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Real Time RT-PCR

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Related Experiment Video

Updated: May 22, 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

Sequence-validated trigger gating for false-positive control in point-of-care isothermal nucleic acid detection.

Gulinaizhaer Abudushalamu¹, Chuankun Yang², Yunpeng Jiang³

¹Center of Clinical Laboratory Medicine, Zhongda Hospital, Medical School of Southeast University, Nanjing, Jiangsu, 210009, China; Department of Laboratory Medicine, Medical School of Southeast University, Nanjing, Jiangsu, 210009, China.

|May 20, 2026

Summary

A new sequence-validated trigger-gating strategy enhances isothermal RNA detection by preventing false positives. This method improves the reliability of point-of-care nucleic acid tests, especially in resource-limited settings.

Keywords:

Isothermal RNA detection Lateral flow assay Point-of-care testing SFTSV Sequence-validated trigger gating

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Last Updated: May 22, 2026

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

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Published on: February 3, 2021

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Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

Published on: June 17, 2022

Area of Science:

Molecular Biology
Diagnostics
Biotechnology

Background:

Nonspecific by-products in isothermal amplification lead to false positives in nucleic acid tests.
Reliability of point-of-care diagnostics is limited by the propagation of these nonspecific amplification products.

Purpose of the Study:

To develop a sequence-validated trigger-gating strategy for catalytic hairpin assembly (CHA) to improve false-positive control in isothermal visual RNA detection.
To enhance the selectivity and reliability of CHA-based visual RNA detection for portable molecular diagnostics.

Main Methods:

Implemented a sequence-validated trigger-gating strategy in a regulated catalytic hairpin assembly platform (RecHA) for severe fever with thrombocytopenia syndrome virus (SFTSV) RNA detection.
Coupled RecHA with lateral flow readout, enabling sequence-dependent validation before trigger strand release and CHA activation.
Tested the assay's performance using SFTSV RNA as a model target and validated it with clinical serum samples against qRT-PCR.

Main Results:

The RecHA assay demonstrated reduced background activation and improved test line discrimination at 34°C.
Achieved a limit of detection of 10 fM for SFTSV RNA and supported direct loading of lysis products.
Clinical validation showed high concordance with qRT-PCR, achieving an AUC of 0.994, sensitivity of 1.000, and specificity of 0.967.

Conclusions:

Sequence-validated trigger gating effectively limits the carry-through of nonspecific amplification products in isothermal visual RNA detection.
The RecHA platform offers a practical strategy for reliable, portable molecular diagnostics in resource-limited settings.
This approach significantly enhances the selectivity and accuracy of CHA-based diagnostic assays.