Real-Time PCR for Ceratocystis platani detection: in-depth validation to assess the diagnostic potential and include additional technical options

Abstract

A high-performing detection method is essential to safeguard those countries that are still unaffected by canker stain, a devastating disease of Platanus spp. caused by Ceratocystis platani. We previously developed EvaGreen and Taqman-based Real-Time PCR to detect this pathogen, but in-depth validation is needed to guarantee users about its effectiveness and promote its utilization. In this work we present a validation study designed according to EPPO standards, focusing on the analytical and diagnostic sensitivity and specificity. We extend its technical application using SYBR Green. By performing standard curves and eight-replication-based experiments, we established the detection limit at 3 fg C. platani gDNA per PCR reaction. The repeatability and the operator-based reproducibility of the Real-Time PCR was demonstrated. Different gDNA extraction events by different operators and different gDNA extraction modalities did not affect the detection limit. The detection limit threshold cycle was earliest with SYBR Green, followed by Taqman, and EvaGreen. Spiking 6 µl DNA extractions of uninfected, necrotized wood with 3 fg C. platani gDNA confirmed the detection limit: 3 fg C. platani gDNA per PCR reaction, i.e., 0.5 fg gDNA per µl of wood extract. The assays tolerated 6 µl of necrotic C. platani-infected wood extracts without inhibition except for long-dead wood samples, while the 2 µl dose consistently allowed for successful detection. Detection of the pathogen in infected samples showed the highest diagnostic sensitivity with the SYBR Green assay. Agarose gel electrophoresis and staining was validated for visualizing amplicons, even at the detection limit. The specificity of the method was tested against 23 isolates representing the diversity of Ceratocystidaceae, and most species were not detected at 5 ng gDNA. However, some South American strains of the C. fimbriata complex were detected at doses as low as 5 fg. The method remains specific for C. platani detection as no other Ceratocystidaceae are known to colonize plane tree and the species within the geographic range of canker stain of plane tree were only detected at 500 pg or more gDNA. This work paves the way for a performance study of inter-laboratory comparisons.

Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystis platani, the Agent of Canker Stain of Platanus spp.

“…The focus of this interlaboratory comparison was the assessment of reproducibility and performance parameters of the Real-Time PCR method based on EvaGreen, Taqman, and SYBR Green variants [ 14 , 17 , 18 ].…”

“…In all the assays the concentration of the primers and, when used, the probe was kept fixed at 0.5 µM and 0.3 µM for each primer and probe, respectively. Use of Bio-Rad master mixes and the CFX96 TM thermocycler implied the integral application of the cycling protocol [ 14 , 17 , 18 ]: iinitial denaturation at 96 °C for 3 min; 40 cycles at 95 °C for 10 s (denaturation) and 66 °C for 20 s (annealing/extension); final extension at 72 °C for 5 min (the latter step was used exclusively for EvaGreen and SYBR Green assays). Slight changes to the amplification program were made according to the operating instructions of the commercial master mix used and to the thermocycler type.…”

“…A cycle threshold (C t ) of ≤37 was suggested by the OL and was applied in the TPS as the upper limit for a positive detection. In fact, it was previously established that 3 fg of C. platani gDNA was the lowest detectable DNA dose per PCR reaction (Limit of Detection, LoD), in the approximate C t range of 33–36, depending on the assay [ 18 ].…”

“…Traditional detection of C. platani has relied on a number of assays—microscopic observations, isolation on nutritive media, moist chamber, carrot test, and baiting test—which are time-consuming and prone to false negative response [ 14 , 17 , 18 ]. Recently Real-Time PCR based methods for C. platani detection were developed [ 7 , 17 ], representing the first rapid and reliable tools for the protection of Platanus spp.…”

“…This method includes three ITS1-targeted assays based on EvaGreen and SYBR Green dyes and a Taqman probe. These assays, validated in depth by in-house comparisons, showed high-level performance on several parameters: amplification efficiency, analytical specificity (inclusivity and exclusivity), diagnostic sensitivity and specificity, analytical sensitivity, enforceability in necrotic wood, and inter-operator, intra-laboratory reproducibility [ 17 , 18 ].…”