The simple and rapid detection of specific PCR products from bacterial genomes using Zn finger proteins.
A novel method of rapid and specific detection of polymerase chain reaction (PCR) products from bacterial genomes using Zn finger proteins was developed. Zn finger proteins are DNA-binding proteins that can sequence specifically recognize PCR products. Since Zn finger proteins can directly detect PCR products without undergoing dehybridization, unlike probe DNA, and can double check the specific PCR amplification and sequence specificity of the PCR products, this novel method would be quick and highly accurate. In this study, we tried to detect Legionella pneumophila using Sp1. It was found that a 49 bp L. pneumophila-specific region containing the Sp1 recognition site is located on the flhA gene of the L. pneumophila genome. We succeeded in specifically detecting PCR products amplified from L. pneumophila in the presence of other bacterial genomes by ELISA, and demonstrated that Sp1 enables the discrimination of L. pneumophila-specific PCR products from others. By fluorescence depolarization measurement, these specific PCR products could be detected within 1 min. These results indicate that the rapid and simple detection of PCR products specific to L. pneumophila using a Zn finger protein was achieved. This methodology can be applied to the detection of other bacteria using various Zn finger proteins that have already been reported.
Osawa
Yuko
Y
Department of Biotechnology and Life Science, Tokyo University of Agriculture & Technology, 2-24-16 Naka-cho, Koganei, 184-8588 Tokyo, Japan.
Ikebukuro
Kazunori
K
Motoki
Hiroaki
H
Matsuo
Takafumi
T
Horiuchi
Michio
M
Sode
Koji
K
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D016680:Genome, Bacterial; D006801:Humans; D016952:Legionella pneumophila / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D016329:Sp1 Transcription Factor / Q000737:chemistry / Q000378:metabolism; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D016329:Sp1 Transcription Factor