Chip-based digital PCR for direct quantification dynamic bacterial load in target organs of tilapia infected with Streptococcus agalactiae, a pathogen causing meningoencephalitis in teleosts

Abstract

To detection of pathogens DNA is considered a promising strategy in sick fish. The current standard for enumeration of pathogens to obtain colony forming units by plate counts has several drawbacks: long time to results, high variability. While qPCR can target genetic identity, its limitations require enumeration to be calculated from a relative standard curve. There is a growing interest in techniques that allows an absolute quantification of pathogens DNA which could be useful for early diagnosis. Recently, digital PCR mainly based on droplets generation, emerged as an affordable technology for precise and absolute quantification of nucleic acids. In this work, we described a new interesting approach for profiling Streptococcus agalactiae DNA in tilapia different tissues samples using a chip-based platform, the Quant Studio 3D digital PCR. A total of 120 tissue samples were analyzed by plant count, qPCR and chip-based dPCR. The cdPCR method had a 5% relative standard deviation (RSD), marking it more precise than plate counts and qPCR with an RSD of 15 % and 10 %. Given its reproducibility and reliability, our approach could be potentially applied for the identification and quantification of pathogens DNA in infected fish. As chip-digital PCR becomes more established, it would be a robust tool for quantitative assessment of S. agalactiae DNA copy number for diagnosis of Streptococcosis.