Table 2. Different real-time LCR protocols tested.

Assay	DNA-Ligase	Duration of ligation	Duration of oligonucleotide denaturation	Temperature of oligonucleotide denaturation	Mean Ct-values of wild type samples (SD)	LODf	LOQf	Efficiency
Protocol-1a,e	Taq-DNA	6 s	30 s	75°C	25.02 (0.32)	0.085	0.35	80%
Protocol- 2a	Taq-DNA	6 s	35 s	75°C	23.71 (0.40)	0.16	0.85	82%
Protocol-3a	Taq-DNA	6 s	40 s	75°C	23.1 (0.38)	0.23	1.13	81%
Protocol-4a	Taq-DNA	12 s	30 s	75°C	23.24 (0.41)	0.15	0.88	83%
Protocol-5a	Taq-DNA	24 s	30 s	75°C	20.49 (0.34)	0.21	0.99	82%
Protocol-6a	Taq-DNA	6 s	30 s	74°C	25.56 (0.55)	0.097	1.14	70%
Protocol- 7a	PFU-DNA	6 s	30 s	75°C	30.10 (0.41)	0.098	0.39	70%
Protocol-8b	Taq-DNA	6 s	30 s	75°C	26.40 (0.32)	0.34	1.07	83%
Protocol- 9c	Taq-DNA	6 s	30 s	75°C	52.12 (0.56)	0.14	0.40	31%
Protocol- 10d	Taq-DNA	6 s	30 s	75°C	37.39 (0.30)	0.11	0.34	51%

Different thermocycling conditions, DNA ligases, length of oligonucleotides, gap modifications were examined. All trials were performed with the Mx3005P QPCR platform. Gap-A and gap-T LCR protocols used Platinum® Taq DNA polymerase.

^aOligonucleotides used LCPR1, LCPR2, LCPR3s, LCPR4s.

^bOligonucleotides used LCPR1, LCPR2, LCPR3L, LCPR4L.

^cOligonucleotides used LCPR1, LCPR2G, LCPR3s, LCPR4s.

^dOligonucleotides used LCPR1, LCPR2, LCPR3G, LCPR4s.

^eOptimal real-time LCR protocol.

^fThe LOD and LOQ values are in % frequencies