A sensitive and compact on-site and real time quantitative detection of foodborne and clinical pathogens by on-chip qPCR

• On-chip qPCR of Staphylococcus aureus can be completed under 20 minutes. • The optimized and validated assay consisted of KAPA2G polymerase with EvaGreen dye. • DoE revealed influential factors and optimized conditions for on-chip qPCR. • Limit of detection is four genome equivalents per reaction (1.8 µL). • Low-cost recipe achieved high PCR efficiency, sensitivity and specificity. Herein we report the quantitative detection of pathogenic bacteria by an on-chip qPCR previously developed for COVID-19 detection, using Staphylococcus aureus as pilot microorganism. A conventional qPCR assay for the rapid and sensitive detection of Staphylococcus aureus harbouring the enterotoxin gene cluster ( egc ) was transferred to a microfluidic platform. Patterned silicon substrates were used as reaction vessels during on-chip qPCR. Reagent optimization was achieved through Design of Experiments (DoE), using multifactorial analysis with fractional factorial and central composite rotatable designs. On-chip thermal cycling and optical inspection were achieved using a compact reader providing real-time monitoring and accurate quantification. Experiments were performed to evaluate optimal assay efficiency, sensitivity, specificity, practicability, and robustness. The assay was tested for probe-based or intercalating dye-based amplification systems as those are fastest and most sensitive. Seven relevant components in the reaction mixture were screened through 16 amplification runs, where cost, speed and amplification quality were assessed. The screening identified the intercalating dye-based system as most promising, with polymerase and primer concentrations as the most influential factors. The following optimization design led to bacterial screening with template concentrations ranging from 4 to 40,000 copies in 1.8 µL of mixture per reaction under 19 minutes, compared to 10 µL of mixture per reaction under 40 minutes of run time using the conventional real time PCR platform. The workflow introduced here simplifies the transfer of qPCR recipes to microfluidic devices targeting faster results when compared with the current gold standard benchtop methods on pathogen detection in real time at on-site locations.