A simple color absorption analysis of colorimetric loop-mediated isothermal amplification for detection of Raillietina spp. in clinical samples using a 3D-printed tube holder coupled with a smartphone camera and notebook screen.

A simple method has been developed for semi-quantitative analysis of the colorimetric output of loop-mediated isothermal amplification (LAMP) using a 3D-printed tube holder with a smartphone and notebook for the detection of Raillietina, which is the cause of Raillietiniasis affecting free-range chicken farming. In this method, a light is directed from a notebook screen to the LAMP products in the tube holder and the color absorption of the LAMP products is measured by using the appropriate smartphone application. It was found that the malachite green dye-coupled LAMP (MaG-LAMP) assay showed the highest sensitivity and specificity for detecting Raillietina without any cross-reaction with other related parasites and hosts. The limit of detection was 10 fg/μL of DNA. A total of 60 fecal samples were infectively confirmed by microscopic examination and the results of microscopy compared with those of MaG-LAMP and triplex PCR assays. Microscopy and MaG-LAMP based on the color absorption demonstrated high agreement in Raillietina detection with kappa = 1. Rapid, simple, cost-effective, and easy interpretation of colorimetric LAMP assays and their high sensitivity make them superior to PCR and morphological investigation, demonstrating the feasibility of this assay in point-of-care screening to support farm management and solve chicken health problems. Our study presents is an alternative diagnostic method using semi-quantitative analysis of colorimetric LAMP based on the differing solution color absorptions between positive and negative reactions for infectious disease diagnosis.