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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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Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

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Multiplex quantitative real-time reverse transcriptase PCR for F+-specific RNA coliphages: a method for use in

Marek Kirs¹, David C Smith

¹Biological Sciences, University of Rhode Island, Kingston, Rhode Island, USA. kirs@email.unc.edu

Applied and Environmental Microbiology

|December 5, 2006

Summary

A new real-time RT-PCR method accurately quantifies F+-specific RNA coliphages, crucial for microbial source tracking in coastal waters. This sensitive assay overcomes limitations of traditional methods, aiding effective water quality management.

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Last Updated: Jul 17, 2026

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Published on: December 3, 2011

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Area of Science:

Environmental microbiology
Molecular biology
Public health

Background:

Microbial contamination in coastal waters threatens human health via recreation and seafood.
Microbial source tracking is vital for managing and remediating contaminated waters.
Current methods for tracking F+-specific RNA coliphages are limited by low concentrations and differential infectivity loss.

Purpose of the Study:

To develop a sensitive, culture-independent method for simultaneous quantification of all four subgroups of F+-specific RNA coliphages.
To improve the accuracy of microbial source tracking in coastal environments.

Main Methods:

A multiplex real-time reverse transcriptase-PCR (RT-PCR) protocol was developed using novel primers and molecular beacons.
The assay was validated for sensitivity, linearity, and performance in survival experiments.
Compared real-time RT-PCR with traditional culture-based methods.

Main Results:

The real-time RT-PCR assay demonstrated high sensitivity (detecting 10 copies of RNA) and linearity over six orders of magnitude.
The assay successfully quantified coliphages in seawater where culture-based methods failed.
Decay constants in seawater did not vary among coliphage subgroups, despite differential infectivity loss.

Conclusions:

The developed real-time RT-PCR method provides accurate in situ quantification of F+-specific RNA coliphages.
This advancement facilitates more effective remediation strategies for coastal water contamination.
Improved microbial source tracking enhances public health protection related to recreational water use and shellfish consumption.