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Real Time RT-PCR

Real Time RT-PCR

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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Updated: Jun 25, 2025

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Published on: May 20, 2020

A new simplified sequence-dependent loop-mediated isothermal amplification (LAMP) detection method.

Yanju Chen¹, Yuanyuan Zhu¹, Jungang Du¹

¹College of Biosystems Engineering and Food Science & ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou, 310058/311215, China.

Analytical and Bioanalytical Chemistry

|May 22, 2024

Summary

This summary is machine-generated.

A new bifunctional probe enhances loop-mediated isothermal amplification (LAMP) for faster, more accurate DNA detection. This method enables both single and multiplexed nucleic acid detection, improving upon traditional single-plex LAMP limitations.

Keywords:

Diagnostics Isothermal amplification LAMP Multiplexed detection Sequence-dependent probe

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Area of Science:

Molecular Biology
Biotechnology
Nucleic Acid Amplification

Background:

Fluorescence dye-based loop-mediated isothermal amplification (LAMP) is a sensitive nucleic acid detection technique.
Current single-plex LAMP methods are limited and can produce non-specific signals.

Purpose of the Study:

To develop a bifunctional probe-based real-time LAMP method for single-plexed or multiplexed nucleic acid detection.
To improve the sensitivity and specificity of LAMP assays while simplifying the process.

Main Methods:

A bifunctional probe was designed by modifying a fluorophore at the 5' end and an internal quencher on a LAMP primer.
The probe integrates into double-stranded DNA amplicons during amplification, leading to fluorescence signal generation.
The method was tested for its ability to detect Vibrio parahaemolyticus DNA and an internal amplification control simultaneously.

Main Results:

The bifunctional probe enabled cumulative exponential fluorescence increase during LAMP amplification.
The method demonstrated simplified and cost-effective operation, aligning with standard LAMP protocols.
The assay was 10 minutes faster than other probe-based LAMP methods and allowed for one-pot multiplexed detection.

Conclusions:

The bifunctional probe-based LAMP method offers a simplified, cost-effective, and faster approach for nucleic acid detection.
This method overcomes limitations of single-plex LAMP, enabling sensitive and specific single or multiplexed detection.
The technique shows significant potential for applications requiring rapid and simultaneous detection of multiple nucleic acid targets.