Generation of PCR-based DNA fragments for specific detection of Streptomyces saraceticus N45.
Streptomyces saraceticus strain N45, a saprophytic Gram-positive bacteria, has been shown to harbor high chitinase activity. Due to its potential use in biological control, the cloning of chitinase genes and the development of methods to quickly and precisely detect its presence have become necessary. In this study, PCR-based random amplified polymorphic DNA (RAPD) and PCR strategies were used to amplify random DNA fragments from the genome of S. saraceticus N45. Three amplified DNA fragments, 417, 523 and 655 bp in length, were further isolated, subcloned and sequenced. Nest primers were designed from terminal ends of these three fragments and used for further PCR reactions. A single specific band was produced from the genomic DNA of S. saraceticus N45 for each nest primer pair. These three single bands were S. saraceticus N45 specific and were not amplified from other species of Streptomyces or bacteria, such as Ralstonia solanacearum, Agrobacterium tumefaciens, E. coli, Bacillus subtilis and Xanthomonas campestris pv. campestris. Through detection of the coexistence of these three fragments in PCR reaction using DNA or bacterial cells directly, the presence of S. saraceticus N45 can be confirmed. Further Southern analysis indicated that these three DNA fragments were specifically present in the S. saraceticus N45 genome in a single copy manner, and therefore, that they can potentially be used as markers for identification of S. saraceticus N45.
Kong
L R
LR
Graduate Institute of Agricultural Biotechnology, National Chung Hsing University, Taichung, Taiwan, ROC.
Tzeng
D D
DD
Yang
C H
CH
D001431:Bacteriological Techniques; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D013302:Streptomyces / Q000235:genetics / Q000302:isolation & purification*
D004269:DNA, Bacterial