TABLE 1.
PCR primer and probe sequences for the development of Acanthamoeba real-time PCR
| Pathogen | Target | Size (bp) | Concn (μM) | Primer (sequence [5′-3′])a and probe |
|---|---|---|---|---|
| Acanthamoeba spp. (Rivière assay) | 18S rDNA | 0.4 | Forward primer TaqAcF1 (CGA CCA GCG ATT AGG AGA CG) | |
| 65 | 0.4 | Reverse primer TaqAcR1 (CCG ACG CCA AGG ACG AC) | ||
| 0.3 | LNA TaqAcP (56-FAM/A +CA +CCA +CCA TCG GCG C/3 BHQ_1) | |||
| Acanthamoeba spp. (Qvarnstrom assay) | 18S rDNA | 0.4 | Forward primer AcantF900 (CCC AGA TCG TTT ACC GTG AA) | |
| 180 | 0.4 | Reverse primer AcantR1100 (TAA ATA TTA ATG CCC CCA ACT ATC C) | ||
| 0.3 | LNA AcantP1000 (5Cy5/TG +C CA +C CGA A +TA +CA/3 BHQ_2) | |||
| VZV (internal control) | ORF 38 | 0.3 | Forward primer (AAG TTC CCC CCG TTC GC) | |
| 82 | 0.3 | Reverse primer (TGG ACT TGA AGA TGA ACT TAA TGA AGC) | ||
| 0.2 | Probe (56-FAM-CCG CAA CAA CTG CAG TAT ATA TCG TCT CA-TAM) | |||
| Acanthamoeba spp. (conventional) | 18S rDNA | 470 | 0.3 | Forward primer JDP1 (GGC CCA GAT CGT TTA CCG TGA A) |
| 0.3 | Reverse primer JDP2 (TCT CAC AAG CTG CTA GGG GAG TCA) | |||
| Acanthamoeba spp. (sequencing primers) | 18S rDNA | 0.0034 | 892 (CCA AGA ATT TCA CCT CTG AC) | |
| 0.0034 | 892C (GTC AGA GGT GAA ATT CTT GG) |
a
+, Position of LNA.