Simple microplate hybridization assay for detection of amplified products of Mycobacterium tuberculosis

We describe a simple microplate hybridization assay for the rapid detection of the IS6110 PCR products of Mycobacterium tuberculosis from clinical cultures and from sputum specimens. The assay is based on the specific detection with a fluorescein-labeled detection probe of biotinylated PCR products which are captured on avidin coated microplate. Hybridized products with fluorescein were identified by using anti-fluorescein antibody, horseradish peroxidase conjugate and colorimetric peroxidase substrate. The specificity of the assay was assessed by analysis of 56 bacterial strains: the assay discriminated perfectly between the positive and negative groups when an OD490 of 0.18 was used as the cut-off point. The assay was sensitive enough to detect as little as 1 pg of M. tuberculosis H37Rv DNA, which is equivalent to approximately three bacilli. To evaluate the assay performance clinically, 190 sputum samples from newly diagnosed TB patients were tested; 79 were classified as TB positive, and 111 were classified as TB negative by culture and acid-fast staining as the 'gold standard'. The sensitivity, specificity and accuracy of the PCR-microplate hybridization assay were 90, 100 and 96%, respectively. The total assay time of hybridization following the PCR was 4 hours. The PCR-microplate hybridization assay is fast, simple and accurate and is suitable for use in the microbiology laboratory or for the analysis of large numbers of samples.