Blue-for-positive: colorimetric LAMP detection on paper-based microfluidic devices using a blue-shift indicator for instrument-free naked-eye readout

Abstract

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that allows the synthesis of large amounts of DNA in a short period of time with high specificity. Its operation at constant temperature, offers great potential for rapid diagnostics outside conventional laboratory settings. These features make LAMP suitable for point-of-care (POC) applications, but despite these promises, current POC detection methods require the use of dedicated equipment and relatively large reaction volumes, compromising the cost per reaction and the applicability. Results This study reports the use of a microfluidic paper-based analytical device (μPAD), which use less than 7 μL of LAMP reaction sample and produce an intense and contrasted blue color for positive samples offering naked-eye detection minimizing the user subjectivity and the cost per sample analysis. Its operation is based on the molybdate blue colorimetric and irreversible reaction happening in a μPAD, specifically optimized for the detection of pyrophosphate, a highly sensitive byproduct of nucleic acid amplification. As a proof of concept, the device was validated using LAMP products amplified from Candidatus Liberibacter asiaticus, the bacterium responsible for Huanglongbing (HLB) disease. μPADs exhibit sensitivity comparable to agarose gel electrophoresis method, detecting as low as 75 pg μL−1 of DNA template, and assays with HLB-infected citrus samples showed similar level of agreement. Significance This work presents a cutting-edge detection platform for molecular diagnosis offering novel, low-cost, and field-deployable μPADs that can be potentially integrated as a detection module in a microfluidic device combining the robustness and sensitivity of LAMP with the simplicity of paper-based colorimetric detection.