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LAMP Visual Readout by pH Indicator (Hydroxynaphthol Blue)

Colorimetric readout of LAMP reactions by pH change. LAMP amplification releases protons as the polymerase extends primers, dropping the reaction's pH. A pH indicator with a transition in the relevant range changes colour only if amplification occurred — so a positive reaction is a tube-visible colour shift, detectable without fluorescence instruments. Ideal for field deployment, DIY-bio settings, and classroom use. Pairs with procedure 77 (LAMP Reaction Setup). Written for a novice audience.

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Version History
Version 0.1.1 Viewing Latest
Effective: 2026-04-20

Catalog fix (pH indicator → t#73 / p#201 HNB) bundled. All `procedure N` references converted to Markdown hyperlinks pointing at https://librebiotech.org/?action=show&id=N — enables in-app click-through to referenced sibling procedures. Text content otherwise preserved.
Version 0.1.0
Effective: 2026-04-20

Initial release. Hydroxynaphthol Blue (HNB) colorimetric LAMP readout (Goto et al. 2009). Pairs with procedure 77. Fresh original prose.

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Procedure Details
Safety & Hazards
  • Very low hazard. pH indicators are dye chemicals at low concentrations; standard gloves sufficient.
  • Hot tubes from 65°C heat block. Brief burn risk.
  • Amplicon contamination. Same LAMP-workflow contamination precautions as procedure 77 — visually positive tubes have massive amplicon content; do not open near future setup area.
  • Indicator choice caveats. Hydroxynaphthol Blue (HNB) is the most common for LAMP because its pKa (~12.4) and transition colour (violet → sky blue) work with the high-pH starting buffer. Phenol red (pKa 7.4) also works but requires weaker buffering. Neutral red, cresol red, and others have been published.
Preparation Notes

Indicator options:

Indicator Transition colour Transition pH Notes
Hydroxynaphthol Blue (HNB) Violet → sky blue ~11 → ~9 Standard for LAMP; 120 µM final; no instrument needed
Phenol red Red/pink → yellow 8.2 → 6.8 Requires weak-buffered reaction (20 µM Tris); sharp transition
Neutral red Red → yellow 7.0 → 6.0 Similar profile to phenol red
Cresol red Red → yellow 8.8 → 7.2 Robust; intermediate buffering tolerance

HNB is the default — no buffer reformulation needed, stable in LAMP reaction mixes, visible colour difference between positive and negative.

Reaction buffer modification (for phenol red / cresol red):

  • Standard LAMP buffer uses 20 mM Tris-HCl (strong buffering) — prevents pH change → pH indicator won't trigger.
  • Weak-buffered variant: 2 mM Tris-HCl at pH 8.8, otherwise unchanged. Allows the ~1-pH-unit drop during amplification.

Comparison reference:

  • Have positive-control tubes (known template) and no-template control (NTC) on every run. Visual comparison against these is more reliable than absolute colour judgment.

Mental model. In LAMP, every incorporated dNTP releases one proton. A 30-minute reaction producing billions of amplicons releases enough protons to shift reaction pH by 1.0–1.5 units even with buffering, more with weak buffering. A pH indicator in the tube reports this change visibly.

Timing
  • Setup (5 min): add indicator to reaction master mix (procedure 77 step 5 equivalent).
  • Readout (<1 min): inspect tubes immediately after LAMP incubation.
  • Photograph tubes against a white background with good lighting for records.
Equipment (Catalog) 2
  • Micropipette Liquid handling
    Specs: P10 for indicator addition
    Filter tips; dedicated pre-LAMP set
  • Heat block Thermal regulation
    Specs: 60–65°C LAMP incubation
    Primary LAMP equipment; same as procedure 77
Materials (Catalog) 2
  • Buffer Reagent
    Qty: 2.5 µL 10× per 25 µL rxn
    Only for phenol red / cresol red readout: 2 mM Tris-HCl pH 8.8 + 10 mM (NH4)2SO4 + 50 mM KCl + 0.1% Tween-20
  • Hydroxynaphthol Blue (HNB) pH indicator
    Qty: 0.3 µL of 10 mM stock per 25 µL rxn
    120 µM final; violet→sky blue transition. Alternative: phenol red (p#202, 50 µM final with weak-buffered LAMP buffer); cresol red (p#203)
Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns
Name Type Required Default Unit Description
indicator_type text — HNB — pH indicator for readout. 'HNB' (Hydroxynaphthol Blue, violet→sky blue, standard buffer compatible) or 'phenol_red' (red→yellow, requires weak-buffered LAMP).
hnb_concentration_uM number — 120 — HNB final concentration. 120 µM standard; lower concentrations give weaker colour contrast.
phenol_red_concentration_uM number — 50 — Phenol red final concentration (only if using phenol-red readout). 50 µM standard.
reading_time_post_incubation_min number — 5 minute (UO:0000031) Time after LAMP incubation to read colour. 0–15 min window; beyond 15 min colour may drift.
Procedure Steps (Version 0.1.1)

Prepare the LAMP master mix per procedure 77, modified to include pH indicator:

For HNB readout: add HNB stock (10 mM in water, filter-sterilised, stored room temperature) to 120 µM final concentration in the master mix (e.g. 0.3 µL per 25 µL reaction). Do NOT reformulate buffer — HNB works with standard Tris-buffered LAMP.

For phenol red / cresol red readout: prepare weak-buffered LAMP buffer (2 mM Tris-HCl pH 8.8 instead of standard 20 mM); add indicator to 50 µM final concentration.

Verify master mix colour: should be strongly violet (HNB) or pink/red (phenol red) before any amplification. If the master mix looks blue or yellow already, the indicator is degraded or pH is off — remake.

Proceed through LAMP reaction per procedure 77 (add template, incubate at 65°C for 30–60 min).

At completion, remove tubes from heat block. Do not wait more than 15 minutes before reading — colour reading is most reliable immediately after incubation.

Inspect tubes against a white background with good lighting (daylight or white LED).

Compare each sample tube against the NTC and positive control:

  • HNB: NTC should be violet; positive should be sky blue.
  • Phenol red: NTC should be pink; positive should be yellow.

Record results: for each sample tube, record 'positive', 'negative', or 'ambiguous'. Ambiguous tubes should be confirmed by agarose gel (procedure 73).

Photograph the tube rack with labels visible. Include NTC and positive control in the frame for reference.

Upload photograph to the LAMP Process record in LibreBiotech as the readout Assay. Annotate each tube's call.

For ambiguous tubes: run 5 µL on a 2% agarose gel (procedure 73) — LAMP gives a characteristic ladder-like band pattern, distinct from single-band PCR products. Present = confirmed positive; absent = confirmed negative.

Completion Notes

Expected outcome.

  • HNB:

    • Negative (NTC, no amplification): violet.
    • Positive (sample or positive control with amplification): sky blue.
    • Ambiguous (pale colour, uncertain): run gel (procedure 73) to confirm.

  • Phenol red:

    • Negative: pink/red.
    • Positive: yellow.
    • Colour transition is sharper than HNB — less ambiguity in marginal cases.

Batch record. Photograph the tube rack under consistent lighting; attach to the LAMP Process record in LibreBiotech. Include the positive control and NTC in every image.

Troubleshooting.

Symptom Likely cause Fix
All tubes show positive colour (including NTC) Cross-contamination; buffer too weak Stricter pre/post-LAMP zone separation; verify buffer strength
All tubes show negative colour (including positive control) Buffer too strong; Bst-LF inactive; primers degraded Switch to HNB + standard buffer if using weak-buffered variant; re-run LAMP QC per procedure 66
Pale / ambiguous tube colour Marginal amplification; insufficient incubation Extend incubation to 60 min; confirm by agarose gel (procedure 73)
NTC shifts colour over time (not immediately) Indicator drift with time; storage Read at 30 min post-incubation; do not leave tubes overnight before reading
References
  1. Goto M, Honda E, Ogura A, Nomoto A, Hanaki K (2009). Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. BioTechniques 46(3):167–72. (HNB method). DOI paper
  2. Tanner NA, Zhang Y, Evans TC Jr (2015). Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. BioTechniques 58(2):59–68. (pH-indicator LAMP methods). DOI paper
  3. LibreBiotech procedure 77 — LAMP Reaction Setup (upstream). Link protocol