Development of the Amplificating Test-System and Equipment for Express-Diagnostics of Diphtheria by Using a Polymerize as Chain Reaction.
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
8 Project completed
Senior Project Manager
Tyurin I A
Research Center of Toxicology and Hygienic Regulation of Biopreparations, Russia, Moscow reg., Serpukhov
- Centers for Disease Control and Prevention (CDC) / National Center for Infectious Diseases / Meningits and Special Patogens Branch, USA, GA, Atlanta
Project summaryDespite of the preventive measures aimed at protection of the popu-lation against diphtheria has been performed, the levels of this infecti-ous disease in Russia and some countrys still remains reasonably high. At present diagnostics of diphtheria is based on microbiological and serologycal methods of examination of clinical material and as a rule it takes 3-5 days to make diagnosis of it. During this period diphtheria toxin can profoundly impair the patient state. As a result,further treatment would require essential efforts of the doctors as well as material inputs, that, unfortunately, does not always terminate with recovery of patient . т For diphtheria liquidation the discovery of germ-carriers is of great impotortence because they are mass and mobile source of dissemination and keeping of epidemic process. At present the discovery of diphtheria microbe carriers is labour-consuming and long.
All mentioned above, in our opinion, has proved the significance and actuality of both fast (for 6-8 hours ) diagnostics of diphtheria and sensitive method for discovery of diphtheria microbe carriers.
In the last years successful using the method of chain polymerase reaction ( PCR ) for express - diagnostics of a number of diseases has been shown. Analysis of the papers dealing with the application of PCRfor an express -- diagnostics of diphtheria has lead to the conclusion that there are no in Russia the developments as to application of PCR method for diagnostic of diphtheria. Among foreign researches are known the work that has been carried out with collection strains and clinical isolates of diphtheria microbe (M. J Fallen. J. Can. Pathol. , v. 44:1025-1026, 1991; M. J. Fallen et al. ,J. Clin. Pathol. ,v. 47: 353-356, 1994) In that work it was shown, that for identification of toxigenic strains it could be used the method of PCR ( duration of analysis is 5-6 hours ) rather than time-consuming (about 24 hours) Elik' s reaction. However, we have failed in looking for publications in which a method of PCR would be applied both to diagnose diphtheria directly with clinical material and to discovery of diphtheria microbe carriers.
The main restrictive factor to use the PCR-teehnology is the absence in Russia serial domestic equipment for PCR-diagnostics. The foreign analogues are expensive and are not available to most medical practice. Our amplificator under development differs from existing analogues in design feature so called "dry" block of the thermic chamber,that permits to lo-wer volume of amplified samples ( up to 10 microliter ) without use of a mineral oil and due to which all samples are in identical temperature conditions, permitting thereby to improve amplification and to lower the expenditure of chemicals. Fast heating and cooling of a block in each cycle and increased number of samples for amplification permit to shortened process time in a whole. Temperature levels, time of samples exposition for each temperature regime and required number of cycles are programmed by the operator through the keys on control panel and are displayed. Operation is automatically performed by microprocessor. Unlike from foreign analogues device under development will use any domestic type of a test-tubes due to much more heat power that is sended to test- tubes. It is supposed to develop for device the special software, that will set up automatically the optimum regime for detecting each concrete infection. The proposed system to process, analyse, and store medical data on PC is important one for performance on large scale analysis of samples in clinical practice. The purpose of the proposed project is the development of clinical express-diagnostics of a toxicogenic diphtheria microbe using the method of polymerase chain reaction ( PCR ), which is able to provide sensitivity of not less than 100 microbial cells and duration of analysis of about 8 hours.To achieve the objective it is necessary to solve the problems as follows:
- Based on available laboratory equipment in model experiments will be performed by using diphtheria toxin (DT) cloned gene to choose the approximate conditions for PCR and to evaluate specificity and sensitivity of the method of PCR at revealing a gene of diphtheria toxin;
- An appropriate laboratory equipment will be modified to adapt it to conditions to make the analysis by PCR method in clinical practice;
- It is supposed to develop technology of express-diagnostic of a diphtheria toxin by PCR method and to determine specificity and sensitivity of this method on collection toxicogenic and non-toxicogenic strains of a diphtheria microbe
- Using the equipment which has been adapted to clinical conditions, it is supposed to develop express-diagnostics of a diphtheria toxin in a clinical material taken both from sick person and supposed carrier of diphtheria microbe.
Technical approach and methodology:
Research activities under the project will involve the following methods : microbiological and serological methods for identification of a diphtheria microbe, immunofermental analysis, electrophoresis of proteins in polyacrylamide gel and nucleic acid electrophoresis in agarose gel, methods of polymerase chain reaction, chromathography, computer programs for choice of various variants of primers, various methods for separation of a DMA and various variants of reaction mixtures for PCR. In development of devices, automated systems of designing and methods of mathematical modelling by PC as well as standard measuring devices and modern microprocessor technology will be used.
PCR-amlification test-system that is intended for express-diagnostics (for 6-12 hours) of diphtheria under clinical conditions will be developed. Design and operation documentation on an equipment adapted to conditions of clinical laboratories will be provided as well as experimental specimens of the specified equipment will be produced.
After completion of research activities under the project, on the basis of the participating institution of the project (SRC TBP) it is supposed to set up manufacturing the develop test-system and equipment for making PCR analysis under clinical conditions, that supposes commercial significance of the project.