A reverse dot blot assay for identification of Pratylenchus spp. has been developed using specific oligonucleotides designed from the sequence of the internal transcribed spacer (ITS) region. The reverse dot blot is a technique which can be especially used for the simultaneous identification of various bacteria. The target fragment was amplified, and labelled with digoxigenine by a polymerase chain reaction (PCR). The amplified fragment was hybridised with the membrane-immobilised oligonucleotide and the hybridization was detected non-radioactively. By this assay, it was possible to identify P. penetrans, P. coffeae, P. vulnus, P. loosi, P. brachyurus, P. crenatus and P. zeae in a single hybridization. Identification rapide et fiable de Pratylenchus spp. a l'aide de l'hybridation retro dot blot - Une technique retro dot blot pour l'identification de Pratylenchus spp. a ete mise au point en utilisant des oligonucleotides specifiques derives de la sequence de l'espaceur transcrit interne (ITS). La technique dot blot est une technique qui peut etre specialement utilisee pour l'identification simultanee de nombreuses bacteries. Le fragment cible a ete amplifie et marque par la digoxigenine a l'aide de la reaction de polymerisation en chaine (PCR). Le fragment amplifie a ete hybride avec l'oligonucleotide immobilise par membrane et l'hybridation detectee non-radioactivement. Grace a cette technique, il a ete possible d'identifier P. penetrans, P. coffeae, P. vulnus, P. loosi, P. brachyurus, P. crenatus et P. zeae en une seule hybridation.
The distribution of virus RNA molecules in tobacco mosaic virus (TMV) infected tobacco protoplasts was examined with time after inoculation using in situ hybridization with strand specific riboprobe labeled with digoxigenin. Analysis of hybridization signals for TMV plus strand RNAs showed very weak signals throughout the cytoplasm of the protoplasts sampled at 2hr post-inoculation (p.i.). Thereafter, intensity of the signals increased in the cytoplasm and reached a maximum level at 12hr p.i. There was then a rapid decrease in the intensity of signals. Weak signals in the in situ hybridization using a minus strand specific probe also were first observed at 2hr p.i. The intensity of signals, then increased and gradually decreased after a maximum level at 12hr p.i. Intensity of the signals at a maximum level, however, was much less than that of plus strand viral RNA. No hybridization signals for plus strand RNAs or minus strand RNAs was demonstrated in the nucleus throughout the period of infection. These results strongly indicated that the site of replication of TMV-RNA is the cytoplasm and that the nucleus is not directly associated with its synthesis.
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