Table 1.
Molecular underpinning of isothermal amplification methods
| Methods | Isothermal temperature1 |
Enzymes2 | Primers3 |
|---|---|---|---|
| Loop mediated isothermal amplification (LAMP) | 60-65°C | DNA polymerase with strand displacement activity, e.g. Bst polymerase | Two inner (forward and reverse), two outer (forward and reverse) primers; optional, two loop primers (forward and reverse) |
| Nucleic acid sequence-based amplification (NASBA) | 41°C | Avian myeloblastosis reverse transcriptase (AMV-RT), RNase H, T7 RNA polymerase | Forward and reverse primers |
| Rolling circle amplification (RCA) | 37-65°C | Ligase, DNA polymerase with strand displacement activity | Forward primer |
| Recombinase polymerase amplification (RPA) | 37-42°C | Recombinase, DNA polymerase | Forward and reverse primers |
| Helicase-dependent amplification (HDA) | 37-65°C | DNA helicase, DNA polymerase | Forward and reverse primers |
1
Assays using double-stranded DNA as a template require prior denaturation step at 94°C
2,3
DNA assays can be amended to amplify RNA template by inclusion of a reverse-transcriptase step utilizing RNA-dependent DNA polymerase and random primer sets