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

Autor: Dolly Sharma, M. Jordan Theodore, Ana Paula Silva de Lemos, Xin Zhao, Leonard W. Mayer, Jenna F. Gritzfeld, Ahmet Soysal, Kathryn E. Arnold, David S. Stephens, Shabnam Jain, Lee H. Harrison, Sarah W. Satola, Cynthia Hatcher, Ana Lucia Andrade, Xin Wang, Kristin B. Linscott, Mustafa Bakir, Juliana Lamaro-Cardoso, Nancy E. Messonnier, Rosemary Hollick, Dara A. Satterfield, Jennifer Dolan Thomas, Raydel Mair, Stephen B. Gordon, Michelle C. Bach, Susanna Schmink
Jazyk: angličtina
Rok vydání: 2011
Předmět:
Bacterial Diseases
Applied Microbiology
Gene Identification and Analysis
lcsh:Medicine
Pathogenesis
Neisseria meningitidis
medicine.disease_cause
Biochemistry
law.invention
Haemophilus influenzae
DNA amplification
law
Genotype
Gram Negative
lcsh:Science
Polymerase chain reaction
0303 health sciences
Multidisciplinary
Reverse Transcriptase Polymerase Chain Reaction
Bacterial Pathogens
Nucleic acids
Infectious Diseases
Real-time polymerase chain reaction
Medical Microbiology
qw_50
Medicine
Research Article
Biotechnology
Molecular Sequence Data
Biology
Microbiology
Molecular Genetics
03 medical and health sciences
Bacterial Proteins
Diagnostic Medicine
wl_200
Bacterial Meningitis
Genetics
medicine
TaqMan
Microbial Pathogens
Gene
030304 developmental biology
Superoxide Dismutase
030306 microbiology
lcsh:R
Bacteriology
DNA
Molecular biology
DNA extraction
lcsh:Q
Zdroj: PLoS ONE
PLoS ONE, Vol 6, Iss 5, p e19361 (2011)
ISSN: 1932-6203
Popis: 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.
Databáze: OpenAIRE
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