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

Oskar Franch, Xiao Han, Lærke Marcussen, Asger Givskov Jørgensen, Marie Bech Andersen, Adwait Anand Godbole, Charlotte Harmsen, Niels Nørskov-Lauritsen, Jonas Thomsen, Finn Skou Pedersen, Yilong Wang, Donglu Shi, Christian Wejse, Lone Pødenphant, Valakunja Nagaraja, Johanna Bertl, Magnus Stougaard, Yi-Ping Ho, Marianne Smedegaard Hede, Rodrigo LabouriauBirgitta Ruth Knudsen

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

Abstract

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.

OriginalsprogEngelsk
TidsskriftNanoscale
Vol/bind11
Nummer2
Sider (fra-til)587-597
Antal sider11
ISSN2040-3364
DOI
StatusUdgivet - 14 jan. 2019

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