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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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Clinical validation of a new multi-Aspergillus species reverse transcriptase quantitative PCR: a diagnostic

Marion Benazra¹, Wicem Mallek¹, Théo Ghelfenstein-Ferreira²

¹Institut Pasteur, Université Paris Cité, National Reference Center for Invasive Mycoses and Antifungals, Translational Mycology Research Group, Mycology Department, Paris, France.

Clinical Microbiology and Infection : the Official Publication of the European Society of Clinical Microbiology and Infectious Diseases

|March 30, 2026

Summary

A new multi-Aspergillus real-time quantitative PCR (qPCR) assay offers improved diagnosis for invasive aspergillosis. This assay accurately detects various Aspergillus species, aiding in patient monitoring and treatment.

Keywords:

Aspergillus fumigatus Aspergillus species Cryptic species Diagnostic Invasive aspergillosis PCR RT-qPCR

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Area of Science:

Mycology and Infectious Diseases
Molecular Diagnostics
Fungal Pathogenesis

Background:

Invasive aspergillosis diagnosis is challenging, relying on multiple criteria.
Standardized Aspergillus real-time quantitative PCR (qPCR) shows promise but lacks a universal assay.
Current methods struggle to detect diverse Aspergillus species causing invasive disease.

Purpose of the Study:

To design, optimize, and validate a novel real-time PCR assay for invasive aspergillosis.
To develop a multi-Aspergillus section-specific assay for broad species detection.
To improve diagnostic accuracy and patient monitoring for invasive aspergillosis.

Main Methods:

Developed a reverse-transcriptase qPCR assay utilizing multi-Aspergillus section-specific primers (MAS-RTqPCR).
Assay designed to detect nucleic acids from six key Aspergillus sections.
Validated using a diagnostic case-control study with 120 patients and 223 plasma samples.

Main Results:

Achieved a limit of detection of five copies/reaction with 100% analytical specificity.
Clinical sensitivity of 0.91 and clinical specificity of 1.00 in patient validation.
MAS-RTqPCR identified non-A. fumigatus infections and showed superiority in patient monitoring.

Conclusions:

The developed MAS-RTqPCR assay is a highly sensitive and specific diagnostic tool.
This assay shows significant promise for diagnosing invasive aspergillosis.
It offers a valuable method for monitoring PCR-positive patients.