Isothermal point mutation detection: toward a first-pass screening strategy for multidrug-resistant tuberculosis

Ng, Benjamin Y. C., Wee, Eugene J. H., Woods, Kyra, Anderson, Will, Antaw, Fiach, Tsang, Hennes Z. H., West, Nicholas P. and Trau, Matt (2017) Isothermal point mutation detection: toward a first-pass screening strategy for multidrug-resistant tuberculosis. Analytical Chemistry, 89 17: 9017-9022. doi:10.1021/acs.analchem.7b01685


Author Ng, Benjamin Y. C.
Wee, Eugene J. H.
Woods, Kyra
Anderson, Will
Antaw, Fiach
Tsang, Hennes Z. H.
West, Nicholas P.
Trau, Matt
Title Isothermal point mutation detection: toward a first-pass screening strategy for multidrug-resistant tuberculosis
Journal name Analytical Chemistry   Check publisher's open access policy
ISSN 1520-6882
0003-2700
Publication date 2017-09-05
Year available 2017
Sub-type Article (original research)
DOI 10.1021/acs.analchem.7b01685
Open Access Status Not yet assessed
Volume 89
Issue 17
Start page 9017
End page 9022
Total pages 6
Place of publication Washington, DC United States
Publisher American Chemical Society
Language eng
Formatted abstract
Point mutations in DNA are useful biomarkers that can provide critical classification of disease for accurate diagnosis and to inform clinical decisions. Conventional approaches to detect point mutations are usually based on technologies such as real-time polymerase chain reaction (PCR) or DNA sequencing, which are typically slow and require expensive lab-based equipment. While rapid isothermal strategies such as recombinase polymerase amplification (RPA) have been proposed, they tend to suffer from poor specificity in discriminating point mutations. Herein, we describe a novel strategy that enabled exquisite point mutation discrimination with isothermal DNA amplification, using mismatched primers in conjunction with a two-round enrichment process. As a proof of concept, the method was applied to the rapid and specific identification of drug-resistant Mycobacterium tuberculosis using RPA under specific conditions. The assay requires just picogram levels of genomic DNA input, is sensitive and specific enough to detect 10% point mutation loading, and can discriminate between closely related mutant variants within 30 min. The assay was subsequently adapted onto a low-cost 3D-printed isothermal device with real-time analysis capabilities to demonstrate a potential point-of-care application. Finally, the generic applicability of the strategy was shown by detecting three other clinically important cancer-associated point mutations. We believe that our assay shows potential in a broad range of healthcare screening processes for detecting and categorizing disease phenotypes at the point of care, thus reducing unnecessary therapy and cost in these contexts.
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID CG-12-07
Institutional Status UQ

 
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Created: Mon, 25 Sep 2017, 01:00:46 EST by Web Cron on behalf of Aust Institute for Bioengineering & Nanotechnology