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Isothermal amplification is a family of nucleic acid amplification methods that copy a target at one constant temperature, or close to it, instead of cycling between temperatures the way PCR does. The best-known methods are LAMP, NASBA, helicase-dependent amplification (HDA), strand displacement amplification (SDA), recombinase polymerase amplification (RPA) and rolling circle amplification (RCA).
Short answer
Isothermal amplification copies DNA or RNA at a constant temperature, so it needs a heating block or water bath rather than a thermal cycler. Strand-displacing polymerases, helicases or recombinases do the work that PCR leaves to heat cycles. The label is not absolute: some methods still use a heat step, and each has its own stated trade-offs.
Definition and scope
In PCR, the reaction is driven through repeated rounds of denaturation, primer annealing and extension, which requires precise cycling between three temperatures. A review of isothermal diagnostic devices in Sensors describes that cycling, and the instrument it needs, as the main reason researchers developed alternatives (see Sources). Isothermal methods replace thermal denaturation with enzyme activity, so the reaction can run on simpler heating equipment.
This guide is a bench-level overview for lab and clinic staff. It describes what the methods are and what the literature says about their trade-offs. It is not a protocol and gives no reaction setup, and nothing here is clinical or diagnostic advice.
How the main methods work
LAMP (loop-mediated isothermal amplification)
LAMP uses a strand-displacing DNA polymerase (Bst polymerase, from a bacterium found in hot springs around 70 °C) and several primers. A virology review describes four core primers (FIP, BIP, F3 and B3) that recognise six regions of the target, with two optional loop primers that, per that review, cut the reaction time by about half. The same review reports a typical temperature of about 63 °C, with RT-LAMP for RNA targets run at 60-65 °C and a reverse transcriptase added. Readouts described in the reviews include gel smears, turbidity from a magnesium precipitate, calcein fluorescence and electrochemical detection. The Sensors review calls primer design “subtle” and notes that it matters for sensitivity and specificity.
NASBA (nucleic acid sequence-based amplification)
NASBA targets RNA and runs mostly at 41 °C, after an initial step at 65 °C for denaturation and primer annealing. It uses reverse transcriptase, RNase H and T7 RNA polymerase with two primers, one of which carries the polymerase promoter, and it produces single-stranded RNA. The virology review notes that because DNA is not denatured, a contaminating DNA genome is not amplified, and it also flags the low reaction temperature as a possible risk factor for specificity. It names amplification inhibitors and RNA integrity as the main concerns when preparing clinical specimens.
HDA (helicase-dependent amplification)
HDA replaces heat denaturation with a helicase that unwinds the double strand, then a DNA polymerase extends two primers. The original system ran at 37 °C. With a thermostable helicase from Thermoanaerobacter tengcongensis (usable from 45 to 65 °C), reactions are now generally run at 65 °C according to the Sensors review. The virology review reports that systems using the UvrD helicase were limited for long targets, and that fused helicase-polymerase systems reached 2.3 kb against an original limit of 400 bp.
SDA (strand displacement amplification)
SDA combines a restriction endonuclease with a strand-displacing polymerase and uses four primers (two with restriction-site overhangs and two bumper primers). The Sensors review points out that it still requires a higher temperature (95 °C) for an initial denaturation step, so it is not isothermal from start to finish.
RPA and RCA
These two have their own pages on this site: recombinase polymerase amplification (RPA) and rolling circle amplification (RCA).
Types compared
The table summarises the two peer-reviewed reviews listed under Sources.
| Method | Temperature as reported | Key enzymes | Primers | Drawback the reviews name |
|---|---|---|---|---|
| LAMP | About 60-65 °C (63 °C typical) | Bst polymerase (plus reverse transcriptase for RNA) | Four core primers recognising six regions; optional two loop primers | Primer design is subtle and critical; sample inhibitors must be removed (saliva is the example given) |
| NASBA | 65 °C initial step, then 41 °C | Reverse transcriptase, RNase H, T7 RNA polymerase | Two | Low temperature flagged as a specificity risk; inhibitors and RNA integrity |
| HDA | 37 °C originally; generally 60-65 °C now | Helicase, DNA polymerase (single-strand binding protein in some systems) | Two | Early UvrD systems limited for long targets |
| SDA | 95 °C initial denaturation step | Restriction endonuclease, strand-displacing polymerase | Four (two with restriction-site overhangs, two bumpers) | Needs the high-temperature denaturation step |
Where it matters for labs and supply decisions
The reviews support a short checklist of what limits an isothermal assay in practice. It describes the literature, not a buying recommendation.
- Heating equipment. One review states these techniques do not require a thermal cycler and can be run with a heating block and/or water bath. The temperature set-point still has to match the chosen method, from 41 °C for NASBA to 65 °C for several others. For background on cycler hardware, see what a thermal cycler is.
- Primer design. LAMP primer design is described as critical to sensitivity and specificity. Primer fundamentals are covered in PCR protocol basics.
- Sample preparation. Inhibitors (saliva for LAMP; inhibitors and RNA integrity for NASBA) and extraction time are the issues the reviews raise. HDA workflows are noted as often keeping sample preparation, amplification and detection as separate steps.
- Contamination control. The Sensors review notes that closed-tube detection reduces contamination risk, which it cites for a NASBA platform.
- Readout. Turbidity, fluorescent dyes such as calcein, gel analysis and electrochemical detection all appear in the reviews. Which one a lab can use depends on the optics it has.
Isothermal assays are often discussed for decentralised testing; see point-of-care testing for how that setting is defined.
Common misunderstandings
- “Isothermal means one temperature.” NASBA uses a 65 °C step before 41 °C, and SDA needs a 95 °C denaturation per the Sensors review.
- “It is just PCR without the cycler.” The mechanisms differ: strand displacement, helicase unwinding, RNA transcription or recombinase-driven invasion each replace thermal denaturation.
- “One method fits every target.” NASBA is built around RNA, while LAMP and HDA are described mainly for DNA, with reverse transcriptase added to LAMP for RNA.
FAQ
Is isothermal amplification as accurate as PCR?
There is no single answer, because accuracy depends on the method, primers and sample. The virology review reports that LAMP was found 10- to 100-fold more sensitive than PCR in the studies it cites, with a detection limit of 0.01-10 pfu of virus, but it also flags specificity risks for NASBA at its low temperature. Treat those as reported results, not guarantees.
Does isothermal amplification need a thermal cycler?
Not for the reaction itself. One review says these techniques can run on a heating block and/or water bath. A cycler is still the standard instrument for qPCR and RT-qPCR.
Sources
- Chang C-C, Chen C-C, Wei S-C, Lu H-H, Liang Y-H, Lin C-W. Diagnostic Devices for Isothermal Nucleic Acid Amplification. Sensors 2012;12(6):8319-8337, doi 10.3390/s120608319. https://pmc.ncbi.nlm.nih.gov/articles/PMC3436031. Checked 2026-10-29.
- Sidoti F, Bergallo M, Costa C, Cavallo R. Alternative Molecular Tests for Virological Diagnosis. Molecular Biotechnology 2012, doi 10.1007/s12033-012-9533-8. https://pmc.ncbi.nlm.nih.gov/articles/PMC7091206. Checked 2026-10-29.
