Novel indicator and stem-loop-primer assisted isothermal amplification for the visual semi-quantitative detection of Toxoplasma gondii

Abstract

This study presents a simple, inexpensive, versatile, pH-dependent colorimetric assay, which for the first time combines the commercial colorimetric indicator bromothymol blue (BTB), and a simpler designed stem-loop-primer-assisted isothermal amplification (SPA) for the detection of Toxoplasma gondii. Compared with other existing indicators, the BTB indicator showed more obvious color change and larger data change, with yellow indicating positive and blue indicating negative. The assay can be applied to the reactions of different volumes (5–20 µL), with the color also obvious at 0.5 µL. The SPA method was further evaluated objectively, and its serious nonspecific amplification problem was resolved. Under optimal parameters, the method tolerates interference from other parasites and genomes of complex samples at extremely high concentrations. A detection limit as low as 0.072 T. gondii/µL was achieved in 90 min and 1 real fecal sample missed by PCR was detected. More accurate results can be obtained by using a smartphone based on the red signal value. Overall, this strategy not only provides a new option for signal output in conventional volume or microfluidic devices, but also the closed-tube naked-eye detection method fundamentally avoids contamination, and enables accurate and rapid detection of Toxoplasma gondii.