Molecular Detection of Citrus Vein Phloem Degeneration
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Date
1997-11
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Perhimpunan Bioteknologi Pertanian Indonesia
Abstract
This research was conducted in order to develop pathogen detection tool using specific monoclonal antibodies and DNA-probes for CVPD or Indonesian citrus greening. Preci pitated protein from the sap of CVPD infected citrus leaf midribs was used to immunize 8 weeks Balb/c mice. Each mouse was injected intraperitonially with 200 pi PBS contai ning 10 pg protein in the present of adjuvant with interval of two weeks and booster injec
tion was given (intra vena) three times everyday after the last injection of the regular immunization. The immune lymphocytes were then fused with NS-1 myeloma cells in the present of PEG 4,000 as fusogen. The hybridomas that producing antibodies were selected against the sap of healthy and CVPD infected citrus leaf midribs by ELISA. The hybridomas producing antibodies reactive to CVPD but not reactive to healthy plants were further cloned and propagated. Total DNA were extracted from CVPD infected citrus leaf midribs using the modification method of SDS-Proteinase K- Lisozyme. The DNA was used as the template for PCR of 16S-rRNA gene using universal primers, the part of the gene using specific oligonucleotides of Liberobacter asiaticum and L africanum, Asian and African greening bacterium. There were 6 clones of hybridoma producing specific monoclonal antibodies against CVPD in vitro, however only clon DW- 0310 that showed consistenly reactive to CVPD. This results indicated that DW 0310 is highly sensitive to CVPD samples originated from many cultivars of citrus, cv. Siem, Keprok, and Manis from different places (Yogyakarta, Magelang, Purworejo, and Bali) and not reactive to any other plant diseases. The universal primers amplified of 1,500 bp DNA fragment that was cut into about 1,300 and 200 bp by Eco Rl, and into 1,250 and 250 bp by Bel I. Using the specific oligonucleotides of L. asiaticum in the fragment of 1,500 bp 16S-rRNA gene, 400 bp fragment was found by amplification, on the other hand, there was no amplification using the specific oligonucleotides of L africanum.
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Keywords
CVPD, ELISA, monoclonal antibodies, DNA-probes.