Comparative evaluation of two high-throughput qPCR systems for environmental and clinical microbiology applications.

High-throughput quantitative PCR (HT-qPCR) is useful for the simultaneous quantification of various genes, such as microbial source tracking (MST) markers, fecal indicator bacteria, pathogens, and antibiotic resistance genes (ARGs) for environmental and clinical studies. While multiple HT-qPCR platforms are commercially available, their performance has rarely been directly compared. In this study, we evaluated and compared two commonly used HT-qPCR platforms, the Takara Bio SmartChip and the Standard BioTools Biomark HD system, using TaqMan probe-based MST assays and intercalating dye-based ARG assays. Despite differences in sample preparation workflows and reaction volumes, the two platforms produced the same detection results for 94.3% of MST sample × assay combinations, and 87.4% of ARG sample × assay combinations. Because HT-qPCR reactions are performed at nanoliter volumes (6.7-100 nL), a specific target amplification (STA) step is often used to improve the detection of low-abundance targets. Comparison of samples before and after STA showed that STA substantially increased detection frequencies on the Biomark HD platform, but had little effect on the SmartChip platform. Although most assays showed strong agreement between pre- and post-STA measurements and between platforms, discrepancies were observed for a subset of assays. Conventional qPCR and digital PCR results indicated that assay-specific amplification efficiency and sample quality contributed to the observed differences. Overall, these results demonstrate that both HT-qPCR platforms can provide reliable quantitative measurements when properly validated. However, consumable costs, workflow complexity, and maintenance requirements differed between platforms. Therefore, platform selection should consider sample throughput, assay requirements, available resources, and study objectives. IMPORTANCE: High-throughput qPCR is a powerful tool to detect and quantify various genes simultaneously. Numerous studies have used HT-qPCR for environmental and clinical microbiology applications; however, it is unclear whether different HT-qPCR platforms (e.g., Biomark and SmartChip) produce comparable results or not, as the HT-qPCR protocols are platform-specific. In particular, the use of pre-amplification reaction, also known as specific target amplification (STA), varies across studies. This study shows that both Biomark and SmartChip platforms provide similar quantitative data for most assays and that the STA reaction is useful to improve the detection of low-abundance targets without major biases. To accurately quantify on HT-qPCR platforms, assays should be carefully selected and validated. To guide current and future users, this study provides a general framework for assay selection in HT-qPCR.