Evaluation of touch-down real-time PCR based on SYBR Green I fluorescent dye for the detection of Neisseria meningitidis in clinical samples.
Antibiotic treatment prior to transport or admission of patients to hospital has reduced the proportion of patients with invasive meningococcal disease (IMD) from whom Neisseria meningitidis can be isolated by standard microbiological techniques. Assays to detect the crgA gene were used to detect meningococcal DNA by both conventional polymerase chain reaction (PCR) and real-time PCR (RTPCR) in relation to microbiological diagnosis of cases over two years between 2002 and 2003. The sensitivity of both PCR assays for culture-confirmed cases was 93% and the specificity was 98.6%. Agreement between the two PCR assays was 96.2%. The inter- and intra-assay variations and effects of different amounts of DNA on the melting temperatures were examined. The touch-down RTPCR based on SYBR Green I fluorescent dye detected and characterized N. meningitidis in clinical samples within one hour.
Kesanopoulos
Konstantinos
K
National Meningococcal Reference Laboratory, National School of Public Health, 196 Alexandras Avenue, Athens, Greece.
Tzanakaki
Georgina
G
Levidiotou
Stamatina
S
Blackwell
Caroline
C
Kremastinou
Jenny
J
D052160:Benzothiazoles; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D003959:Diamines; D005456:Fluorescent Dyes; D006801:Humans; D008589:Meningococcal Infections / Q000175:diagnosis / Q000382:microbiology; D009345:Neisseria meningitidis / Q000235:genetics* / Q000302:isolation & purification*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D011804:Quinolines; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I