For real-time PCR both intercalator based (no internal probe) and probe based (e.g. TaqMan probes) reporter system work well with 5' TINA modified primers(1). When used in multiplex Real-time PCR assays, the 5' TINA modified primers ensures more even PCR performance between targets compared to conventional DNA primers. Furthermore, the TINA molecule increases freedom in primer design due to increased assay robustness and thereby increased probability of enhanced analytical and clinical assay sensitivity. The TINA molecule additionally works well with other frequently used real-time technologies such as MGB modified probes. For specific advices on how to optimize your real-time PCR assay using 5' TINA modified primers, please refer to the Real-time PCR section on this site.
TETRAPLEX REAL-TIME PCR TARGETING METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS
A tetraplex real-time PCR assay targeting the methicillin resistant genes mecA and mecC and the Staphylococcus aureus specific genes nuc and femA was used to compare unmodified DNA primers and 5' TINA modified primers.
|Primer name||Cprimer (nM)
|femA FP||80 nM||5’-(Z)TCATTTTGCCGGAAGTTATGC-3’,|
|femA RP||300 nM||5’-(Z)AACGGTCAATGCCATGATTTAAT-3’,|
|nuc FP||600 nM||5’-(Z) GCATATGTATGGCAATCGTTTCA-3’,|
|nuc RP||600 nM||5’-(Z) CGTATTGCCCTTTCGAAACATT-3’,|
|mecA FP||300 nM||5’-(Z) ACTGATTAACCCAGTACAGATCCTTTC-3’,|
|mecA RP||300 nM||5’-(Z) TCCAAACTTTGTTTTTCGTGTCTTT-3’,|
|mecC FP||100 nM||5’-(Z) GCAAGCAATAGAATCATCAGACAAC-3’,|
|mecC RP||100 nM||5’-(Z) TCTTGCATACCTTGCTCAAATTTT-3’,|
Cprimer is the finale primerconcentration, FP is a forward primer, RP is a reverse primer, (Z) is ortho-TINA, underlined nucleobases were removed to target a Tm of 56°C. Clinical isolates of MRSA and MSSA strains were evaluated in a Fast Advanced Master Mix (ABI7500) on the ABI7500 Fast real-time PCR system using a thermal profile of 50°C for 2 minutes, 95°C for 10 minutes followed by 42 cycles of 95°C for 15 seconds and 60°C for 60 seconds.
Data between multiple strains were consistent and in concordance with the data for the M2711 isolate (mecC positive and mecA negative MRSA strain). The cycle of quantification (Cq) for femA/nuc was lowered from 26.5 ± 0.3 for unmodified primers to 21.15 ± 0.05 for TINA modified primers, whereas Cq for mecC was lowered from 30.95 ± 0.35 unmodified primers to 22.15 ± 0.05 for TINA modified primers. When the melting temperatures for the primer sequences were lowered to target 56 °C the difference in Cq between unmodified and TINA modified primers was even more pronounce. For femA/nuc the Cq was lowered from 31.4 ± 0.4 for unmodified primers to 22.0 ± 0.1 for TINA modified primers, whereas Cq for mecC for unmodified primers could not be determined as the amplification dropped out and increased to 34.05 ± 0.15 for TINA modified primers.
Figure legend: Amplification of femA/nuc in the left picture and mecC in the right picture with standard primer lengths. For both targets the primers were changed from unmodified primers (higher Cq) to 5' TINA modified primers (lower Cq) keeping all other parameters constant. Figure legend: Amplification of femA/nuc in the left picture and mecC in the right picture with shortened primer lengths. For both targets the primers were changed from unmodified primers (higher Cq) to 5' TINA modified primers (lower Cq) keeping all other parameters constant. (1) Schneider UV, Mikkelsen ND, Lindqvist A, Okkels LM, Jøhnk N, Lisby G (2012) Improved Efficiency and Robustness in qPCR and Multiplex End-Point PCR by Twisted Intercalating Nucleic Acid Modified Primers. PLoS ONE, 7(6): e38451.