Detection of cariogenic bacterial genes by microchip electrophoresis.
Allele-specific PCR primers were designed, based on the dextranase (dex) gene, to identify Streptococcus mutans and Streptococcus sobrinus in dental plaque; subsequently, PCR products were detected via microchip electrophoresis (ME). In order to amplify the dex gene fragment of S. mutans and S. sobrinus, the following two PCR methods were established. Duplex allele-specific PCR primers were designed on a region of low DNA homology; furthermore, 211 and 126-bp fragments were amplified for S. mutans and S. sobrinus, respectively. Common PCR primer for single allele-specific PCR was designed so as to sandwich a region exhibiting high homology and amplify PCR product of different DNA size due to deletion of small DNA fragment in two dex genes. S. mutans and S. sobrinus were amplified, leading to the generation of 202 and 226-bp products, respectively. Analysis of DNA base size by ME in order to achieve efficient separation employed a polymer mixture consisting of hydroxypropyl methylcellulose (HPMC) and polyethylene oxide (PEO). In the presence of a polymer mixture of 0.125% PEO/0.6% HPMC, two PCR products were obtained, displaying degree of separation of 226 bp/202 bp of 2.67 (Rs). Reproducibility (CV%, n = 7) was 0.3%; additionally, separation time was approximately 85 s. This method was applied to the detection of S. mutans and S. sobrinus in dental plaque. Detection of the dex genes of S. mutans and S. sobrinus characterized by quickness, precision and high sensitivity was possible.
Karasawa
Koji
K
Department of Analytical Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan.
Arakawa
Hidetoshi
H
Igarashi
Takeshi
T
Goto
Nobuichi
N
Maeda
Masako
M
D000483:Alleles; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D003731:Dental Caries / Q000382:microbiology*; D003910:Dextranase / Q000235:genetics*; D004586:Electrophoresis; D005798:Genes, Bacterial / Q000235:genetics*; D006801:Humans; D007202:Indicators and Reagents; D008838:Microcomputers; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013295:Streptococcus mutans / Q000737:chemistry / Q000235:genetics*; D016986:Streptococcus sobrinus / Q000737:chemistry / Q000235:genetics*; D013698:Templates, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial; D007202:Indicators and Reagents; D003910:Dextranase