Specific detection and differentiation of two subspecies of Fusobacterium necrophorum by PCR.
The phylogenic relationships of two subspecies of Fusobacterium necrophorum were investigated by randomly amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR). With each of the 12 random primers, the DNA fingerprints generated were subjected to cluster analysis for dendrograms. The analysis indicated that twelve strains were organized into two major clusters, and that all strains of each subspecies were confined to one cluster. Furthermore, two of the random primers examined each generated a unique band in F. n. necrophorum strains. We cloned these specific bands and determined the nucleotide sequences. A search for amino acid sequence homologies revealed that the two specific fragments had significant homology to the rpoB gene of Lactococcus lactis subsp. lactis and the hemagglutinin-related protein gene of Ralstonia solanacearum, respectively. New specific primers designed for the rpoB gene were able to amplify 900bp fragments from both subspecies. However, the specific primers designed for the hemagglutinin-related protein gene amplified only a 250bp fragment of the genome of the F. n. necrophorum strains, suggesting that this gene is unique to F. n. necrophorum. These results were further confirmed by dot blot hybridization. Finally, a one-step duplex PCR technique in a single tube for the rapid detection and differentiation of the F. necrophorum subspecies was developed.
Narongwanichgarn
Wonganun
W
Department of Veterinary Microbiology, Faculty of Agriculture, Miyazaki University, 1-1 Gakuen Kibanadai Nishi, 889-2192, Miyazaki, Japan.
Misawa
Naoaki
N
Jin
Jing Hua
JH
Amoako
Kingsley Kwaku
KK
Kawaguchi
Emi
E
Shinjo
Toshiharu
T
Haga
Takeshi
T
Goto
Yoshitaka
Y
D001483:Base Sequence; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D005675:Fusobacterium necrophorum / Q000737:chemistry / Q000145:classification* / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000662:veterinary; D019105:Random Amplified Polymorphic DNA Technique / Q000662:veterinary; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004269:DNA, Bacterial