Production and evaluation of non-radioactive probes for the detection of the two « Candidatus Liberobacter» species associated with citrus huanglongbing (greening)
Identifieur interne : 003326 ( Main/Exploration ); précédent : 003325; suivant : 003327Production and evaluation of non-radioactive probes for the detection of the two « Candidatus Liberobacter» species associated with citrus huanglongbing (greening)
Auteurs : Agnès Hocquellet [France] ; Joseph-Marie Bové [France] ; Monique Garnier [France]Source :
- Molecular and Cellular Probes [ 0890-8508 ] ; 1997.
English descriptors
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Abstract
The production and evaluation of non-radioactive probes for the detection of «CandidatusLiberobacter asiaticum» and «CandidatusLiberobacter africanum», the two bacterial species associated with citrus huanglongbing (greening) disease is described. Two DNA fagments, In 2·6 and AS 1·7, obtained previously from the β operons of «CandidatusLiberobacter asiaticum» and «CandidatusLiberobacter africanum», respectively, were the starting materials for production of the two non-radioactive probes. These digoxigenin (DIG)-labelled probes were generated by PCR incorporation of DIG-11-dUTP, yielding In 1·7-DIG and AS 1·7-DIG. Probe In 1·7-DIG was hybridized with DNAs extracted from 24 field-collected samples in Bali (Indonesia). The membrane on which the DNAs were blotted was first hybridized with radioactive probe32P-In 2·6. After the hybridization results were recorded, the radioactive probe was removed, and the membrane hybridized with DIG-labelled probe In 1·7-DIG. Identical results were obtained for 23 samples. One sample was positive with the DIG-labelled probe and negative with the32P-labelled probe. However, cross-hybridization of In 1·7-DIG with DNA from L. africanum was higher than that obtained with the radioactive probe. This cross-hybridization could be eliminated by raising the temperature of the stringent washing step. No field samples from Africa being available, probe AS 1·7-DIG was dot-blot hybridized against DNAs extracted from leaves of greenhouse-kept citrus plants from different geographical origins and infected with one or other Liberobacter species. The data showed that AS 1·7-DIG hybridized with L. africanum with a sensitivity equivalent to that of the radioactive probe.
Url:
DOI: 10.1006/mcpr.1997.0140
Affiliations:
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Two DNA fagments, In 2·6 and AS 1·7, obtained previously from the β operons of «CandidatusLiberobacter asiaticum» and «CandidatusLiberobacter africanum», respectively, were the starting materials for production of the two non-radioactive probes. These digoxigenin (DIG)-labelled probes were generated by PCR incorporation of DIG-11-dUTP, yielding In 1·7-DIG and AS 1·7-DIG. Probe In 1·7-DIG was hybridized with DNAs extracted from 24 field-collected samples in Bali (Indonesia). The membrane on which the DNAs were blotted was first hybridized with radioactive probe32P-In 2·6. After the hybridization results were recorded, the radioactive probe was removed, and the membrane hybridized with DIG-labelled probe In 1·7-DIG. Identical results were obtained for 23 samples. One sample was positive with the DIG-labelled probe and negative with the32P-labelled probe. However, cross-hybridization of In 1·7-DIG with DNA from L. africanum was higher than that obtained with the radioactive probe. This cross-hybridization could be eliminated by raising the temperature of the stringent washing step. No field samples from Africa being available, probe AS 1·7-DIG was dot-blot hybridized against DNAs extracted from leaves of greenhouse-kept citrus plants from different geographical origins and infected with one or other Liberobacter species. The data showed that AS 1·7-DIG hybridized with L. africanum with a sensitivity equivalent to that of the radioactive probe.</div></front></TEI><affiliations><list><country><li>France</li></country><region><li>Aquitaine</li><li>Nouvelle-Aquitaine</li></region></list><tree><country name="France"><region name="Nouvelle-Aquitaine"><name sortKey="Hocquellet, Agnes" sort="Hocquellet, Agnes" uniqKey="Hocquellet A" first="Agnès" last="Hocquellet">Agnès Hocquellet</name></region><name sortKey="Bove, Joseph Marie" sort="Bove, Joseph Marie" uniqKey="Bove J" first="Joseph-Marie" last="Bové">Joseph-Marie Bové</name><name sortKey="Garnier, Monique" sort="Garnier, Monique" uniqKey="Garnier M" first="Monique" last="Garnier">Monique Garnier</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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