Visible label-free detection of bacterial DNA using flocculation of sterically stabilised cationic latexes

Abstract

The current gold standard diagnostic for bacterial infections is the use of culture, which can be time consuming and can take up to five days for results to be reported....

Bridging Flocculation of a Sterically Stabilized Cationic Latex as a Biosensor for the Detection of Microbial DNA after Amplification via PCR

“…These positive results expand upon previous demonstrations from our group showing flocculation in response to amplified DNA from Pseudomonas aeruginosa, a Gram-negative bacterium. 30 Importantly, the PEGMA-P2VP latexes did not show any visual signs of flocculation on the addition of either the DNA extraction reagents or the HSV-2 Mastermix (Figure SI3). Bridging flocculation requires macromolecules (DNA in this case) to be of a certain molecular weight, and the dNTPs, primers and salts present in these mixtures are not of a sufficient molar mass to induce flocculation of the PEGMA-P2VP latex despite having some negative charge or inducing charge screening.…”

“…Lightly cross-linked, PEGMA-P2VP with a mean diameter of approximately 700 nm was prepared by conventional emulsion polymerization to yield latex particles with a nonionic steric stabilizer and cationic core (Figure SI1). This latex was used in all subsequent studies reported herein …”

“…When combined with slower, established methods such as bacterial culture to confirm a true positive result, this gives confidence in a positive result and benefits patients within the clinical setting by providing a quicker time to result for commencement or cessation of antimicrobial treatment. As discussed previously, the response depends on DNA concentration, which can be dependent on a number of factors, including application parameters, amplicon size, and DNA yield …”

“…As discussed previously, the response depends on DNA concentration, which can be dependent on a number of factors, including application parameters, amplicon size, and DNA yield. 30 ■ CONCLUSIONS This paper demonstrates a method of detecting the presence of amplified DNA from bacterial and viral samples using a sterically stabilized, cationic polymer latex and widely available equipment, providing an accessible alternative DNA detection method. If the targeted DNA is present in a sample, successful PCR results in the obtained amplified DNA inducing flocculation of the cationic polymer latex, followed by rapid sedimentation and visible clear supernatant.…”

“…28,29 A new approach to detect amplified DNA from conventional PCR using the flocculation of sterically stabilized cationic latexes was recently reported by our group. 30 Specifically, poly(ethylene glycol methacrylate)-stabilized poly(2-vinylpyridine) (PEGMA-P2VP) latexes flocculated in the presence of negatively charged amplified DNA from Pseudomonas aeruginosa, showing visible sedimentation of the latex particles in 30 min without the use of fluorescent labels (Scheme 1). This allowed for the visual detection of amplified DNA without the use of gel electrophoresis, labels, or DNA probes.…”