DNA, mRNA and microRNA are released and circulate in blood. Recent clinical studies have revealed the presence and extent of cell-free nucleic acid (cfNAs) in peripheral blood correlates with tumor aggressiveness and clinical outcome. Comparing with cancer protein blood biomarkers, cfNAs can be more informative, specific and accurate. Therefore, detection of cell-free nucleic acid is becoming a powerful tool for cancer diagnosis and prognosis, managing during therapy, and for personalized medicine. Traditional methods for cfNAs analysis including qPCR, gene array and sequencing for gene mutations, methylation, DNA integrity, microsatellite alterations, and viral DNA detection in blood. However, these methods are complex, expensive and time consuming. In particular, a crucial factor in cfNAs detection is cfNAs extraction which results in major batch-to-batch variations. Here, we describe a procedure allowing highly sensitive detection of cfNAs in a quantitative manner directly from serum with minimal sample preparation and pretreatment. The method is based on using a droplets-based microfluidic system for compartmentalization of sample and droplet digital nucleic acid amplification (DDNAA) to detect cfNAs in serum. Our central hypothesis is that the confinement of cfNAs in pico-litter droplets significantly increases sensitivity and selectivity for cfNAs detection in a rapid fashion. Since compartmentalization of blood within picoliter droplets, minimal blood components can be encapsulated in droplets with single cfNAs and this can minimize the interference effects from blood components such as protein, and other NAs. We demonstrate the accurate and sensitive quantification platform technology for cfNAs detection in serum without cfNAs extraction, which solves one of the biggest issue in cfNAs analysis field. Furthermore, our rapid cell free nucleic acid detection system can be applied to monitor various targets such as circulating tumor cells, bacteria, virus and fungus.

Rapid cell-free nucleic acid detection using micro-encapsulation system

248th National Meeting (2014)

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