Direct Detection of Bacterial Genomic DNA at Sub-Femtomolar Concentrations Using Single Molecule Arrays

Abstract

We report a method for the sensitive measurement of genomic DNA based on the direct detection of single molecules of DNA in arrays of femtoliter wells. The method begins by generating short fragments of DNA from large, double-stranded molecules of genomic DNA using either restriction enzymes or sonication. Single-stranded fragments are then generated by melting the duplex, and these fragments are hybridized to complementary biotinylated detection probes and capture probes on paramagnetic beads. The resulting DNA complexes are then labeled with an enzyme (streptavidin-β-galactosidase), and single enzymes associated with these complexes on beads are detected in single molecule arrays (Simoa). DNA concentration is quantified by determining the average number of enzymes per bead via Poisson statistics (digital) or the average bead intensity (analog). The Simoa DNA assay was used to detect genomic DNA purified from S. aureus with an average limit of detection (LOD) of 0.07 fM, or 2100 DNA molecules per 50 μL sample. We used this assay to detect S. aureus spiked into (a) whole blood, with an average LOD of 1100 bacteria per 25 μL sample (0.074 fM), and (b) water from the Charles River, with an LOD of 1300 bacteria per 50 μL sample (0.042 fM). Bacteria were detected in river water without prior purification of DNA. The Simoa DNA assay, which directly detects target DNA molecules without molecular replication, is an attractive alternative to existing sensitive DNA detection technologies that rely on amplification using polymerases, such as the polymerase chain reaction (PCR).

Advancing the speed, sensitivity and accuracy of biomolecular detection using multi-length-scale engineering

“…This single-molecule-array strategy can also be used to detect synthetic nucleic acids present at femtomolar concentrations without the need for target amplification 71 , and was also adapted to detect bacterial DNA at attomolar levels 72 . The approach has also been used to detect tau protein in samples from patients with Alzheimer disease 73 and brain injury 74 .…”

Novel Signal-Enhancing Approaches for Optical Detection of Nucleic Acids—Going beyond Target Amplification

“…Major applications of enzyme labeling include enzyme-linked immunosorbent assays (ELISAs), immunocytochemical and immunohistochemical analyses, and nucleic acid hybridization assays. Protein enzymes are applied in ELISA, immunocytochemical and immunohistochemical analyses, whereas DNA manipulating enzymes find applications in nucleic acid hybridization assays [5,6].…”

“…One particular technique, the Simoa assay, demonstrates a LOD of approximately 70 aM [6]. The principle of Simoa is to bind the target DNA to magnetic particles by using specific capture DNA sequences.…”

“…Then, a second pool of probes containing biotin labels is then used to detect the target sequences. The hybridized biotin-labeled probes bind streptavidin-conjugated enzyme β-galactosidase, and the β-galactosidase converts a colorless fluorescein substrate into the purple oxidized derivative detected by fluorometry [6] ( Figure 1A). …”

Immunomagnetic nanoparticle-based assays for detection of biomarkers

“…Specifically, the capture moieties detect the desired target (ie, protein biomarker, DNA, RNA) for subsequent separation from the remaining solution via a simple magnet, and consequently, for various methods of analyses. [2][3][4][5][6] The noninvasiveness and simple nature of this method continues to enable the widespread use of magnetic particles. Furthermore, the high surface-area-to-volume ratio of the particles confers a correspondingly high probability of interaction with target biomarkers, and essentially increases the efficiency of the system.…”