PCR-Based Assay for Detection of Neisseria meningitidis Capsular Serogroups 29E, X, and Z

Abstract

PCR-based assays for the identification of Neisseria meningitidis serogroups 29E, X, and Z by detection of specific regions of the ctrA gene are described. The specificities of these assays were confirmed using serogroups A, B, C, 29E, H, W135, X, Y, and Z and nongroupable meningococcal isolates.

International Circumpolar Surveillance Interlaboratory Quality Control Program for Serotyping Haemophilus influenzae and Serogrouping Neisseria meningitidis, 2005 to 2009

“…Although serogroups X, Z, and 29E are rare causes of invasive meningococcal disease, they have been reported to cause disease (16,41) or even outbreaks (17). The detection and identification of the relatively rare serogroups X, Z, and 29E based on a PCR assay that targets the sequence variations at the 5= ends of their ctrA genes have been described (5). With common serogroups (serogroups A, C, Y, and W135) being controlled by vaccination, rare serogroups may become relatively more common causes of IMD than before, and therefore, laboratories may wish to adopt and update their laboratory protocols to be able to detect these unusual organisms.…”

Consecutive Use of Two Multiplex PCR-Based Assays for Simultaneous Identification and Determination of Capsular Status of Nine Common Neisseria meningitidis Serogroups Associated with Invasive Disease

“…Strains of serogroups D, I, K, and L have rarely been detected and are not thought to be disease associated. An additional problem is that some meningococcal strains lack genes encoding a capsule and harbor sequences that have replaced the capsular biosynthesis and transport loci, referred to as the capsule null locus (cnl) (1,5,8,27). Such serologically nongroupable (NG) acapsulate strains have frequently been isolated from healthy carriers (5, 27; D. E. Bennett, A. D. Stack, and M. T. Cafferkey, unpublished data).…”

“…Each 25-l volume of PCR mix comprised of 1 unit of Taq DNA polymerase (Gibco); 7.5 mM MgCl 2 ; 2.5 l of 10ϫ buffer (1ϫ buffer contains 10 mM Tris-HCl [pH 9 at 25°C], 50 mM KCl, and 0.1% Triton X-100); 1.25 mM each of primers ctrA 1F and 1R, orf-2 F and orf-2 R, siaD CF and siaD CR, siaD W135 F, siaD YF, and siaD YWR; 2 mM each of primers siaD BF, siaD BR, and Sg XF3; 0.5 mM each of the porA 2F and porA 15R primers; and 0.2 mM deoxynucleoside triphosphates (dATP, dGTP, dCTP, and dTTP), to which 1 l of N. meningitidis DNA (100 to 300 ng) purified using MicroLysis solution (Microzone Ltd., United Kingdom) was added. This assay was subjected to the same cycling conditions as described by Bennett et al (1).…”

“…Control strains used in this study were as previously described (1). The N. meningitidis isolates were 2 isolates each of serogroups A and H; 20 isolates each of serogroups B, C, W135, and Y; 10 isolates each of serogroups 29E, X, and Z; and 10 isolates designated NG.…”

“…The N. meningitidis isolates were 2 isolates each of serogroups A and H; 20 isolates each of serogroups B, C, W135, and Y; 10 isolates each of serogroups 29E, X, and Z; and 10 isolates designated NG. The capsular status of each isolate was determined using conventional slide agglutination (24) and the individual PCR genogrouping assays as documented previously (1,13,27,29). A two-step multiplex PCR approach was designed to detect and determine the capsular type of each N. meningitidis isolate, using the primers listed in Table 1.…”