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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: Feb 28, 2026

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

Published on: June 15, 2018

Clostridium difficile PCR Cycle Threshold Predicts Free Toxin.

Fiona Senchyna¹, Rajiv L Gaur¹, Saurabh Gombar¹

¹Department of Pathology, Stanford University School of Medicine, Stanford, California, USA.

Journal of Clinical Microbiology

|June 16, 2017

Summary

This summary is machine-generated.

The Clostridium difficile PCR cycle threshold (Ct) can predict toxin status in stool samples. This molecular diagnostic approach offers a sensitive and rapid method for detecting C. difficile toxins.

Keywords:

Clostridium difficile EIA PCR cycle threshold free toxin free toxins

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Last Updated: Feb 28, 2026

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

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Published on: June 15, 2018

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

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Published on: October 18, 2019

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Published on: December 10, 2016

Area of Science:

Clinical microbiology
Molecular diagnostics
Infectious disease diagnostics

Background:

Accurate detection of Clostridium difficile toxins is crucial for guiding patient treatment.
Current diagnostic methods for C. difficile toxins can be slow or lack sensitivity.
There is a need for rapid and sensitive stand-alone diagnostics for fecal free toxins.

Purpose of the Study:

To evaluate the performance of the Clostridium difficile PCR cycle threshold (Ct) for predicting free toxin status in stool samples.
To determine the accuracy of Ct values in identifying toxin-positive and toxin-negative samples.
To assess the utility of PCR-based methods as a complementary diagnostic tool.

Main Methods:

Analysis of 312 stool samples positive for toxigenic C. difficile by PCR.
Testing samples with rapid membrane enzyme immunoassay (RMEIA), cell cytotoxicity neutralization assay (CCNA), and tgcBIOMICS ELISA.
Measuring the accuracy of Ct values at different cutoffs using RMEIA, CCNA, and ELISA as reference methods.

Main Results:

A Ct cutoff of 26.35 showed 96.0% sensitivity and 65.9% specificity for detecting toxin-positive samples using RMEIA.
Incorporating CCNA into the reference method improved Ct specificity to 78.0%.
Intercartridge lot Ct variability was low (2.8%), and standardizing stool volume did not enhance specificity.

Conclusions:

Clostridium difficile PCR Ct values can accurately predict free toxin status in stool samples.
On-demand PCR offers a sensitive method for detecting toxigenic C. difficile and predicting toxin-negative results.
This approach can provide additional valuable information to guide therapeutic decisions in C. difficile infections.