[The use of real time PCR for quantitative determination of some propionic bacteria residing on human skin].

A test system has been developed for determination of propionic bacterial species residing on human skin. This system developed in the real time PCR format is applicable for quantitative determination and also detection of genomes of the following Propionibacterium species: P. acnes, P. granulosum and P. avidum. This system was used for analysis of wash samples from the skin of 17 pentathlon sportsmen and 16 students. All three species of propionic bacteria were found in all skin wash samples. However, contamination with P. acnes was two times higher in control group than in the group of pentathlon sportsmen.

[The molecular genetic characteristic of RNA of human enterovirus detected in bio-test from child with serous meningitis].

The article considers molecular genetic characteristic of RNA of human enterovirus detected in bio-test from child with serous meningitis. The nucleotide sequence of genome DNA is analyzed. In 98% it is identical to corresponding nucleotide sequences of strains of human enterovirus A serotype 71 detected in China.

[The use of real time PCR for quantitative determination of some propionic bacteria residing on human skin].

A test system has been developed for determination of propionic bacterial species residing on human skin. This system developed in the real time PCR format is applicable for quantitative determination and also detection of genomes of the following Propionibacterium species: P. acnes, P. granulosum and P. avidum. This system was used for analysis of wash samples from the skin of 17 pentathlon sportsmen and 16 students. All three species of propionic bacteria were found in all skin wash samples. However, contamination with P. acnes was two times higher in control group than in the group of pentathlon sportsmen.

[The detection of RhD antigen of the rhesus system by the real time polymerase chain reaction].

A method for the detection of RhD using a DNA preparation and the newly-developed test-system based on the real time polymerase chain reaction is described. The study including a large variety of specimens has demonstrated the applicability of the novel system to forensic medical examination.

[The development of a test-system for the quantitative and qualitative evaluation of DNA content in criminalistic objects by the real-time polymerase chain reaction].

An original test-system for the preliminary quantitative and qualitative evaluation of isolated DNA is proposed by the polymerase chain reaction in real time (PCR-RT) based on the TaqMan technology. This test-system permits to simultaneously measure the amount of DNA in the sample, identify the genetic gender, and detect PCR inhibitors. The method has been approbated in the practical work of forensic medical experts.

[The diagnostic value of RNA oncomarkers in evaluation of malignant breast tumors].

The levels of the RNA oncomarkers, telomerase (hTERT), cytokeratin-19 (CK-19) and mammaglobin (MAM) have been investigated in capillary blood of female patients with mammary ductal carcinoma. The study revealed overexpression of all three factors in patients with this pathology. This overexpression was not found in healthy donors and female patients with mammary fibroadenoma. Levels of the RNA oncomarkers return to the normal level within 10 days after successful tumor resection. These results have been used for the development of diagnostic kits, which may be applicable for differential diagnostics, screening and postoperation monitoring of patients with malignant breast tumors

[Using real-time polymerase chain reaction for detection of Legionella in objects of the environment].

AIM: To assess efficacy of using the method of quantitative detection of Legionella in objects of the environment by real-time polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: For the development of the assay, genus-specific primers from gene coding 16S rRNAas well as species-specific primers for detection of Legionella pneumophila on the basis of mip gene sequence. For quantitative detection of L. pneumophila calibration samples of pGEM plasmid containing fragment of the mip gene in known concentration were used. Samples of water and biofilms obtained from cooling stacks of production plants, systems of autonomic water supply, humidification blocks of centralized systems of air conditioning were studied. RESULTS: Correlation of results obtained with RT-PCR and bacteriologic methods was shown during monitoring of potentially dangerous water objects as well as during epidemic outbreak of Legionella infection. Importance of samples preparation stage, during which considerable losses of DNA and inhibition of reaction could occur, is underlined. Disinfection measures on the studied objects significantly influenced on the results of the RT-PCR and can lead to false positive results. CONCLUSION: Obtained results confirm usefulness of testing of potentially dangerous water objects on the presence of Legionella based on the preliminary screening with RT-PCR for the 24 hours followed by bacteriologic testing of samples for 8 - 12 days.

[Polymerase chain reaction to determine and control drug-resistant Mycobacterium tuberculosis strains].

Real-time polymerase chain reaction was used to develop a one-stage procedure for molecular genetic analysis of Mycobacterium tuberculosis (MBT) DNA in order to determine mutations associated with drug resistance to the antituberculous agents: isoniazid and rifampicin. To analyze the spread of drug-resistance of the causative agent of tuberculosis in Russia, two thousand MBT strains were studied in 24 regions of all the federal districts. Testing 1406 MBT strains isolated by first detected and untreated patients revealed multidrug resistance (MDR) in 21.9% of cases. MRD was detected in 58.5% of the previously treated patients with MDR. The agreement of molecular genetic analysis of drug resistance with the results of cultural tests of 1096 strains was 94%.

[Synthesis of nucleotide derivatives containing 1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-2,3-propanediol for photoaffinity modification of proteins and nucleic acid]. / Sintez oligonukleotidnykh proizvodnykh, soderzhashchikh 1-(4-(3-(triftorometil)-3H-diazirin-3-il)benzamido)-2,3-propandiol, dlia fotoaffinnoi modifikatsii belkov i nukleinovykh kislot.

1-(4-(3-(Trifluoromethyl)-3H-diazirin-3-yl)benzamido)-3-O-(4,4'- dimethoxytrityl)-2,3-propanediol phosphoramidite was synthesized and used as a modified unit in the automatic synthesis of oligodeoxyribonucleotides. Pentadecathymidylates with various numbers of 2,3-propanediol moieties substituted with aryl(trifluoromethyl)diazirinyl (ATFMD) were obtained, and the thermal stability of their duplexes with (dA)15 were studied. One ATFMD-propanediol residue was shown to reduce the thermal stability of the duplex by 8-9 degrees C. The irradiation of the ATFMD-containing duplexes by UV light with the wavelength of 350 nm was found to cause the cross-linking reaction of the ATFMD-containing strand with the complementary strand and the formation of the cross-linked duplexes. The photomodification efficiency was independent of the oligonucleotide sequence, with each ATFMD group providing for 5% cross-linking. The irradiation of an ATFMD-containing duplex, a substrate of the HIV-1 integrase, in the presence of this enzyme resulted in the covalent DNA-protein complex. The oligonucleotides with the 1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-2,3-propanediol moiety in their chains can be used for the photoaffinity modification of both nucleic acids and proteins that recognize them. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

[Pyrimidine oligodeoxyxylonucleotides form triplexes with purine DNA in neutral media]. / Sposobnost' pirimidinovykh oligodezoksiksilonukleotidov obrazovyvat' tripleksy s pyurinovoi DNK v neitral'noi srede.

Interactions of oligonucleotides comprising (1-beta-D-2'-deoxy-threo-pentafuranosyl)thymine and (1-beta-D-2'-deoxy-threo-pentafuranosyl)cytosine residues (oligodeoxyxylonucleotides or OXNs) with complementary single-stranded DNA fragments were investigated. Using nondenaturing gel electrophoresis, footprinting, and melting assays, pyrimidine OXNs were shown to form triplexes with the purine DNA template, which are stable at neutral pH and comparable in heat stability with the corresponding natural polypurine-polypyrimidine DNA duplexes. In such triplexes, the N3 of cytosines in one of the OXNs are protonated. As revealed by CD spectroscopy in the 210-340 nm range, the form of the triple helix depends on the nucleotide composition and sequence of the DNA template, and is intermediate between A and B.

[Interaction of RNAase H from E. coli with modified hybrid duplexes. I. Duplexes modified at the carbohydrate residue]. / Vzaimodeistvie RNKazy H iz E. coli s modifitsirovannymi gibridnymi dupleksami. I. Dupleksy, soderzhashchie modifikatsii po uglevodnomu ostatku.

The influence of oligodeoxyribonucleotide probes containing 1-(D-beta-2'-deoxythreo-pentofuranosyl)thymine or 1-(D-beta-2'-deoxy-2'-fluoro-pentofuranosyl)uracil on the ability of the hybrid duplexes to interact with RNase H from E. coli was studied. A kinetic approach was used to measure of the modification effect. The hybrid duplex, prA18/d(TTflU)6TT, was shown not to interact with RNase H, whereas prA18/d(xTTT)6 inhibited the RNase H activity (Ki = 0.67 mkM). The thermostability of the modified duplexes was estimated. The present technique may lead to the use of some modified oligonucleotides as antisences.