Open AccessEvaluation of touch‐down real‐time PCR based on SYBR Green I fluorescent dye for the detection of Neisseria meningitidis in clinical samples
Author(s) -
Kesanopoulos Konstantinos,
Tzanakaki Georgina,
Levidiotou Stamatina,
Blackwell Caroline,
Kremastinou Jenny
Publication year - 2005
Publication title -
fems immunology & medical microbiology
Language(s) - English
Resource type - Journals
eISSN - 1574-695X
pISSN - 0928-8244
DOI - 10.1016/j.femsim.2004.10.011
Subject(s) - sybr green i , neisseria meningitidis , polymerase chain reaction , biology , real time polymerase chain reaction , microbiology and biotechnology , melting curve analysis , bacteria , virology , gene , genetics
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.