Nucleotide sequence analysis of the sialyltransferase genes of meningococcal serogroups B, C, Y and W135.
A rapid method for serogrouping meningococci is essential for the characterization of phenotypically non-groupable meningococcal isolates and clinical samples, particularly for public health management purposes. The Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) provides serogrouping results of meningococcal isolates and clinical samples using a PCR assay which detects restriction fragment length polymorphisms in meningococcal serogroups B, C, Y and W135. Although this PCR system was invaluable when first introduced, it has several drawbacks and lacks the required sensitivity for detecting DNA in clinical samples. Due to the recent introduction of the meningococcal group C conjugate vaccine and an impending group B vaccine, a more robust and informative method for serogroup determination is required. A protocol was devised allowing PCR amplification of the siaD gene of serogroup B, C, Y and W135 meningococci. This system was multiplexed and allowed serogroup differentiation between serogroups B and C and also between B/C and Y/W135 by product size analysis. A nested stage was incorporated into the system for enhanced detection of meningococci in clinical samples, and finally a sequencing protocol was designed allowing detection of any nucleotide changes within the siaD gene. This system allows rapid serogrouping results for use within an agarose gel system as well as more informative results when used for sequencing within the siaD gene.
Lewis
C
C
Scottish Centre for Infection and Environmental Health, Scottish Meningococcus and Pneumococcus Reference Laboratory, Stobhill Hospital, Glasgow, UK.
Diggle
M A
MA
Clarke
S C
SC
D015373:Bacterial Typing Techniques; D017931:DNA Primers; D006801:Humans; D008585:Meningitis, Meningococcal / Q000382:microbiology; D009345:Neisseria meningitidis / Q000145:classification* / Q000201:enzymology / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012703:Serotyping; D012799:Sialyltransferases / Q000235:genetics*
D017931:DNA Primers; D012799:Sialyltransferases