TY - JOUR
T1 - SodC-based real-time PCR for detection of neisseria meningitidis
AU - Dolan Thomas, Jennifer
AU - Hatcher, Cynthia P.
AU - Satterfield, Dara A.
AU - Theodore, M. Jordan
AU - Bach, Michelle C.
AU - Linscott, Kristin B.
AU - Zhao, Xin
AU - Wang, Xin
AU - Mair, Raydel
AU - Schmink, Susanna
AU - Arnold, Kathryn E.
AU - Stephens, David S.
AU - Harrison, Lee H.
AU - Hollick, Rosemary A.
AU - Andrade, Ana Lucia
AU - Lamaro-Cardoso, Juliana
AU - de Lemos, Ana Paula S.
AU - Gritzfeld, Jenna
AU - Gordon, Stephen
AU - Soysal, Ahmet
AU - Bakir, Mustafa
AU - Sharma, Dolly
AU - Jain, Shabnam
AU - Satola, Sarah W.
AU - Messonnier, Nancy E.
AU - Mayer, Leonard W.
PY - 2011
Y1 - 2011
N2 - 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.
AB - 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.
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U2 - 10.1371/journal.pone.0019361
DO - 10.1371/journal.pone.0019361
M3 - Article
C2 - 21573213
AN - SCOPUS:79955874796
SN - 1932-6203
VL - 6
JO - PloS one
JF - PloS one
IS - 5
M1 - e19361
ER -