[The detection and study of the dynamics of the count of nonculturable forms of Yersinia pseudotuberculosis in the environment by using the polymerase chain reaction]. / Vyiavlenie i izuchenie dinamiki chislennosti nekul'tiviruemykh form Yersinia pseudotuberculosis vo vneshnei srede pri ispol'zovanii polimeraznoi tsepnoi reaktsii.

On the basis of the polymerase chain reaction (PCR) a test system permitting the detection of Y.pseudotuberculosis in different environmental objects was developed. On a model approximating natural conditions the dynamics of the amount of these bacteria in sterile soil extract were studied. The comparison of the amount of Y.pseudotuberculosis, determined in terms of colony forming units and by the detection of their DNA marker fragment with the use of PCR, showed the possibility of the transition of Y.pseudotuberculosis cells into the noncultivated state during their persistence in the aqueous soil extract.

[Quantitative PCR-analysis: development of a system for determining the level of an amplified DNA fragment]. / Kolichestvennyi PTsR-analiz: razrabotka sistemy opredeleniia soderzhaniia amplifitsirovannogo fragmenta DNK.

A new technique is elaborated for quantitative evaluation of the polymerase chain reaction (PCR) results based on a system for yopA gene identification in Yersinia pseudotuberculosis fragment located on p45 plasmid. The analysis schedule includes amplification of the studied DNA sequence under the conditions when the reverse primer carries chemically bound biotin label on the 5'-end, hybridization of the labelled amplified fragment with the probe immobilized on the microplate surface, the probe being complementary to the inner portion of specific DNA, visualization of the label, and calculation of bound labelled DNA quantity. The lower threshold of analysis sensitivity is some picograms. It makes possible the analysis and detection of PCR product in the period of exponential increase of its content, i.e. in the initial period of reaction when the quantity of amplified DNA in the reaction mixture is not enough for reliable identification by ethidium bromide staining.

[Use of the polymerase chain reaction for studying the processes of Salmonella typhimurium cells to an noncultivated state]. / Ispol'zovanie metoda polimeraznoi tsepnoi reaktsii dlia izucheniia protsessa perekhoda kletok Salmonella typhimurium v nekul'tiviruemoe sostoianie.

The possibility to identify noncultivating forms of Salmonella by the polymerase chain reaction (PCR) has been shown. To do it the technique for Salmonella identification was elaborated, based on amplification of a 500 bp fragment of araC gene. Time course of populations of two Salmonella typhimurium strains during prolonged incubation in water was studied by the techniques of serial dilutions on solid nutrient media, acridine orange staining, and PCR. The strains differed in pathogenicity levels and genetic characteristics. Cells of nonvirulent strain were shown to loose gradually in the process of incubation the ability to grow on solid nutrient media and transform into noncultivating forms whose ability to proliferation can be restored under definite conditions. No difference in the dynamics identified by PCR or traditional microbiological techniques was found for population of virulent Salmonella typhimurium incubated in water indicating the possibility of fast degradation of cells having lost the ability to divide.

[Test-systems of polymerase chain reaction for determining the tuberculosis pathogen]. / Test-sistemy na osnove polimeraznoi tsepnoi reaktsii dla identifikatsii vozbuditelia tuberkulëza.

Two polymerase chain reaction-based test systems were used to identify Mycobacteria tuberculosis by using a clinical material for practical health purposes. The former test system strictly specifically enables M. tuberculosis to be detected. The latter also allows one to differentiate a BCG vaccine strain from the M. tuberculosis. The test systems were tested by using 11 clinical sputum samples, three of them were taken from patients with fibrocavernous tuberculosis, two from those with focal tuberculosis and one from that with infiltrative one, 5 from those with non-specific pulmonary diseases (chronic bronchitis and asthma). In addition, one serum sample from a patient with fibrocavernous tuberculosis was examined. All tests obtained from patients with tuberculosis were positive, whereas those from patients with non-specific pulmonary diseases were negative. This suggests that differentiation of BCG vaccine strain from M. tuberculosis can be used both in model experiments and in the study of clinical material lysates.

[A polymerase chain reaction method for studying host persistence of pathogenic leptospira]. / Metod polimeraznoi tsepnoi reaktsii v izuchenii gostal'noi persistentsii patogennykh leptospir.

Polymerase chain reaction has for the first time been shown to be applicable to indication of Leptospira interrogans in the organs of infected animals with acute or chronic leptospirosis (on the model of golden syrian hamsters). Polymerase chain reaction is superior to microscopic and bacteriological analyses in identification of leptospirae in organ suspensions. The sensitivity of the technique is 1-10 cells per sample in studies of kidney or brain suspensions or 100-1000 cells in studies of liver suspensions.

[Development of a test system for detecting Leptospira interrogans using the polymerase chain reaction]. / Razrabotka test-sistemy dlia vyiavleniia Leptospira interrogans metodom polimeraznoi tsepnoi reaktsii.

Based on polymerase chain reaction a test-system has been elaborated permitting one to identify the leptospirae of the most common serogroups (Icterohaemorrhagiae, Canicola, Javanica, Ballum, Pyrogenes, Pomona, Habdomadis, Sejroe, Tarassovi) of the species Leptospira interrogans. Sensitivity of the technique is 1-10 cells in a sample. The specificity of the system has been shown to depend on the temperature of the primers annealing. The elaborated system exceeds all other systems for leptospiral identification in sensitivity. It is prospective for leptospiral identification in biological liquids aimed at early diagnosis of leptospiroses and in the studies of leptospiral persistence in host organisms in the saprophitic phase of life cycle.

[The use of a polymerase chain reaction method for the diagnosis of Mycoplasma pneumonia]. / Primenenie metoda polimeraznoi tsepnoi reaktsii dlia diagnostiki mikoplazmennoi pnevmonii.

The assay system for the diagnosis of Mycoplasma pneumoniae infections, based on polymerase chain reaction (PCR), has been developed. The sensitivity of the detection of M. pneumoniae DNA in analysis of different clinical samples has been established. The study has shown the possibility of using the newly developed assay system for the detection of M. pneumoniae in the material obtained from patients with atypical forms of pneumonia and from Mycoplasma carriers.

[Design of a chromosomal DNA probe species specific for Yersinia pestis]. / Konstruirovanie vidospetsifichnogo dlia Yersinia pestis khromosomnogo DNA-zonda.

A 14.8 kb DNA fragment from the chromosome of Yersinia pestis TWJ was cloned and the restriction map constructed. The fragment designated as T16 and its subfragments were tested in dot-hybridization with strains of Yersinia genus and other members of Enterobacteriaceae. A species-specific DNA probe (designated MK) was constructed on the basis of the T16 fragment. As judged from restriction analysis, blot-hybridization experiments and, partially, sequencing, significant homology exists between the MK DNA probe and this one developed by Bardarov et. al. (1990). A repeated sequence in two copies was discovered in the MK fragment.

[Self-inactivation of NADH-oxidase from Mycoplasma cells during reaction]. / Samoinaktivatsiia NADH-oksidazy kletok mikoplazmy v protsse reaktsii.

The feasibility of self-inactivation of NADH-oxidase by plasma membranes of Acholeplasma laidlawii cells was investigated. It was demonstrated that the rate of NADH oxidation in a flow reactor upon stirring diminishes with time. This decrease of the reaction rate is not coupled with the presence in the reaction mixture of the reaction products--NAD+ and H2O2, and is irreversible. In the absence of NADH the enzyme activity is unaffected. The data obtained suggest that NADH-oxidase is inactivated in the course of the catalytic reaction. Under the stipulation that the enzyme obtained from plasma membranes of aged (60 hrs of inoculation) cells has identical values of Km and ki but lower values as compared to young cells (24 hrs of inoculation) of Vmax and v0, it is concluded that the decrease of the NADH-oxidase activity upon ageing of cultures is due to the decrease in the amount of active molecules of AND-oxidase in mycoplasm cell plasma membranes.

[Dependence of membrane protein turnover in Mycoplasma cells on the age of the culture]. / Zavisimost' obnovleniia membrannykh belkov kletok mikoplazm ot vozrasta kul'tury.

To understand the molecular mechanisms of damages appearing in biological membranes in the process of cellular aging, changes in the rate of catabolic processes in Mycoplasma cells have been studied. This study has revealed that the aging of Acholeplasma laidlawii culture is accompanied by a decrease in the activity of such catabolic enzymes as DNA-ase, RNA-ase, cathepsin D and beta-glucosidase. A considerable increase in the duration of the half-life of membrane proteins has been registered, which is indicative of a decrease in their turnover rate. The electrophoretic separation of membrane proteins has revealed essential changes in their properties. Such decline in the functional activity of the plasma membrane of Mycoplasma cells at the stationary phase is probably due to the inactivation of membrane enzymes and to the decreased rate of their turnover.