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.
Highlights
There is currently no widely available optimal assay for diagnosing patients with enteric fever
Enteric fever control has been complicated by the lack of sensitive and specific diagnostic assays that could be used for clinical management and surveillance programs to assess disease burden within a community and measure effectiveness of various intervention strategies[2]
Individuals presenting to the International Centre for Diarrhoeal Disease Research, Bangladesh hospital or the Kamalapur field site of the icddr,b with clinical symptoms of enteric fever were eligible for enrollment if they were 1–59 years of age and had a fever (≥38 °C) of 3–7 days duration without an obvious focus of infection or alternate diagnosis
Summary
There is currently no widely available optimal assay for diagnosing patients with enteric fever. 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. We have included three types of signal amplification during the procedure: asymmetric PCR amplification of the target nucleic acid, enrichment of target sequences by bead capture, and magnetic amplification (i.e. a single magnetic nanoparticle can have an effect on billions of surrounding water particles)[9]. This assay is a promising platform for the rapid and culture-free diagnosis of enteric fever and non-typhoidal Salmonella bacteremia
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