Programmable Clostridium perfringens Argonaute-Based, One-Pot Assay for the Multiplex Detection of miRNAs

Abstract

Assays for the molecular detection of miRNAs are typically constrained by the level of multiplexing, especially in a single tube. Here, we report a general and programmable diagnostic platform by combining mesophilic Clostridium perfringens Argonaute (CpAgo) with exponential isothermal amplification (EXPAR), which is a dual-signal amplification strategy, allowing for the rapid and sensitive detection of multiple miRNAs with single-nucleotide discrimination in one pot. The CpAgo-based One-Pot (COP) assay achieved a limit of detection of 1 zM miRNA within 30 min of turnaround time and a wide concentration range. This COP assay was applied to simultaneously detect four miRNAs in a single tube from clinical serum samples, showing superior analytical performance in distinguishing colorectal cancer patients from healthy individuals. This programmable, one-pot, multiplex, rapid, and specific strategy offers great promise in scientific research and clinical applications.

Multiplex Signal Transduction and Output at Single Recognition Interface of Multiplexed Photoelectrochemical Sensors

“…Second, the problem of multiplex detection signal outputs on the single recognition surface of the sensor and avoiding mutual interference of detection signals between multiplex signal pathways necessitate the development of more sophisticated methods or techniques. During the multiplex detection process, various targets to be measured exist simultaneously in one reaction system; thus, cross-interference easily occurs . The previously mentioned strategies for the transduction and output of multiplex detection signals on the single recognition interfaces of PEC sensors involve complex electrode modifications.…”

Binding Activity of Prokaryotic Argonaute for Background-Suppressed Exponential Isothermal Amplification

“…Herein, we explored the effect of pAgos on the enhancement of specificity of EXPAR based on its natural binding affinity with single-stranded nucleic acids. The principle of background-suppressed EXPAR is illustrated in Scheme ; TtAgo was incorporated in the isothermal amplification system to bind amplification templates but not to conduct its cleavage activity at 55 °C. , Such binding affinity could effectively prevent mismatched hybridization between templates. In the presence of the target, Vent (exo-) DNA polymerase binds to the double strand structure formed between the target and corresponding template and extends along the 3′ terminus of the target.…”

Smartphone-Based Fluorescent Profiling of Quaternary MicroRNAs in Urine for Rapid Diagnosis of Urological Cancers Using a Multiplexed Isothermal Exponential Amplification Reaction

“…Some label-free strategies have been developed for the accurate probing of miRNAs in urine. However, those methods either require high sample consumption 16,17 and standard bulk instrumentation 18 or lack the multiplexing capability for profiling miRNA mixtures in a single detection medium. 19−22 Gold standard techniques for miRNA analysis include the quantitative reverse transcription polymerase chain reaction (RT-qPCR), 23 capillary electrophoresis, 24 and nucleic acid microarrays.…”

“…Some label-free strategies have been developed for the accurate probing of miRNAs in urine. However, those methods either require high sample consumption , and standard bulk instrumentation or lack the multiplexing capability for profiling miRNA mixtures in a single detection medium. − …”