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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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Related Experiment Video

Updated: Jun 5, 2025

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

Published on: October 18, 2019

A quantitative PCR to detect non-toxigenic Clostridioides difficile.

Khurshida Begum¹, Hubert C Chua¹, M Jahangir Alam¹

¹Department of Pharmacy Practice and Translational Research, University of Houston College of Pharmacy, Houston, Texas, USA.

Microbiology Spectrum

|December 11, 2024

Summary

A new qPCR assay can detect non-toxigenic Clostridioides difficile (NTCD) strains, which may protect against C. difficile infection (CDI). This assay identified NTCD colonization in 7% of hospitalized patients, aiding diagnostics and antimicrobial stewardship.

Keywords:

Clostridioides difficile diagnostics non-toxigenic stewardship

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Last Updated: Jun 5, 2025

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

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Published on: October 18, 2019

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

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Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

Published on: November 17, 2018

Area of Science:

Microbiology
Molecular Diagnostics
Infectious Diseases

Background:

Current diagnostics for Clostridioides difficile infection (CDI) do not identify non-toxigenic C. difficile (NTCD) strains.
NTCD colonization may confer protection against CDI, but its prevalence is unknown due to diagnostic limitations.

Purpose of the Study:

To develop and validate a molecular assay for the specific detection of NTCD.
To assess the prevalence of NTCD colonization in hospitalized patients undergoing testing for CDI.

Main Methods:

A quantitative PCR (qPCR) assay was designed using specific primers and probes targeting unique regions of NTCD.
The assay's analytical sensitivity and specificity were validated using reference strains.
95 clinical stool samples were analyzed to determine NTCD prevalence in patients tested for CDI.

Main Results:

The developed qPCR assay demonstrated high analytical sensitivity (linear between 3x10^1 and 3x10^6 gDNA, R^2=0.999).
NTCD was detected in 20% of samples negative for GDH enzyme and 8.7% of samples positive for GDH enzyme.
The assay identified NTCD colonization in 7% of the overall hospitalized patient cohort.

Conclusions:

A novel qPCR assay effectively identifies NTCD strains, enabling their detection in clinical samples.
NTCD colonization is present in a notable percentage of hospitalized patients, suggesting a potential protective role against CDI.
This assay has significant implications for improving CDI diagnostics, infection control, and antimicrobial stewardship strategies.