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 Labouriau; Birgitta Ruth Knudsen

doi:10.1039/c8nr07850e

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

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

13 Citations (Scopus)

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.

Original language	English
Journal	Nanoscale
Volume	11
Issue	2
Pages (from-to)	587-597
Number of pages	11
ISSN	2040-3364
DOIs	https://doi.org/10.1039/c8nr07850e
Publication status	Published - 14 Jan 2019

Access to Document

10.1039/c8nr07850e

Cite this

Franch, O., Han, X., Marcussen, L., Jørgensen, A. G., Andersen, M. B., Godbole, A. A., Harmsen, C., Nørskov-Lauritsen, N., Thomsen, J., Pedersen, F. S., Wang, Y., Shi, D., Wejse, C., Pødenphant, L., Nagaraja, V., Bertl, J., Stougaard, M., Ho, Y.-P., Hede, M. S., ... Knudsen, B. R. (2019). A new DNA sensor system for specific and quantitative detection of mycobacteria. Nanoscale, 11(2), 587-597. https://doi.org/10.1039/c8nr07850e

@article{ff8a6e5fa5f2467094873cc9e2f6206e,

title = "A new DNA sensor system for specific and quantitative detection of mycobacteria",

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.",

author = "Oskar Franch and Xiao Han and L{\ae}rke Marcussen and J{\o}rgensen, {Asger Givskov} and Andersen, {Marie Bech} and Godbole, {Adwait Anand} and Charlotte Harmsen and Niels N{\o}rskov-Lauritsen and Jonas Thomsen and Pedersen, {Finn Skou} and Yilong Wang and Donglu Shi and Christian Wejse and Lone P{\o}denphant and Valakunja Nagaraja and Johanna Bertl and Magnus Stougaard and Yi-Ping Ho and Hede, {Marianne Smedegaard} and Rodrigo Labouriau and Knudsen, {Birgitta Ruth}",

year = "2019",

month = jan,

day = "14",

doi = "10.1039/c8nr07850e",

language = "English",

volume = "11",

pages = "587--597",

journal = "Nanoscale",

issn = "2040-3364",

publisher = "Royal Society of Chemistry",

number = "2",

}

Franch, O, Han, X, Marcussen, L , Jørgensen, AG, Andersen, MB, Godbole, AA, Harmsen, C, Nørskov-Lauritsen, N, Thomsen, J, Pedersen, FS, Wang, Y, Shi, D, Wejse, C, Pødenphant, L, Nagaraja, V, Bertl, J, Stougaard, M, Ho, Y-P, Hede, MS, Labouriau, R & Knudsen, BR 2019, 'A new DNA sensor system for specific and quantitative detection of mycobacteria', Nanoscale, vol. 11, no. 2, pp. 587-597. https://doi.org/10.1039/c8nr07850e

TY - JOUR

T1 - A new DNA sensor system for specific and quantitative detection of mycobacteria

AU - Franch, Oskar

AU - Han, Xiao

AU - Marcussen, Lærke

AU - Jørgensen, Asger Givskov

AU - Andersen, Marie Bech

AU - Godbole, Adwait Anand

AU - Harmsen, Charlotte

AU - Nørskov-Lauritsen, Niels

AU - Thomsen, Jonas

AU - Pedersen, Finn Skou

AU - Wang, Yilong

AU - Shi, Donglu

AU - Wejse, Christian

AU - Pødenphant, Lone

AU - Nagaraja, Valakunja

AU - Bertl, Johanna

AU - Stougaard, Magnus

AU - Ho, Yi-Ping

AU - Hede, Marianne Smedegaard

AU - Labouriau, Rodrigo

AU - Knudsen, Birgitta Ruth

PY - 2019/1/14

Y1 - 2019/1/14

N2 - 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.

AB - 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.

U2 - 10.1039/c8nr07850e

DO - 10.1039/c8nr07850e

M3 - Journal article

C2 - 30556557

SN - 2040-3364

VL - 11

SP - 587

EP - 597

JO - Nanoscale

JF - Nanoscale

IS - 2

ER -

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

Abstract

Access to Document

Fingerprint

Cite this