A new DNA sensor system for specific and quantitative detection of mycobacteria

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DOI

  • Oskar Franch
  • Xiao Han, School of Materials Science and Engineering, Tongji University, Shanghai, PR China,
  • Lærke Marcussen
  • Asger Givskov Jørgensen,
  • Marie Bech Andersen
  • Adwait Anand Godbole, Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India, Charlotte Harmsen,
  • Niels Nørskov-Lauritsen
  • Jonas Thomsen,
  • Finn Skou Pedersen
  • Yilong Wang, The Institute for Translational Nanomedicine, Shanghai East Hospital, the Institute for Biomedical Engineering & Nano Science, Tongji University School of Medicine, Shanghai, P. R. China, Materials Science and Engineering Program, Department of Mechanical and Materials Engineering, College of Engineering and Applied Science, University of Cincinnati, Cincinnati, USA, Donglu Shi, The Institute for Translational Nanomedicine, Shanghai East Hospital, the Institute for Biomedical Engineering & Nano Science, Tongji University School of Medicine, Shanghai, P. R. China, Materials Science and Engineering Program, Department of Mechanical and Materials Engineering, College of Engineering and Applied Science, University of Cincinnati, Cincinnati, USA,
  • Christian Wejse
  • Lone Pødenphant, Klinisk Mikrobiologisk afdeling, Skejby Sygehus, DenmarkValakunja Nagaraja, Department of Microbiology and Cell Biology, Indian Institute of Science & Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India,
  • Johanna Bertl
  • Magnus Stougaard
  • Yi-Ping Ho, Marianne Smedegaard Hede,
  • Rodrigo Labouriau
  • Birgitta Ruth Knudsen

In the current study, we describe a novel DNA sensor system for specific and quantitative detection of mycobacteria, which is the causative agent of tuberculosis. Detection is achieved by using the enzymatic activity of the mycobacterial encoded enzyme topoisomerase IA (TOP1A) as a biomarker. The presented work is the first to describe how the catalytic activities of a member of the type IA family of topoisomerases can be exploited for specific detection of bacteria. The principle for detection relies on a solid support anchored DNA substrate with dual functions namely: (1) the ability to isolate mycobacterial TOP1A from crude samples and (2) the ability to be converted into a closed DNA circle upon reaction with the isolated enzyme. The DNA circle can act as a template for rolling circle amplification generating a tandem repeat product that can be visualized at the single molecule level by fluorescent labelling. This reaction scheme ensures specific, sensitive, and quantitative detection of the mycobacteria TOP1A biomarker as demonstrated by the use of purified mycobacterial TOP1A and extracts from an array of non-mycobacteria and mycobacteria species. When combined with mycobacteriophage induced lysis as a novel way of effective yet gentle extraction of the cellular content from the model Mycobacterium smegmatis, the DNA sensor system allowed detection of mycobacteria in small volumes of cell suspensions. Moreover, it was possible to detect M. smegmatis added to human saliva. Depending on the composition of the sample, we were able to detect 0.6 or 0.9 million colony forming units (CFU) per mL of mycobacteria, which is within the range of clinically relevant infection numbers. We, therefore, believe that the presented assay, which relies on techniques that can be adapted to limited resource settings, may be the first step towards the development of a new point-of-care diagnostic test for tuberculosis.

Original languageEnglish
JournalNanoscale
Volume11
Issue number2
Pages (from-to)587-597
Number of pages11
ISSN2040-3364
DOIs
Publication statusPublished - 14 Jan 2019

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