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

• Published in: Sensors and actuators. B, Chemical Chemical, 2022-12, Vol.372, p.132544, Article 132544
• Main Authors: Zhang, Suying, Lin, Shenghao, Zhu, Longjiao, Du, Zaihui, Li, Jiale, Wang, Lei, Xu, Wentao
• Format: Article
• Language: English
• Subjects: Amplification; Bromothymol blue (BTB); Closed-tube; Color; Colorimetry; Genomes; Isothermal; Microfluidic devices; Protozoa; Toxoplasma gondii detection; Universality; Visualization
• ISSN: 0925-4005; 1873-3077
• DOI: 10.1016/j.snb.2022.132544

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 (5-20µL), with the color also obvious at 0.5µL. 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 0.072T. gondii/µL was achieved in 90minutes and 1 real stool 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. [Display omitted] •BTB was first applied to establish a universal colorimetric detection method with naked eye observation and quantifying RGB with a smartphone will be more obvious.•Universal one-step closed-tube detection based on commercial consumables to avoid contamination.•A preliminary study on the causes and solutions of non-specific amplification of SPA is discussed.•0.071 Toxoplasma gondii/µL can be semi-quantitated by naked eye within 90min under isothermal conditions.•Excellent anti-interference ability and detection ability of real stool samples.