Ultra-fast and sensitive detection of non-typhoidal Salmonella using microwave-accelerated metal-enhanced fluorescence ("MAMEF")

Abstract

Certain serovars of Salmonella enterica subsp. enterica cause invasive disease (e.g., enteric fever, bacteremia, septicemia, meningitis, etc.) in humans and constitute a global public health problem. A rapid, sensitive diagnostic test is needed to allow prompt initiation of therapy in individual patients and for measuring disease burden at the population level. An innovative and promising new rapid diagnostic technique is microwave-accelerated metal-enhanced fluorescence (MAMEF). We have adapted this assay platform to detect the chromosomal oriC locus common to all Salmonella enterica subsp. enterica serovars. We have shown efficient lysis of biologically relevant concentrations of Salmonella spp. suspended in bacteriological media using microwave-induced lysis. Following lysis and DNA release, as little as 1 CFU of Salmonella in 1 ml of medium can be detected in <30 seconds. Furthermore the assay is sensitive and specific: it can detect oriC from Salmonella serovars Typhi, Paratyphi A, Paratyphi B, Paratyphi C, Typhimurium, Enteritidis and Choleraesuis but does not detect Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae or Acinetobacter baumanii. We have also performed preliminary experiments using a synthetic Salmonella oriC oligonucleotide suspended in whole human blood and observed rapid detection when the sample was diluted 1:1 with PBS. These pre-clinical data encourage progress to the next step to detect Salmonella in blood (and other ordinarily sterile, clinically relevant body fluids).

Figure 1. Lysis of S. Enteritidis.

(A) Gold lysing triangles with bowtie configuration. (B) Lysis of biologically relevant concentrations of CVD 1920 (pGEN206). 2 CFU/ml to 1.3×10⁴ CFU/ml were lysed using gold lysing triangles and heated for 13 s on high power in a GE microwave Model No. JE2160BF01. (C) Agarose gel of DNA released from lysed CVD 1920 (pGEN206). An overnight culture of bacteria (1.9×10¹⁰ CFU/ml) was lysed using gold lysing triangles and microwave radiation. Lane 1, 1 kb Plus DNA ladder (Invitrogen); 2, lysed bacteria; 3, unlysed bacteria. (D) Transmission electron micrographs of lysed and unlysed CVD 1920 (pGEN206). Bar = 500 nm.

Figure 2. Schematic representation of anchor and fluorescent probes binding to Salmonella oriC target DNA.

Figure 3. Detection of various concentrations of a synthetic oriC oligonucleotide by MAMEF.

(A) The graph shows the increase in intensity as the concentration of oligonucleotide increases. (B) The actual fluorescent signal produced at each concentration. Excitation = 532 nm, Emission = 575 nm.

Figure 4. Detection of DNA released from microwave-lysed CVD 1920 (pGEN206) suspended in bacteriological media.

Serial dilutions from two independent samples were examined: A) 10-fold serial dilutions of a 10³ CFU/ml suspension and B) 2-fold serial dilutions of a 12 CFU/ml suspension.

Figure 5. MAMEF-based detection of a synthetic oriC oligonucleotide suspended in whole human blood.

Whole human blood was spiked with the oriC oligonucleotide (250 nM) and tested by MAMEF or diluted 1∶1 with PBS and then tested by MAMEF.