Rapid Sanger sequencing of the 16S rRNA gene for identification of some common pathogens

• Published in: PloS one Vol. 9; no. 2; p. e88886
• Main Authors: Chen, Linxiang, Cai, Ying, Zhou, Guangbiao, Shi, Xiaojun, Su, Jianhui, Chen, Guanwu, Lin, Kun
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 14.02.2014; Public Library of Science (PLoS)
• Subjects: Bacteria; Bacteria - genetics; Base Sequence; Biochemical tests; Biology; Coliforms; Deoxyribonucleic acid; DNA; DNA sequencing; E coli; Electrophoresis, Agar Gel; Escherichia coli; Escherichia coli - genetics; Escherichia coli - isolation & purification; Ethidium - metabolism; Gene amplification; Gene sequencing; Genes, Bacterial; Humans; Identification; Identification methods; Laboratories; Methods; Molecular biology; Molecular Sequence Data; Nucleic Acid Denaturation - genetics; Pathogenic microorganisms; Pathogens; Polymerase Chain Reaction; Preventive medicine; Pseudomonas aeruginosa; Pseudomonas aeruginosa - genetics; Pseudomonas aeruginosa - isolation & purification; Quarantine; RNA; RNA, Ribosomal, 16S - genetics; rRNA 16S; Sequence Analysis, RNA - methods; Shigella; Staphylococcus; Staphylococcus aureus - genetics; Staphylococcus aureus - isolation & purification; Statistical analysis; Statistical methods; Strains (organisms)
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0088886

Conventional Sanger sequencing remains time-consuming and laborious. In this study, we developed a rapid improved sequencing protocol of 16S rRNA for pathogens identification by using a new combination of SYBR Green I real-time PCR and Sanger sequencing with FTA® cards. To compare the sequencing quality of this method with conventional Sanger sequencing, 12 strains, including three kinds of strains (1 reference strain and 3 clinical strains, which were previously identified by biochemical tests), which have 4 Pseudomonas aeruginosa, 4 Staphyloccocus aureus and 4 Escherichia coli, were targeted. Additionally, to validate the sequencing results and bacteria identification, expanded specimens with 90 clinical strains, also comprised of the three kinds of strains which included 30 samples respectively, were performed as just described. The results showed that although statistical differences (P<0.05) were found in sequencing quality between the two methods, their identification results were all correct and consistent. The workload, the time consumption and the cost per batch were respectively light versus heavy, 8 h versus 11 h and $420 versus $400. In the 90 clinical strains, all of the Pseudomonas aeruginosa and Staphyloccocus aureus strains were correctly identified, but only 26.7% of the Escherichia coli strains were recognized as Escherichia coli, while 33.3% as Shigella sonnei and 40% as Shigella dysenteriae. The protocol described here is a rapid, reliable, stable and convenient method for 16S rRNA sequencing, and can be used for Pseudomonas aeruginosa and Staphyloccocus aureus identification, yet it is not completely suitable for discriminating Escherichia coli and Shigella strains.