Development of RT-qPCR and loop-mediated isothermal amplification (LAMP) assays for the rapid detection of Rahnella aquatilis in fish.

Rahnella aquatilis is widely recognized as an opportunistic pathogen of fish, but molecular detection and diagnostic techniques for it remain to be developed. In a previous study, we isolated the pathogenic R. aquatilis strain KCL-5 from diseased crucian carp Carassius auratus and identified it as a new fish pathogen causing enteritis and septicemia. Here, we describe novel real-time quantitative PCR (RT-qPCR) and loop-mediated isothermal amplification (LAMP) assays for the rapid and sensitive detection of R. aquatilis. Specific RT-qPCR and LAMP primers were developed for the outer membrane protein (OmpA) gene. Their reaction systems were optimized using the recombinant plasmid pMD18-T containing the OmpA gene and their specificities were confirmed using various bacterial fish pathogens including Aeromonas hydrophila , A. veronii , A. allosaccharophila , Yersinia ruckeri , Vibrio vulnificus , V. alfacsensis, Providencia rettgeri , and R. aquatilis. The sensitivities of the RT-qPCR and LAMP assays were further evaluated using serial dilutions of the positive recombinant plasmid pMD18-T/ OmpA. Both the RT-qPCR and LAMP assays were able to reliably detect the presence of R. aquatilis with high accuracy and a specificity of 100%. The lowest detection limits of the RT-qPCR and LAMP assays were 2.3 copies/μL and 2.3 × 101 copies/μL, respectively. To the best of our knowledge, this is the first report of RT-qPCR and LAMP methods for R. aquatilis detection, and provides a means for its rapid detection and diagnosis in fish. • Development of qPCR assay for detection of R. aquatilis in fish. • Establishment of LAMP assay for detection of R. aquatilis with MDL of 2.3 × 101 copies/μL. • The qPCR and LAMP methods were firstly established for the rapid detection of R. aquatilis. • Analysis of bacterial loads was performed for R. aquatilis infection in fish. [ABSTRACT FROM AUTHOR]