sodC-Based Real-Time PCR for Detection of Neisseria meningitidis

• Type of Material: Article
• Authors: Jennifer Dolan Thomas, Centers for Disease Control and Prevention; Cynthia P. Hatcher, Centers for Disease Control and Prevention; Dara A. Satterfield, Centers for Disease Control and Prevention; M. Jordan Theodore, Centers for Disease Control and Prevention; Michelle C. Bach, Centers for Disease Control and Prevention; Kristin B. Linscott, Centers for Disease Control and Prevention; Xin Zhao, Centers for Disease Control and Prevention; Xin Wang, Centers for Disease Control and Prevention; Raydel Mair, Centers for Disease Control and Prevention; Susanna Schmink, Centers for Disease Control and Prevention; Kathryn E. Arnold, Georgia Department of Community Health; David Stephens, Emory University; Lee H. Harrison, Johns Hopkins Bloomberg School of Public Health; Rosemary A. Hollick, Johns Hopkins Bloomberg School of Public Health; Ana Lucia Andrade, Universidade Federal de Goiás; Juliana Lamaro-Cardoso, Universidade Federal de Goiás; Ana Paula S. de Lemos, Instituto Adolfo Lutz Registro; Jenna Gritzfeld, University of Liverpool; Stephen Gordon, University of Liverpool; Ahmet Soysal, Marmara University; Mustafa Bakir, Marmara University; Dolly Sharma, Emory University; Shabnam Jain, Emory University; Sarah Satola, Emory University; Nancy E. Messonnier, Centers for Disease Control and Prevention; Leonard W. Mayer, Centers for Disease Control and Prevention
• Language: English
• Date: 2011-05-05
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
• License: Creative Commons Creative Commons CC0 1.0 Universal
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.pone.0019361
• Title of Journal or Parent Work: PLoS ONE
• ISSN: 1932-6203
• Volume: 6
• Issue: 5
• Start Page: e19361
• End Page: e19361
• Grant/Funding Information: APS de Lemos was supported by the Fogarty International Center Global Infectious Diseases Research Training Program grant from the National Institutes of Health.; JG and SG thank the NIHR Biomedical Research Centre in Microbial Diseases and the Northwest Regional Development Agency for infrastructural support.; ALA was supported by the Brazilian National Council for Scientific and Technological Development (CNPq grant # 309196/2007-8) and was supported by the National Institute of Science and Technology for Health Technology Assessment/IATS.
• Keywords: Science & Technology; SEQUENCE; BACTERIAL-MENINGITIS; Multidisciplinary Sciences; CEREBROSPINAL-FLUID; MENINGOCOCCAL CARRIAGE; Science & Technology - Other Topics; STREPTOCOCCUS-PNEUMONIAE; HAEMOPHILUS-INFLUENZAE; IDENTIFICATION; MULTIDISCIPLINARY SCIENCES; DIAGNOSIS; GENES; SUPEROXIDE-DISMUTASE

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 (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.