Use of 32P-labelled nucleic acid probes for sensitive and specific detection of coconut cadang-cadang viroid (CCCVd)
1994
Rodriguez, M.J.B.
The conditions that assure high sensitivity, specificity, and reliability of 32P-labelled probes in hybridization assay for CCCVd were determined. Two types of probe were prepared. Complementary DNA (cDNA) probe was synthesized by primer extension on purified CCCVd 246 while cRNA probe was prepared by in vitro transcription of the CCCVd 246 insert in a PSP64 plasmid. These probes gave nearly the same sensitivity in several tests done with a detection limit of approximately 2 pg. Their specificity to CCCVd was likewise assessed by comparing results of dot-blot and northern-blot hybridization of the probes to other nucleic acids present in the samples was distinguished and appropriate steps to eliminate this problem were incorporated in the protocol. Nucleic acid hybridization has recently been the method of choice worldwide for routine indexing of plant pathogens. Previous methods are either not sensitive enough or are inadequate for large-scale testing
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