Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples
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PLANT METHODS
METHODOLOGY
Open Access
Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples Nejc Rački*, Tanja Dreo, Ion Gutierrez-Aguirre, Andrej Blejec and Maja Ravnikar
Abstract Background: Detection and quantification of plant pathogens in the presence of inhibitory substances can be a challenge especially with plant and environmental samples. Real-time quantitative PCR has enabled high-throughput detection and quantification of pathogens; however, its quantitative use is linked to standardized reference materials, and its sensitivity to inhibitors can lead to lower quantification accuracy. Droplet digital PCR has been proposed as a method to overcome these drawbacks. Its absolute quantification does not rely on standards and its tolerance to inhibitors has been demonstrated mostly in clinical samples. Such features would be of great use in agricultural and environmental fields, therefore our study compared the performance of droplet digital PCR method when challenged with inhibitors common to plant and environmental samples and compared it with quantitative PCR. Results: Transfer of an existing Pepper mild mottle virus assay from reverse transcription real-time quantitative PCR to reverse transcription droplet digital PCR was straight forward. When challenged with complex matrices (seeds, plants, soil, wastewater) and selected purified inhibitors droplet digital PCR showed higher resilience to inhibition for the quantification of an RNA virus (Pepper mild mottle virus), compared to reverse transcription real-time quantitative PCR. Conclusions: This study confirms the improved detection and quantification of the PMMoV RT-ddPCR in the presence of inhibitors that are commonly found in samples of seeds, plant material, soil, and wastewater. Together with absolute quantification, independent of standard reference materials, this makes droplet digital PCR a valuable tool for detection and quantification of pathogens in inhibition prone samples. Keywords: PCR amplification, Inhibition, qPCR, Droplet digital PCR, Environmental samples
Background At present, real-time quantitative PCR (qPCR) is the method of choice for detection and quantification of many pathogens and other DNA and RNA targets [1-3]. However, absolute quantification using qPCR depends on the use of standards; i.e., reference materials with known target concentrations, which are rarely commercially available. Even when reference materials are available, quantification accuracy is highly dependent on the amplification efficiencies between the samples and the reference material [4].
* Correspondence: [email protected] Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, SI-1000, Ljubljana, Slovenia
One of the factors that can significantly influence amplification efficiency is the presence of inhibitors in samples for PCR amplification. Such inhibitors are either coextracted with nucleic acids from the samples, or can arise from the extraction p
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