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Direct detection of mycobacterium tuberculosis complex using gold nanoparticles
Tuberculosis (TB) is still one of the most significant causes of morbidity and mortality worldwide. According to WHO, TB causes 2 million deaths and more than 9 million new cases annually; the overwhelming majority of TB cases occur in developing countries where accurate diagnosis of TB remains a ch...
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AUC Knowledge Fountain
2011
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| Summary: | Tuberculosis (TB) is still one of the most significant causes of morbidity and mortality worldwide. According to WHO, TB causes 2 million deaths and more than 9 million new cases annually; the overwhelming majority of TB cases occur in developing countries where accurate diagnosis of TB remains a challenge. This work aims to develop a rapid nano-gold assay for specific detection of mycobacterium tuberculosis complex (MTBC). In the first version of the assay, DNA was extracted from clinical isolates grown on LJ media. 16s rDNA regions were amplified by PCR then the genus and species of MTB were confirmed by semi-nested PCR. Spherical gold nanoparticles (AuNPs; 13 nm) were synthesized by citrate reduction method of HAuCl4 and characterized by spectrophotometry and SEM. In the first assay, the 16srDNA amplicons were denatured (95 oC, 30 s) then allowed to anneal (48 oC, 30 s) with genus- and species-specific oligotargeters in a hybridization buffer contaning NaCl (40 nM). This was followed by the addition of unmodified AuNPs (14 nM). In case of a positive specimen, the AuNPs aggregated and the solution color changed from red to blue. The solution retained red color in case of negative specimen. This assay was further optimized to specificially differentiate MTBC from other mycobacterial strains. In the second version of the assay, MTBC was directly detected in the extracted genomic DNA. Species-specific oligotargeter was added to genomic DNA and denatured for 3 min at 95 oC followed by annealing at 48 oC for 1 min. AuNPs were added and solution color changed from red to blue in case of MTBC-positive specimens. The assay detection limit was 1 ng for PCR product and 40 ng for genomic DNA. The assay showed 100% sensitivity and specificity (n = 27) as compared with automated liquid culture system (MGIT) and semi-nested PCR. Following DNA extraction according to standard procedures, the assay turnaround time is about 1 hour. In conclusion, we have developed a nano-gold assay prototype for direct detection of MTBC as a low cost alternative to current amplification-based detection platforms. The developed assay is simple, sensitive, rapid, and shows a great potential in the clinical diagnosis of TB especially in developing countries with low resource settings. |
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