STUDY WORKFLOW
Sample Collection 1 Blood Culture 2 & ddPCR Testing 3
Blood samples (5 mL) were collected for both blood culture and ddPCR testing. Samples were stored in cell-free DNA preservation tubes and processed within 24 hours. Two blood culture samples were collected per patient for aerobic and anaerobic culture. Antibiotic susceptibility tests were performed manually according to Clinical and Laboratory Standards Institute (CLSI) guidelines. ddPCR was performed using a 7-fluorescent-channel fully-automated droplet digital PCR system AD3207 (Pilot Gene Technology Co., Ltd., Hangzhou, China), an optimized platform for clinical infectious disease testing. DNA was extracted from plasma and analyzed using multiplex ddPCR panels (Pilot Gene, Hangzhou, China) targeting specific pathogens and antimicrobial resistance genes. The platform enables rapid, multiplexed, and absolute quantitative detection with robust performance on complex blood-derived samples, supporting early sepsis diagnosis and dynamic pathogen load monitoring in clinical settings. Synthesized DNA fragments were used as positive controls, while DNase-free water or blood samples from three healthy subjects served as negative controls. The entire ddPCR workflow required approximately 2.5 hours, delivering actionable clinical results within the same timeframe.
Plasma DNA Extraction
STUDY RESULTS Compared with blood culture, the ddPCR panel demonstrated an overall sensitivity of 75.0% and specificity of 68.7%, yielding an additional detection rate of 17.0% for sepsis cases, with a turnaround time of 2.5 h [ Figure 1 ]. Antimicrobial resistance genes were identified in 13 samples. Whenever ddPCR detected the ß -lactamase—Klebsiella pneumoniae carbapenemase (blaKPC) or ß -lactamase—New Delhi metallo (blaNDM) genes, these findings corresponded to the cultivation of carbapenem- resistant Gram-negative bacteria. Dynamic ddPCR monitoring revealed a consistent correlation between quantitative ddPCR results and trends in C-reactive protein and procalcitonin levels [ Figure 2 ].
This study also highlighted the versatility of Meridian's chemistry: one mix, multiple applications, including digital PCR. Digital platforms require finely-tuned reagent formulations to ensure precise droplet partitioning and consistent amplification without inhibition. Meridian's Lyo-Ready 1-Step RT-qPCR Mix (MDX024) demon- strated strong compatibility with ddPCR workflows and robust performance in this case study, confirming its flexibility across diverse molecular testing workflows.
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