Isothermal and Signal Amplification

The idea

PCR separates strands with heat. Isothermal methods do it with enzymes, at one constant temperature, so they need only a heat block - or a heated pouch in a handheld device ¹. They’re fast and suit point-of-care and high-volume testing.

TMA and NASBA

Transcription-mediated amplification and nucleic acid sequence-based amplification route the target through an RNA intermediate ¹:

1. A primer carrying a T7 promoter binds target RNA; reverse transcriptase copies it into DNA.
2. RNase H activity degrades the RNA strand of the hybrid; a second primer makes the DNA double-stranded with a working promoter.
3. T7 RNA polymerase transcribes 100-1,000 RNA copies from each template.
4. Every new RNA copy re-enters the cycle ².

Output grows enormously in under an hour, and RNA targets go straight in. TMA is the engine of widely used high-volume Chlamydia trachomatis / Neisseria gonorrhoeae and HIV/HCV assays; targeting rRNA, present in thousands of copies per cell, boosts sensitivity further.

LAMP

Loop-mediated isothermal amplification uses a strand-displacing polymerase and four to six primers recognizing six to eight regions of the target ¹. Two primers carry tails that fold back into loops, giving other primers new places to bind, so synthesis cascades into long looped products without any denaturation.

• Specific: several independent sites must all match.
• Fast: positive in 15-60 minutes.
• Readable by eye: pyrophosphate from massive synthesis clouds the tube (turbidity), or a pH or fluorescent dye changes color ¹.
• Drawbacks: complex primer design, and nonspecific amplification can produce false positives if reactions run too long; huge product amounts mean contamination must be managed carefully.

LAMP spread widely in home and point-of-care SARS-CoV-2 tests.

Amplifying signal instead of target

Buckingham sorts amplification into three kinds: target amplification (PCR, TMA, LAMP), probe amplification (copying the probe, as in ligase chain reaction), and signal amplification, which makes each detected target produce more signal ¹:

Because no target copies are made, signal methods have less amplicon contamination risk and measure target directly, but are usually less sensitive than target amplification ¹.

What to take from this

Isothermal methods replace heat with enzymes. TMA and NASBA cycle through RNA with reverse transcriptase and T7 RNA polymerase at about 41 °C - ideal for RNA and rRNA targets. LAMP uses a strand-displacing polymerase and four to six primers at 60-65 °C, read by turbidity or color in under an hour. Signal methods (hybrid capture, bDNA, Invader) amplify the readout, not the target, trading some sensitivity for less contamination risk.