PCR-enzyme-linked immunosorbent assay and partial rRNA gene sequencing: a rational approach to identifying...

A PCR-enzyme-linked immunosorbent assay (ELISA) for amplification and rapid identification of mycobacterial DNA coding for 16S rRNA was developed. The PCR selectively targeted and amplified part of the 16S rRNA gene from all mycobacteria while simultaneously labelling one strand of the amplified product with a 5' fluorescein-labelled primer. The identity of the labelled strand was subsequently determined by hybridization to a panel of mycobacterial species-specific capture probes, which were immobilized via their 5' biotin ends to a streptavidin-coated microtiter plate. Specific hybridization of a 5' fluorescein-labelled strand to a species probe was detected colorimetrically with an anti-fluorescein enzyme conjugate. The assay was able to identify 10 Mycobacterium spp. A probe able to hybridize to all Mycobacterium species (All1) was also included. By a heminested PCR, the assay was sensitive enough to detect as little as 10 fg of DNA, which is equivalent to approximately three bacilli. The assay was able to detect and identify mycobacteria directly from sputa. The specificities of the capture probes were assessed by analysis of 60 mycobacterial strains corresponding to 18 species. Probes Avi1, Int1, Kan1, Xen1, Che1, For1, Mal1, Ter1, and Gor1 were specific. The probe Tbc1 cross-hybridized with the Mycobacterium terrae amplicon. Analysis of 35 strains tested blind resulted in 34 strains being correctly identified. This method could be used for rapid identification of early cultures and may be suitable for the detection and concurrent identification of mycobacteria within clinical specimens.