A magneto-DNA nanoparticle system for the rapid and sensitive diagnosis of enteric fever

Abstract

There is currently no widely available optimal assay for diagnosing patients with enteric fever. Here we present a novel assay designed to detect amplified Salmonella nucleic acid (mRNA) using magneto-DNA probes and a miniaturized nuclear magnetic resonance device. We designed primers for genes specific to S. Typhi, S. Paratyphi A, and genes conserved among Salmonella enterica spp. and utilized strongly magnetized nanoparticles to enhance the detection signal. Blood samples spiked with in vitro grown S. Typhi, S. Paratyphi A, S. Typhimurium, and E. coli were used to confirm the specificity of each probe-set, and serial 10-fold dilutions were used to determine the limit of the detection of the assay, 0.01-1.0 CFU/ml. For proof of principle, we applied our assay to 0.5 mL blood samples from 5 patients with culture-confirmed enteric fever from Bangladesh in comparison to 3 healthy controls. We were able to detect amplified target cDNA in all 5 cases of enteric fever; no detectable signal was seen in the healthy controls. Our results suggest that a magneto-DNA nanoparticle system, with an assay time from blood collection of 3.5 hours, may be a promising platform for the rapid and culture-free diagnosis of enteric fever and non-typhoidal Salmonella bacteremia.

Figure 1. A point-of care nuclear magnetic resonance system for the rapid diagnosis of enteric fever.

Schematic representation of detecting pathogen-specific nucleic acid from patients infected with S. Typhi or S. Paratyphi A using a magneto-DNA nanoparticle system. Total RNA is extracted from a whole blood sample of a patient with suspected enteric fever; the RNA is converted to cDNA and amplified by asymmetric PCR. The amplified target single-stranded DNA is then captured by beads conjugated to capture probes, hybridized with biotinylated detection probes, and then labeled with strepavidin coated MNPs to form a magnetic sandwich complex. Samples are then analyzed using a miniaturized micro-NMR (μNMR) system.

Figure 2. Assay validation using in vitro culture.

(A) Selected primer sets for each target sequence (red). Complimentary oligonucleotide sequences used for the bead capture probe (5′ end of target sequence) and MNP detection (3′end of target sequence) are listed in blue. (B) Limit of detection of each primer set in 10 fold serial dilutions of pure in vitro grown bacteria.

Figure 3. Detection of Salmonella spiked into human blood.

(A) Limit of detection of each primer set in spiked blood samples. (B) Specificity of each primer set demonstrated in blood samples spiked with different bacteria.

Figure 4. Detection of Salmonella in patients with culture-confirmed infection.

Detection of S. Typhi and S. Paratyphi in Bangladeshi patients with blood culture confirmed S. Typhi (n = 3) and S. Paratyphi A (n = 2) infection and 3 non-endemic healthy controls (North American).