Crop loss due to plant pathogens has provoked renewed interest in bacteriophages as a feasible biocontrol strategy of plant diseases. Phage cocktails in particular present a viable option for broadening the phage host range, limiting the emergence of bacterial resistance while maintaining the lytic activity of the phages. It is therefore important that the design used to formulate a phage cocktail should result in the most effective cocktail against the pathogen. It is also critical that certain factors are considered during the formulation and application of a phage cocktail: their stability, the production time and cost of complex cocktails, the potential impact on untargeted bacteria, the timing of phage application, and the persistence in the plant environment. Continuous monitoring is required to ensure that the efficacy of a cocktail is sustained due to the dynamic nature of phages. Although phage cocktails are considered as a plausible biocontrol strategy of phytobacteria, more research needs to be done to understand the complex interaction between phages and bacteria in the plant environment, and to overcome the technical obstacles.

Droplet digital PCR (ddPCR) is a third generation of PCR that was recently developed to overcome the challenges of real-time fluorescence-based quantitative PCR (qPCR) in absolute quantification of pathogens. Few studies have been done on tuberculosis (TB) detection and quantification using ddPCR despite its many advantages over qPCR. From the few studies, none explores a single dye duplex assay for the detection and quantification of TB. In this study, steps toward developing and evaluating a duplex single dye (FAM) assay for detecting two targets (IS6110 and IS1081) are clearly described using simplex and duplex experiments. To achieve this, various parameters are investigated, including annealing temperature, primer and probe concentration, sensitivity and specificity, sample concentration, and inter/intra-assay variability. From the results, primer and probe concentration, annealing temperature, and sample concentration have an effect on the position and separation of droplets in both simplex and duplex assays. The copies of target genes in a duplex assay can be estimated accurately using the threshold tool with little inter-assay (CV <1%) and intra-assay (CV <6%) variability when compared to simplex assays. The ddPCR assay specificity and sensitivity are both 100% when compared to qPCR. This work shows steps toward the detection and quantification of two targets in a single channel, enabling higher multiplexing to include more targets in future works.