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Bacteriology

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

C. E. Corless, M. Guiver, R. Borrow, V. Edwards-Jones, A. J. Fox, E. B. Kaczmarski
C. E. Corless
Meningococcal Reference Unit, Manchester Public Health Laboratory, Withington Hospital, Manchester M20 2LR, and
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M. Guiver
Meningococcal Reference Unit, Manchester Public Health Laboratory, Withington Hospital, Manchester M20 2LR, and
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R. Borrow
Meningococcal Reference Unit, Manchester Public Health Laboratory, Withington Hospital, Manchester M20 2LR, and
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V. Edwards-Jones
Department of Biological Sciences, Manchester Metropolitan University, Manchester M1 5GD, United Kingdom
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A. J. Fox
Meningococcal Reference Unit, Manchester Public Health Laboratory, Withington Hospital, Manchester M20 2LR, and
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E. B. Kaczmarski
Meningococcal Reference Unit, Manchester Public Health Laboratory, Withington Hospital, Manchester M20 2LR, and
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DOI: 10.1128/JCM.39.4.1553-1558.2001
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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. pneumoniaewere 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The ctrAprimers 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 thebexA primers amplified H. influenzaetypes 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.

  • Copyright © 2001 American Society for Microbiology
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Simultaneous Detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in Suspected Cases of Meningitis and Septicemia Using Real-Time PCR
C. E. Corless, M. Guiver, R. Borrow, V. Edwards-Jones, A. J. Fox, E. B. Kaczmarski
Journal of Clinical Microbiology Apr 2001, 39 (4) 1553-1558; DOI: 10.1128/JCM.39.4.1553-1558.2001

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Simultaneous Detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in Suspected Cases of Meningitis and Septicemia Using Real-Time PCR
C. E. Corless, M. Guiver, R. Borrow, V. Edwards-Jones, A. J. Fox, E. B. Kaczmarski
Journal of Clinical Microbiology Apr 2001, 39 (4) 1553-1558; DOI: 10.1128/JCM.39.4.1553-1558.2001
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KEYWORDS

ATP-Binding Cassette Transporters
bacteremia
DNA-Binding Proteins
Haemophilus influenzae
Meningitis, Bacterial
Neisseria meningitidis
Streptococcus pneumoniae
Transcription Factors

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