Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles Emmanouil Liandris a , Maria Gazouli b , Margarita Andreadou a , Mirjana Čomor c , Nadica Abazovic c , Leonardo A. Sechi d , John Ikonomopoulos a, ⁎ a Faculty of Animal Science, Laboratory of Anatomy–Physiology, Agricultural University of Athens, Iera Odos 75,11855 Votanikos, Athens, Greece b Department of Biology, School of Medicine, University of Athens, Athens, Greece c Laboratory for Radiation Chemistry and Physics, Vinča Institute of Nuclear Sciences, Belgrade, Serbia d Dipartimento di Scienze Biomediche, Sezione di Microbiologia Sperimentale e Clinica, Universita` degli Studi di Sassari, Sassari, Italy abstract article info Article history: Received 24 February 2009 Received in revised form 1 June 2009 Accepted 4 June 2009 Available online 17 June 2009 Keywords: Mycobacteria 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 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 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. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Pathogens of the genus Mycobacterium are a major cause of morbidity and mortality worldwide. Concerning humans, in addition to tuberculosis, exposure to mycobacteria has been linked to the pathogenesis of sarcoidosis and Crohn's disease which affect millions of people in Europe alone (Gazouli et al., 2005; Sechi et al., 2001). Non- human primates can also be infected by Mycobacterium tuberculosis (Lin et al., 2008), while cattle and other ruminants are natural hosts of the closely-related species M. bovis, which causes substantial financial loss in many regions worldwide (Serrano-Moreno et al., 2008). The M. avium complex affects birds and mammals, including both livestock and dogs (Primm et al., 2004). Finally, paratuberculosis is another chronic infectious disease caused by a member of Mycobacterium spp., namely M. avium subsp paratuberculosis (MAP) that affects mainly ruminants. Diagnostic investigation of mycobacterial infections is hampered by the difficulty to detect in a specific manner low populations of mycobacteria, or the immunology markers associated with the infec- tions they cause. The laboratory diagnostic investigation of mycobacter- ial diseases relies on light microscopy of hematoxylin–eosin (H&E) or Ziehl–Neelsen (ZN) stained sections, mycobacterial culture, ELISA test and DNA amplification techniques. Each of these methods has certain advantages and limitations but in general, those with high specificity and low minimum detection limit are usually complex and expensive (Bancroft and Stevens, 1990; Brisson et al., 1991; Cousins et al., 1992; Pandey and Talib, 1993; Ravva and Stanker, 2005). In most cases the reliable application of these diagnostic methods requires highly trained personnel and very often, dedicated equipment that can be of considerable cost. Therefore, the development of a new diagnostic assay that would be easily applicable even by non-specialized personnel, and would allow specific and sensitive detection and identification of mycobacteria directly from clinical samples without the need for high- Journal of Microbiological Methods 78 (2009) 260–264 ⁎ Corresponding author. School of Animal Science, Department of Anatomy– Physiology, Agricultural University of Athens, Iera Odos 75, 11855 Votanikos, Athens, Greece. Tel.: +30 2105294383. E-mail address: ikonomop@aua.gr (J. Ikonomopoulos). 0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.06.009 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth