Direct and rapid identification and genogrouping of meningococci and porA amplification by LightCycler PCR.
Invasive meningococcal infections are usually diagnosed by culture. This approach is far from optimal due to, e.g., treatment with precollection antibiotics. Molecular-genetics methods are therefore recognized as important tools for optimal laboratory confirmation of meningococcal infections as well as characterization of meningococci (Mc). The aims of the present study were to develop real-time PCRs for identification and genogrouping (A, B, C, Y, and W-135) of Mc and porA amplification that further can be used for subsequent genosubtyping. In a first run Mc were identified. In a second run they were genogrouped and porA genes were amplified. All the Mc isolates (n = 71) but one and cerebrospinal fluid samples (n = 11) tested gave the expected positive results. The specificity, inter- and intra-assay variations, and effects of different amounts of DNA on the melting temperatures were also explored. The LightCycler PCR system was found to effectively detect and characterize Mc in a few hours. This testing, including the DNA sequencing of the porA gene to reveal the genosubtype, does not take more than a working day, and the results can be compared to those from culturing with phenotypic analysis, which takes at least a few days.
Mölling
Paula
P
National Reference Laboratory for Pathogenic Neisseria at the Department of Clinical Microbiology, Orebro University Hospital, Orebro, Sweden. paula.molling@orebroll.se
Jacobsson
Susanne
S
Bäckman
Anders
A
Olcén
Per
P
D015373:Bacterial Typing Techniques; D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism; D005838:Genotype; D006801:Humans; D008585:Meningitis, Meningococcal / Q000097:blood / Q000134:cerebrospinal fluid / Q000382:microbiology; D008589:Meningococcal Infections / Q000097:blood / Q000382:microbiology*; D009345:Neisseria meningitidis / Q000145:classification* / Q000235:genetics*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D018272:Porins / Q000235:genetics*; D011804:Quinolines; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013696:Temperature; D013997:Time Factors
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D018272:Porins; D011804:Quinolines; C057699:porin protein, Neisseria; C098022:SYBR Green I