Detection and genotyping of meningococci using a nested PCR approach.
An effective vaccine against Neisseria meningitidis serogroup B is required. Outer-membrane protein vaccines have been developed, which may provide protection against common circulating serotypes and serosubtypes in some countries. However, limited genosubtyping data are available because most laboratories use mAbs directed against a limited number of specific serotypes and serosubtypes and laboratories do not genosubtype directly from body fluids due to the lack of a sensitive PCR method. A nested PCR was therefore developed that enables the amplification of the porA gene directly from clinical samples and has the required sensitivity for nucleotide sequencing of the three main variable regions, VR1, VR2 and VR3. Data were compared with those from culture-based nucleotide sequencing, and the use of this method increased the availability of genosubtype information by 45 %, thereby indicating the impact that this methodology has on the data provided and the implications for vaccine design.
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Scottish Meningococcus and Pneumococcus Reference Laboratory, North Glasgow University Hospitals NHS Trust, Stobhill Hospital, Balornock Road, Glasgow G21 3UW, UK 2Faculty of Biomedical and Life Sciences, University of Glasgow, UK.
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Scottish Meningococcus and Pneumococcus Reference Laboratory, North Glasgow University Hospitals NHS Trust, Stobhill Hospital, Balornock Road, Glasgow G21 3UW, UK 2Faculty of Biomedical and Life Sciences, University of Glasgow, UK.
D005798:Genes, Bacterial / Q000235:genetics; D005838:Genotype; D006801:Humans; D008589:Meningococcal Infections / Q000097:blood / Q000134:cerebrospinal fluid / Q000175:diagnosis / Q000382:microbiology; D009345:Neisseria meningitidis / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D018272:Porins / Q000032:analysis / Q000235:genetics
D018272:Porins; C057699:porin protein, Neisseria