A genus-based qPCR probe design for rapid and accurate detection of Aeromonas spp., Pseudomonas spp., Vibrio spp. and Mycobacterium spp.

The popularity of home aquaria has increased, increasing the demand and trade of fish worldwide. With this increased trade comes a corresponding amplification and expansion of pathogens. The genera Mycobacterium, Aeromonas, Pseudomonas and Vibrio encompass numerous common bacterial species associated with disease in both aquarium and cultured fish species worldwide. Typical disease diagnoses rely on a post-mortem necropsy collecting samples for culture, isolation, and subsequent biochemical or molecular analyses. These techniques are often implausible for smaller fish, so there is an urgent need for sensitive and specific assays that can rapidly identify bacterial pathogens. This manuscript describes four qPCR assays targeting conserved genes within the Aeromonas, Pseudomonas, Vibrio and Mycobacterium genera. A total of 67 housekeeping gene regions for Pseudomonas, 12 regions for Aeromonas, 22 regions for Vibrio and 48 regions for Mycobacterium were analyzed in detail for homogeneity The sequences include about 602 sequences for Aeromonas (gyrA), 3983 sequences for Pseudomonas (rpoD), 929 sequences for Vibrio (atpA) and 1009 sequences for Mycobacterium (hsp65). The Aeromonas assay targets the gyrA gene and utilities three forward primers, one reverse primer, and one probe sequence and was capable of detecting 3.65 x 104 cfu/ml (colony forming units), (73 cfu/reaction). The Pseudomonas qPCR targets the rpoD gene and uses five forward, three reverse primers and a single probe and was capable of detecting the concentration of 2329 cfu/ml (4.66 cfu/reaction). For Vibrio detection, the rpoA gene was targeted and utilized seven forward, eight reverse primers and 5 probes and was capable of detecting 68 x 107 cfu/ml (2.72 x 106 cfu/reaction). The Mycobacterium assay targeted the hsp65 gene using nine forward, four reverse primers and three probes and was capable of detecting concentrations as low as 105cfu/ml (200 cfu/ reaction). A total of 190, 167, 80, and 53 isolates from Aeromonas, Pseudomonas, Vibrio and Mycobacterium genera, respectively, were examined for specificity additional 500 isolates representing other relevant genera. This study sheds light on the structure of these four sensitive and specific qPCR assays, providing ease of use a powerful tool for rapidly detecting pathogens, contributing practical applicability to the reduction of losses and increasing animal welfare by non-invasive disease detection method. A powerful degenerated primer and probe designing tool was also an innovation provided in this study for the first time.