A Robust Amplification Switch for Biomarker Detection in Limited-Resource Settings
Abstract
The first step in treating any disease is an accurate and speedy diagnosis. Since modern medicine increasingly relies on molecular markers rather than just a physician s judgement for diagnosis, it is important to develop new molecular markers for diseases where the tests do not exist, where the current tests are inaccurate, or where the current tests require expensive equipment and specialized skills to evaluate them. Small regulatory RNAs called microRNA (miRNA) are relatively recently discovered components of cellular function, which likely regulate >60% of our protein-encoding genes. These molecules are stable and ubiquitous in the bloodstream. A rapidly growing body of evidence shows that specific miRNA molecules change concentration in the blood of patients with a wide variety of ailments such as infectious disease, major depressive disorder, many cancers, and heart disease. Counting these small RNA in a simple blood draw will indicate the presence of a disease and thus can serve as a marker for the disease. Of particular interest are miRNA involved in regulation of the immune system, which show a unique fingerprint of the infection type, location, and severity. Several studies have found unique miRNA profiles in tuberculosis patients. This is an exciting development that can aid diagnosis in individuals who have difficulty producing sputum, such as children and HIV-positive patients. These markers also appear differently in patients with active infections when compared to patients with latent (inactive) infections. Malaria also leaves a unique fingerprint in the circulating miRNA profile. Current malaria diagnostics and monitoring is notoriously difficult and inaccurate in the field since identification of the particular Plasmodium strain is done by visual observation of blood smears, and the parasites may not be freely circulating in the blood. Our project aims to develop and validate a new detection method of miRNA in the blood. The main challenges we want to address are (1) specificity: ability to detect a particular miRNA of interest (i.e., indicating presence of tuberculosis infection) in the presence of many other similar miRNAs; (2) robustness: ability to give the same result in repeated experiments and varying conditions (i.e., ambient temperature, humidity); (3) speed: delivering results in minutes, rather than days, which will be essential in field situations where malaria patients travel long distances to clinics; and (4) low technological overhead: the method should not require extensive lab equipment, nor a specialized set of skills to administer. While the current detection methods rely on standard DNA amplification strategies that require complex equipment and hence access to a high-end laboratory with specialized technical personnel, our method uses a different method that works at a single temperature and converts target miRNA into many signal molecules within minutes. There is a trade-off for this simplicity: signal molecules can be produced at a low rate without the presence of the target miRNA marker of interest, and the rate at which these molecules are produced varies with environmental conditions. To address the specificity and robustness goals, the new method will use competition: the signal molecule will be removed from the reaction at a low rate, such that we will only observe signal molecule produced at a high rate by our marker. We will study the necessary rates of reaction to rapidly produce a signal only in the presence of the marker of interest. While the new detection technology, when fully developed, can be used for many diseases, our focus in this project is on tuberculosis and malaria diagnosis. Current widely used tests for tuberculosis detection only detect past exposure to the tuberculosis bacteria, but to diagnose acute illness, the bacteria from the sample need to be grown in the laboratory. The new detection test may also give more rapid and accurate d
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Aug 07, 2017
- Source ID
- W81XWH1710319
Entities
People
- Stephanie McCalla
Organizations
- Montana State University
- United States Army