PCR-based assays for detecting Xanthomonas axonopodis pv. allii in onion seed
Bacterial blight of onion is an emerging disease threatening world onion production, and causing damage to other Allium crops. The causal agent, Xanthomonas axonopodis pv. allii (Xaa) has been listed on the EPPO A1 list of pests recommended for regulation as quarantine pests, since 2009. A duplex nested-PCR assay, targeting two markers specific to Xaa has been recently developed (Robène-Soustrade et al., 2010). A triplex quantitative real-time PCR assay (Taqman® technology) was developed targeting the same Xaa-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Xaa strains were detected by the amplification of one or both of the two specific markers. The internal control signal validates both the extraction process and the reaction itself. Several successive steps have to be performed before detection from seed: seed maceration for 48h at 4°C, followed by homogenization of the seed macerate with a stomacher® and DNA extraction using DNeasy® Plant mini kit (Qiagen). This assay is currently being validated following the European standard EN ISO 16140: 2003 and the EPPO standard PM7/98 (1). The performance of Nested-PCR and real-time PCR assays are discussed. These PCR-based tools could be useful for the international sanitary surveillance of seed exchanges.
| Main Authors: | , , , , |
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| Format: | conference_item biblioteca |
| Language: | eng |
| Published: |
s.n.
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| Subjects: | H20 - Maladies des plantes, |
| Online Access: | http://agritrop.cirad.fr/565544/ http://agritrop.cirad.fr/565544/1/document_565544.pdf |
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| Summary: | Bacterial blight of onion is an emerging disease threatening world onion production, and causing damage to other Allium crops. The causal agent, Xanthomonas axonopodis pv. allii (Xaa) has been listed on the EPPO A1 list of pests recommended for regulation as quarantine pests, since 2009. A duplex nested-PCR assay, targeting two markers specific to Xaa has been recently developed (Robène-Soustrade et al., 2010). A triplex quantitative real-time PCR assay (Taqman® technology) was developed targeting the same Xaa-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Xaa strains were detected by the amplification of one or both of the two specific markers. The internal control signal validates both the extraction process and the reaction itself. Several successive steps have to be performed before detection from seed: seed maceration for 48h at 4°C, followed by homogenization of the seed macerate with a stomacher® and DNA extraction using DNeasy® Plant mini kit (Qiagen). This assay is currently being validated following the European standard EN ISO 16140: 2003 and the EPPO standard PM7/98 (1). The performance of Nested-PCR and real-time PCR assays are discussed. These PCR-based tools could be useful for the international sanitary surveillance of seed exchanges. |
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