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. 1997 Mar;35(3):714–718. doi: 10.1128/jcm.35.3.714-718.1997

An enzyme-linked immunosorbent assay to detect PCR products of the rfbS gene from serogroup D salmonellae: a rapid screening prototype.

J M Luk 1, U Kongmuang 1, R S Tsang 1, A A Lindberg 1
PMCID: PMC229656  PMID: 9041418

Abstract

We describe a digoxigenin-based enzyme-linked immunosorbent assay (DIG-ELISA) following a PCR to detect the amplified lipopolysaccharide rfbS gene as a means for rapid screening of serogroup D salmonellae in stool specimens. For pure bacterial cultures, the sensitivity of the PCR DIG-ELISA was approximately 10 bacteria. In the presence of stool materials, the salmonellae were first isolated by an immunomagnetic separation technique with an O9-specific monoclonal antibody. MATy-O9, followed by PCR and DIG-ELISA. The corresponding sensitivity was about 10 to 100 bacteria. To evaluate the assay performance clinically, 203 stool samples from patients with diarrhea were subjected to the routine culture techniques and the PCR ELISA method with overnight enrichment. The conventional culture method identified 145 salmonellae (31 serogroup B, 27 serogroup C, 83 serogroup D, and 5 serogroup E isolates) and 58 non-salmonella bacteria. The PCR ELISA method correctly identified all 82 serogroup D salmonellae (A405 by ELISA, 2.54 +/- 0.74) but was negative for the other Salmonella serogroups (A405, 0.26 +/- 0.08; n = 63) and non-Salmonella isolates (A405, 0.16 +/- 0.04; n = 58). In order to obtain a visible result, the assay takes approximately 6 h (PCR, 4 h; ELISA, 2 h), along with brief enrichment cultivation of the samples (from 4 to 16 h). Thus, the PCR DIG-ELISA offers a fast, accurate, semiquantitative means of detecting infectious agents such as salmonellae, and future robotic automation is possible.

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Selected References

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  1. Bobo L., Coutlee F., Yolken R. H., Quinn T., Viscidi R. P. Diagnosis of Chlamydia trachomatis cervical infection by detection of amplified DNA with an enzyme immunoassay. J Clin Microbiol. 1990 Sep;28(9):1968–1973. doi: 10.1128/jcm.28.9.1968-1973.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chevrier D., Popoff M. Y., Dion M. P., Hermant D., Guesdon J. L. Rapid detection of Salmonella subspecies I by PCR combined with non-radioactive hybridisation using covalently immobilised oligonucleotide on a microplate. FEMS Immunol Med Microbiol. 1995 Feb;10(3-4):245–501. doi: 10.1111/j.1574-695X.1995.tb00039.x. [DOI] [PubMed] [Google Scholar]
  3. Coutlée F., Saint-Antoine P., Olivier C., Voyer H., Kessous-Elbaz A., Berrada F., Bégin P., Giroux L., Viscidi R. Evaluation of infection with human immunodeficiency virus type 1 by using nonisotopic solution hybridization for detection of polymerase chain reaction-amplified proviral DNA. J Clin Microbiol. 1991 Nov;29(11):2461–2467. doi: 10.1128/jcm.29.11.2461-2467.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. De Beenhouwer H., Liang Z., De Rijk P., Van Eekeren C., Portaels F. Detection and identification of mycobacteria by DNA amplification and oligonucleotide-specific capture plate hybridization. J Clin Microbiol. 1995 Nov;33(11):2994–2998. doi: 10.1128/jcm.33.11.2994-2998.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. De Wit D., Steyn L., Shoemaker S., Sogin M. Direct detection of Mycobacterium tuberculosis in clinical specimens by DNA amplification. J Clin Microbiol. 1990 Nov;28(11):2437–2441. doi: 10.1128/jcm.28.11.2437-2441.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Diamandis E. P., Christopoulos T. K. The biotin-(strept)avidin system: principles and applications in biotechnology. Clin Chem. 1991 May;37(5):625–636. [PubMed] [Google Scholar]
  7. Fluit A. C., Widjojoatmodjo M. N., Verhoef J. Detection of Salmonella species in fecal samples by immunomagnetic separation and PCR. J Clin Microbiol. 1995 Apr;33(4):1046–1047. doi: 10.1128/jcm.33.4.1046-1047.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gibellini D., Zerbini M., Musiani M., Venturoli S., Gentilomi G., La Placa M. Microplate capture hybridization of amplified parvovirus B19 DNA fragment labelled with digoxigenin. Mol Cell Probes. 1993 Dec;7(6):453–458. doi: 10.1006/mcpr.1993.1067. [DOI] [PubMed] [Google Scholar]
  9. Kongmuang U., Luk J. M., Lindberg A. A. Comparison of three stool-processing methods for detection of Salmonella serogroups B, C2, and D by PCR. J Clin Microbiol. 1994 Dec;32(12):3072–3074. doi: 10.1128/jcm.32.12.3072-3074.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lebech A. M., Hansen K. Detection of Borrelia burgdorferi DNA in urine samples and cerebrospinal fluid samples from patients with early and late Lyme neuroborreliosis by polymerase chain reaction. J Clin Microbiol. 1992 Jul;30(7):1646–1653. doi: 10.1128/jcm.30.7.1646-1653.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Liu D., Verma N. K., Romana L. K., Reeves P. R. Relationships among the rfb regions of Salmonella serovars A, B, and D. J Bacteriol. 1991 Aug;173(15):4814–4819. doi: 10.1128/jb.173.15.4814-4819.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Luk J. M., Kongmuang U., Reeves P. R., Lindberg A. A. Selective amplification of abequose and paratose synthase genes (rfb) by polymerase chain reaction for identification of Salmonella major serogroups (A, B, C2, and D). J Clin Microbiol. 1993 Aug;31(8):2118–2123. doi: 10.1128/jcm.31.8.2118-2123.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Luk J. M., Lindberg A. A. Rapid and sensitive detection of Salmonella (O:6,7) by immunomagnetic monoclonal antibody-based assays. J Immunol Methods. 1991 Mar 1;137(1):1–8. doi: 10.1016/0022-1759(91)90387-u. [DOI] [PubMed] [Google Scholar]
  14. Luk J. M., Zhao C. R., Karlsson K. M., Lindberg A. A. Specificity of monoclonal antibodies binding to the polysaccharide antigens (Vi, O9) of Salmonella typhi. FEMS Microbiol Lett. 1992 Oct 1;76(1-2):173–178. doi: 10.1016/0378-1097(92)90382-x. [DOI] [PubMed] [Google Scholar]
  15. Luk M. C., Tsang R. S., Ng M. H. Murine monoclonal antibody specific for lipopolysaccharide of Salmonella serogroup A. J Clin Microbiol. 1987 Nov;25(11):2140–2144. doi: 10.1128/jcm.25.11.2140-2144.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Morrison D. C., Ryan J. L. Endotoxins and disease mechanisms. Annu Rev Med. 1987;38:417–432. doi: 10.1146/annurev.me.38.020187.002221. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Rietschel E. T., Brade H. Bacterial endotoxins. Sci Am. 1992 Aug;267(2):54–61. doi: 10.1038/scientificamerican0892-54. [DOI] [PubMed] [Google Scholar]
  19. Saiki R. K., Walsh P. S., Levenson C. H., Erlich H. A. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6230–6234. doi: 10.1073/pnas.86.16.6230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Verma N. K., Quigley N. B., Reeves P. R. O-antigen variation in Salmonella spp.: rfb gene clusters of three strains. J Bacteriol. 1988 Jan;170(1):103–107. doi: 10.1128/jb.170.1.103-107.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Young K. K., Resnick R. M., Myers T. W. Detection of hepatitis C virus RNA by a combined reverse transcription-polymerase chain reaction assay. J Clin Microbiol. 1993 Apr;31(4):882–886. doi: 10.1128/jcm.31.4.882-886.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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