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

Abstract

ABSTRACT 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 Hae III-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.