Highly-sensitive detection of Salmonella typhi in clinical blood samples by magnetic nanoparticle-based enrichment and in-situ measurement of isothermal amplification of nucleic acids

• Published in: PloS one Vol. 13; no. 3; p. e0194817
• Main Authors: Kaur, Avinash, Kapil, Arti, Elangovan, Ravikrishnan, Jha, Sandeep, Kalyanasundaram, Dinesh
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 28.03.2018; Public Library of Science (PLoS)
• Subjects: Analysis; Biology and Life Sciences; Engineering and Technology; Gene amplification; Medicine and Health Sciences; Nucleic acids; Research and Analysis Methods; Salmonella typhi
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0194817

Enteric fever continues to be a major cause of mortality and morbidity globally, particularly in poor resource settings. Lack of rapid diagnostic assays is a major driving factor for the empirical treatment of enteric fever. In this work, a rapid and sensitive method 'Miod' 'has been developed. Miod includes a magnetic nanoparticle-based enrichment of target bacterial cells, followed by cell lysis and loop-mediated isothermal amplification (LAMP) of nucleic acids for signal augmentation along with concurrent measurement of signal via an in-situ optical detection system. To identify positive/negative enteric fever infections in clinical blood samples, the samples were processed using Miod at time = 0 hours and time = 4 hours post-incubation in blood culture media. Primers specific for the STY2879 gene were used to amplify the nucleic acids isolated from S. typhi cells. A limit of detection of 5 CFU/mL was achieved. No cross-reactivity of the primers were observed against 106 CFU/mL of common pathogenic bacterial species found in blood such as E. coli, P. aeruginosa, S. aureus, A. baumanni, E. faecalis, S. Paratyphi A and K. pneumonia. Miod was tested on 28 human clinical blood samples. The detection of both pre-and post-four-hours incubation confirmed the presence of viable S. typhi cells and allowed clinical correlation of infection. The positive and negative samples were successfully detected in less than 6 hours with 100% sensitivity and specificity.