Experts in quantifying selected microbial species by qPCR

An effective tool to estimate microbial community composition and function

The development of microbial quality indicators applicable to soil and aquatic environments has been largely hampered by the limitations associated with traditional culture-based methods.

Culture-independent molecular methods, based on the direct analysis of total DNA extracted from environmental samples, now allow for better assessments of the composition and diversity of microbial communities. Among these methods, quantitative real-time PCR (qPCR) has become increasingly popular due to its high sensitivity, speed, and cost-effectiveness. Moreover, contrary to other PCR-based methods such as DGGE and T-RFLP, qPCR can be applied not only to detect the presence of target sequences but also to quantify their abundance. Actually, the use of qPCR for the estimation of abundance of selected microbial gene sequences in soil quality assessment has been recently standardised (ISO 17601:2016).

At AllGenetics, we perform custom-tailored qPCR assays, with two main applications:

  • Inferring the abundance of selected microbial taxa (bacteria, archaea, eukaryotes) in environmental samples.
  • Quantifying key functional genes involved in biogeochemical and biodegradation processes, which may be used as indicators for the bioremediation potential of a microbial community.

Please note that the abundance of target DNA sequences estimated by qPCR may not correlate with the number of microorganisms under certain experimental designs (e.g. when the target is located within a plasmid which can be in variable copy number in the cell). The technique does not distinguish between living and dead cells.

How we work

qPCR workflow

Step 1: We isolate DNA from your environmental samples. We have adapted different DNA isolation protocols, depending on the starting biological material.

Step 2: We perform qPCR to amplify and quantify target DNA sequences using in-house developed primers pairs and fluorescent probes specific for the microbial taxa under study. A pilot qPCR experiment is carried out beforehand in order to check the specificity and efficiency of the assay to set up the qPCR conditions.

Step 3: We analyse the results of qPCR to confirm the presence/absence of the taxa of interest and, if present, the abundace of the target sequences in each of the samples analysed.

Please contact us for further information and pricing.

What you receive

A report with a summary of the methods followed.

The raw qPCR data.

The results of the analysis including:

  • A table with the estimated abundance of selected target sequences.
  • A report of assay performance characteristics (melting curve analysis, PCR amplification efficiency, linear dynamic range, LOD, and Cq values of non-template negative controls).

If you require additional analyses, please let us know and we will do our best to meet your needs.

At no additional cost, our project specialists will be available to you after project delivery. We offer up to five 60-min sessions per project, depending on the project size.

What we need

Your environmental samples appropriately preserved depending on the sample type (ethanol, silica gel, frozen, or another suitable preservation method). If required, we provide sampling kits and sampling collection guidelines to ensure that your samples arrive at our lab in optimal conditions.

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