July 14, 2022 | PCR Solutions | Digital PCR

Your microbial DNA detection questions answered

Pathogen detection by digital PCR

Bacteria, fungi, viruses and parasitic metazoans are ubiquitous in the environment and are part of all aspects of human life, from human health to food production. For example, the human microbiome, found on and in our bodies, is estimated to include about ten times more microbes than cells in the human body. In turn, each microbe can trigger various harmful or beneficial effects on humans. This makes the specific detection and monitoring of microbes important for understanding their biological function, especially in infection or colonization of the human body.

In this webinar, scientific expert Ronny Kellner presented DNA-based methods for detecting and identifying microbial species and microbial genes, focusing on hydrolysis probe-based assays and digital PCR. He discussed setting up a dPCR microbial DNA detection experiment, analyzing multiple targets in one reaction and tackling viral RNA targets. The new assays enable rapid profiling and identification of microbial species, antibiotic resistance genes and virulence genes from diverse samples.

To give you a glimpse, here are some of the frequently asked questions during the live Q&A session. If you couldn’t attend but would like to view the recording, you can sign up to watch it here.

What is a good amplicon size for dPCR?
It is the same as for qPCR. Amplicon sizes below 200 bp are recommended for optimal dPCR efficiency. 

What is the possibility of dPCR detecting live bacteria from dead?
So far we haven’t tested this with dPCR. However, to distinguish DNA from living and dead bacteria using digital PCR, the DNA of dead bacteria has to be inactivated or removed before amplification. One possibility is the pre-treatment of the sample with propidium monoazide (PMA). This DNA intercalating dye enables the selective masking of DNA from dead, membrane-compromised cells immediately before DNA extraction. Visible light then induces a photoreaction that will lead to a covalent bond of PMA and the double-stranded DNA. Thus, it is no longer available for downstream amplification in the PCR reaction and only DNA from living cells will be amplified.

Is it possible to correlate the presence of targets in the same cell?
For DNA samples extracted from >1 cell, this is only possible if both target loci are located on the same DNA molecule. This way, both targets co-segregate into partitions and can be detected with individual assays using individual fluorescent dyes. In case both linked targets are present, the partition will show two signals, one for each target. For targets that are not linked or that are very distant from each other, only single-cell extracts will show the presence of >2 targets in one cell.

How many data points are needed for LOB, LOD and LOQ evaluation?
A recommended practical number of LOB and LOD samples to be used to establish these parameters is 60, while verification of a given LOD (and possibly the LOB) is 20. Once established, we recommend running >2 replicates for detecting low abundant targets. This reduces the subsampling error and allows the calculation of the standard variation of the measured copies/µl. The subsampling error increases toward low amounts of target molecules in the sub-sample.

Is there a multi-target multiplex dPCR assay for quantifying multiple Bacteroidales as well as Enterococcus faecalis, the water quality indicator, for performing microbial source tracking assays using recreational and drinking water source samples?
The assay portfolio comprises assays for Enterococcus faecalis as well as a selection of different Bacterioidales species. A summary of all target species is listed in the technical note here.

Are your pan-bacteria assays also suitable for microbiome studies in animals, for example in birds? Are there already any data available?
The microbiomes of birds and humans will differ significantly for certain taxonomic groups. We haven't tested the pan-bacteria assays with samples from bird microbiomes. However, given the broad specificity of the pan assays towards different taxonomic families and species within bacteria, we speculate that these assays will also cover a broader spectrum of bacterial species in the microbiomes of birds. 

How to avoid the "rain phenomenon" (namely, positive and negative parts could not be distinguished) when reviewing the 1D scatterplots? 
In the case of very “rainy” assays, the addition or reduction of the number of cycles can improve the signal-to-noise separation. Also, adapting the cycling conditions like annealing temperature can improve rainy assays.

Is LOB for NTC calculated only at the time of validation? Should it be calculated with every batch of samples?
It is only necessary to establish that at the time of validation.

Can you use the pan bacteria assay to identify the detected bacteria and divide them into phyla? 
Both pan bacteria assays are designed for broad specificity towards the major bacterial groups, e.g., Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and more. The assay is designed to capture the amount of bacteria in a sample rather than distinguish individual taxonomic groups. This can, for example, be used to quantify the bacterial load of a sample and, thereby, can be used for the normalization of species-specific assays run on the same samples.

Can this be used on environmental samples – for example, soil DNA that is usually more difficult to analyze? 
In principle, the assays can be used for any environmental sample. Some samples like soil are known to contain higher levels of inhibitory substances which might interfere with the PCR reaction. Although more robust towards inhibitors, digital PCR can also be negatively affected by such inhibitors. It is therefore recommended to use extraction kits that are optimized for the sample to minimize the content of sample-specific inhibitors as much as possible like the DNeasy PowerSoil Pro Kits using Inhibitor Removal Technology that yields inhibitor-free DNA for unbiased use in downstream assays. Alternatively, a dilution of the input template can reduce the concentration of inhibitory substances in a dPCR reaction.

What is the upper limit of detection?
The upper limit of detection is set by the limits of Poisson Statistics used for the calculation of the absolute number of target molecules in the reaction. This calculation requires a certain amount of negative partitions which limits the average maximum number of copies per partition (λ) to a recommended value of 5. This adds up to 1,30,000 copies in 26,000 partitions (Nanoplate 26K) which corresponds to a total volume of about 24 µl. This results in an upper limit of detection of about 5400 copies/µl reaction for the Nanoplate 26K. 

To perform multiplexing, do I need to order the assay separately or can I use GeneGlobe to pick and mix it in a single tube or a bundle pack? 
Assays are shipped in separate tubes in separate boxes. In GeneGlobe assays of interest can be selected either individually or in bundles from a list of wet-lab tested assay bundles. 

Will you add more resistance genes to the assay?
At the moment it is not planned to extend the assay portfolio by including additional resistance gene targets. However, any existing qPCR assay not covered by the portfolio can potentially be adapted for use with digital PCR.

The QIAcuity comes with preassigned dyes to a channel. Can we customize this option and add a dye of our choice?
No, this is not possible. Only one of the five FAM, HEX, TAMRA, ROX and Cy5 can be selected.

1D scatter plot for Pan Bacteria 1- the threshold for 3 dilutions of gDNA is different. Can we compare the results within the same assay with different thresholds?
The software sets an auto-threshold based on distinct criteria which leads to differences in the thresholds between the wells. To account for that the average threshold of all replicates can be applied instead, which can be set manually in the software. 

Does the number of partitions affect the sensitivity of the limit of detection or % of confidence?
Yes, the more partitions, the higher the sensitivity. The Nanoplate 26K 24-well is more sensitive than the Nanoplate 8.5K 96-well because it has more partitions and can be loaded with more sample (up to 26 µl) compared to the Nanoplate 8.5K (up to 7.8 µl).

How is the thermocycling protocol set up when quantifying DNA and RNA targets simultaneously?
 Using the OneStep Advanced Probe Master Mix, the cycling protocol is: 40 minutes of reverse transcription at 50°C, followed by a 2-minute RT enzyme inactivation at 95°C, followed by 40 cycles (2-step cycling) of 5-second denaturation at 95°C, followed by a 30-second annealing/extension at 60°C.

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