Towards Point of Care Diagnostics: Visual Detection of Meningitis Pathogens Directly from Cerebrospinal Fluid

Abstract

Rapid and cost‐efficient methods for diagnostics of infectious diseases can expedite prescription of adequate treatment, thus shortening the patients’ recovery and reducing adverse side effects. In this study, we developed a procedure for visual detection of bacterial pathogens Gram‐negative Neisseria meningitidis and Gram‐positive Streptococcus pneumoniae directly from human samples of cerebrospinal fluid (CSF). The assay uses hybridization probes that can efficiently recognize folded single‐stranded DNA analytes due to their split design. The assay includes the following stages: PCR amplification of N. meningitidis and S. pneumoniae targeted sequences; λ exonuclease treatment of the polymerase chain reaction (PCR) amplicons, and detection of the amplicons by hybridization probes with visual signal output. The assay requires 2.0 h with hands‐on time of about 40 min, and a regular PCR thermal cycler. It detects down to 10 bacteria in 2 μL of cerebrospinal fluid. The assay works for both gram‐positive and gram‐negative bacteria and does not require optimization of PCR conditions. This development creates a basis for the point‐of‐care diagnostics of bacterial meningitis with visual signal output.

DNA nanomachine for visual detection of structured RNA and double stranded DNA

“…13 For the analysis of stem-loop folded RNA or ssDNA, the following biPxD design was proposed. 17,18 The analyte binding arm of strand F (Scheme 1A) is long and binds one side of the stem, the loop, and several nucleotides of the other side of the stem. This arm binds the analyte strongly and unwinds its secondary structure.…”

“…We recently applied biPxD technology for detecting PCR amplicons of two pathogenic bacteria. 18 The detection scheme required l exonuclease treatment of dsDNA amplicons to convert them to ssDNA by the biPxD probe. The l exonuclease treatment stage required 15 min.…”

“…Earlier, we developed a probe that enables both (i) visual signal output and (ii) high selectivity at room temperature. 13,18 In this study, we put the probe in the context of a nanostructure to enable the detection of structurally constrained amplicons of human pathogens at room temperature without the need for extra stages requiring equipment or reagents. PxDm takes advantage of the two-analyte binding arms linked to a common dsDNA scaffold.…”

“…The average values of the three independent experiments are presented. (C) The complex between the analyte-binding arms of the biPxD probe predicted by UNAfold 18. (D) L.m._PxDm (L.m._M-7, L.m._T1_ PxDm, L.m._T2_PxDm) in the complex with synthetic DNA analyte L.m._DNA.…”

DNA-nanomachines for nucleic acid detection

“…We report here two DNM variants: (i) DNAzyme-based DNM with fluorescent output [1] (Fig. 1) and (ii) G-quadruplex based DNM with visual output [2] (Fig. 2).…”