1,721,000 research outputs found
Detection and genotyping of meningococci using a nested PCR approach
No full textAn 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.</p
Semi-automation of the polymerase chain reaction for laboratory confirmation of meningococcal disease
No full textDemand for accurate high-throughput detection and characterisation of medically important bacteria has increased dramatically within research and clinical laboratories. Liquid-handling robots have been developed to achieve high levels of accuracy and reproducibility. Assay automation can play a key role in the modern diagnostic laboratory and the data presented here shows that automated PCR is comparable with manual methods. Importantly, automation is preferred when high-quality results cannot be guaranteed using manual methods. This is particularly important when results are required quickly for public health management.</p
Rapid assignment of nucleotide sequence data to allele types for multi-locus sequence analysis (MLSA) of bacteria using an adapted database and modified alignment program
No full textA novel database and modified alignment program is described which provides a fast and accurate procedure for assigning nucleotide sequences to allele types for multi-locus sequence analysis (MLSA). The database has between 40 and 160 alleles per organism including Neisseria meningitidis, Streptococcus pneumoniae, Staphylococcus aureus and Haemophilus influenzae. The database directly compares the query nucleotide sequence against all alleles within the database and this system reduces the time taken for the analysis of nucleotide sequence data and assignment of alleles for subsequent sequence analysis.</p
Automated PCR/sequence template purification
No full textA commercially available filtration method is described for the purification of polymerase chain reaction (PCR) templates and sequence-labeled products. The methodology is described for the automation of this application and its use on a high-throughput liquid handling robot and capillary-based automated DNA sequencer. The application provides good-quality DNA, is relatively cheap, and can be used in either 96- or 384-well format.</p
Truncated xpt gene present in invasive Streptococcus pneumoniae may have implications for MLST schemes
No full textA serotype 1 disease-causing pneumococcus possessing a truncated xanthine phosphoribosyltransferase (xpt) housekeeping gene is described. The deletion is within the gene region used for multi-locus sequence typing (MLST) and may have occurred through genetic transformation or capsule switch between clones. The identification of this deletion in a clinical isolate therefore warrants highlighting due to potential errors that may ensue in isolate characterization and due to the fact that deletions may occur in other genes in this or other species characterized by MLST.</p
Automated non-culture-based sequence typing of meningococci from body fluids
No full textIn recent years, the polymerase chain reaction has been used for the non-culture diagnosis of meningococcal disease, and sequence-based typing takes this further by providing the full characterisation normally only available by culture. In this study, porA gene sequencing was used to perform non-culture-based sequence typing of Neisseria meningitidis strains direct from body fluids. Non-culture porA gene sequencing provided the serosubtype of the infecting organism, and proved to be a useful method as N. meningitidis was not isolated from any of the patients in this study. In conclusion, porA gene sequencing is a very useful tool for the non-culture characterisation of meningococci and provides important information for public health management of cases and contacts.</p
Nucleotide sequence analysis of the sialyltransferase genes of meningococcal serogroups B, C, Y and W135
No full textA 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.</p
A single nucleotide polymorphism identification assay for the genotypic characterisation of <i>Neisseria meningitidis</i> using MALDI-TOF mass spectrometry
No full textThe ability of matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF) to identify virulent clones of meningococci quickly and accurately is investigated. A single nucleotide polymorphism (SNP) within the fumC gene which differentiates between the hypervirulent ET-15 strain and other ET-37 complex strains is used to determine the usefulness of this method. In this study, MALDI-TOF proved to be a fast, effective alternative to traditional DNA sequencing for the identification of an individual nucleotide.</p
Genetic relatedness of antibiotic-resistant pneumococci isolated during case clusters
No full textMultilocus sequence typing of Streptococcus pneumoniae associated with two case clusters of disease is reported here for the first time. Isolates from the first cluster were serotype 19F, resistant to penicillin and erythromycin, and were characterized as ST 320. Isolates from the second cluster were serogroup 4, resistant to ciprofloxacin, and were characterized as ST 206. Therefore, the isolates from these clusters were antibiotic-resistant, of serotypes infrequently isolated, and of uncommon sequence types.</p
- …
