Article pubs.acs.org/ac
Direct Detection of Bacterial Genomic DNA at Sub-Femtomolar Concentrations Using Single Molecule Arrays Linan Song,† Dandan Shan,† Mingwei Zhao,† Brian A. Pink,† Kaitlin A. Minnehan,† Lyndsey York,† Melissa Gardel,† Sean Sullivan,† Aaron F. Phillips,‡ Ryan B. Hayman,‡ David R. Walt,‡ and David C. Duffy*,† †
Quanterix Corporation, 113 Hartwell Avenue, Lexington, Massachusetts 02421, United States Department of Chemistry, Tufts University, 62 Talbot Avenue, Medford, Massachusetts 02155, United States
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S Supporting Information *
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).
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a result, PCR requires careful sample preparation to purify DNA in order for the amplification reaction to be performed. DNA purification makes automation of PCR more complex, with each type of sample often requiring a different preparation method.10 Second, PCR can be prone to false positive results from erroneous amplification of nontarget sequences.16 Third, PCR is relatively expensive compared to other sensitive analytical techniques, such as immunoassays. Fourth, PCR amplification of positive samples generates high concentrations of target molecules that can easily lead to cross-contamination of negative samples. These limitations provide incentives to develop new methods for detecting NAs. Methods that do not rely on the replication of DNA, i.e., that directly detect the target molecules without polymerases, are promising alternatives to PCR. For example, the branched DNA (bDNA) method relies on the capture of NA molecules on a solid substrate followed by labeling via a sandwich with highly branched NA sequences containing many alkaline phosphatase molecules that turn over a chemiluminescent
he sensitive measurement of nucleic acids (NAs) is important in a number of fields such as clinical diagnostics,1 environmental monitoring,2 detection of biological threats,3 and food safety.4 The detection and quantification of NAs at low concentrations from viruses and bacteria are especially important for the early diagnosis of infectious diseases5 and testing of blood supplies.6 Here, we report the development of a method based on the direct detection of single DNA molecules derived from genomic DNA that enables sensitive measurements suitable for these applications. The gold standard method for detecting NA is the polymerase chain reaction (PCR).7 PCR is now capable of routinely detecting
