Rapid and sensitive recombinase polymerase amplification combined with lateral flow strips for detecting Candida albicans

Owing to modern lifestyles and increasing amounts of medical intervention, clinical infections caused by conditionally pathogenic fungi are becoming increasingly serious. Among these, Candida albicans is the most common. Therefore, the rapid and accurate detection of this pathogenic fungus is important to guiding the selection of clinical therapeutic agents. Recombinase polymerase amplification (RPA) combined with lateral flow strips (LFS) is a promising molecular detection method with the advantages of rapidity, simplicity of operation and high sensitivity. However, this simplicity brings with it the inherent and non-negligible risk of false-positive signals from primer-dimers. In this study, primer-dependent artifacts were eliminated by using probes in the RPA reaction, introducing specific base substitutions to the primer and probe sequences and analyzing and screening the formation of primer-probe complexes. These measures were rigorously tested for efficacy, leading to the creation of an improved RPA-LFS system. The standardized method enabled the specific detection of C. albicans within 25 min at 37 degrees C without interference. The system had a detection limit of 1 CFU per reaction without DNA purification or 102 fg genomic DNA/50 mu L. The detection sensitivity was not affected by the presence of other fungal DNA. The RPA-LFS method can therefore be used to detect clinical samples, and the results are accurate and consistent in comparison with those obtained using quantitative PCR. This study provides a paradigm for eliminating the risk of false-positive primer dimers in isothermal amplification assays and establishes a simple and easy method for the detection of C. albicans.

Automated summary

Due to modern lifestyles and increasing medical intervention, clinical infections caused by conditionally pathogenic fungi, especially Candida albicans, are becoming more serious. Recombinase polymerase amplification (RPA) combined with lateral flow strips (LFS) is a promising method for rapid and accurate detection of this pathogenic fungus. However, the simplicity of the method brings the risk of false-positive signals from primer-dimers. This study successfully eliminated primer-dependent artifacts in the RPA reaction by using probes, introducing specific base substitutions, and analyzing primer-probe complexes, leading to an improved RPA-LFS system for specific detection of C. albicans within 25 minutes without interference.