Dual-target recombinase polymerase amplification-lateral flow strip (RPA-LFS) for rapid detection of HSV-1 and Fusarium keratoplasticum in infectious keratitis.

This study focuses on the challenge of pathogen detection in keratitis, aiming to develop a dual target detection strip using recombinant enzyme polymerase amplification (RPA) combined with lateral chromatography strip (LFS) technology, and to detect the specific DNA sequences of Herpes simplex virus type 1 (HSV-1) and Fusarium keratoplasticum (F. keratoplasticum). To test the detection system in different ocular surface samples for keratitis pathogens diagnosis. The RPA primers with good specificity for HSV-1 and F. keratoplasticum were designed and screened separately, and further modified into probes for a dual target RPA-LFS detection system. This LFS test strip can simultaneously detect HSV-1 and F. keratoplasticum, and distinguish them by the positive bands at different test lines. The detection limit of this test strip for two targets is 203.69 copies/reaction for HSV-1 and 84.91 copies/reaction for F. keratoplasticum. There is no cross reactivity with DNA of other common pathogenic microorganisms of ocular surface except for Candida albicans. In clinical sample testing, the dual target detection system has shown reliable detection performance for both eye swaps and cornea tissue. The dual targets RPA-LFS detection system in this study could be completed within 20 min with visualized results, the coincidence rate with qPCR was 93.3%, indicating its broad clinical application prospects for the clinical diagnosis and treatment of infectious keratitis.