Table 2.
Design of 13 candidate qPCR assays in ascending order by template copy number
| qPCR assay | Fluorescence | Forward primer (FP), reverse Primer (RP) and Probe sequences (5′ → 3′) |
|---|---|---|
| crt | MGB TaqMan |
FP: GACACCGAAGCTTTAATTTAC RP: GCAGAAGAACATATTAATAGGAA Probe: 5′-HEX/TTAGATGCCTGTTC/3′-MGBEc/ |
|
crt76K crt76T |
MGB TaqMan |
FP: TGGTAAATGTGCTCATGTGTTT RP: AGTTTCGGATGTTACAAAACTATAGT Probe(K76): 5′-HEX/TGTGTAATGAATAAAATTTTTGCTAA/3′-MGBEc/ Probe(T76): 5′-6-FAM/TGTGTAATGAATACAATTTTTGCTAA/3′-MGBEc/ |
| ldh(a) | Standard TaqMan |
FP: ACGATTTGGCTGGAGCAGAT RP: TCTCTATTCCATTCTTTGTCACTCTTTC Probe: 5′-HEX/AGTAATAGTAACAGCTGGATTTACCAAGGCCCCA/3′-IABkFQ/ |
| ldh(b) | MGB TaqMan |
FP: TGGTCATATTAAGAAGAATTGTC RP: CTGAGATATGTAATACTTCAATC Probe: 5′-HEX/CCATAACATCTACT/3′-MGBEc/ |
| 18SrRNA(a) | Standard TaqMan |
FP: CTTTTGAGAGGTTTTGTTACTTTGAGTAA RP: TATTCCATGCTGTAGTATTCAAACACAA Probe: 5′-HEX/TGTTCATAACAGACGGGTAGTCATGATTGAGTTCA/3′-IABkFQ/ |
| 18SrRNA(b) | MGB TaqMan |
FP: ATTGCTTTTGAGAGGTTTTGTTACTTT RP: GCTGTAGTATTCAAACACAATGAACTCAA Probe: 5′-HEX/CATAACAGACGGGTAGTCAT/3′-MGBEc/ |
| r364(a) | PET |
FP: ACCCCTCGCCTGGTGTTTTT RP/Probe: 5′-HEX/aggcgcatagcgcctggTCGGGCCCCAAAAATAGGAA |
| cytB | LNA TaqMan |
FP: TACTAACTTGTTATCCTCTATTCCAGTAGC RP: CCTTTAACATCAAGACTTAATAGATTTGGA Probe: 5′-HEX/+ GTG + CTA + CCA + TGT + AAA + TGTAA/3′-IABkFQ/ |
| coxI | MGB TaqMan |
FP: GTCACGCAATATCAATATACTG RP: CGATCTCTTGTATGGTAATAGG Probe: 5′-HEX/ATAGAACTCCAGGC/3′-MGBEc/ |
| varATS | MGB TaqMan |
FP: CCCATACACAACCAAYTGGA RP: TTCGCACATATCTCTATGTCTATCT Probe: 5′-HEX/TRTTCCATAAATGGT/3′-MGBEc/ |
| r364(b) | MGB TaqMan |
FP: AGTCCATTTTCCCCTAGC RP: GACCATATAGTAAGTGACCCA Probe: 5′-HEX/AATTGACATGCACT/3′-MGBEc/ |
| TARE-2 | dsDNA-dye |
FP: CTATGTTGCACTTACATGCAYAAT RP: TGACCTAAGAAGTAVAATAATGATGA |
Information provided in this table includes the abbreviations for each of the 13 qPCR assays, the source of the fluorescent signal in each assay and the sequences of the forward and reverse primers and probes for each assay (beginning on the left with the 5′ end and concluding on the right with the 3′ end for each primer and probe)
MGB, minor groove binder; PET, photo-induced electron transfer; LNA, locked nucleic acid; dsDNA, double-stranded DNA; 6-FAM, 6-carboxyfluorescein fluorescent dye; HEX, hexachloro-fluorescein fluorescent dye; IABkFQ, Iowa Black fluorescent quencher; MGBEc, MGB Eclipse® fluorescent quencher