Application of electrochemical LAMP-MB signal evaluation using screen-printed graphene electrodes for quantitative detection of paramphistome egg DNA in faeces sample

LAMP is a highly sensitive technique and is a useful and applicable tool for DNA detection. This study presents and compares alternative evaluations of the PAR-LAMP for paramphistome DNA detection using electrochemical signal measurements of methylene blue (MB) on screen-printed graphene electrodes (SPGEs) among the Other LAMP applications. Two LAMP-MB signal evaluations are (i) the dropping LAMP-MB mixture and (ii) MB-DNA probe on SPGEs. These assays revealed a decrease in the current change (∆I) for positive result using square wave voltammetry (SWV). The dropping LAMP-MB mixture evaluation showed a higher fold current change difference (∆∆I/I₀) than the Other evaluation and showed that the positive and negative results can be significantly discriminated. The analytical specificity assay revealed that the target paramphistome DNAs were detectable by the dropping LAMP-MB mixture assay, leading to an increase of the ∆∆I, which was significantly higher than the negative LAMP (P < 0.05). For analytical sensitivity, the gradient DNA concentrations of two paramphistomes were used to construct calibration curves and standard linear regression equations, and these revealed the lowest detected DNA compared with the Other LAMP applications, including Agarose gel electrophoresis and colorimetry. The electrochemical evaluation can detect a paramphistome egg, as well as the contaminated egg in the host's faeces. In addition, the estimated DNA for a paramphistome egg was calculated using a faeces-interfered factor. This is the first known application of electrochemical assay for Parasite egg detection and the DNA quantification in faeces. Therefore, the application of the electrochemical LAMP-MB measurement using SPGEs, particularly the dropping LAMP-MB mixture assay, presented an effective diagnostic tool for DNA quantification in faeces as clinical specimens.

Electrochemical sensor; Loop-mediated isothermal amplification (LAMP); Methylene blue (MB); Rumen fluke; Screen-printed graphene electrode (SPGE).