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Related Concept Videos

Real Time RT-PCR02:57

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
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Amplification-Free Ribonucleic Acid Detection via Cas13-Csm6-Horseradish Peroxidase Cascade with Single-Molecule

Huimin Zhong1,2, Xin Zhang2,3, Jiayan Zhou1,2

  • 1College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.

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|February 26, 2026
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Summary

We developed a novel assay for single-molecule RNA detection, offering rapid, amplification-free results. This breakthrough advances point-of-care diagnostics and surveillance capabilities.

Keywords:
Amplification-free RNA assayCas13–Csm6–HRP cascadeColorimetric biosensorSingle-molecule sensitivitySynthetic biologyViral RNA

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Diagnostics

Background:

  • Single-molecule RNA detection is crucial for point-of-care diagnostics but remains challenging.
  • Existing methods often require complex amplification steps and lengthy procedures.

Purpose of the Study:

  • To develop an amplification-free assay for sensitive, rapid RNA detection at the single-molecule level.
  • To enable point-of-care RNA diagnostics with a simple colorimetric readout.

Main Methods:

  • A three-step enzymatic cascade assay (CCHP) utilizing Cas13, Csm6, and horseradish peroxidase.
  • Colorimetric readout at 450 nm within 1 hour under standard conditions.
  • Physics-guided minimal modeling to inform assay design and sensitivity.

Main Results:

  • CCHP achieved amplification-free RNA detection with a dynamic range of 10-10^7 copies per reaction.
  • Demonstrated strong agreement with RT-qPCR (r ≈ 0.95) and perfect concordance in a feasibility study (κ = 1.0).
  • Detected single-molecule RNA inputs for H1N1 and SARS-CoV-2 in clinical matrices without purification.

Conclusions:

  • CCHP offers a sensitive, rapid, and simple method for point-of-care RNA detection.
  • The assay advances RNA diagnostics and surveillance by enabling ultralow-copy detection.
  • This technology holds promise for infectious disease monitoring and diagnostics.