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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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A methodology to detect and quantify five pathogens causing potato tuber decay using real-time quantitative

Z K Atallah, W R Stevenson

|October 24, 2008

Summary

This study developed a real-time quantitative-polymerase chain reaction method to detect five major potato tuber diseases. The method accurately identifies pathogens like Phytophthora infestans and Fusarium sambucinum, even without visible symptoms.

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Area of Science:

Plant Pathology
Molecular Biology
Agricultural Science

Background:

Potato tuber diseases caused by Phytophthora infestans, Phytophthora erythroseptica, Pythium ultimum, Fusarium sambucinum, and Erwinia carotovora pose significant global storage challenges.
Accurate and early detection of these pathogens is crucial for predicting potato storability and minimizing post-harvest losses.

Purpose of the Study:

To develop and validate a real-time quantitative-polymerase chain reaction (qPCR) methodology for the simultaneous detection and quantification of five key potato tuber pathogens.
To establish a sensitive and specific molecular tool for identifying causal agents of late blight, pink rot, leak, dry rot, and soft rot in potato tubers.

Main Methods:

Design of six specific primer pairs targeting single-copy protein-coding genes of the five pathogens and the potato host.
Utilizing real-time quantitative-polymerase chain reaction (qPCR) for pathogen DNA detection and quantification.
Testing primer specificity and amplification efficiency using pure culture DNA and spiked potato tuber samples.

Main Results:

All primer pairs demonstrated high specificity, accurately detecting their intended pathogenic species.
Amplification efficiencies ranged from 95-100% across a five-log dilution series, unaffected by high concentrations of host DNA.
The method achieved a detection limit of 0.5 pg of target DNA and successfully identified pathogens in potato tubers with or without visible symptoms, even at low DNA concentrations.

Conclusions:

The developed qPCR methodology provides a reliable tool for detecting and quantifying major potato tuber pathogens.
This molecular approach is the first step towards a comprehensive system for predicting potato tuber storability.
The ability to detect pathogens regardless of symptom presence offers significant advantages for early disease management in stored potatoes.