Ratiometric Fluorescence Coding Method for Multiplex Nucleic Acid Amplification Assays

Unmet Need
Nucleic acid amplification testing (NAAT) is a powerful analytic method that involves the amplification of genetic material, DNA or RNA, for the detection and identification of infectious microbial and viral pathogens like HIV, hepatitis B and C, as well as, oncogene mutations and genetic disorders.  One example of its global importance is that more than 53 million units of blood are screened worldwide for presence of pathogens using NAAT.  However, NAAT testing remains expensive and is not readily available at point-of-care sites for at-risk populations who are susceptible to many infectious diseases.  As such, there is a need for new approaches that can expand the “one target per test” limitation of current NAAT assays.
 
Technology Overview
Johns Hopkins researchers recently developed a multiple-target detection approach termed, Ratiometric Fluorescence Coding (RFC), in which a nucleic acid target can be probed by a unique relative ratio of two or more color fluorophores.  This fluorescent color ratio technique facilitates a high-degree of multiplexing (e.g., >40-plex using only 3 fluorophores with distinct colors) and can be implemented in a variety of assays and readout formats, e.g., standard plate reader, microfluidic digital detection and single-molecule detection modalities.  RFC is capable of simultaneously detecting DNA targets from six infectious diseases using only 2 color probes, with the possibility of additional color-labeled probes to expand its multiplexing potential.  With further development, Ratiometric Fluorescence Coding has the potential to increase the number of nucleic acid targets via highly multiplexed fluorescent probes into one single assay facilitating molecular pathogen identification and diagnosis of diseases.
 
Stage of Development
In vitro data is available.

Publications
Anal Chem. 2018 Oct 16;90(20):12180-12186.
 

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