Simultaneous Detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in Suspected Cases of Meningitis and Septicemia Using Real-Time PCR

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ID: 113293
2001
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Abstract
A single-tube 5′ nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae from clinical samples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsular transport ( ctrA ) , capsulation ( bexA ), and pneumolysin ( ply ) gene targets specific for N. meningitidis, H. influenzae, and S. pneumoniae, respectively, were selected. Using sequence-specific fluorescent-dye-labeled probes and continuous real-time monitoring, accumulation of amplified product was measured. Sensitivity was assessed using clinical samples (CSF, serum, plasma, and whole blood) from culture-confirmed cases for the three organisms. The respective sensitivities (as percentages) for N. meningitidis, H. influenzae, and S. pneumoniae were 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The ctrA primers amplified meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z; the ply primers amplified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and the bexA primers amplified H. influenzae types b and c. Coamplification of two target genes without a loss of sensitivity was demonstrated. The multiplex assay was then used to test a large number ( n = 4,113) of culture-negative samples for the three pathogens. Cases of meningococcal, H. influenzae, and pneumococcal disease that had not previously been confirmed by culture were identified with this assay. The ctrA primer set used in the multiplex PCR was found to be more sensitive ( P < 0.0001) than the ctrA primers that had been used for meningococcal PCR testing at that time.
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Authors C. E. Corless;M. Guiver;R. Borrow;V. Edwards-Jones;A. J. Fox;E. B. Kaczmarski;C. E. Corless;M. Guiver;R. Borrow;V. Edwards-Jones;A. J. Fox;E. B. Kaczmarski;
Journal Journal of clinical microbiology
Year 2001
DOI
10.1128/JCM.39.4.1553-1558.2001
URL
http://dx.doi.org/10.1128/JCM.39.4.1553-1558.2001
Keywords
National Center for Biotechnology Information NCBI NLM MEDLINE humans pubmed abstract nih national institutes of health national library of medicine research support non-u.s. gov't DNA sensitivity and specificity Evaluation Study Taq Polymerase / metabolism Molecular Sequence Data Polymerase Chain Reaction / methods culture media meningitis pneumococcal / microbiology streptococcus pneumoniae / genetics streptococcus pneumoniae / isolation & purification* bacterial / diagnosis bacterial / microbiology* neisseria meningitidis / isolation & purification* bacterial / blood bacterial proteins / genetics bacterial / cerebrospinal fluid dna primers streptolysins / genetics pneumococcal / diagnosis haemophilus / diagnosis meningococcal / diagnosis neisseria meningitidis / genetics bacteremia / microbiology* haemophilus influenzae / genetics meningococcal / microbiology pmid:11283086 pmc87969 doi:10.1128/jcm.39.4.1553-1558.2001 c e corless m guiver e b kaczmarski atp-binding cassette transporters* bacteremia / diagnosis dna-binding proteins* haemophilus influenzae / isolation & purification* haemophilus / microbiology transcription factors*

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