Abstract
PCR has proved to be a reliable tool for the detection of the diphtheria toxin gene, tox, and its use has allowed for the rapid differentiation between toxigenic and nontoxigenic strains. In this study, this PCR was further developed, evaluated, and standardized to detect this gene directly from clinical specimens. Optimal conditions for collection, transport, and storage of the clinical specimens and isolation and purification of DNA from the clinical specimens were defined. With two sets of primers that detect the A and B subunits of the diphtheria toxin gene, sensitivity levels of 50 and 500 CFU/PCR mixture, respectively, were achieved. This PCR was evaluated with 162 clinical samples collected from patients with diphtheria and other upper respiratory tract infections, as well as from healthy individuals.
Full Text
The Full Text of this article is available as a PDF (1.8 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bollet C., Gevaudan M. J., de Lamballerie X., Zandotti C., de Micco P. A simple method for the isolation of chromosomal DNA from gram positive or acid-fast bacteria. Nucleic Acids Res. 1991 Apr 25;19(8):1955–1955. doi: 10.1093/nar/19.8.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dawson J. E., Stallknecht D. E., Howerth E. W., Warner C., Biggie K., Davidson W. R., Lockhart J. M., Nettles V. F., Olson J. G., Childs J. E. Susceptibility of white-tailed deer (Odocoileus virginianus) to infection with Ehrlichia chaffeensis, the etiologic agent of human ehrlichiosis. J Clin Microbiol. 1994 Nov;32(11):2725–2728. doi: 10.1128/jcm.32.11.2725-2728.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hauser D., Popoff M. R., Kiredjian M., Boquet P., Bimet F. Polymerase chain reaction assay for diagnosis of potentially toxinogenic Corynebacterium diphtheriae strains: correlation with ADP-ribosylation activity assay. J Clin Microbiol. 1993 Oct;31(10):2720–2723. doi: 10.1128/jcm.31.10.2720-2723.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klouche M., Lühmann D., Kirchner H. Low prevalence of diphtheria antitoxin in children and adults in northern Germany. Eur J Clin Microbiol Infect Dis. 1995 Aug;14(8):682–685. doi: 10.1007/BF01690874. [DOI] [PubMed] [Google Scholar]
- Maple P. A., Efstratiou A., George R. C., Andrews N. J., Sesardic D. Diphtheria immunity in UK blood donors. Lancet. 1995 Apr 15;345(8955):963–965. doi: 10.1016/s0140-6736(95)90705-x. [DOI] [PubMed] [Google Scholar]
- Mikhailovich V. M., Melnikov V. G., Mazurova I. K., Wachsmuth I. K., Wenger J. D., Wharton M., Nakao H., Popovic T. Application of PCR for detection of toxigenic Corynebacterium diphtheriae strains isolated during the Russian diphtheria epidemic, 1990 through 1994. J Clin Microbiol. 1995 Nov;33(11):3061–3063. doi: 10.1128/jcm.33.11.3061-3063.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakao H., Mazurova I. K., Glushkevich T., Popovic T. Analysis of heterogeneity of Corynebacterium diphtheriae toxin gene, tox, and its regulatory element, dtxR, by direct sequencing. Res Microbiol. 1997 Jan;148(1):45–54. doi: 10.1016/S0923-2508(97)81899-2. [DOI] [PubMed] [Google Scholar]
- Nakao H., Pruckler J. M., Mazurova I. K., Narvskaia O. V., Glushkevich T., Marijevski V. F., Kravetz A. N., Fields B. S., Wachsmuth I. K., Popovic T. Heterogeneity of diphtheria toxin gene, tox, and its regulatory element, dtxR, in Corynebacterium diphtheriae strains causing epidemic diphtheria in Russia and Ukraine. J Clin Microbiol. 1996 Jul;34(7):1711–1716. doi: 10.1128/jcm.34.7.1711-1716.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pallen M. J., Hay A. J., Puckey L. H., Efstratiou A. Polymerase chain reaction for screening clinical isolates of corynebacteria for the production of diphtheria toxin. J Clin Pathol. 1994 Apr;47(4):353–356. doi: 10.1136/jcp.47.4.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pallen M. J. Rapid screening for toxigenic Corynebacterium diphtheriae by the polymerase chain reaction. J Clin Pathol. 1991 Dec;44(12):1025–1026. doi: 10.1136/jcp.44.12.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Popovic T., Kombarova S. Y., Reeves M. W., Nakao H., Mazurova I. K., Wharton M., Wachsmuth I. K., Wenger J. D. Molecular epidemiology of diphtheria in Russia, 1985-1994. J Infect Dis. 1996 Nov;174(5):1064–1072. doi: 10.1093/infdis/174.5.1064. [DOI] [PubMed] [Google Scholar]
- Popovic T., Wharton M., Wenger J. D., McIntyre L., Wachsmuth I. K. Are we ready for diphtheria? A report from the Diphtheria Diagnostic Workshop, Atlanta, 11 and 12 July 1994. J Infect Dis. 1995 Apr;171(4):765–767. doi: 10.1093/infdis/171.4.765. [DOI] [PubMed] [Google Scholar]
- Rappuoli R., Podda A., Giovannoni F., Nencioni L., Peragallo M., Francolini P. Absence of protective immunity against diphtheria in a large proportion of young adults. Vaccine. 1993;11(5):576–577. doi: 10.1016/0264-410x(93)90235-p. [DOI] [PubMed] [Google Scholar]
- de Lamballerie X., Zandotti C., Vignoli C., Bollet C., de Micco P. A one-step microbial DNA extraction method using "Chelex 100" suitable for gene amplification. Res Microbiol. 1992 Oct;143(8):785–790. doi: 10.1016/0923-2508(92)90107-y. [DOI] [PubMed] [Google Scholar]