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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1994 Nov;32(11):2738–2744. doi: 10.1128/jcm.32.11.2738-2744.1994

Detection of bacterial DNA in cerebrospinal fluid by an assay for simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and streptococci using a seminested PCR strategy.

P Rådström 1, A Bäckman 1, N Qian 1, P Kragsbjerg 1, C Påhlson 1, P Olcén 1
PMCID: PMC264152  PMID: 7852565

Abstract

Primers specific to conserved and variable regions in the 16S rRNA sequence were selected from the partially sequenced 16S rRNA genes of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, and Staphylococcus epidermidis. The PCR assay was divided into two DNA amplifications. The first resulted in a general bacterial amplicon, and the second resulted in a species-specific amplicon. The high specificity of the PCR assay was documented after testing bacteria of 28 different species (133 strains). A total of 304 clinical cerebrospinal fluid samples, including 125 samples from patients with bacterial meningitis, were assayed to investigate the diagnostic sensitivity and specificity for bacterial meningitis. The assay showed high sensitivity (0.94) and specificity (0.96) with the clinical samples, although some false results were obtained, the reasons for which are discussed. With agarose gel electrophoresis for detection of the PCR products, the detection limit for meningococci in cerebrospinal fluid was 3 x 10(2) CFU/ml.

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Selected References

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  1. Berman N. S., Siegel S. E., Nachum R., Lipsey A., Leedom J. Cerebrospinal fluid endotoxin concentrations in gram-negative bacterial meningitis. J Pediatr. 1976 Apr;88(4 Pt 1):553–556. doi: 10.1016/s0022-3476(76)80004-2. [DOI] [PubMed] [Google Scholar]
  2. Brosius J., Palmer M. L., Kennedy P. J., Noller H. F. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4801–4805. doi: 10.1073/pnas.75.10.4801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Eisenstein B. I. The polymerase chain reaction. A new method of using molecular genetics for medical diagnosis. N Engl J Med. 1990 Jan 18;322(3):178–183. doi: 10.1056/NEJM199001183220307. [DOI] [PubMed] [Google Scholar]
  5. Gray M. W., Sankoff D., Cedergren R. J. On the evolutionary descent of organisms and organelles: a global phylogeny based on a highly conserved structural core in small subunit ribosomal RNA. Nucleic Acids Res. 1984 Jul 25;12(14):5837–5852. doi: 10.1093/nar/12.14.5837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Greenwood B. M., Whittle H. C., Dominic-Rajkovic O. Counter-current immunoelectrophoresis in the diagnosis of meningococcal infections. Lancet. 1971 Sep 4;2(7723):519–521. doi: 10.1016/s0140-6736(71)90439-9. [DOI] [PubMed] [Google Scholar]
  7. Greisen K., Loeffelholz M., Purohit A., Leong D. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clin Microbiol. 1994 Feb;32(2):335–351. doi: 10.1128/jcm.32.2.335-351.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kristiansen B. E., Ask E., Jenkins A., Fermer C., Rådstrøm P., Skøld O. Rapid diagnosis of meningococcal meningitis by polymerase chain reaction. Lancet. 1991 Jun 29;337(8757):1568–1569. doi: 10.1016/0140-6736(91)93262-8. [DOI] [PubMed] [Google Scholar]
  9. Kristiansen B. E., Rådström P., Jenkins A., Ask E., Facinelli B., Sköld O. Cloning and characterization of a DNA fragment that confers sulfonamide resistance in a serogroup B, serotype 15 strain of Neisseria meningitidis. Antimicrob Agents Chemother. 1990 Nov;34(11):2277–2279. doi: 10.1128/aac.34.11.2277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lane D. J., Pace B., Olsen G. J., Stahl D. A., Sogin M. L., Pace N. R. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6955–6959. doi: 10.1073/pnas.82.20.6955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  12. Nato F., Mazie J. C., Fournier J. M., Slizewicz B., Sagot N., Guibourdenche M., Postic D., Riou J. Y. Production of polyclonal and monoclonal antibodies against group A, B, and C capsular polysaccharides of Neisseria meningitidis and preparation of latex reagents. J Clin Microbiol. 1991 Jul;29(7):1447–1452. doi: 10.1128/jcm.29.7.1447-1452.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Olcén P. Serological methods for rapid diagnosis of haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae in cerebrospinal fluid: a comparison of Co-agglutination, immunofluorescence and immunoelectroosmophoresis. Scand J Infect Dis. 1978;10(4):283–289. doi: 10.3109/inf.1978.10.issue-4.05. [DOI] [PubMed] [Google Scholar]
  14. Persing D. H. Polymerase chain reaction: trenches to benches. J Clin Microbiol. 1991 Jul;29(7):1281–1285. doi: 10.1128/jcm.29.7.1281-1285.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Porter-Jordan K., Rosenberg E. I., Keiser J. F., Gross J. D., Ross A. M., Nasim S., Garrett C. T. Nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented DNA. J Med Virol. 1990 Feb;30(2):85–91. doi: 10.1002/jmv.1890300202. [DOI] [PubMed] [Google Scholar]
  16. Rådström P., Fermér C., Kristiansen B. E., Jenkins A., Sköld O., Swedberg G. Transformational exchanges in the dihydropteroate synthase gene of Neisseria meningitidis: a novel mechanism for acquisition of sulfonamide resistance. J Bacteriol. 1992 Oct;174(20):6386–6393. doi: 10.1128/jb.174.20.6386-6393.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  18. Salih M. A., Ahmed H. S., Hofvander Y., Danielsson D., Olcén P. Rapid diagnosis of bacterial meningitis by an enzyme immunoassay of cerebrospinal fluid. Epidemiol Infect. 1989 Oct;103(2):301–310. doi: 10.1017/s0950268800030661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Salih M. A., Danielsson D., Bäckman A., Caugant D. A., Achtman M., Olcén P. Characterization of epidemic and nonepidemic Neisseria meningitidis serogroup A strains from Sudan and Sweden. J Clin Microbiol. 1990 Aug;28(8):1711–1719. doi: 10.1128/jcm.28.8.1711-1719.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Saunders N. B., Zollinger W. D., Rao V. B. A rapid and sensitive PCR strategy employed for amplification and sequencing of porA from a single colony-forming unit of Neisseria meningitidis. Gene. 1993 Dec 31;137(2):153–162. doi: 10.1016/0378-1119(93)90001-j. [DOI] [PubMed] [Google Scholar]
  22. Shine J., Dalgarno L. Determinant of cistron specificity in bacterial ribosomes. Nature. 1975 Mar 6;254(5495):34–38. doi: 10.1038/254034a0. [DOI] [PubMed] [Google Scholar]
  23. Woese C. R., Gutell R., Gupta R., Noller H. F. Detailed analysis of the higher-order structure of 16S-like ribosomal ribonucleic acids. Microbiol Rev. 1983 Dec;47(4):621–669. doi: 10.1128/mr.47.4.621-669.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  25. van Ketel R. J., de Wever B., van Alphen L. Detection of Haemophilus influenzae in cerebrospinal fluids by polymerase chain reaction DNA amplification. J Med Microbiol. 1990 Dec;33(4):271–276. doi: 10.1099/00222615-33-4-271. [DOI] [PubMed] [Google Scholar]

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