Date Published: July 18, 2017
Publisher: Public Library of Science
Author(s): Dheeraj Pal, Nongthombam Boby, Satish Kumar, Gurpreet Kaur, Syed Atif Ali, Julien Reboud, Sameer Shrivastava, Praveen K. Gupta, Jonathan M. Cooper, Pallab Chaudhuri, Aristides Docoslis.
Brucellosis is a bacterial disease, which, although affecting cattle primarily, has been associated with human infections, making its detection an important challenge. The existing gold standard diagnosis relies on the culture of bacteria which is a lengthy and costly process, taking up to 45 days. New technologies based on molecular diagnosis have been proposed, either through dip-stick, immunological assays, which have limited specificity, or using nucleic acid tests, which enable to identify the pathogen, but are impractical for use in the field, where most of the reservoir cases are located. Here we demonstrate a new test based on hybridization assays with metal nanoparticles, which, upon detection of a specific pathogen-derived DNA sequence, yield a visual colour change. We characterise the components used in the assay with a range of analytical techniques and show sensitivities down to 1000 cfu/ml for the detection of Brucella. Finally, we demonstrate that the assay works in a range of bovine samples including semen, milk and urine, opening up the potential for its use in the field, in low-resource settings.
Brucellosis is a bacterial disease caused by Brucella species (facultatively intracellular coccobacilli) and remains the world’s most common zoonoses. Ten species have so far been identified and out of these, four classical species (Brucella melitensis, Brucella suis, Brucella abortus and Brucella canis) have been reported to be responsible for human infection.[2,3] Because of its non-specific clinical features and the lack of efficient diagnostic methods, the number of undetected cases remains high. Brucellosis is also linked to a number of serious challenges in the economy of developing countries, including India. The control of brucellosis in humans first requires its control in animals, leading to large programmes of vaccinations. However, this strategy cannot succeed without the ability to detect the reservoir animals in a robust and efficient way, so that they can be removed from the herd. Given the local infrastructure around many farms in rural India, there is a need to be able to provide a test that is sensitive and which can be detected visually. For example, rural veterinary clinics often do not have access to essential lab equipment necessary to run current gold standard tests, while access to expertise (for the preparation of reagents, assay processing and result interpretation) can also be lacking.
Current techniques for the determination of Brucella are not wholly appropriate for the needs of rural counties, particularly in LMICs, where there may be reduced access to infrastructure. In this study, we have demonstrated a simple and sensitive AuNP based detection of Brucella spp. with high specificity. Our test relies upon the plasmonic colour change when specific DNA hybridization events occur at a metal nanoparticle, thus needing only visual inspection of the results. The hybridization reaction does not require more than a heater (no thermal cycling, no extensive control required) or technical expertise beyond the addition of the sample. In terms of cost and convenience, the assay can be implemented readily in the field. We show that this assay can be used for detection of bacteria from milk, semen and urine which are considered the most relevant samples for the diagnosis of reproductive diseases, including brucellosis.