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,Maja Ravnikar

doi:10.1186/s13007-014-0042-6

Abstract

BackgroundDetection 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.ResultsTransfer 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-014-0042-6) contains supplementary material, which is available to authorized users.

Highlights

Detection and quantification of plant pathogens in the presence of inhibitory substances can be a challenge especially with plant and environmental samples
We have previously reported greater tolerance of reverse transcriptase (RT)-Droplet digital PCR (ddPCR) to inhibitors from wastewater, in comparison to RT-quantitative PCR (qPCR) [15]; there are limited data available on the performance of RT-ddPCR in the presence of inhibitors common to diverse plant and environmental samples
RT-ddPCR typically showed better precision than RT-qPCR, especially for the dilutions with lower target concentrations (Figure 1B). Both assays showed good linearity of amplification, which was seen as high correlation coefficients (R2) obtained by linear regression of the same dilutions (RT-qPCR, 0.998; RT-ddPCR, 0.989) (Figure 1A)

Summary

Introduction

Detection and quantification of plant pathogens in the presence of inhibitory substances can be a challenge especially with plant and environmental samples. 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, 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. 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 procedure itself [5,6,7]. Interference with cell lysis, sequestration or degradation of nucleic acids, and hindrance of polymerase activity are all common mechanisms of PCR inhibition [8]

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Conclusion