Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles.
Mycobacterial infections have a high economic, human and animal health impact. Herein, we present the development of a colorimetric method that relies on the use of gold nanoparticles for fast and specific detection of Mycobacterium spp. dispensing with the need for DNA amplification. The result can be recorded by visual and/or spectrophotometric comparison of solutions before and after acid induced AuNP-probe aggregation. The presence of a complementary target prevents aggregation and the solution remains pink, whereas in the opposite event it turns to purple. The application of the proposed method on isolated bacteria produced positive results with the mycobacterial isolates and negative with the controls. The minimum detection limit of the assay was defined at 18.75 ng of mycobacterial DNA diluted in a sample-volume of 10 microl. In order to obtain an indication of the method's performance on clinical samples we applied the optimized assay to the detection of Mycobacterium avium subsp. paratuberculosis DNA in faeces, in comparison with real-time PCR. The concordance of the two methods with connection to real-time PCR positive and negative sample was defined respectively as 87.5% and 100%. The proposed method could be used as a highly specific and sensitive screening tool for the detection of mycobacteria directly from clinical samples in a very simple manner, without the need of high-cost dedicated equipment. The technology described here, may develop into a platform that could accommodate detection of many bacterial species and could be easily adapted for high throughput and expedite screening of samples.
Liandris
Emmanouil
E
Faculty of Animal Science, Laboratory of Anatomy-Physiology, Agricultural University of Athens, Iera Odos 75, 11855 Votanikos, Athens, Greece.
Gazouli
Maria
M
Andreadou
Margarita
M
Comor
Mirjana
M
Abazovic
Nadica
N
Sechi
Leonardo A
LA
Ikonomopoulos
John
J
D000818:Animals; D003124:Colorimetry / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D005243:Feces / Q000382:microbiology; D006041:Goats; D006046:Gold / Q000737:chemistry; D009161:Mycobacterium / Q000235:genetics / Q000302:isolation & purification*; D053758:Nanoparticles / Q000737:chemistry; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D006046:Gold