TY - JOUR
T1 - Profiling system of oral microbiota utilizing polymerase chain reaction-restriction fragment length polymorphism analysis
AU - Sano, Hiroto
AU - Wakui, Anna
AU - Kawachi, Miho
AU - Washio, Jumpei
AU - Abiko, Yuki
AU - Mayanagi, Gen
AU - Yamaki, Keiko
AU - Tanaka, Kaori
AU - Takahashi, Nobuhiro
AU - Sato, Takuichi
N1 - Funding Information:
This study was supported in part by Grants-in-Aid for Scientific Research (17H04420, 17K11698, 17K12006, 20K10241, 21H03151, 21H03247, and 21K10266) from the Japan Society for the Promotion of Science, Tokyo, Japan.
Funding Information:
This study was supported in part by Grants-in-Aid for Scientific Research ( 17H04420 , 17K11698 , 17K12006 , 20K10241 , 21H03151 , 21H03247 , and 21K10266 ) from the Japan Society for the Promotion of Science , Tokyo, Japan.
Publisher Copyright:
© 2021 Japanese Association for Oral Biology
PY - 2021/9
Y1 - 2021/9
N2 - Objectives: Profiling of oral microbiota has traditionally been performed using conventional methods. These methods are relatively time-consuming and labor-intensive. Metagenomic analysis of oral microbiota using high-speed next-generation sequencing is a highly promising technology. However, it is expensive. This study sought to develop a simple and cost-effective profiling method for oral microbiota using 16S rRNA gene polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of PCR-amplified 16S ribosomal RNA genes. Methods: Oral isolates of 59 bacterial species from human saliva, including Streptococcus, Actinomyces, and Veillonella, were cultured anaerobically on CDC Anaerobe 5% sheep blood agar plates. Genomic DNA was extracted from single colonies and 16S rRNA genes were PCR-amplified using the 27F and 1492R universal primers. The PCR products were purified and characterized by single digestion with HpaII restriction endonuclease. 16S rRNA gene sequences were obtained from the GenBank database, and the expected restriction profiles were compared with the RFLP patterns obtained from agarose gel electrophoresis. Results: Sixty-five RFLP patterns were obtained from 27 genera and 59 species. The expected fragment sizes of these species were calculated based on GenBank 16S rRNA gene sequences. Fifty-nine patterns were obtained from the analysis of GenBank sequences. The RFLP patterns produced with HpaII distinguished many oral bacterial species. RFLP patterns enabling identification of oral bacteria were generated. The 16S rRNA gene PCR-RFLP analysis did not require expensive equipment and reagents and was cost-effective. Conclusion: PCR-RFLP analysis based on 16S rRNA genes could be an alternative method for oral microbiota analysis in smaller laboratories.
AB - Objectives: Profiling of oral microbiota has traditionally been performed using conventional methods. These methods are relatively time-consuming and labor-intensive. Metagenomic analysis of oral microbiota using high-speed next-generation sequencing is a highly promising technology. However, it is expensive. This study sought to develop a simple and cost-effective profiling method for oral microbiota using 16S rRNA gene polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of PCR-amplified 16S ribosomal RNA genes. Methods: Oral isolates of 59 bacterial species from human saliva, including Streptococcus, Actinomyces, and Veillonella, were cultured anaerobically on CDC Anaerobe 5% sheep blood agar plates. Genomic DNA was extracted from single colonies and 16S rRNA genes were PCR-amplified using the 27F and 1492R universal primers. The PCR products were purified and characterized by single digestion with HpaII restriction endonuclease. 16S rRNA gene sequences were obtained from the GenBank database, and the expected restriction profiles were compared with the RFLP patterns obtained from agarose gel electrophoresis. Results: Sixty-five RFLP patterns were obtained from 27 genera and 59 species. The expected fragment sizes of these species were calculated based on GenBank 16S rRNA gene sequences. Fifty-nine patterns were obtained from the analysis of GenBank sequences. The RFLP patterns produced with HpaII distinguished many oral bacterial species. RFLP patterns enabling identification of oral bacteria were generated. The 16S rRNA gene PCR-RFLP analysis did not require expensive equipment and reagents and was cost-effective. Conclusion: PCR-RFLP analysis based on 16S rRNA genes could be an alternative method for oral microbiota analysis in smaller laboratories.
KW - Microbiota
KW - Polymerase chain reaction
KW - Restriction fragment length polymorphism
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U2 - 10.1016/j.job.2021.05.003
DO - 10.1016/j.job.2021.05.003
M3 - Article
C2 - 34111508
AN - SCOPUS:85108542571
VL - 63
SP - 292
EP - 297
JO - Journal of Oral Biosciences
JF - Journal of Oral Biosciences
SN - 1349-0079
IS - 3
ER -