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Journal of Clinical Microbiology, May 2000, p. 1772-1776, Vol. 38, No. 5
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A PCR-Colorimetric Microwell Plate Hybridization Assay for Detection of Mycobacterium tuberculosis and M. avium from Culture Samples and Ziehl-Neelsen-Positive Smears

Maria Cristina Rossi,* Andrea Gori, Gianguglielmo Zehender, Giulia Marchetti, Giulio Ferrario, Chiara De Maddalena, Lidia Catozzi, Alessandra Bandera, Anna Degli Esposti, and Fabio Franzetti

Institute of Infectious Diseases and Tropical Medicine, Luigi Sacco Hospital, University of Milan, Milan, Italy

Received 29 September 1999/Returned for modification 4 December 1999/Accepted 3 February 2000

Differentiation between Mycobacterium tuberculosis and M. avium is essential for the treatment of mycobacterial infections. We have developed an easy and rapid detection assay for the diagnosis of mycobacterial diseases. This is a PCR-hybridization assay based on selective amplification of a 16S rRNA gene sequence using pan-Mycobacterium primers followed by hybridization of the amplification products to biotinylated M. tuberculosis and M. avium-specific probes. A total of 55 mycobacterial isolates were tested. For all isolates, results concordant with those of conventional identification methods were obtained. Moreover, we developed a method for extraction of DNA from Ziehl-Neelsen-positive smears which allows the recovery of intact target DNA in our PCR-hybridization assay. Our method was able to confirm all culture results for 59 Ziehl-Neelsen-positive smears from clinical specimens (35 sputum, 11 lymph node biopsy, 6 stool, 4 pus, 2 urine, and 1 pericardial fluid specimens). These data suggest that our PCR-hybridization assay, which is simple to perform and less expensive than commercial probe methods, may be suitable for the identification of M. tuberculosis and M. avium. It could become a valuable alternative approach for the diagnosis of mycobacterial infections when applied directly to DNA extracted from Ziehl-Neelsen-positive smears as well.

* Corresponding author. Mailing address: Clinic of Infectious Diseases, Luigi Sacco Hospital, University of Milan, Via G.B. Grassi, 74, 20157 Milan, Italy. Phone: 39 02 35799676. Fax: 39 02 3560805. E-mail: andrea.gori{at}unimi.it.

Journal of Clinical Microbiology, May 2000, p. 1772-1776, Vol. 38, No. 5
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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