Automated on-chip analysis of tuberculosis drug-resistance mutation with integrated DNA ligation and amplification

Autor:	Bo Tian, Gabriel Antonio S. Minero, Mikkel Fougt Hansen, Francesca Garbarino, Martina Bagnasco, Jeppe Fock
Rok vydání:	2020
Předmět:	DNA Bacterial 02 engineering and technology 01 natural sciences Biochemistry Analytical Chemistry Limit of Detection Lab-On-A-Chip Devices Tuberculosis Multidrug-Resistant Polymerase chemistry.chemical_classification Detection limit DNA ligase biology Chemistry 010401 analytical chemistry Mycobacterium tuberculosis 021001 nanoscience & nanotechnology Operation temperature Molecular biology 0104 chemical sciences Rolling circle replication Mutation biology.protein Mycobacterium tuberculosis DNA 0210 nano-technology Ligation Nucleic Acid Amplification Techniques
Zdroj:	Analytical and Bioanalytical Chemistry. 412:2705-2710
ISSN:	1618-2650 1618-2642
DOI:	10.1007/s00216-020-02568-x
Popis:	Detection of a single base mutation in Mycobacterium tuberculosis DNA can provide fast and highly specific diagnosis of antibiotic-resistant tuberculosis. Mutation-specific ligation of padlock probes (PLPs) on the target followed by rolling circle amplification (RCA) is highly specific, but challenging to integrate in a simple microfluidic device due to the low temperature stability of the phi29 polymerase and the interference of phi29 with the PLP annealing and ligation. Here, we utilized the higher operation temperature and temperature stability of Equiphi29 polymerase to simplify the integration of the PLP ligation and RCA steps of an RCA assay in two different strategies performed at uniform temperature. In strategy I, PLP annealing took place off-chip and the PLP ligation and RCA were performed in one pot and the two reactions were clocked by a change of the temperature. For a total assay time of about 1.5 h, we obtained a limit of detection of 2 pM. In strategy II, the DNA ligation mixture and the RCA mixture were separated into two chambers on a microfluidic disc. After on-disc PLP annealing and ligation, the disc was spun to mix reagents and initiate RCA. For a total assay time of about 2 h, we obtained a limit of detection of 5 pM. Graphical abstract.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::5a84cfd5456f4bfdf3a3db02cc9e9669 https://doi.org/10.1007/s00216-020-02568-x Zobrazit plný text záznamu Plný text ve formátu PDF Plný text ve formátu HTML
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