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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: Mar 19, 2026

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

Published on: March 9, 2016

Optimization of Quantitative PCR Methods for Enteropathogen Detection.

Jie Liu¹, Jean Gratz¹, Caroline Amour²

¹Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, United States of America.

|June 24, 2016

Summary

This summary is machine-generated.

Accurate enteropathogen quantification is key for diagnosing diarrhea. This study optimized methods for stool sample collection and nucleic acid extraction, improving pathogen detection and quantification in clinical laboratories.

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Published on: July 9, 2012

Area of Science:

Microbiology
Molecular Diagnostics
Infectious Disease Epidemiology

Background:

Accurate detection and quantification of enteropathogens in stool are crucial for diagnosing infectious diarrhea.
Current molecular methods face technical challenges impacting reliable quantification.
Optimizing specimen collection and nucleic acid extraction is essential for improving diagnostic accuracy.

Purpose of the Study:

To evaluate key determinants of enteropathogen quantification in stool specimens.
To compare pathogen detection and quantification in rectal swabs versus whole stool samples.
To assess the efficiency of a simplified total nucleic acid (TNA) extraction method.

Main Methods:

Paired flocked rectal swabs and whole stool samples were collected from 129 children with diarrhea.
Molecular detection and quantification of pathogens were performed.
A simplified total nucleic acid (TNA) extraction procedure was compared against separate DNA and RNA extractions.
A novel quantification scheme adjusting for extraction and amplification efficiency was developed.

Main Results:

Rectal swabs yielded higher quantification cycle (Cq) values but detected 80% of pathogens found in stool.
The TNA extraction method demonstrated high sensitivity (92%) and specificity (98%) with no significant difference in Cq values compared to separate extractions.
The developed quantification scheme provided a more accurate estimate of pathogen quantity in spiked specimens than raw Cq values.

Conclusions:

Optimized methods for stool sample collection and nucleic acid extraction enhance enteropathogen quantification.
The simplified TNA extraction is a viable and efficient alternative for molecular diagnostics.
These advancements will aid laboratories in studying enteric diseases and improving diarrhea diagnosis.