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.

Here we report on the first assessment of droplet digital PCR (ddPCR) for detection and absolute quantification of two quarantine plant pathogenic bacteria that infect many species of the Rosaceae and Solanaceae families: Erwinia amylovora and Ralstonia solanacearum. An open-source R script was written for the ddPCR data analysis. Analysis of a set of samples with known health status aided the assessment and selection of different threshold settings (QuantaSoft analysis, definetherain pipeline and manual threshold), which led to optimal diagnostic specificity. The interpretation of the E. amylovora ddPCR was straightforward, and the analysis approach had little influence on the final results and the concentrations determined. The sensitivity and linear range were similar to those for real-time PCR (qPCR), for the analysis of both bacterial suspensions and plant material, making ddPCR a viable choice when both detection and quantification are desired. With the R. solanacearum ddPCR, the use of a high global threshold was necessary to exclude false-positive reactions that are sometimes observed in healthy plant material. ddPCR significantly improved the analytical sensitivity over that of qPCR, and improved the detection of low concentrations of R. solanacearum in potato tuber samples. Accurate and rapid absolute quantification of both of these bacteria in pure culture was achieved by direct ddPCR. Our data confirm the suitability of these ddPCR assays for routine detection and quantification of plant pathogens and for preparation of defined in-house reference materials with known target concentrations.

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Background: Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available.

Specific and sensitive TaqMan real-time PCR assays were developed targeting chromosomal DNA of Erwinia amylovora (amsC gene and ITS region). These assays increased the reliability of detection of E. amylovora strains, regardless of their plasmid profile, and have the ability to differentiate between Erwinia spp. strains from Hokkaido, Erwinia pyrifoliae and Erwinia spp. isolated from necrotic pear blossoms in Spain. The assays were used for testing the efficiency of three different extraction methods to remove plant-based PCR inhibitors. Combined with an automated DNA-extraction method based on magnetic beads (QuickPick TM ), the real-time PCR assays reliably detected at least 10 3 cells mL )l (c. four cells per reaction) of the pathogen from blighted woody plant material. In testing of symptomless samples, absolute quantification of E. amylovora before and after enrichment in liquid media provided proof of E. amylovora viability and its ability to multiply, including in cases when subsequent isolation in pure culture was unsuccessful.