platinum Taq DNA polymerase (Life Technologies, Invitrogen, Frederick, MD, USA). §§SNP from (7).