Internal Control Fails Due to Sample pH Incompatibility (LAMP (Loop-mediated Isothermal Amplification))

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The internal control reaction fails to turn yellow after incubation at 65°C when spiked into sample, though it works in water, indicating sample matrix interference. Common causes: 1) Sample nucleic acid extraction buffer has acidic pH (<7.0) incompatible with colorimetric LAMP pH indicator 2) Sample contains chelating agents (e.g., EDTA >2 mM) that sequester Mg²⁺ required for polymerase activity 3) High salt concentration in sample (>150 mM NaCl equivalent) inhibits enzyme function 4) Residual organic solvents (phenol, ethanol >1%) from extraction inhibit LAMP amplification Solutions: 1) Dilute sample 1:10 or 1:20 in nuclease-free water to reduce inhibitor concentration below threshold 2) Adjust sample pH to ~8.0 using 1 M Tris-HCl pH 8.0 prior to addition to the reaction (add <10% of sample volume) 3) Re-extract nucleic acid using LAMP-compatible extraction method or cleanup columns to remove inhibitors 4) Use RNA/DNA purification kits with neutral elution buffers (pH 7.5-8.5) instead of acidic or basic buffers 5) Perform ethanol precipitation and resuspend in nuclease-free water or TE buffer pH 8.0