The limit of detection is reported as 2 CFU/mL in the abstract, yet experimental data in Table 1 and Section 2.8 suggest detection at 1 CFU/mL. Could the authors elaborate on the statistical method used to derive the exact LOD? Was probit analysis or another model applied, and how was the potential for stochastic effects at the single-copy level addressed?
In the spiked food experiments, no internal amplification control was included to monitor inhibition in complex matrices. Given that digital amplification can be affected by inhibitors in meat, milk, and eggs, how can false negatives at low spike levels be confidently ruled out? Were any mitigation steps taken, such as dilution or pre-treatment, to reduce matrix interference?
Figure 3b shows fluorescence images for serial dilutions, but the 10⁴ CFU/mL image appears unexpectedly sparse in positive wells. Could the authors provide the raw counts of positive wells for each dilution to allow independent verification of the Poisson-based quantification and the reported 5-log dynamic range?
Specificity was evaluated against only 10 non-target strains. Considering the genetic diversity within Staphylococcus and Salmonella genera, especially species commonly co-occurring in livestock and aquatic products, how were the primers and probes validated for cross-reactivity? Is there data on inclusivity across relevant serovars and strains?
The discordant result in sample 4 (cdLAMP-positive, BAM- and qPCR-negative) is attributed to VBNC state. However, if VBNC cells were present, their DNA should still be detectable by qPCR. This discrepancy raises the possibility of cdLAMP false positivity due to contamination, non-specific amplification, or amplicon carryover. Could the authors discuss alternative explanations and whether negative control reactions throughout the workflow were consistently blank?
