One of the biggest challenges of the COVID-19 pandemic has been accessible, low-cost, and rapid detection systems for tracking the spread of the virus. At the Engelhart and Adamala lab, we developed a detection system called Apta-NASBA that can detect SARS-CoV-2, as well as other pathogens, using a different fluorescent colour output per pathogen.
The most common test to detect pathogens is the polymerase chain reaction (PCR) which is highly sensitive.. However, PCR requires cycling between high and low temperatures and skilled technicians who know how to run the tests.
Techniques that avoid high and low thermal cycling, termed isothermal techniques, use various enzymes and probes that allow amplification at constant temperatures, making them suitable for low-resource environments, field applications and laboratories lacking expensive PCR equipment.
However, many isothermal techniques aren’t perfect either. They often face the problem of false positives, where the test falsely indicates pathogen presence. Our work modifies the system and introduces a competitive reaction that is able to fully suppress false positives. With Apta-NASBA, we show successful detection and differentiation of various genetic components of E.Coli, a diarrhea-causing bacteria.
Apta-NASBA uses a one-pot reaction system, where the whole NASBA procedure (Nucleic-Acid Sequence-Based Amplification) occurs in one tube. The main component of this platform is a fluorogenic aptamer -- a short, single-stranded sequence of DNA or RNA that is designed to be amplified alongside the pathogen that fluoresces upon binding to its corresponding dye. If the reaction system is coupled to a fluorescence reader, real-time detection of the fluorescence level directly corresponds to the amount of pathogen in the sample (as seen for the E.Coli aggR template in the graph below). Reactions with no pathogen will have no fluorescence. Lauren Aufdembrink, the lead researcher, designed a Python program to quantify fluorescence using a low-cost, credit card-sized computer called Raspberry Pi and a cell phone camera.
We also enabled multiple reactions to take place at the same time.Fluorescent aptamers of diverse sequences and structures can generate different fluorescent colour readouts to detect presence of multiple pathogens in the same tube. We showed successful detection and differentiation between two different strains of E.Coli- estP and estH. In a single reaction, one aptamer detected estP and another detected estH, allowing simultaneous, real-time fluorescence readout for each pathogen.
The methods developed in our work offer a powerful means to suppress undesired false positives in isothermal disease detection methods. Requiring low-cost reagents and minimal training, Apta-NASBA is more accessible than many other pathogen detection systems. At a time when the SARS-CoV-2 virus is pushing the limits of testing kits, this study is valuable and promising for the development of accessible, low-cost, and rapid detection techniques.
Aufdembrink, Lauren M, et al. “Highly Specific, Multiplexed Isothermal Pathogen Detection with Fluorescent Aptamer Readout.” RNA, 2020, doi:10.1261/rna.075192.120.