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ENVIRONMENTAL SPECIFIC AND NON- SPECIFIC INDICATION OF MICROORGANISMS

E. N. Chramov

State Research Center the Federal State unitary enterprise State scientifically - research institute of biological instrument of the RF Ministry of Industry, Science and Technology

The problem of biomonitoring at threat of the terrorist attack defines necessity of development of high-sensitivity and selective methods of indication of microorganisms and creation on that basis technical means, suitable for protective measures.

It is known, that reliability of protection of the population with other things being equal is in direct dependence on efficiency of each of parts of biological protection (BP): the biological control, emergency preventive maintenance (common and special), isolation-restrictive and other measures on liquidation of consequences of application of the bioagents, continuity, coordination of actions of divisions and parts of ABC (anti-Atomic, Biological, Chemical defense) - Ministry of Emergency Situations, Home office, armies ABC , divisions of a medical service of Ministry of Defense, Ministry of Health and others.

Prime tasks facing to the biological control, are detection of the fact of application of the bioagent, establishment of a nature the applied microorganism, borders of infection both moment of application and removal of means of protection.

In the usual system of the biological control now for the decision of a task of detection of the fact of application will carry out non- specific(NS) express analysis - such as: increase of a protein background, enzyme activity in analyzed sample of air, water, etc. On that stage samples a taken for subsequent specific indication(SI) of bioagent.

Generally BP includes:

From listed above the decisive importance belongs to the biological control. Received data form the basis for an estimation of biological conditions, preparation preventive measures against the bioagents with the indication of character, volumes and terms of performance in each concrete case, liquidation of consequences of its application.

Application of individual and collective means of protection, disinfecting, common and special preventive maintenance, or quarantine also will be carried out on the data of the biological control (non- specific biological indication and specific indication).

Thus it is obvious, that than those or other measures will be commissioned earlier, the more their efficiency, including economic.

On the other hand, operative use of separate elements of a complex of measures of protection from the bioagents should be carried out on the basis of the authentic and complete information on biological conditions.

From here essential feature of process ABC is, that all subsequent measures are carried out by results of the analysis of biological conditions. Thus the system of the biocontrol including NSI and SI, is the primary factor, which realizes potential opportunities of available means and methods of protection from the bioagents.

At the decision of a problem ABC it is necessary to mean, that potential danger of application of the biological agents some states now is kept, since there are no reliable mechanisms of the control behind observance of the Convention of 1972 and difficulty to differentiate works between the solved activity in the field of protection and researches on perfection of biological objects. Besides, recently special urgency was got by a problem of bioterrorism. The analysis of consequences of application biological weapon, carried out by the American experts, shows, that for cities with the population 100 thousand people the material damage can make from 470 million up to 22 billion USD depending on a kind used bioagent, the number of dead people can reach 35 thousand. Thus it is necessary especially to emphasize, that effective detection of the biological agents and accordingly organization of protection is possible at presence of the information about probable ways and means of application bioagents, that in itself is very difficult task.

Consider, that the most probable way of infection of objects at application bioagents is aerosol way of distribution. For maintenance of measures NSI and SI structure of system of the biocontrol should include the following complexes of means:

For coordination measures BP there should be also channels of communication for transfer to the automated control systems, and the hardware should have the necessary interfaces for transfer and reception of signals. The hardware should settle down on the appropriate vehicles ensuring conducting of the biocontrol, delivery of tests, realization of the analyses (intelligence machine ABC, helicopters, medical and sanitary - epidemic laboratories).

Now existing devices for BP (the ASP- detector and optical microscope MLD), being a material basis of existing system of the biocontrol, can not solve in sufficient volume all tasks facing BP. Proceeding from this, the creation of new more modern means is the important urgent task.

The development of high-sensitivity and specific (selective) methods of indication pathogenic microorganisms , and creation is necessary for the decision of tasks of biomonitoring on the basis of these methods of modern means.

At the same time, now there are no regulating documents and requirements to system of biological monitoring and its components.

At the same time at the end of 90-th the experts of the interested ministries and departments of the country together with Gos NII BP in the initiative order developed the project of a document under name " System of the general technical requirements. Means of system of biological and ecological monitoring of an environment ".

According to the given document depending on purpose and principle of action of a means of system of biological and ecological monitoring of an environment should include the following classification groups:

In offered system automatic detectors of biological aerosols should carry out the constant control (monitoring) air attached to Earth surface, providing detection biological aerosol in a cloud at passage it through a controllable point.

The automatic analyzers of biological means in a mode of periodic functioning should provide differentiation of biological objects which are taking place in an aerosol condition, on following taxonomic groups: viruses, ricketsia, bacterium (separately vegetative and capsulated forms), toxins of the bacterial nature.

The high-efficiency devices of biological aerosols are intended for selection from air of representative tests of the biological agents and their storage in conditions ensuring the maximal preservation of viability.

The automatic (semi-automatic) devices of indication of the biological agents should provide the express - indication (establishment of presence within the limits of taxonomic groups of microorganisms) biological agents and products of microbiological synthesis in samples selected by high-efficiency sampling devices of biological aerosols.

The equipment, devices, sets, complete sets both devices of detection and the identification of the biological agents should provide an establishment typical belonging of activators of infectious illnesses and bacterial toxins in primary and enriched tests of air, ground, waters and other objects of an environment.

Prime task for offered system and its elements is the establishment of the fact of biological infection.

For the decision of this task at the first stage will carry out non-specific detection, to which assign also selection of samples for SI. Further, at the second stage will carry out SI.

In our country for the first time in the world the development of a mean of detection bioobjects in an aerosol was carried out. Created ASP-detector is based on chemiluminescent method of the analysis. ASP-detects presence of bioaerosol in a bottom layer of an atmosphere on the basis of comparison of an electronic threshold of operation with an analytical signal registered with the help of the photoelectric multiplier during course of reaction of luminol chemiluminescence , catalyzed by aerosol particles having peroxidase enzymatic activity. The excess of amplitude of an analytical signal above an electronic threshold actually forms the basis for the notification about application bioagents.

Being by the most mass device of non- specific indication of biological objects, ASP-detector has a number of the following essential lacks lowering its efficiency on detection of the fact of application:
The limited spectrum of found out bioaerosols in view of a low level of peroxidase activity or its absence at a number(line) biological agents;

Deterioration of a threshold of sensitivity at detection in a dusty atmosphere.

It is necessary also to note, that continuous work within day needs four liters dispensing chemicals from a reagent kit device.

The further perfection of these devices is expedient for carrying out in a direction of reduction of the mass-dimensional characteristics and energy consumption at the expense of use of the newest technologies of detecting luminescence and light scattering of particles and modern electronic element base (photoreceptor device, compact semi-conductor laser systems of optical sounding and microprocessor engineering).

Detectors are supplied with sampling devices intended for selection of aerosol of the biological agents with the purpose of the subsequent analysis by methods to specific indication and realization of measures on biological protection.

For ASP-detector this device is executed as the built - in cyclone. The volumetric charge of air through a cyclone makes 160 … 220 l/mines. Aspirated particles have an average diameter more than 2,5 microns.

Now is developed high-efficiency sampling device, which design as an obligatory element includes the concentrator for a aerosol fraction sized from 1 up to 10 microns. Thus its productivity should make not less than 1,5-2 м3/mines.

The basic characteristics existing and developed of sampling devices are given in tab. 1.


Table 1. Aerosol Sampling Devices for Specific Indication

Type of a sampling device, principle of
work

PAB 20/50

Sampling device
to ASP-detector
Air sampling device to ASP detector
  Inertial sedimentation Centrifugal sedimentation Inertial sedimentation, concentration
Volumetric capacity,
l.min-1

20-50

160-220 1500
Efficacy of capture, % 85 80 85
Power consumption, W 40 80 250
Viability, % 20…85 40…60 20…85
Weight, kg 4,5 1…5,5 15…20

Alongside with development automatic detectors in 80-90-th Gos NII BP carried out development of remote devices of indication of biological aerosols. Based on a LIDAR principle the ground based complexes detected on distance up to several kilometers emissions of protein aerosol at the Kirishi and Svetlogorsk biochemical factories.

For not specific detection of biological objects in samples are used field portable kits. They are intended for the analysis of samples with the purpose of their primary sorting. The basic task at use of such kits - reception fast, during 10-15 mines, preliminary answer about presence and group belonging of biological materials.

For this purpose the set colorimetric and enzymatic reactions and biochemical tests, characteristic for various groups of bioobjects is used.

With use of the specified reactions and tests Gos NII BP the reagent kits for the analysis of samples KSP-11 and KSAP were developed. The kits are intended for detection and group indication of microorganisms - activators of infectious diseases in field conditions.

Reagent kits include : filter papers, moistening solutions, reagents, solvents, tools and the materials provide selection, preparation and realization of the analysis of unknown sample and microorganisms..

The complete set KSAP provides partial group indication of microorganisms (with reference of found out microorganisms to viruses-ricketsiae and vegetative bacterial groups) in objects of an environment in an interval of temperatures from a minus 20 up to 30 0С. The complete set is served by one operator.

Modernization of a complete set KSAP in a direction of expansion of its functionality's on reference of determined microorganisms in addition to virus and bacterial capsulated forms now will be carried out.

Now for the decision of tasks of SI the following methods and means of the microbiological express train - analysis are used: a method fluorescing antibodies (MFA), reaction indirect haemoagglutination (RIHA), solids phase enzyme analysis (ELISA).

The sensitivity of MFA and RIHA makes 10 5 … 10 6 bacterial cells/ml.

SI assumes also biological enrichment and further research by MFA and RIHA methods. Laboratory animals used also, the researches procedure occupy about 2-3 day. At reception of the negative answer will carry out the complete microbiological analysis. It can occupy time about 36 days and more. Such terms of realization of the analysis any more are not acceptable now.

Basis for practically all methods used for SI, are immunochemistry. The insufficient efficiency of the classical biological analysis has served as driving force of development immunochemical and amplification (hybridization of nucleic acids) methods of SI and diagnostics of activators of infectious diseases.

In particular, in Gos NII BP the further development was received with methods immunofuorescent analysis of biomaterials. One of them - method universal polyphase concentration of complexes of the bioagents (viruses, microorganisms, toxins) with FITC labeled antibodies. The basic lacks of this method are: low stability to influence of the large concentration of interfering impurities and strong influence antibody purity on results of the analysis. These lacks mentioned to be overcome by use of solutions with various specific density as biphase system. Thus in a zone of equilibrium density the peak luminescence, caused by formation immune complex of the bioagent with fluorescent labeled Ig is observed. At the same time high concentration of impurity are allocated in one of phases according to their physical density and do not prevent right analyses.

The perfection of methods - indication on a basis solid phase luminescent immunoanalysis is directed on search of new luminescent labels and methodical receptions ensuring essential increase a signal / background ratio. The high level of sensitivity 10-13 … 10-14 М (in recalculation on concentration of a label) was achieved of use of labels having is abnormal long luminescence, and special way of cutting of a background short lived component. Such approach was realized in DELFIA (delayed lanthanide fluoroimmunoassay). As labels were used chelate compounds of Eu, Tb, Dy, Sm, covalent bound with antibodies. The given method well has recommended itself at diagnostics of virus infections. At the same time DELFIA has appeared insufficiently effective for realization of the high-sensitivity multicomponent analysis of bacterial infections. It is connected with the reduction of sensitivity of a method at use instead of Eu others rare earth ions as labels having weaker long-lived luminescence.

The molecular-genetic analysis has the greatest sensitivity from all known methods of express SI. The application of traditional fluorescent labels for detection of the bioagents with the help DNA - probes allows to reveal 102 - 103 bacterial cells in a sample . Amplification of a genetic material by a method of the polimerase chain reaction (PCR) of nucleic acids reduces this threshold up to individual cells. The development PCR methodology nowadays is examined as one of major directions for creation of sensitive and specific methods of indication and identification of bioagents not having equal among methods of laboratory diagnostics on sensitivity and specificity of the analysis.

At the same time, as has shown experience of practical application PCR in the set, created by us in 2001, KPBK-1U for express SI of activators essential restrictions of a method are its duration and false positive results from contamination by products of reaction. In this connection one of directions in perfection of PCR methodology for the purposes of SI are: development of luminescent probes of new generation allowing it is essential to lower number of amplification cycles. As well as in DELFIA, use is most perspective of long -lived luminescence labels on a basis lanthanide chelates or porfirine metalchelates.

Now in practical work the traditional techniques based on FITC -labeled antibodies dominate. At the same time the modern base luminescent microscopy allows using a small quantities of fluorolabels, differing on the spectral-luminescent characteristics, that can supply much more reliably identification of bioagents. The functionality's of immunoluminescent microscopy extends when long -lived luminescent labels are used and registration of a signal is made in a mode of phosphorometry with cutting off a background luminescence. The bi- photon excitation is also used.

Apparently, combination of modern technologies of the ultramicroanalysis, used in luminescent microscopy, such, as the laser excitation luminescence of DNA-probes with registration in a mode of bi-photone excitation, with computer recognition of images will allow to exclude amplification stages and to supervise presence of the bioagents in sample directly on a level of a signal of luminescence. However complexity and high cost of the equipment necessary for realization of such type of the analysis cause an opportunity of its introduction only at the large diagnostic centers.

One of directions in development of SI is the creation luminescent immunosensors. In this area the methodical approaches prevail the use of long -lived luminescent labels with registration in a time delayed mode. At the same time, the analysis given of the scientific and technical literatures shows, that semi-conductor, electrochemical immunosensors, capillary and optic-fiber imunoluminescent sensors now has not left a stage of scientific - methodical and technological development.


The submitted results testify to an opportunity of perfection of means of express SI on the basis of a combination of modern methods imunochemical and molecular-genetic analysis with the newest development in the field of creation of luminescent labels, engineering of registration of superweak light flows in a mode of the account of single photons and laser spectroscopy . The tasks of express SI of the biological agents now and in the nearest future, can not be solved with the help of one universal method.

For researches connected to necessity of fast detection of activators and toxins, optimal are homogeneous immunoassay and immunosensor technology.

For stationary laboratories the major importance is got by such characteristics of methods, as sensitivity, specificity, productivity, whereas the factor of time of the analysis is less important. In this situation the methods of nucleic acid hybridization and multicomponent solid phase immunoassay with registration of products of biospecific linkage in a mode of the delayed luminescence , mutually supplementing each other, apparently, will dominate above others.

As perspective method of indication of biological agents in an environment certainly it is necessary to recognize a method of the analysis on immunochromatographic test - strips with use as antibody labels colloid gold or graphite. In the given direction Gos NII BP the intensive researches are conducted.

Production-technological base for "dry" chemistry was created earlier. It was linked with the problem of test-strip diagnostics of sugar diabetes.

In the conclusion it is necessary to note, that the complexity of tasks and complex approach of problems of bioindication requires constant perfection of organizational structure, which in turn can be reformed basing on real successes in development of methods and means of indication. In view of the usual organizational structure and specific tasks of various departments it is possible to allocate a line of key directions in development of means of indication:

Thus it is necessary constantly to remember that despite of necessity of development of methods of diagnostics of infectious diseases, where as the role of the detector of bioagent human being plays, essential reduction of consequences acts at probable application biological agents, including bioterrorism, it is possible only duly detection of the given biological materials, that is reached by wide application of all means of indication.

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Proceedings of First Russian Workshop on Biological Security
Copyright © Committee of Scientists for Global Security and Arms Control