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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.
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Related Experiment Video

Updated: Jan 11, 2026

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Published on: February 3, 2021

One-Pot Isothermal Linear Amplification and Cas12a-Based Nucleic Acid Detection.

Selma Sinan¹, Remy M Kooistra², Karunya Rajaraman^2,3

¹Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, United States.

ACS Synthetic Biology

|November 17, 2025

Summary

This summary is machine-generated.

We developed CATNAP, a novel CRISPR-based diagnostic method. This integrated approach simplifies nucleic acid detection for point-of-care use, enhancing accessibility in resource-limited settings.

Keywords:

genotyping molecular diagnostics strand displacement amplification

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Last Updated: Jan 11, 2026

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Area of Science:

Molecular Biology
Biotechnology
Diagnostics

Background:

CRISPR-based diagnostics offer point-of-care potential but require preamplification, hindering use in low-resource settings.
Nucleic acid preamplification is a critical yet complex step for sensitive molecular diagnostics.

Purpose of the Study:

To develop an integrated nucleic acid amplification and detection method for CRISPR diagnostics.
To overcome the limitations of preamplification in resource-limited settings for point-of-care diagnostics.

Main Methods:

Developed CATNAP (Cas trans-nuclease detection of amplified products), integrating isothermal linear DNA amplification with Cas12a detection in a single reaction.
Utilized a nicking enzyme and DNA polymerase for continuous single-stranded DNA generation, activating Cas12a's trans-cleavage activity.
Optimized enzyme combinations, buffer conditions, and target selection for high catalytic efficiency.

Main Results:

CATNAP successfully distinguished between high- and low-risk HPV strains.
Detected HPV-16 in crude cell lysates of cervical cancer cells with minimal equipment.
Demonstrated advantages over traditional PCR-based approaches in simplicity and equipment requirements.

Conclusions:

CATNAP bridges the sensitivity gap in CRISPR diagnostics while maintaining simplicity.
The method enhances the accessibility of accurate disease detection in resource-limited settings.
CATNAP represents a significant advancement for point-of-care molecular diagnostics.