Rapid visualization in the specific detection of Flavobacterium columnare, a causative agent of freshwater columnaris using a novel recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) assay

Abstract

Flavobacterium columnare is the causative agent of columnaris, a serious disease affecting numerous freshwater fish species worldwide. Although several molecular protocols have been established to detect this pathogen, there still remains a need to develop simpler on-field applicable techniques. Here, we report a more practical and efficient assay to detect F. columnare based on the combination of recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD). The assay, as performed for 30 min at 37 °C for RPA, followed by 2 min at ambient temperature for LFD, revealed a comparable detection limit (200 fg total DNA and 0.4 CFU) to the PCR assay developed by Mabrok et al. in 2020 but required only ~35 min to complete tests (i.e. ~four times faster). Cross-reactivity against other bacteria and false results on 20 clinical samples were not observed, indicating its extremely high specificity and accuracy. The robust performance of the assay to crude tissue samples and clinical specimens reflect its potential use in field application/low-resource settings. The use of a fast and affordable DNA extraction kit is imperative as part of our assay to make the detection of F. columnare more feasible.