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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f1: 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.

Mentions: New point-of-care technologies that can overcome some of these limitations are needed. Here we describe the use of a rapid magneto-DNA nanoparticle assay that can be used to detect Salmonella mRNA directly in the blood of infected individuals (Fig. 1). To increase the sensitivity of our assay, we chose genes within operons that have previously been demonstrated to be expressed in the blood of infected individuals with enteric fever78. In addition, 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. Thus, this assay is a promising platform for the rapid and culture-free diagnosis of enteric fever and non-typhoidal Salmonella bacteremia.


A magneto-DNA nanoparticle system for the rapid and sensitive diagnosis of enteric fever
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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015101&req=5

f1: 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.
Mentions: New point-of-care technologies that can overcome some of these limitations are needed. Here we describe the use of a rapid magneto-DNA nanoparticle assay that can be used to detect Salmonella mRNA directly in the blood of infected individuals (Fig. 1). To increase the sensitivity of our assay, we chose genes within operons that have previously been demonstrated to be expressed in the blood of infected individuals with enteric fever78. In addition, 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. Thus, this assay is a promising platform for the rapid and culture-free diagnosis of enteric fever and non-typhoidal Salmonella bacteremia.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus