Hocquellet A, Bové J M, Garnier M
Laboratoire de Biologie Cellulaire et Moléculaire, Institut National de la Recherche Agronomique, Villenave d'Ornon, France.
Mol Cell Probes. 1997 Dec;11(6):433-8. doi: 10.1006/mcpr.1997.0140.
The production and evaluation of non-radioactive probes for the detection of 'Candidatus Liberobacter asiaticum' and 'Candidatus Liberobacter 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 beta operons of 'Candidatus Liberobacter asiaticum' and 'Candidatus Liberobacter 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 probe 32P-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 the 32P-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.
本文描述了用于检测与柑橘黄龙病（黄化病）相关的两种细菌——亚洲韧皮杆菌（‘Candidatus Liberobacter asiaticum’）和非洲韧皮杆菌（‘Candidatus Liberobacter africanum’）的非放射性探针的制备及评估。之前分别从亚洲韧皮杆菌和非洲韧皮杆菌的β操纵子中获得的两个DNA片段In 2.6和AS 1.7，是制备这两种非放射性探针的起始材料。这些地高辛（DIG）标记的探针通过在PCR反应中掺入DIG - 11 - dUTP生成，得到In 1.7 - DIG和AS 1.7 - DIG。探针In 1.7 - DIG与从印度尼西亚巴厘岛采集的24个田间样本中提取的DNA进行杂交。首先将DNA印迹的膜与放射性探针32P - In 2.6杂交。记录杂交结果后，去除放射性探针，然后用DIG标记的探针In 1.7 - DIG与膜杂交。23个样本得到了相同的结果。一个样本用DIG标记的探针呈阳性，而用32P标记的探针呈阴性。然而，In 1.7 - DIG与非洲韧皮杆菌DNA的交叉杂交高于放射性探针。通过提高严格洗涤步骤的温度可以消除这种交叉杂交。由于没有来自非洲的田间样本，探针AS 1.7 - DIG与从不同地理来源且感染了一种或另一种韧皮杆菌属细菌的温室柑橘植物叶片中提取的DNA进行斑点杂交。数据表明，AS 1.7 - DIG与非洲韧皮杆菌杂交的灵敏度与放射性探针相当。
