Objective – development and production of domestic PCR test kits for detection and differentiation of Brucella species in Kazakhstan according to their generic and specific markers.
Expected results:
1. Development of primer system for detection of generic and specific genetic markers of bacteria belonging to Brucella genus;
2. Construction of test kits for detection, identification and differentiation of Brucella species by the method of PCR in multiplex mode;
3. Obtaining of industrial batch of the developed PCR test kits.
Polymerase chain reaction (PCR) is one of the most effective methods for detection of infectious agents. The method has high sensitivity and specificity in comparison with most of the currently existing methods of diagnostics. The use of so-called "multiplex mode", i.e. simultaneous detection of two or more targets in the genome of a pathogenic agent, can increase the diagnostic accuracy and relevance of the PCR method.
Moreover, the efficiency of PCR diagnostics can be improved by applying of ready-to-use test kits, which are sets of reagents required to perform the PCR procedure, including a system of primers complementary to the marker (genus- and species-specific) regions of the genomes of infectious agents. An instruction is supplied with the test kit indicating the parameters of the amplification and the rules of reaction mixture preparation.
Application of test kits can reduce time of analysis and risk of mistakes, and makes the PCR tests more simple and standard.
These diagnostic sets for detection, identification and differentiation of bacteria belonging to Brucella genus will be developed and prepared for production in the Republic of Kazakhstan for the first time.
The tests will be based on detection and identification of genus-specific and species-specific genetic markers of bacteria belonging to Brucella genus.
The diagnostic preparations (PCR test kits) will be produced on the basis of the developed and synthesized primers by technology that meets the modern international standards. The present level of production and quality control of preparations ensure their relevance and competitiveness in the market of diagnostic products.
The developed PCR test kits will provide quick and precise detection, identification and differentiaton of bacterial agents causing brucellosis, as well as an intraspecific differentiation of Brucella populations circulating in Kazakhstan.
The following provisions substantiate the practical significance of the project:
1. Organization of an industrial base for production of primers and PCR test kits for detection, identification and genotyping of the causative agent of brucellosis;
2. Availability of professionals mastered in technology of production and in methodology of scientific, operational and pedagogical application of molecular-genetic research methods;
3. Improvement of preventive and anti-epidemic measures in foci of dangerous infectious diseases;
4. Improvement of biosafety system;
5. Development of curricula and training systems for specialists.
The project manager is a young scientist, PhD, who passed internships in a number of foreign institutions:
1. Jacob Blaustein’s Institute of Desert Research, Ben-Gurion University, Sde Boker, Israel. November 2005 – May 2007.
2. Department of Molecular Medicine of the Atomic Bomb Disease Institute, Nagasaki University, Japan. January 2009 and March 2010.
3. “Hygiene of Production and Microbiology” laboratory, V.M. Gorbatov’s All-Russian Research Institute of Meat Industry, Moscow, Russia. September 2013
4. “BioTeZ Berlin-Buch GmbH” oligonucleotide producing company, Berlin, Germany. September 2014 г.
5. Department of Microbiology and Immunology, University of Texas Medical Branch (UTMB), Galveston, USA. October-December 2014.
The researchers participating in the project have great experience in study of molecular and genetic aspects of epizootiology, epidemiology, laboratory diagnostics, and phylogenetics of especially dangerous infectious (EDI). This confirmed by their participation in various international programs and projects including those of ISTC:
ISTC K-1817 "Training on Biosafety and Biosecurity in the Kazakh Scientific Center for Quarantine and Zoonotic Diseases (KSCQZD)";
ISTC K-2081p "Design and manufacture of fluorescent antibodies for rapid plague diagnostics"; biological threat reduction program of the Ministry of Defense;
KZ-28 "Genetic features of especially dangerous zoonotic pathogens (Yersinia pestis, Francisella tularensis, Bacillus anthracis) of Kazakhstan";
TAP-6 project "Serodiagnostics of Anti-Plague Vaccinated People In Kazakhstan»;
TAP-1 project "Evaluating of the effectiveness of real-time PCR test kits";
ISTC K-2058r "Development and production of primers and PCR test kits for detection of Salmonella and Shigella".
Moreover, the members of the Project workgroup participate in international conferences and workshops, including the conferences on biosafety risk assessment, organized by the Biosafety Association for Central Asia & the Caucasus (BACAC).
Implementation of the proposed project will contribute to the development of international scientific and technical cooperation in the field of epidemiology and molecular genetics of brucellosis and create long-term prospects for the control of infection in the framework of the International Scientific Association.
The foreign collaborator of the project will be Yuriy Fofanov, PhD, Professor of the Department of Pharmacology and Toxicology, director of the Genomics and Bioinformatics Lab, Sealy Center for Structural Biology and Molecular Biophysics at University of Texas Medical Branch, Galveston, USA.
Duties of the foreign collaborator will include the following activities:
- Analysis of the technological process at every stage of work,
- Correction of the project work plan,
- Consultation on theoretical foundations, methodology and technology of the project,
- Participation in selection of generic and specific genetic markers of Brucella spp.,
- Training on the basic sections of the project implementation,
- Evaluation of the results of each project task,
- Control of work plan and schedule implementation,
- Final evaluation of the project plan implementation and the work of the project participants.
