Genotypic characterization of Neisseria meningitidis using Pyrosequencing.
Pyrosequencing involves the synthesis of single-stranded deoxyribonucleic acid leading to rapid and accurate analysis of nucleotide sequences. This article describes the development of typing assays for the characterization of Neisseria meningitidis using Pyrosequencing. This involved developing methods for the nucleotide sequence analysis of important variable regions contained on a major capsule gene and on outer membrane protein genes that are used for grouping, typing, and subtyping meningococci. To achieve this, primers were designed for amplification of three genes, siaD, porB, and porA from the four main serogroups B, C, Y, and W135. To facilitate throughput and reproducibility, the method was also automated. Data from 717 isolates have shown that Pyrosequencing can be used for the single nucleotide polymorphism and sequence-analysis characterization of meningococci.
Scottish Meningococcus and Pneumococcus Reference Laboratory, Department of Microbiology, House on the Hill, Stobhill Hospital, Balornock Road, Glasgow, G21 3UW, Scotland.
Matthew A
MA
Diggle
Stuart C
SC
Clarke
D015373:Bacterial Typing Techniques; D004277:DNA, Single-Stranded / Q000737:chemistry; D005838:Genotype; D009345:Neisseria meningitidis / Q000235:genetics*; D018272:Porins / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*
D004277:DNA, Single-Stranded; D018272:Porins; C057699:porin protein, Neisseria