Overview
Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains one of the world's most serious public health problems. Although tuberculosis is curable and the treatment success rate is high, it is still the second most common cause of death from infectious disease. Most of the deaths occur for the lack of effective identification of those in need of therapy. Case detection is currently the rate-limiting step in TB control.
The currently widely-used TB detection methods all have their own problems. Nucleic Acid Detection (NAD) is a safer, faster, and more sensitive detection method. However, its shortcoming is critical: the high false-positive rate from non-specific amplification, and the requirement of extremely expensive clumsy instruments; these make NAD not common for TB diagnosis.
To obviate such shortcoming, Peking iGEM developed a novel detection system, paired dCas9 (PC) reporter, that converts the sequence-specific information of pathogenic bacteria's genome (in our case, M. tuberculosis, MTB) into easily readable signal including bioluminescence, pigment, or electrical. Our PC Reporter was successfully applied to the detection of real pathogenic M.Tuberculosis H37Rv (isolated genomic DNA that is absolutely safe, prepared by our collaborator, not by us). Combined with our work in multiple-marker array (using MTB-specific markers extracted from the entire genome of MTB) and hardware development, our PC reporter system is expected to be a powerful tool in TB diagnosis, with a huge potential for various applications and extensions.
Paired dCas9 Reporter
With two fragments of split firefly luciferase separately fused to dCas9 protein, we successfully designed and built a paired dCas9 reporter (PC reporter) that can convert specific sequence information into light signal. We finished the characterization and optimization. Most importantly, we successfully demonstrated the PC reporter with isolated genomic DNA from a pathogenic MTB.
Multi-marker array
To deal with challenges from clinical practices, such as strain mutations, sample variations, and other uncontrollable environmental factors, we designed an array to extract more sequence information for our PC Reporter to test multiple sites on the target genome at one time to improve the reliability. With statistics analysis, we can analyze read-outs in a quantitative way.
Hardware
Noticing that most TB cases occur in developing areas, we designed and built a device (prototype), which was portable, affordable, and can be easily used by medical workers.
Specific isothermal amplification
In order to popularize our detection system to developing areas, we invented two novel isothermal amplification methods that can use one pair of easily designed primers to achieve specific amplification.