Ratiometric Fluorescence Coding for Multiplex Nucleic Acid Amplification Testing

Abstract

Although nucleic acid amplification testing (NAAT) has become the cornerstone for molecular diagnosis of diseases, expanding the multiplexed detection capacity of NAAT remains an important objective. To this end, encoding each nucleic acid target with a specific fluorescently labeled probe has been the most mature approach for multiplexed detection. Unfortunately, the number of targets that can be differentiated via this one-target-one-fluorophore multiplexed detection approach is restricted by spectral overlaps between fluorophores. In response, we present herein a new multiplexed detection approach termed ratiometric fluorescence coding, in which we encode each nucleic acid target with a specific ratio between two standard fluorophores. In ratiometric fluorescence coding, we employ the padlock probe chemistry to encode each nucleic acid target with a specific number of binding sites for two probes labeled with different fluorophores. Coupling the padlock probes with either rolling circle amplification (RCA) or hyperbranched rolling circle amplification (HRCA), we transform each nucleic acid target into a specific template that allows hybridization with the fluorescently labeled probes at predesigned ratios, thereby achieving multiplexed detection. For demonstration, we detected DNA targets from six infectious diseases and demonstrated the potential for further expanding the multiplexing capability of our approach. With further development, ratiometric fluorescence coding has the potential to enable highly multiplexed detection of nucleic acid targets and facilitate molecular diagnosis of diseases.

Composable Microfluidic Plates (cPlate): A Simple and Scalable Fluid Manipulation System for Multiplexed Enzyme-Linked Immunosorbent Assay (ELISA)

“…Since many diseases are complex and multifactorial, results from a single biomarker testing are not always adequate for the diagnosis or prognosis of a disease. − Therefore, multiplexed ELISA, which simultaneously detects different biomarkers from a single specimen, is highly desired to offer more reliable diagnostic results . To address the issues in conventional ELISA, microfluidics is a powerful platform due to its superior capability of manipulating small amount of fluids.…”

Bridging the gap between development of point-of-care nucleic acid testing and patient care for sexually transmitted infections

“…The authors detected DNA targets from six infectious diseases, including HIV, CT, NG, and TP. 207 Despite the demonstrated multiplexing capacity, this ratiometric fluorescence coding assay still must be simplified into a single-step assay to facilitate POC use.…”

Ligation-Enabled Fluorescence-Coding PCR for High-Dimensional Fluorescence-Based Nucleic Acid Detection

“…25,26 More recently, we also introduced a novel multiplexing scheme that addresses many of the primary issues associated with the use of multiple sets of probes. 27 Here, multiplex nucleic acid detection is achieved by coupling padlock probe chemistry 28 with rolling circle amplification (RCA) or hyperbranched RCA (HRCA) 29,30 to generate specific fluorescence ratios by concurrent hybridization of multiple fluorescently labeled probes.…”

“…Another reported strategy is to encode each target with different fluorescence intensities within the same channel. , With this approach, different endpoint fluorescence intensities are generated by varying the relative concentrations of different fluorescently labeled probes. Drawbacks of this approach include the need to fine-tune numerous primer and probe sequences and concentrations in order to obtain the desired endpoint fluorescence intensities, while limiting nonspecific amplification and amplification biases. , More recently, we also introduced a novel multiplexing scheme that addresses many of the primary issues associated with the use of multiple sets of probes . Here, multiplex nucleic acid detection is achieved by coupling padlock probe chemistry with rolling circle amplification (RCA) or hyperbranched RCA (HRCA) , to generate specific fluorescence ratios by concurrent hybridization of multiple fluorescently labeled probes.…”