Sensitive detection of the IS 6110 sequence of Mycobacterium tuberculosis complex based on PCR-magnetic bead ELISA

Abstract

Tuberculosis (TB) is ranked as the top killer among infectious diseases worldwide. Early and accurate diagnosis of the disease is crucial to end the global TB epidemic. The current commercially available molecular tests are still unaffordable by most TB affected communities. Herein, we developed a novel rapid and sensitive diagnostic method to detect the IS6110 sequence of Mycobacterium tuberculosis (M. tuberculosis) complex using PCR-magnetic bead ELISA. PCR amplification ofa 123 bp repetitive sequence of the IS6110 gene was performed by using digoxigenin (DIG) and biotin-labelled primers. Streptavidin-conjugated magnetic beads were used to collect the dual-labelled amplicons and subsequently, colourimetric detection was done by using horseradish peroxidase (HRP)-conjugated anti-DIG antibody. This method is able to detect M. tuberculosis DNA down to 0.5 fg per reaction within 3 hours. The sensitivity of IS6110 PCR detection by magnetic bead ELISA is 100 times higher than that of conventional agarose gel electrophoresis. The assay specificity was determined using a panel of DNA extracted from 10 common bacteria causing lower respiratory tract infections. No cross-reactivity was detected from those bacteria by IS6110 PCR-magnetic bead ELISA. Thus, the novel highly sensitive and specific, reduced assay time and simplicity of this PCR-magnetic bead ELISA for the detection of the specific gene of M. tuberculosis complex makes it an attractive diagnostic tool for large-scale screening of tuberculosis in standard clinical laboratories.

Fig. 1. Schematic illustration of PCR-magnetic bead ELISA. The component enclosed in the dashed balloon was elaborated in the lower panel.

Fig. 2. Optimization of magnetic bead ELISA. Different concentration of magnetic beads (5, 10 and 15 μg) and PCR amplicons (5% and 10%) were incubated with 50 μl of anti-DIG-HRP (20 ng ml⁻¹) for 30 min and colourimetric detection was done to determine the optimal concentrations of magnetic beads and PCR amplicons. One nanogram of M. tuberculosis (H37Rv) DNA and molecular biology grade water were used as template in the PCR amplification of positive and negative control samples respectively.

Fig. 3. (A) Agarose gel electrophoresis of PCR amplicons obtained from 10-fold serial dilution of H37Rv DNA template starting from 0.5 ng to 5 ag (M = marker, 1 = NTC, 2 = 0.5 ng, 3 = 50 pg, 4 = 5 pg, 5 = 0.5 pg, 6 = 50 fg, 7 = 5 fg, 8 = 0.5 fg, 9 = 50 ag and 10 = 5 ag). The lowest detection limit of agarose gel electrophoresis (marked with *) is 50 fg which is 100 times higher than the 0.5 fg detection limit of magnetic bead ELISA (**). (B) An intensity plot obtained from triplicate analysis of agarose gel images. (C) Sensitivity testing of magnetic bead ELISA. The same set of PCR amplicons used in the agarose gel electrophoresis were tested by magnetic bead ELISA. Error bars were obtained from 3 independent experiments. The lowest concentration of M. tuberculosis DNA above the cutoff value was 0.5 fg. (D) A representative photograph showing the colour intensity of magnetic bead ELISA from various concentration of M. tuberculosis template and no template control (NTC).

Fig. 4. Specificity testing of (A) magnetic bead ELISA. PCR amplification was performed with 1 ng of DNA samples from each non-Mycobacterium bacteria and M. tuberculosis clinical isolate. Error bars were obtained from two independent experiments. (B) Agarose gel electrophoresis of PCR amplicons from specificity testing. M = Marker, 1 = NTC, 2 = M. tuberculosis, 3 = K. pneumoniae, 4 = S. pneumoniae, 5 = P. aerugniosa, 6 = E. coli, 7 = C. diphtheriae, 8 = A. baumannii, 9 = A. lwoffii, 10 = E. aerogenes, 10 = M. catarrhalis and 12 = S. aureus. IS6110 specific 123 bp amplicon was detected only in DNA sample extracted from M. tuberculosis.