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Journal of Clinical Microbiology, December 2002, p. 4713-4719, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4713-4719.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Development of a Real-Time Fluorescence PCR Assay for Rapid Detection of the Diphtheria Toxin Gene

Elizabeth A. Mothershed, Pamela K. Cassiday, Kevin Pierson, Leonard W. Mayer, and Tanja Popovic*

Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333

Received 8 July 2002/ Returned for modification 17 August 2002/ Accepted 3 September 2002

We developed and evaluated a real-time fluorescence PCR assay for detecting the A and B subunits of diphtheria toxin (tox) gene. When 23 toxigenic Corynebacterium diphtheriae strains, 9 nontoxigenic C. diphtheriae strains, and 44 strains representing the diversity of pathogens and normal respiratory flora were tested, this real-time PCR assay exhibited 100% sensitivity and specificity. It allowed for the detection of both subunits of the tox gene at 750 times greater sensitivity (2 CFU) than the standard PCR (1,500 CFU). When used directly on specimens collected from patients with clinical diphtheria, one or both subunits of the tox gene were detected in 34 of 36 specimens by using the real-time PCR assay; only 9 specimens were found to be positive by standard PCR. Reamplification by standard PCR and DNA sequencing of the amplification product confirmed all real-time PCR tox-positive reactions. This real-time PCR format is a more sensitive and rapid alternative to standard PCR for detection of the tox gene in clinical material.

* Corresponding author. Mailing address: Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, CDC, MS G34, 1600 Clifton Rd., NE, Atlanta, GA 30333. Phone: (404) 639-1730. Fax: (404) 639-3172. E-mail: txp1{at}cdc.gov.

Journal of Clinical Microbiology, December 2002, p. 4713-4719, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4713-4719.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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