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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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Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

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Published on: June 19, 2015

Quantitative multiplex real-time PCR for detecting class 1, 2 and 3 integrons.

O Barraud¹, M C Baclet, F Denis

¹EA3175, Univ Limoges, Faculté de Médecine, 2 rue du Dr Marcland, 87025 Limoges, France.

The Journal of Antimicrobial Chemotherapy

|June 15, 2010

Summary

A new real-time PCR method can rapidly detect the three main classes of integrons, which are key markers for antibiotic resistance in Gram-negative bacteria. This sensitive technique aids in screening bacterial isolates and clinical samples for potential multidrug resistance.

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Area of Science:

Microbiology
Molecular Biology
Genetics

Background:

Integrons are mobile genetic elements found in Gram-negative bacteria.
Integrons play a crucial role in the dissemination of antibiotic resistance genes.
Existing methods for integron detection can be limited in scope or sensitivity.

Purpose of the Study:

To develop a novel, highly specific, and sensitive real-time PCR method.
To enable simultaneous detection of the three major classes of integrons.
To facilitate rapid screening for potential multidrug resistance.

Main Methods:

Development of a TaqMan probe-based real-time PCR assay.
Utilized three distinct primer-probe pairs for simultaneous detection.
Validated sensitivity using target gene mixtures from 10 to 10^8 copies.
Assessed specificity with known integron-containing and integron-free strains.
Applied the method to clinical samples.

Main Results:

The PCR method demonstrated high specificity.
Achieved a sensitivity of 10^2 copies for all three integron classes.
The assay was quantitative for gene targets ranging from 10^3 to 10^7 copies.
Successfully detected integrons directly in biological samples.

Conclusions:

A rapid, quantitative, specific, and sensitive real-time PCR method for integron detection was successfully developed.
This method can be valuable for the initial screening of Gram-negative bacteria.
The assay aids in identifying clinical samples with likely multidrug resistance.