Development of a Multiplex PCR Assay for Detection and Genogrouping of Neisseria meningitidis

Abstract

Neisseria meningitidis is a leading pathogen of epidemic bacterial meningitis and fulminant sepsis worldwide. Twelve different N. meningitidis serogroups have been identified to date based on antigenic differences in the capsular polysaccharide. However, more than 90% of human cases of N. meningitidis meningitis are the result of infection with just five serogroups, A, B, C, W135, and Y. Efficient methods of detection and genogrouping of N. meningitidis isolates are needed, therefore, in order to monitor prevalent serogroups as a means of disease control and prevention. The capsular gene complex regions have been sequenced from only seven out of the 12 serogroups. In this study, the capsular gene complexes of the remaining five serogroups were sequenced and analyzed. Primers were designed that were specific for N. meningitidis species and for the 12 individual serogroups, and a multiplex PCR assay using these specific primers was developed for N. meningitidis detection and genogrouping. The assay was tested using 15 reference strains covering all 12 serogroups, 143 clinical isolates, and 21 strains from closely related species or from species that cause meningitis. The assay could detect N. meningitidis serogroups and was shown to be specific, with a detection sensitivity of 1 ng of genomic DNA (equivalent to ϳ4 ؋ 10 5 genomes) or 3 ؋ 10 5 CFU/ml in noncultured mock cerebrospinal fluid (CSF) specimens. This study, therefore, describes for the first time the development of a molecular protocol for the detection of all N. meningitidis serogroups. This multiplex PCR-based assay may have use for the clinical diagnosis and epidemiological surveillance of N. meningitidis.

The Capsule Polymerase CslB of Neisseria meningitidis Serogroup L Catalyzes the Synthesis of a Complex Trimeric Repeating Unit Comprising Glycosidic and Phosphodiester Linkages

“…However, considering the fact that both glycosidic as well as phosphodiester linkages are present in the complex CPSL repeating unit (33)-␤-D-GlcNAc-(133)-␤-D-GlcNAc-(133)-␣-D-GlcNAc-(13 OPO 3 3) n , we speculated that CslA acts in concert with a glycosyltransferase. Congruent with this assumption, region A of the chromosomal locus cps (capsular polysaccharide synthesis) of N. meningitidis serogroup L comprises three open reading frames (ORFs), of which cslB was predicted to encode a bifunctional glycosyltransferase and cslC an acetyltransferase (7,43). Therefore, cslA and cslB were cloned, expressed as soluble proteins, and functionally characterized.…”

Changing epidemiology of Infant Meningococcal Disease after the introduction of meningococcal serogroup C vaccine in Italy, 2006–2014

“…Clinical data and isolates and/or clinical samples are collected from hospitalized patients throughout the country. Specifically, IMD cases are reported to the local health authorities, and samples are sent to the national reference laboratory at the Istituto Superiore di Sanità for further characterization and strains storage at −80 • C. In particular, serogroup identification is obtained by agglutination with commercial antisera (Remel Europe, Ltd, UK) or by multiplex PCR [9]. DNA was extracted from clinical samples with commercial kit (Qiagen, Hilden, Germany).…”

Pan-PCR, a Computational Method for Designing Bacterium-Typing Assays Based on Whole-Genome Sequence Data

“…In general, PCR-based typing assays, such as those designed for Streptococcus pneumoniae (13), Neisseria meningitidis (14), and Acinetobacter baumannii (15), are desirable because of their inherent advantages of low cost and high speed. The expense comprises the cost of standard PCR reagents, and the turnaround time consists of only the few hours needed to prepare DNA and run the PCR.…”

“…Several groups are attempting to take advantage of existing genome sequence data to design sequence-and PCR-based typing assays. In the last year, PCR-based typing assays have been published for organisms such as Streptococcus pneumoniae (13), Neisseria meningitidis (14), and Acinetobacter baumannii (15).…”