Rapid and sensitive identification of Pratylenchus spp. using reverse dot blot hybridization

Abstract

AbstractA 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.