Molecular characterization of Neisseria meningitidis isolates using a resequencing DNA microarray.
Neisseria meningitidis is a major cause of both meningitis and septicemia. Typically, isolates are characterized by using a combination of immunological phenotyping, using monoclonal and polyclonal antisera, and Sanger nucleotide sequencing of epitope-encoding variable regions, although these methods can be both time-consuming and limited by reagent availability. Herein, we describe and evaluate a novel microarray to define the porB and porA serotypes of N. meningitidis by the resequencing of variable regions in a single hybridization reaction. PCR products for each gene were amplified, pooled in equimolar concentrations, hybridized to the microarray, and analyzed using Affymetrix GeneChip DNA Analysis Software. Resequencing of the microarray data was then validated by comparison with sequencing data. Molecular profiles were generated for 50 isolates that were combinations of phenotypically typeable (ie, PorA and PorB) and non-typeable (PorB only) isolates. Microarray-generated profiles from isolates with a PorB phenotype were concordant with predicted profiles compared with a previously described typing scheme. In addition, 42% (8 of 19) of previously non-typeable samples were assigned a PorB type when tested using the microarray. The remaining isolates were novel types for which no typing antisera are currently available. The porA data were 97% concordant with Sanger nucleotide sequencing. These results suggest that that microarray resequencing may be a useful tool for the characterization of meningococci, particularly for those isolates that cannot be phenotyped, offering an alternative to conventional sequencing methods.
Corless
Caroline E
CE
Meningococcal Reference Unit, Health Protection Agency Manchester Laboratory, Manchester Royal Infirmary, Manchester, UK. caroline.corless@hpa.org.uk
Kaczmarski
Edward
E
Borrow
Ray
R
Guiver
Malcolm
M
D015373:Bacterial Typing Techniques; D006801:Humans; D016130:Immunophenotyping; D008589:Meningococcal Infections; D009345:Neisseria meningitidis / Q000145:classification / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods; D012703:Serotyping / Q000379:methods*