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Real Time RT-PCR

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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.
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Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

Published on: November 5, 2020

Data on the optimization of an archaea-specific probe-based qPCR assay.

Malin Bomberg¹, Hanna Miettinen¹

¹VTT Technical Research Centre of Finland Ltd., Tietotie 2, FIN-02150 Espoo, Finland.

|May 24, 2021

Summary

Optimizing fluorescent DNA dye assays improves archaeal quantification. This enhanced method increases specificity, overcoming challenges posed by bacterial DNA contamination in environmental samples.

Keywords:

Bacteria Bioinformatics High throughput sequencing Microbial metabolism Microorganisms in mine processing water

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Area of Science:

Microbiology
Molecular Biology
Environmental Science

Background:

Estimating archaeal populations using fluorescent DNA binding dyes is difficult due to cross-reactivity with bacterial 16S rRNA genes, especially in mixed microbial communities.
High bacterial abundance can interfere with accurate archaeal quantification, necessitating more specific detection methods.

Purpose of the Study:

To optimize a fluorescent probe-based assay for specific and accurate quantification of archaeal 16S rRNA genes.
To enhance the specificity of archaeal detection in the presence of bacterial DNA.

Main Methods:

Optimization of a fluorescent probe-based assay using established archaeal primers and probes.
Testing the assay on genomic DNA from pure bacterial and archaeal cultures.
Utilizing PCR amplicons of archaeal 16S rRNA genes to improve assay performance.

Main Results:

The optimized assay demonstrated high specificity for archaeal strains, with only slight amplification observed in bacterial strains.
Amplification levels varied between archaeal strains due to differences in genome size and 16S rRNA gene copy number.
Using PCR-amplified archaeal 16S rRNA gene fragments resulted in good amplification, though efficiency still varied among strains.

Conclusions:

The developed fluorescent probe-based quantitative PCR (qPCR) assay offers improved specificity for archaeal enumeration.
This optimized assay is suitable for estimating archaeal numbers in complex environmental samples, such as industrial process water.
The method will be applied in future microbiological analyses within the H2020 ITERAMS project.