A duplex quantitative real-time PCR assay for the detection and quantification of Xanthomonas phaseoli pv. dieffenbachiae from diseased and latently infected anthurium tissue

Jouen, Emmanuel; Chiroleu, Frédéric; Maillot-Lebon, Véronique; Chabirand, Aude; Merion, Sabine; Boyer, Claudine; Pruvost, Olivier; Robène, Isabelle

doi:10.1016/j.mimet.2019.03.003

Cited by 4 publications

(10 citation statements)

References 43 publications

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“…citri concentrations ≤1 × 10 4 CFU ml − 1 . The plant signal was inhibited when higher bacterial concentrations were present in the extracts, which is consistent with previously published data [42]. Importantly, the plant control always yielded positive reactions in the absence of a bacterial signal.…”

Section: Discussionsupporting

confidence: 91%

“…The analytical sensitivity of the XAC1051-2qPCR and the XAC1051-F/R PCR were estimated by determining the 95% limit of detection (LOD95%), i.e., the concentration at which a detection probability of 95% is expected [ 55 – 57 ] as explained previously [ 42 ].…”

Section: Methodsmentioning

confidence: 99%

“…A ROC (Receiver operating characteristic) was used in order to determine the Ct cut-off value, i.e., the PCR cycle number above which signals are no longer interpreted as positive [52,53]. This analysis, based on the determination of the Youden J index, which considers false positives and negatives [42,54], was performed on Ct values obtained (see § 2.5. above) for samples with a priori positive status, i.e., citrus spiked samples with different bacterial concentrations (n = 135) and for samples with a priori negative status, i.e., NTC (n = 110). Samples with Ct values higher than the Ct cut-off value were then considered negative.…”

Section: Cut-off Ct Value and Limit Of Detection (Lod)mentioning

confidence: 99%

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Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants

et al. 2020

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Background Asiatic Citrus Canker, caused by Xanthomonas citri pv. citri, severely impacts citrus production worldwide and hampers international trade. Considerable regulatory procedures have been implemented to prevent the introduction and establishment of X. citri pv. citri into areas where it is not present. The effectiveness of this surveillance largely relies on the availability of specific and sensitive detection protocols. Although several PCR- or real-time PCR-based methods are available, most of them showed analytical specificity issues. Therefore, we developed new conventional and real-time quantitative PCR assays, which target a region identified by comparative genomic analyses, and compared them to existing protocols. Results Our assays target the X. citri pv. citri XAC1051 gene that encodes for a putative transmembrane protein. The real-time PCR assay includes an internal plant control (5.8S rDNA) for validating the assay in the absence of target amplification. A receiver-operating characteristic approach was used in order to determine a reliable cycle cut-off for providing accurate qualitative results. Repeatability, reproducibility and transferability between real-time devices were demonstrated for this duplex qPCR assay (XAC1051-2qPCR). When challenged with an extensive collection of target and non-target strains, both assays displayed a high analytical sensitivity and specificity performance: LOD95% = 754 CFU ml− 1 (15 cells per reaction), 100% inclusivity, 97.2% exclusivity for XAC1051-2qPCR; LOD95% = 5234 CFU ml− 1 (105 cells per reaction), 100% exclusivity and inclusivity for the conventional PCR. Both assays can detect the target from naturally infected citrus fruit. Interestingly, XAC1051-2qPCR detected X. citri pv. citri from herbarium citrus samples. The new PCR-based assays displayed enhanced analytical sensitivity and specificity when compared with previously published PCR and real-time qPCR assays. Conclusions We developed new valuable detection assays useful for routine diagnostics and surveillance of X. citri pv. citri in citrus material. Their reliability was evidenced through numerous trials on a wide range of bacterial strains and plant samples. Successful detection of the pathogen was achieved from both artificially and naturally infected plants, as well as from citrus herbarium samples, suggesting that these assays will have positive impact both for future applied and academic research on this bacterium.

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Section: Discussionsupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Section: Cut-off Ct Value and Limit Of Detection (Lod)mentioning

confidence: 99%

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Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants

et al. 2020

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“…Primers based on these genes have been used in conventional PCR or qPCR, following simplex or multiplex approaches, allowing to distinguish between pathogenic and non-pathogenic Xanthomonas strains isolated from Prunus spp. [31,[87][88][89][90]. For X. arboricola pv.…”

Section: Pcr-based Methodsmentioning

confidence: 99%

“…Finally, on X. arboricola, a duplex PCR assay, using primers designed and based on ftsX and qumA genes, were used to identify X. arboricola pv. corylina [90,92].…”

Section: Pcr-based Methodsmentioning

confidence: 99%

Trends in Molecular Diagnosis and Diversity Studies for Phytosanitary Regulated Xanthomonas

et al. 2021

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Bacteria in the genus Xanthomonas infect a wide range of crops and wild plants, with most species responsible for plant diseases that have a global economic and environmental impact on the seed, plant, and food trade. Infections by Xanthomonas spp. cause a wide variety of non-specific symptoms, making their identification difficult. The coexistence of phylogenetically close strains, but drastically different in their phenotype, poses an added challenge to diagnosis. Data on future climate change scenarios predict an increase in the severity of epidemics and a geographical expansion of pathogens, increasing pressure on plant health services. In this context, the effectiveness of integrated disease management strategies strongly depends on the availability of rapid, sensitive, and specific diagnostic methods. The accumulation of genomic information in recent years has facilitated the identification of new DNA markers, a cornerstone for the development of more sensitive and specific methods. Nevertheless, the challenges that the taxonomic complexity of this genus represents in terms of diagnosis together with the fact that within the same bacterial species, groups of strains may interact with distinct host species demonstrate that there is still a long way to go. In this review, we describe and discuss the current molecular-based methods for the diagnosis and detection of regulated Xanthomonas, taxonomic and diversity studies in Xanthomonas and genomic approaches for molecular diagnosis.

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Molecular characterization of Xanthomonas species isolated from Araceae and the development of a triplex TaqMan assay for detection of Xanthomonas phaseoli pv. dieffenbachiae

et al. 2022

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In total 58 Xanthomonas strains isolated from Araceae worldwide, together with 13 other phylogenetically-related Xanthomonas strains, were characterized using multilocus sequence analysis based on concatenated sequences of seven single copy orthologous genes, extracted from whole genome sequences. The analysis revealed a monophyletic clade of 48 strains, 44 isolated from Anthurium, identified as X. phaseoli pv. dieffenbachiae (Xpd) confirmed by nucleotide identity analysis. The other strains from aroids were identified as Xanthomonas euvesicatoria (2 strains), X citri (5 strains) and Xanthomonas sacchari (3 strains). Two TaqMan assays were designed for specific detection of Xpd, one targeting sequences of a hypothetical protein and one targeting a type I restriction endonuclease subunit S. The two assays showed similar reaction kinetics and were merged with an assay comprising an amplification and extraction control into a triplex assay. The assay was able to detect minimally 100 copies of a target sequence delivered as a gBlock, 100 fg of genomic DNA and 104 cells per mL in an Anthurium leaf extract.

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A duplex quantitative real-time PCR assay for the detection and quantification of Xanthomonas phaseoli pv. dieffenbachiae from diseased and latently infected anthurium tissue

Cited by 4 publications

References 43 publications

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants

Trends in Molecular Diagnosis and Diversity Studies for Phytosanitary Regulated Xanthomonas

Molecular characterization of Xanthomonas species isolated from Araceae and the development of a triplex TaqMan assay for detection of Xanthomonas phaseoli pv. dieffenbachiae

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