2019 / 01

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DOI: 10.24075/brsmu.2019.007

ORIGINAL RESEARCH

Detection of Ser450Leu mutation in rpoB gene of Mycobacterium tuberculosis by allele-specific loop-mediated isothermal DNA amplification method

Filipenko ML1, Oscorbin IP1, Khrapov EA1, Shamovskaya DA1, Cherednichenko AG2, Shvartz YaSh2
About authors

1 Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia

2 Novosibirsk Tuberculosis Research Institute, Novosibirsk, Russia

Correspondence should be addressed: Maxim L. Filipenko
Lavrentyev Prospect 8/2, Novosibirsk, 630090; moc.liamg@oknepiliflm

About paper

Funding: the study was done with the financial support of the basic budgetary financing project № VI.62.1.5 «Synthetic biology: development of tools for the genetic material manipulation and creation of promising drugs for therapy and diagnostics» (0309-2018-0003).

Author contribution: Filipenko ML created а general concept of the study, planned experiments, analyzed the results and participated in the writing of this article; Oscorbin IP participated in the writing of this article; Khrapov EA conducted experiments; Shamovskaya DV conducted experiments; Cherednichenko AG analyzed the experiments results; Shvartz YaSh was involved in planning and analyzed the experiments results.

Received: 2018-12-07 Accepted: 2019-02-25 Published online: 2019-03-09
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Fig. 1. Schematic illustration of the different principles of allele-specific isothermal loop amplification (AS-LAMP): allele-specific primer FIP (А), allele-specific primer F3 (Б)
Fig. 2. Fluorescence accumulation curves for AS-LAMP products. Curves marked by circles correspond to FIP-AS-LAMP, marked by crosses — to F3-AS-LAMP
Fig. 3. Detection of TCG/TTG (S450L) mutation of the rpoB gene by AS-LAMP method with visualization of amplification products by adding the SYBR Green I intercalating dye in natural light (А) and with the 280 nm UV illumination (B). Samples 1, 4, 5 contain а mutation, 2 and 3 — contain no mutation, they were amplified with mutation-specific primer, tubes marked with «К» were amplified with norm-specific primer
Table 1. List of oligonucleotide primers and fluorescently labeled probes used in present study
Table 2. The range of rpoB gene mutations of the Mycobacterium tuberculosis isolates detected by Sanger sequencing and the results of a TCG/TTG (S450L) mutation detection using AS-LAMP