Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR.
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.
Meningococcal Reference Unit, Manchester Public Health Laboratory, Withington Hospital, Manchester M20 2LR, United Kingdom.
C E
CE
Corless
M
M
Guiver
R
R
Borrow
V
V
Edwards-Jones
A J
AJ
Fox
E B
EB
Kaczmarski
D018528:ATP-Binding Cassette Transporters; D016470:Bacteremia / Q000175:diagnosis / Q000382:microbiology*; D001426:Bacterial Proteins / Q000235:genetics; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial / Q000097:blood / Q000134:cerebrospinal fluid; D004268:DNA-Binding Proteins; D006193:Haemophilus influenzae / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016920:Meningitis, Bacterial / Q000175:diagnosis / Q000382:microbiology*; D008583:Meningitis, Haemophilus / Q000175:diagnosis / Q000382:microbiology; D008585:Meningitis, Meningococcal / Q000175:diagnosis / Q000382:microbiology; D008586:Meningitis, Pneumococcal / Q000175:diagnosis / Q000382:microbiology; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D013301:Streptolysins / Q000235:genetics; D019914:Taq Polymerase / Q000378:metabolism; D014157:Transcription Factors
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C097706:CtrA protein, Caulobacter; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D013301:Streptolysins; D014157:Transcription Factors; C032290:plY protein, Streptococcus pneumoniae; C068101:bexA protein, Haemophilus influenzae; D019914:Taq Polymerase