20 research outputs found
sodC-Based Real-Time PCR for Detection of Neisseria meningitidis
Real-time PCR (rt-PCR) is a widely used molecular method for detection of
Neisseria meningitidis (Nm). Several rt-PCR assays for Nm
target the capsule transport gene, ctrA. However, over
16% of meningococcal carriage isolates lack ctrA,
rendering this target gene ineffective at identification of this sub-population
of meningococcal isolates. The Cu-Zn superoxide dismutase gene,
sodC, is found in Nm but not in other
Neisseria species. To better identify Nm, regardless of
capsule genotype or expression status, a sodC-based TaqMan
rt-PCR assay was developed and validated. Standard curves revealed an average
lower limit of detection of 73 genomes per reaction at cycle threshold
(Ct) value of 35, with 100% average reaction efficiency
and an average R2 of 0.9925. 99.7% (624/626) of Nm isolates
tested were sodC-positive, with a range of average
Ct values from 13.0 to 29.5. The mean sodC
Ct value of these Nm isolates was 17.6±2.2 (±SD).
Of the 626 Nm tested, 178 were nongroupable (NG) ctrA-negative
Nm isolates, and 98.9% (176/178) of these were detected by
sodC rt-PCR. The assay was 100% specific, with all
244 non-Nm isolates testing negative. Of 157 clinical specimens tested,
sodC detected 25/157 Nm or 4 additional specimens compared
to ctrA and 24 more than culture. Among 582 carriage specimens,
sodC detected Nm in 1 more than ctrA and
in 4 more than culture. This sodC rt-PCR assay is a highly
sensitive and specific method for detection of Nm, especially in carriage
studies where many meningococcal isolates lack capsule genes