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

Analytical and Bioanalytical Chemistry
Gabriel Antonio S MineroMikkel Fougt Hansen

Abstract

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 detec...Continue Reading

References

Nov 4, 1998·Nucleic Acids Research·J BanérU Landegren
Feb 6, 2008·Nano Letters·Mattias StrömbergMaria Strømme
Dec 9, 2014·Biosensors & Bioelectronics·Rebecca S BejhedMattias Strömberg
Mar 16, 2017·Physical Chemistry Chemical Physics : PCCP·Jeppe FockMikkel Fougt Hansen
Dec 8, 2017·Biosensors & Bioelectronics·Iván Hernández-NeutaMats Nilsson
Apr 13, 2018·Analytical and Bioanalytical Chemistry·Qian Ma, Zhiqiang Gao
Aug 2, 2018·Analytical and Bioanalytical Chemistry·Zhihua GaoZhifa Shen
May 7, 2019·Frontiers in Microbiology·Thi Ngoc Anh NguyenThi Van Anh Nguyen
Jul 13, 2019·Mikrochimica Acta·Gabriel Antonio S MineroMikkel Fougt Hansen
Jul 14, 2019·Biosensors & Bioelectronics·Francesca GarbarinoMikkel Fougt Hansen
Jul 31, 2019·Analytical and Bioanalytical Chemistry·Amanda MoyanoM Carmen Blanco-López
Nov 2, 2019·Methods in Molecular Biology·Gabriel Antonio S MineroMikkel F Hansen

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