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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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Patrick Hardinge¹

¹School of Biosciences, Cardiff University, Cardiff, Wales, UK. hardingep@cardiff.ac.uk.

Methods in Molecular Biology (Clifton, N.J.)

|July 13, 2022

Summary

This study details a rapid molecular diagnostic method combining loop-mediated isothermal amplification (LAMP) with the bioluminescent assay in real time (BART). This LAMP-BART assay enables sensitive detection of nucleic acid amplification for applications like genetically modified maize testing.

Keywords:

Bioluminescence (BL)Bioluminescent assay in real time (BART)DNA Isothermal nucleic acid amplification technology (iNAAT)Loop-mediated isothermal amplification (LAMP)Low copy number Molecular diagnostics Optimization Quantification

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Area of Science:

Molecular diagnostics
Biotechnology
Biochemistry

Background:

The bioluminescent assay in real time (BART) is a molecular diagnostic tool that detects nucleic acid amplification via light output.
A key component of BART is a thermostable luciferase from the firefly Photinus pyralis.
Combining BART with loop-mediated isothermal amplification (LAMP) offers rapid molecular diagnostics with simple equipment.

Purpose of the Study:

To introduce a protocol for amplifying and detecting genetic markers using LAMP with BART.
To provide optimization strategies for LAMP assays to enhance sensitivity and specificity.
To address and eliminate false positive results in molecular assays.

Main Methods:

Utilizing the bioluminescent assay in real time (BART) for light output detection.
Employing loop-mediated isothermal amplification (LAMP) for isothermal nucleic acid amplification.
Applying the LAMP-BART method to microliter and nanoliter microfluidic assays.
Optimizing genetically modified (GM) maize detection by targeting specific genetic sequences (NOSt and 35Sp).

Main Results:

Demonstration of a protocol for LAMP-BART assay implementation.
Guidance on optimizing LAMP assays for improved sensitivity and specificity.
Strategies for minimizing false positive results in molecular detection.
Successful application of LAMP-BART for detecting specific genetic markers in GM maize.

Conclusions:

LAMP-BART is a robust, quantitative, closed-tube molecular diagnostic method.
The protocol and optimization advice facilitate sensitive and specific nucleic acid detection.
This method is suitable for various assay volumes, including microfluidic applications.
The approach is effective for identifying specific genetic sequences, such as those in GM maize.