Gazouli, Maria and Liandris, Emmanouil and Andreadou, Margarita and Sechi, Leonardo Antonio and Masala, Speranza and Paccagnini, Daniela and Ikonomopoulos, John (2010) Specific detection of unamplified mycobacterial DNA using fluorescent semiconductor quantum dots and magnetic beads. Journal of Clinical Microbiology, Vol. 48 (8), p. 2830-2835. eISSN 1098-660X. Article.
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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. Cadmim selenite QDs conjugated with streptavidin and species specific probes were used to produce 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 on isolated bacteria produced the expected result in all cases. The minimum detection limit of the assay was defined at 12.5 ng of DNA diluted in a sample volume of 20 μl. In order to obtain an indication of the method's performance on clinical samples we applied the optimized assay to the detection of Mycobacterium tuberculosis in DNA isolated from bronchoalveolar lavage of patients with tuberculosis, and Mycobacterium avium subsp. paratuberculosis in faeces and paraffin embedded tissues, in comparison with culture, Ziehl-Neelsen stain and Real Time PCR. The concordance of these methods compared to the proposed with connection to positive and negative samples varied between 53.84% - 87.23% and 84.61%-100% respectively. The overall accuracy of the QD assay compared to Real Time PCR was 70-90% 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.
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