Simple and cost-effective SNP genotyping method for discriminating subpopulations of the fish pathogen, Nocardia seriolae

Nocardia seriolae has caused significant fish losses in Asia and the Americas in recent decades, including in Vietnam, which has witnessed devastating economic and social impacts due to this bacterial pathogen. Surveillance strategies are urgently needed to mitigate N. seriolae dissemination in Vietnamese aquaculture and mariculture industries. Whole-genome sequencing (WGS) offers the highest level of resolution to discriminate closely related strains and to determine their putative origin and transmission routes. However, WGS is impractical for epidemiological investigations and pathogen surveillance due to its time-consuming and costly nature, putting this technology out-of-reach for many industry end-users. To overcome this issue, we targeted two previously characterised, phylogenetically informative single-nucleotide polymorphisms (SNPs) in N. seriolae that accurately distinguish: i) Vietnamese from non-Vietnamese strains, and ii) the two Vietnamese subclades. Using the mismatch amplification mutation assay (MAMA) format, we developed assays that genotype strains based on differences in amplicon melting temperature (melt-MAMA) and size (agarose-MAMA). Our MAMA assays accurately genotyped strains both from culture and fish tissues at low cost, using either real-time (~AUD$1/per sample) or conventional (~AUD$0.50/per sample) PCR instrumentation. Our novel assays provide a rapid, reproducible, and cost-effective tool for routine genotyping of this pathogen, allowing faster identification and treatment of nocardiosis-effected permit fish within Vietnamese aquaculture/mariculture facilities, an essential step in mitigating N. seriolae-associated losses.