ORIGINAL RESEARCH
Detection of Ser450Leu mutation in rpoB gene of Mycobacterium tuberculosis by allele-specific loop-mediated isothermal DNA amplification method
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
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.
To identify genetic mutations a rather time-consuming and expensive method of polymerase chain reaction (PCR) is widely used. The aim of the present work was to evaluate the possibility of using the two schemes of the method of allele-specific isothermal loop amplification (LAMP) to detect the TCG/TTG (S450L) mutation in the rpoB gene of Mycobacterium tuberculosis. 48 clinical isolates of M. tuberculosis and 11 samples of sputum were used, randomized and obtained in the microbiological laboratory of the city of Novosibirsk from incident patients. It is shown that the use of an analysis scheme using the allele-specific primer FIP compared to F3 has the best resolution: the difference between the amplification time of the mutation and the wild type allele was 22 ± 2,4 versus 13 ± 4,1 minutes (p = 0,0011). When using 100 DNA genomic equivalents a true positive signal (amplification of the rpoB gene with a mutation using the corresponding allele-specific primer) was detected after 29,4 ± 3,4 minutes. A positive signal was visualized after adding SYBR Green I to the reaction, both when illuminated with daylight and when using a UV transilluminator. Using the developed method the DNA sample of 20 RIFR isolates from M. tuberculosis was analyzed containing the Ser450Leu mutation in the rpoB gene, 10 RIFR isolates containing other mutations in the rpoB gene and 18 RIFs isolates without any mutations; the presence of mutations in the samples was determined using classical Sanger sequencing. The sensitivity and specificity of LAMP for detecting a Ser450Leu mutation in the rpoB gene was 100%. This approach allows the use of crude lysates of mycobacteria as DNA, which reduces the total analysis time to 1,5 hour.
Keywords: Mycobacterium tuberculosis, drug resistance, rifampicin, rpoB gene, isothermal loop amplification, LAMP