Evaluation of external control for inhibitory response in loop-mediated isothermal amplification

Abstract

The loop-mediated isothermal amplification (LAMP) method is used for detecting novel coronaviruses (e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)). However, the effect of reaction inhibitors in specimens has not been evaluated. We evaluated external controls developed for the LAMP assay targeting SARS-CoV-2. The specimens included nasopharyngeal specimens (20 were SARS-CoV-2-positive and 80 were SARS-CoV-2 negative). Threshold time (Tt) was determined by the LAMP method using standard kits and external controls. For the external control, distilled water (SARS-CoV-2-negative sample) was measured, and the Dt ([Tt of specimen] − [Tt of distilled water]) was calculated. We evaluated Tt and Dt of the external control. All the specimens (n = 100) showed amplification of the external control. No specimen was suspected of having an obvious inhibitory reaction. The median Dt values were 1.4 for SARS-CoV-2-positive specimens and 1.3 for SARS-CoV-2-negative specimens, with a maximum value of 4.0. The median Tt of the external control was not significantly different between the SARS-CoV-2-positive (15.9) and -negative (15.8) specimens (p = 0.75). In conclusion, external controls showed normal amplification reactions in all the specimens. The result of the specimen that showed the maximum Dt value of 4.0 min could be determined without the influence of reaction inhibition. Further evaluation of external controls is needed using specimens and sputum that show response inhibition.