Comparison of real-time PCR and droplet digital PCR for the detection of Xylella fastidiosa in plants

Abstract

14Xylella fastidiosa (Xf) is a quarantine plant pathogen bacterium originating from the Americas 15 and that has emerged in Europe in 2013. Xf can be detected directly on plant macerate using 16 molecular methods such as real-time PCR, which is a sensitive technique. However, some 17 plants may contain components that can act as PCR reaction inhibitors, which can lead to false 18 negative results or an underestimation of the bacterial concentration present in the analyzed 19 plant sample. Droplet digital PCR (ddPCR) is an innovative tool based on the partitioning of 20 the PCR reagents and the DNA sample into thousands of droplets, allowing the quantification 21 of the absolute number of target DNA molecules present in a reaction mixture, or an increase 22 of the detection sensitivity. In this study, a real-time PCR protocol, already used for Xf detection 23 in the framework of official surveys in the European Union, was transferred and optimized for 24Xf detection using ddPCR. This new assay was evaluated and compared to the initial real-time 25 PCR on five plant matrices artificially inoculated and on naturally infected plants. In our 26 conditions, this new ddPCR enabled the detection of Xf on all artificially inoculated plant 27 macerates with a similar limit of detection, or a slight benefit for Quercus ilex. Moreover, 28 ddPCR improved diagnostic sensitivity as it enabled detection of Xf in samples of Polygala 29 myrtifolia or Q. ilex that were categorized as negative or close to the limit of detection using 30 the real-time PCR. Here, we report for the first time a ddPCR assay for the detection of the 31 bacterium Xf. 32

Current and emerging trends in techniques for plant pathogen detection

“…Recent examples of ddPCR methods developed for plant pathogen detection include a ddPCR assay for detection of Xylella fastidiosa (Dupas et al, 2019), Acidovorax citrulli (Lu et al, 2020), Tilletia controversa (Liu et al, 2020), and an RT-ddPCR for detection of peach latent mosaic viroids extracted from infected peach leaves (Lee et al, 2021). ddPCR is also suitable for multiplexing applications although there are only few examples described in literature focusing on plant pathogen detection, including a multiplex ddPCR assay for detection of Candidatus Liberibacter asiaticus and Spiroplasma citri (Maheshwari et al, 2021).…”