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Journal of Clinical Microbiology, December 2002, p. 4705-4712, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4705-4712.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Novel Algorithm Identifies Species in a Polymycobacterial Sample by Fluorescence Capillary Electrophoresis-Based Single-Strand Conformation Polymorphism Analysis

Tomotada Iwamoto,1* Toshiaki Sonobe,1 and Kozaburo Hayashi2

Department of Bacteriology,1 Department of Parasitic Agents, Kobe Institute of Health, 4-6 Minatojima-nakamachi, Chuo-ku, Kobe 650-0046, Japan2

Received 10 June 2002/ Returned for modification 5 August 2002/ Accepted 17 September 2002

An algorithm to directly identify multiple mycobacterial species in a sample by using fluorescence capillary electrophoresis (FCE)-based single-strand conformation polymorphism (SSCP) analysis was developed. Part of the 16S-23S ribosomal DNA internal transcribed spacer (ITS) region in 37 reference strains and 73 clinical isolates representing 19 mycobacterial species and Mycobacterium tuberculosis complex was PCR amplified with a fluorescence-labeled mycobacterium-specific primer, 6-carboxyfluorescein-labeled primer Sp1f, and 5-hexachlorofluorescein-tagged Sp2r. FCE-SSCP analysis was applied to both undigested PCR products and the corresponding HaeIII-digested restriction fragments (RF) from each strain. The 23 resultant SSCP patterns distinguished all 19 species and M. tuberculosis complex. The technique is applicable for the detection of multiple mycobacterial species in a sample. It was demonstrated by analyzing two model mycobacterial communities consisting of five species with both rapidly and slowly growing species (model A) and four species commonly encountered in clinical practice (model B). The sensitivity study with spiked sputum samples with different amounts of M. tuberculosis H37Rv, M. avium, and M. intracellulare cells indicated that up to 25% of the amount of each mycobacterium could be detected relative to the two other species. Fifty-one sputum specimens analyzed by FCE-RF-SSCP were compared with the Amplicor assay (Roche Diagnostics GmbH). Species identified by both assays were always the same. Moreover, FCE-RF-SSCP could identify M. abscessus and M. kansasii, which are not targeted by Amplicor. FCE-RF-SSCP of sputum obtained from a patient with mixed M. avium and M. intracellulare infection gave SSCP patterns corresponding to these two species.

* Corresponding author. Mailing address: Department of Bacteriology, Kobe Institute of Health, 4-6 Minatojima-nakamachi, Chuo-ku, Kobe 650-0046, Japan. Phone: 81 78 302 6251. Fax: 81 78 302 0894. E-mail: kx2t-iwmt{at}asahi-net.or.jp.

Journal of Clinical Microbiology, December 2002, p. 4705-4712, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4705-4712.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.





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