Detecting Multi-Drug Resistant Tuberculosis using Rolling Circle Amplification and MinION Sequencing
Tuberculosis (TB) is one of the most common infectious diseases in the world, with approximately one third of the world population with latent TB and 9.6 million with active TB. TB is caused by Mycobacterium tuberculosis, a bacterial species known to infect lung tissues, which is typically spread via small droplets when an infected individual coughs or sneezes. Multi-drug resistant TB (MDR-TB) is a growing problem worldwide. Conventional culture based methods for detecting drug resistant TB are problematic due to slow growth rates (e.g. 2-6 weeks) while microscopy (e.g. acid-fast staining) is often unreliable and molecular based methods often miss many DNA mutations linked to resistance. These conditions make it difficult to properly treat drug resistant TB because resistant subpopulations are undetected and allowed to grow in the presence of some drug treatments. Rapid drug susceptibility testing (RDST) based on DNA sequence analysis is a desirable method for detecting drug resistance.
While several DNA sequencing platforms exist, Oxford Nanopore Technologies’ MinION Sequencer looks especially promising for MDR-TB detection in clinical samples as it is a handheld sequencer that can read hundreds of kilobases in real-time and can be used in a variety of conditions as it simply requires a USB connection to run on a laptop computer. Our objective was to modify TGen’s existing MDR-TB detection system (based on PCR and DNA sequencing) for use with this DNA sequencer. One requirement was to produce long DNA fragments using rolling circle amplification (RCA) to create a template for drug susceptibility testing. RCA is an isothermal method of gene amplification that results in long, tandem repeats of the same gene. We used two methods for RCA to detect MDR-TB linked mutations: circular ligation of PCR fragments and pre-designed “padlock probes” from published literature. Circular ligation of PCR products has proven difficult, and multiple methods of phosphorylation, ligation, and preventative supercoiling methods have been attempted. The padlock probes were successful on synthetic DNA, however their use on genomic DNA has proven more difficult.
Once RCA is successfully performed, we plan on sequencing the RCA products on the MinION Sequencer using a quick adapter ligation sequencing kit. The overall goal is to incorporate this new approach into the tool set for clinicians fighting MDR-TB in traditionally low income countries.