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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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Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Published on: May 13, 2019

Novel method for estimating viable Salmonella cell counts using real-time PCR.

Hiroshi Fujikawa¹, Yukako Shimojima, Kazuyoshi Yano

¹Department of Microbiology, Tokyo Metropolitan Institute of Public Health: 3-24-1, Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan.

Shokuhin Eiseigaku Zasshi. Journal of the Food Hygienic Society of Japan

|September 21, 2006

Summary

This study introduces a new method using real-time PCR to count viable Salmonella Enteritidis cells. The technique tracks DNA amplification kinetics, offering a reliable way to estimate bacterial concentrations in samples.

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Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

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Published on: October 25, 2018

Area of Science:

Microbiology
Molecular Biology
Food Safety

Background:

Accurate enumeration of viable bacteria is crucial for food safety and clinical diagnostics.
Traditional methods for bacterial quantification can be time-consuming and labor-intensive.
Real-time PCR offers a sensitive platform for molecular detection, but quantifying viable cells requires specific approaches.

Purpose of the Study:

To develop and validate a novel method for estimating viable Salmonella Enteritidis cell counts.
To utilize the kinetics of target DNA amplification (invA gene) during bacterial growth for quantification.
To establish a correlation between DNA amplification parameters and initial viable cell concentration.

Main Methods:

Development of a 5'-nuclease real-time PCR assay targeting the invA gene of Salmonella.
Monitoring the threshold cycle (CT) values over time as Salmonella cells grow in buffered peptone water at 39°C.
Analyzing the sigmoidal curve generated by CT values against time to determine the slope.
Correlating the CT value derived from the slope with initial Salmonella cell concentrations.
Employing selective media to mitigate interference from non-target bacteria.

Main Results:

A clear inverse relationship was observed between the initial viable Salmonella cell concentration and the CT value derived from the amplification curve slope.
Higher initial cell concentrations resulted in lower CT values.
The slope of the CT curve remained constant across different initial cell densities.
Incubation in selective media effectively reduced deviations in CT curves caused by other bacterial species.

Conclusions:

The developed real-time PCR method provides a novel and potentially rapid approach for estimating viable Salmonella Enteritidis cell counts.
The method leverages the kinetics of invA gene amplification during growth, offering quantitative insights.
This approach shows promise for application to other cultivable microorganisms, broadening its potential utility in microbial analysis.