Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot

Abstract

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.

Droplet Digital PCR for Absolute Quantification of Pathogens

“…As a practical example, we describe a recently published ddPCR based protocol for the quantifi cation of the DNA quarantine bacterial pathogen E. amylovora [ 18 ] (Table 4 and Fig. 2 ).…”

“…Quantifi cation of the Quarantine Pathogen E. amylovora Parameters were calculated using an R script [ 18 ] for the positive samples shown in Fig. 2 and include parameters listed in the minimum information for the publication of dPCR experiments as previously described [ 9 ].…”

“…Moreover dPCR can allow overcoming problems associated with the presence of inhibitors associated with certain plant materials and other diffi cult matrices (e.g., soil). Studies describing the application of dPCR to the quantifi cation of pathogens are increasing [ 10 , 14 -16 ], among them also fi rst examples dealing with plant pathogens, such as phytoplasma [ 17 ], Erwinia amylovora and Ralstonia solanacearum [ 18 ]. In our laboratory, pathogen quantifi cation was successfully undertaken using droplet digital PCR (ddPCR).…”

“…In summary, dPCR in general and in particular ddPCR have great potential for practical use in plant pathogen detection: (1) for quality control of in-house reference materials to be used in qPCR and other PCR-based diagnosis methods, (2) for quality control of materials used in test performance studies, (3) in optimizing and assessing qPCR and DNA/RNA extraction methods [ 18 ], and fi nally (4) for routine quantifi cation of nucleic acid target in a wide variety of samples. While running costs of ddPCR currently remain slightly above qPCR, this is compensated by an improved performance (i.e., lower susceptibility to inhibition), ease of interpretation, and absolute quantifi cation independent of reference materials.…”

Nucleic-acid analysis in new fields of metrology

“…Other qPCR methods that have been developed at the NIB can detect plant pathogenic viruses (e.g., potato virus Y isolates, Pepino mosaic virus [27]) and bacteria (e.g., Erwinia amylovora), and Flavescence dorée and Bois noir phytoplasma in grapevine [28,29]. Recently, methods using dPCR for detection and quantification of microorganisms have been published [30,31]. Additionally, LAMP isothermal method was developed for rapid and cost-effective detection of microorganisms [32,33].…”