Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA.
In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.
Guiver
M
M
Meningococcal Reference Unit, Withington Hospital, Nell Lane, Manchester, UK. mguiver@nw.phls.nhs.uk
Borrow
R
R
Marsh
J
J
Gray
S J
SJ
Kaczmarski
E B
EB
Howells
D
D
Boseley
P
P
Fox
A J
AJ
D001426:Bacterial Proteins / Q000235:genetics; D017931:DNA Primers; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis; D004268:DNA-Binding Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008585:Meningitis, Meningococcal / Q000097:blood / Q000134:cerebrospinal fluid / Q000382:microbiology*; D009345:Neisseria meningitidis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012799:Sialyltransferases / Q000235:genetics; D014157:Transcription Factors
D001426:Bacterial Proteins; C097706:CtrA protein, Caulobacter; D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D012799:Sialyltransferases