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  1. Mohammadi MR, Vadamalai G, Joseph H
    PMID: 21534490
    Coconut cadong-cadong viroid (CCCVd) causes the Lethal cadang-cadang disease of coconut palms in the Philippines and it is recently reported to be associated with the orange spotting disease on oil palm in Malaysia. The low concentration of the viroid RNA in oil palm as well as the high content of polyphenols and polysaccharides in this plant which interfere with the purification steps makes it difficult to extract and detect this viroid from oil palm. A previously described method was modified and optimized for extraction and detection of CCCVd from infected oil palms. Briefly, 7 g of leaf material was homogenized in a mortar or a blender using liquid nitrogen. 10 ml of extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA) along with 100 mM 2-mercaptoethanol and 10 ml water saturated phenol was added to the frozen powder. After centrifuging at 4 degrees C, 4000 g for 30 min, the aqueous phase was extracted once more with phenol then once with chloroform-isoamyl alcohol (24:1). After adding sodium acetate, pH 5.6 to 200 mM, the mixture was precipitated with 2.5 vol ethanol overnight in -20 freezer and then the pellet was washed with 70% ethanol and air-dried. One milliliter of 8 M LiCl was added to the dried pellet and after shaking overnight at 4 degrees C and another centrifugation step the supernatant was collected and precipitated again with ethanol and then the resulting pellet was washed and air-dried. To carry out northern blotting, samples equivalent to 40 g of plant tissue were mixed with formamide buffer and loaded onto a 12% polyacrylamide gel containing 7 M urea and after separation by electrophoresis, were electroblotted onto membrane and fixed by UV cross-linking. Pre-hybridization and hybridization using hybridization buffer (50% formamide, 25%SSPE, 0.1% Ficol and PVP, 0.1 % SDS, 0.02 % DNA (5mg/ml)) was carried out at 45 degrees C for 90 min and 16 h, respectively followed by two low stringency washes (0.5 X SSC, 0.1% SDS, at room temperature for 5 min) and one high stringency wash (0.1X SSC, 0.1% SDS at 60 degrees C for 1 hour). In vitro synthesized DIG-labeled full-length CCCVd(-) RNA probe was used in hybridization step. DIG Nucleic Acid Detection Kit (Roche) instructions were followed for detection procedure and as a result the blue bands corresponding to the position of the viroid were appeared on the membrane. The result of this study showed the ability of DIG labeled probe in detection of the viroid and also provided a suitable extraction and hybridization method for the detection of CCCVd from oil palm.
    Matched MeSH terms: Viroids/isolation & purification*
  2. Vadamalai G, Hanold D, Rezaian MA, Randles JW
    Arch Virol, 2006 Jul;151(7):1447-56.
    PMID: 16470341
    Variants of Coconut cadang-cadang viroid have been identified in a plantation oil palm growing in Malaysia. Three size classes are described, comprising 297, 293, and 270 nt. Compared with the 296-nt form of coconut cadang-cadang viroid (CCCVd), all variants substituted C31 --> U in the pathogenicity domain and A175 --> C in the right-hand terminus. Other mutations and deletions accounted for the different sizes. These are the first sequences reported for variants of Coconut cadang-cadang viroid in a species other than coconut palm, and this is the first evidence that variants closely related to CCCVd occur outside the Philippines.
    Matched MeSH terms: Viroids/isolation & purification*
  3. Thanarajoo SS, Kong LL, Kadir J, Lau WH, Vadamalai G
    J Virol Methods, 2014 Jun;202:19-23.
    PMID: 24631346 DOI: 10.1016/j.jviromet.2014.02.024
    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60 min at 60 °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd.
    Matched MeSH terms: Viroids/isolation & purification*
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