Novel SCAR primers for specific and sensitive detection of Agrobacterium vitis strains.

ABSTRACT

An Agrobacterium vitis-specific DNA fragment (pAVS3) was generated from PCR polymorphic bands amplified by primer URP 2R. A. vitis specificity of this fragment was confirmed by Southern hybridization with genomic DNA from different Agrobacterium species. Sequence-characterized amplified region (SCAR) markers were developed for A. vitis specific detection, using 24-mer oligonucleotide primers designed from the flanking ends of the 670bp insert in pAVS3. The SCAR primers amplified target sequences only from A. vitis strains and not from other Agrobacterium species or other bacterial genera. First round PCR detected bacterial cells between 5x10(2) and 1x10(3)cfu/ml and the detection sensitivity was increased to as few as 2cfu/ml by nested PCR. This PCR protocol can be used to confirm the potential presence of infectious A. vitis strains in soil and furthermore, can identify A. vitis strains from naturally infected crown galls.