Specific Detection of Unamplified Mycobacterial DNA by Use of Fluorescent Semiconductor Quantum Dots and Magnetic Beads

Abstract

Here we present the development of a specific DNA detection method using fluorescent semiconductor quantum dots (QDs) and magnetic beads (MBs) for fast detection of Mycobacterium spp., dispensing with the need for DNA amplification. Two biotinylated oligonucleotide probes were used to recognize and detect specific complementary mycobacterial target DNA through a sandwich hybridization reaction. Cadmium selenite QDs conjugated with streptavidin and species-specific probes were used to produce a fluorescent signal. MBs conjugated with streptavidin and a genus-specific probe were used to isolate and concentrate the DNA targets. The application of the proposed method to isolated bacteria produced the expected result in all cases. The minimum detection limit of the assay was defined as 12.5 ng of DNA diluted in a sample volume of 20 l. In order to obtain an indication of the method's performance with clinical samples, we applied the optimized assay to the detection of Mycobacterium tuberculosis in DNA isolated from bronchoalveolar lavage specimens from patients with tuberculosis and Mycobacterium avium subsp. paratuberculosis in DNA isolated from feces and paraffin-embedded tissues in comparison with culture, Ziehl-Neelsen staining, and real-time PCR. The concordance of these methods compared to the proposed method with regard to positive and negative samples varied between 53.84% and 87.23% and between 84.61% and 100%, respectively. The overall accuracy of the QD assay compared to real-time PCR was 70 to 90% depending on the type of clinical material. The proposed diagnostic assay offers a simple, rapid, specific, and cost-effective method for direct detection and identification of mycobacterial DNA in clinical samples.

Nanomaterials in the Prevention, Diagnosis, and Treatment of Mycobacterium Tuberculosis Infections

“…These hybrid detection systems utilize TB-specific molecular probes to aid in TB detection. [19] These probes include MYCO4 (specifically binds 23S rRNA in Mycobacterium), which were coated onto magnetic beads, and MAP3 probes that specifically recognize IS900 conserved sequence in Mycobacterium paratuberculosis, which were coated on sulforous acid chromium quantum dots. Following hybridization with target gene sequences, which were isolated from bronchoalveolar lavage (BAL) samples, the resultant quantum dot-magnetic bead conjugate was then exposed to ultraviolet (UV) light, emitting red fluorescence visible to the naked eye.…”

Nanodiagnostics for Tuberculosis

“…Gazouli and coworkers developed and evaluated a detection assay for specific DNA sequences combining fluorescent semiconductor QDs with magnetic beads allowing for a fast identification of two members of the Mycobacterium genus (M. tuberculosis and M. avium subsp. paratuberculosis) without the need for DNA amplification (Gazouli et al, 2010). The assay involves two biotinylated oligonucleotide probes to recognize and detect specific complementary mycobacterial target DNA through a sandwich like hybridization.…”

“…Given that the capture and detection probes of the QD assay are complementary to different genes of the mycobacterial genome, the chances of false-negative results due to DNA fragmentation are inevitably increased. Nonetheless, this weakness can be circumvented by the use of a different set of DNA probes that anneal closer to each other, allowing an assessment that minimizes falsepositive results associated with low specificity (Gazouli et al, 2010). Additionally, the method avoids the drawback of PCR-based diagnostic assays that are prone to falsenegative results generated by inhibitors commonly found in clinical samples such as feces.…”

Molecular Biomarkers for Ancient Tuberculosis

“…Quantum dots conjugated with streptavidin and specific probes are used to produce a fluorescent signal. Magnetic beads, conjugated with streptavidin and a genusspecific probe are used to isolate and concentrate the DNA targets (Gazouli et al, 2010). Surface primer extension reactions may also be used to quantitatively detect unamplified, double-stranded genomic DNA (Martins et al, 2010).…”