Detection of Mycobacterium tuberculosis in clinical samples using insertion sequences IS6110 and IS990.

To detect Mycobacterium tuberculosis in clinical samples, we used the M. tuberculosis-complex specific insertion sequence IS990 as the target in a simple DIG-PCR ELISA assay, as this element is present as a single copy in all strains of M. tuberculosis we have examined to date. The IS990 test was compared with a similar PCR that utilizes IS6110 as target. For detection of PCR product, digoxigenin-11-dUTP (DIG-dUTP) was incorporated into the product. After amplification, the PCR product was hybridized with biotinylated capture probe, which was complementary to the inner part of the amplicon. The hybrid was captured onto streptavidin-coated microtiter plate and DIG-labeled PCR product was detected using a peroxidase-conjugated antibody to DIG. We evaluated DIG-PCR ELISA for the detection of M. tuberculosis DNA in 265 respiratory and non-respiratory specimens taken from patients with known and suspected tuberculosis disease or from controls. The sensitivity and specificity of both IS990-based test and IS6110-based test was 96.5% and 95.3% respectively, comparable to the sensitivity and specificity of the IS6110-based test. The results demonstrate that the IS990 PCR ELISA test is a rapid and sensitive tool for the detection and identification of M. tuberculosis in clinical samples, and may have advantages to the more widely used IS6110-based tests, particularly in areas where IS6110-negative strains are found.

[Detection of tubercle bacilli in clinical samples using genetic methods(PCR)compared with Lowenstein-Jensen culture methods]. / Wykrywanie pratków gruzlicy w materialach klinicznych przy pomocy metody genetycznej (PCR) w porównaniu z metoda hodowli na podlozu löwensteina-jensena.

The aim of this work was to compare data obtained using PCR assay for detection of Mycobacterium tuberculosis with culture. We report a test for detection of tubercle bacilli by PCR and identification at species level by capture plate hybridisation and enzyme-linked immunoassay. 222 clinical samples obtained from patients with confirmed and suspected tuberculosis were analysed. These specimens were tested parallelly by conventional culture on Löwenstein-Jensen slants and PCR test. For 205 samples a complete agreement between these methods was observed.