Table 1 Summary of modifications to RT-qPCR components for efficient multiplex amplification.

Binding protein gp2.5

The concentration of binding protein was raised to achieve 0.2 µg per pmol of primers and probes to sequester the additional amount of primer pairs and TaqMan probes in multiplex reaction

0.51 µg/µL

0.95 µg/µL

Taq polymerase

The quantity of Taq polymerase was increased for efficient amplification of multiple targets^54,56

0.3 U/µL

0.6 U/µL

KCl

KCl facilitates annealing of DNA molecules and increase in KCl increases product yield of shorter amplicons⁵⁵

55 mM

90 mM

MgCl₂

Facilitates annealing of DNA molecules and acts as a cofactor of Taq polymerase⁵¹

2.0 mM

2.6 mM

dNTP

More dNTPs are required to produce more amplification products. As increasing amounts of dNTPs can bind to Mg²⁺ electrostatically, reduce the amount of Mg²⁺ available and inhibit the reaction⁵⁵, concentrations of MgCl₂ and dNTPs were tested in a factorial experimental design

400 μM

500 μM

Primers and Taqman probes

Primers and Taqman probes were redesigned and verified by gel electrophoresis to minimize dimer formation and off-target amplification

See Supplementary Table S1 for sequences

See Supplementary Table S1 for sequences