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sodC-based real-time PCR for detection of Neisseria meningitidis
Jennifer Dolan Thomas1, Cynthia P Hatcher, Dara A Satterfield
1Meningitis and Vaccine Preventable Diseases Branch, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
Abstract:
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 (C(t)) value of 35, with 100% average reaction efficiency and an average R(2) of 0.9925. 99.7% (624/626) of Nm isolates tested were sodC-positive, with a range of average C(t) values from 13.0 to 29.5. The mean sodC C(t) 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.
Insights
A new real-time PCR (rt-PCR) assay targeting the sodC gene effectively detects Neisseria meningitidis (Nm), including strains lacking the ctrA gene. This highly sensitive and specific method improves Nm identification in various sample types.
Area of Science:
- Molecular Biology
- Microbiology
- Infectious Diseases
Background:
- Real-time PCR (rt-PCR) is a standard method for detecting Neisseria meningitidis (Nm).
- Existing rt-PCR assays often target the capsule transport gene (ctrA).
- A significant proportion of Nm carriage isolates (over 16%) lack the ctrA gene, limiting detection accuracy.
Purpose of the Study:
- To develop and validate a novel TaqMan rt-PCR assay for Neisseria meningitidis detection.
- To utilize the Cu-Zn superoxide dismutase gene (sodC) as a target for improved identification.
- To ensure accurate Nm detection regardless of capsule genotype or expression.
Main Methods:
- Development of a sodC-based TaqMan rt-PCR assay.
- Validation using standard curves to determine sensitivity and efficiency.
- Testing against a panel of Neisseria meningitidis isolates and non-Neisseria species.
- Evaluation of the assay's performance on clinical and carriage specimens.
Main Results:
- The sodC rt-PCR assay demonstrated a high average reaction efficiency (100%) and R-squared value (0.9925).
- The lower limit of detection was approximately 73 genomes per reaction.
- The assay successfully identified 99.7% of Neisseria meningitidis isolates, including 98.9% of ctrA-negative strains.
- 100% specificity was observed, with no non-Neisseria isolates testing positive.
- Compared to ctrA, the sodC assay detected additional Nm cases in both clinical (4 additional) and carriage (1 additional) specimens.
Conclusions:
- The sodC-based rt-PCR assay is a highly sensitive and specific tool for Neisseria meningitidis detection.
- This assay overcomes the limitations of ctrA-targeting methods, particularly for identifying Nm in carriage studies.
- The sodC assay offers improved detection capabilities for Neisseria meningitidis across diverse sample types.
