WarmStart colorimetric loop-mediated isothermal amplification for the one-tube, contamination-free and visualization detection of Shigella flexneri

Abstract

Objectives: Shigella flexneri (S. flexneri) is prevalent worldwide and the most common Shigella in many countries, causing highly contagious diarrhea, which seriously threatens public health. This study aimed to develop a colorimetric loop-mediated isothermal amplification (LAMP) for the rapid, accurate, and visualization detection of S. flexneri. Methods: According to the screened specific genes of S. flexneri, three groups of LAMP primers were designed and evaluated, and the colorimetric LAMP reaction volume was optimized. The specificity of the colorimetric LAMP was validated by 20 S. flexneri and 96 non-S. flexneri clinical isolates. In addition, the sensitivity of the developed assay was evaluated by the serial 10-fold dilutions of plasmid DNA. Results: A colorimetric LAMP assay was developed based on the specific S. flexneri hypothetical protein gene (Accession: AE014073 Region: 4170556.4171068). The colorimetric LAMP method had good specificity for detecting S. flexneri and enabled detection of S. flexneri within 30 minutes, with a plasmid detection limit of 7*10° copies/μL. The results of amplification could be easily identified by color. Conclusions: This colorimetric LAMP assay could be used for rapid and accurate diagnosis of S. flexneri infection, especially in remote hospitals and laboratories with under-equipped medical facilities, and in situations where an urgent diagnosis is needed.

Keywords

• Loop-mediated isothermal amplification
• Shigella flexneri
• Hypothetical protein gene
• Colorimetric detection
• Point-of-care testing