Search

Periprosthetic infection (PPI) after arthroplasty of large joints is the third (among the main causes of unsatisfactory results of surgical treatment) a serious threat to the health of patients. The «gold standard» for the diagnosis of PPI is the bacteriological examination of samples of periprosthetic tissues and synovial fluid. In 10-30% of cases, it is impossible to isolate microorganisms, which is explained by the difficulty of cultivation and taking antibiotics before sampling. The purpose of study is to demonstrate the diagnostic value of PCR diagnostics for identifying the genetic material of an infectious pathogen of a culture-negative periprosthetic infection. Material of the study is a description of a clinical case of a culture-negative periprosthetic infection that caused a second two-stage revision of the hip joint prosthesis In the first episode of PPI that occurred 3 years after hip replacement, a microbiological examination of the puncture of the trochanteric zone of the operated joint revealed a massive increase in methicillin-resistant Staphylococcus epidermidis (MRSE). A two-stage revision joint replacement was performed. 5 years after the revision, the patient was hospitalized with clinical and radiological signs of PPI, while examining the puncture of the joint revealed characteristic PPI cytosis. Microbiological examination of punctate and intraoperative aspirate at the first stage of the repeated two-stage revision endoprosthesis replacement did not reveal aerobic and anaerobic microorganisms. In PCR studies, the DNA of methicillin-sensitive Staphylococcus aureus (MSSA) was detected in washouts from the removed components of the endoprosthesis; no resistance marker (mecA gene) was found. Given the concomitant oncological disease, this result determined the appointment of pathogenetic antibiotic therapy, the effectiveness of which was confirmed after 8 weeks at the II stage of revision. The PCR study of joint and trochanteric punctures (before surgery), flushing from the removed spacer components (after ultrasound treatment) and intraoperative aspirate from the joint did not reveal Staphylococcus aureus DNA and resistance marker (mecA gene). In some cases of periprosthetic infection, traumatologists and orthopedists deal with culturally negative results of a microbiological study of the patient's biomaterial and swabs from the components of endoprostheses in the presence of clinical manifestations of PPI, confirmed by laboratory diagnostics and X-ray examination. According to the literature, such clinical situations are observed in 10-30% of cases and are caused by previous antibiotic therapy in the early stages of an infectious complication. After surgical treatment of PPI for the selection of adequate antibiotic therapy, such patients need to at least indirectly determine the type of infection pathogen, which is achieved by the use of additional diagnostic methods, such as a PRC study. In the case described by us, after a course of antibiotic therapy, prescribed according to the results of the first PCR study, the patient's body does not contain DNA traces of the desired infectious agent. Thus, the repeated PCR not only confirmed the accuracy of the initial diagnosis of the source of infection, but also further illustrated the success of the rehabilitation of the periprosthetic infection using a correctly selected antibacterial drug at the previous stage of the study. The use of the PCR method made it possible to diagnose the pathogen and prescribe adequate antibiotic therapy for culture-negative periprosthetic infection., Competing Interests: The authors declare no conflict of interest.

Gastroenterocolitis caused by Campylobacter bacteria are the most common acute infectious zoonotic foodborne diseases. One of the important factors for the transmission of infection is contaminated dairy products, so the assessment of contamination of raw milk with Campylobacter is necessary to develop effective measures to suppress the growth of the pathogen and ensure the safety of products. The aim of the study was to assess the microbial characteristics of raw milk samples and the nature of their contamination with thermophilic bacteria of the Campylobacter genus. Material and methods . A total of 60 samples of raw milk from the central regions of the Russian Federation and 48 experimentally infected samples of raw milk were studied. To assess the microbial contamination of milk, the number of extraneous microflora was determined, including coliform bacteria (CFB). The identification and quantification of bacteria of the genus Campylobacter was carried out by cultural methods in comparison with quantitative PCR assay. For PCR, primers were used that detected the speciesspecific sequence of C. jejuni 16s rRNA, the presence of the cytotoxic toxin gene cdtB and the invasion gene ciaB. Results and discussion . A significant part of the samples of raw milk (31.6%) was characterized by high levels of microbial contamination exceeding 10 6 CFU/cm 3 . Gram-negative bacteria were the dominant type of bacterial microflora, their levels were comparable with the detected values of the total number of microorganisms. Coliform bacteria were found in all studied samples, and their content in 90% of the samples reached 10 5 CFU/cm 3 , and in some samples - 10 7 CFU/cm 3 . Campylobacter spp. detection rate in raw milk was 8.3%, and their number ranged from 0.1 to 100 CFU/cm 3 (average 2.0×10 CFU/cm 3 ). The isolated strains of campylobacters were identified as a C. jejuni species. In the study of the microbial background of the examined samples of raw milk, a comparative analysis of their contamination by campylobacters by rti-PCR was simultaneously carried out. The majority of samples (over 60%) were positive for the presence of 16s rRNA genomic sequence, and they were characterized by the highest values of total bacterial contamination and the amount of coliforms. The use of a multi-primer approach (simultaneous testing for the presence of 16s rRNA and the gene of cytoletal toxin cdtB C. jejuni) reduced the number of positive cases of Campylobacter DNA detection to 16.6%, which suggests a greater informative value of the cdtB gene for the detection of viable, including uncultivated cells. An indicative assessment of the results in a quantitative format showed levels of 10 4 -10 6.5 genomic equivalents of the DNA in 1 cm 3 , suggesting the possible presence of viable Campylobacter cells in the tested probes with a significantly greater frequency than that established by cultural method. Conclusion . At low levels of Сampylobacter contamination the microbiological methods do not provide reliable detection of the pathogen due to massive contamination of raw milk by extraneous microflora. Campylobacter spp. were detected by the culture method in 8.3% of cases, while the use of multi-primer PCR assay with cdtB and ciaB genes can double the detection of C. jejuni in raw milk samples., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

A modification of the enzymatic method for the preparation of combinatorial random DNA libraries, which combines amplification in isolated microvolumes with the simultaneous incorporation of modified nucleotides and subsequent separation of DNA strands, was developed. Deoxyuridine triphosphate with hydrophobic substituents such as structural analogues of amino acid side chains in the C5 position of the pyrimidine ring was used to introduce modifications into DNA. To prevent competitive amplification, which reduces the representativeness of combinatorial libraries, PCR in inverse emulsion was used. The separation of the strands of PCR products was carried out. There were six single-stranded DNA libraries with complete substitution of deoxythymidine via modified analogues with various functional groups. These DNA libraries are suitable for generating aptamers to protein targets through additional hydrophobic interactions from the introductions of appropriate modifications, and are completely compatible with the SELEX aptamer selection methodology.

Expert evaluation of genetically engineered organisms (GMO) identification methods is aimed at confirmation their adequacy with the tool and methodological base used in the institutions of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing to control market turnover and labelling of genetically engineered food. The primer system's specificity was experimentally confirmed in studies with other GM potato lines, as well as with the results of the BLAST-analysis. The efficiency, linearity and correctness of the method meet the requirements of the European Union Reference Laboratory for GM Food and Feed. Limit of detection and limit of quantification of GM potato line PH05-026-0048 genomic DNA were 0.019% (11 copies of the GM potato genomic DNA) and 0.06% (36 copies of the GM genomic DNA of the potato) per 100 ng of total DNA, respectively., (Copyright© GEOTAR-Media Publishing Group.)

The detection of somatic mutations in the 9 exon of the calreticulin gene (CALR) is regulated by the clinical recommendations as a diagnostic criterion for chronic Ph-negative myeloproliferative neoplasms (MPN). Some methods of nucleic acids testing are used to identify CALR gene mutations with different requirements for special skills of personnel and expensive equipment. The purpose of this work is to compare the results of the detection of CALR gene mutations in venous blood samples by allele-specific RT-PCR with subsequent electrophoresis, fragment analysis and Sanger- or pyro- sequencing. We used 1284 blood samples of patients with suspected MPN and 20 blood donor samples. Mutations in the CALR gene of the I and II type were identified using PCR-RT with the original primers and TaqMan probes. Also, all samples were tested for mutations in the CALR gene by electrophoretic detection of PCR results in an agarose gel. The use of allele-specific RT-PCR followed by electrophoretic detection made it possible to determine clinically significant mutations in the CALR gene in 81 venous blood samples of JAK2- and MPL-negative patients, including 42 cases of type I mutation, 33 cases of type II mutation and 8 rare CALR mutations. Mutations in the 9 exon of the CALR gene were not detected in any of the 20 blood donor samples or in 121 blood samples of patients with polycythemia vera. In randomly selected 20 negative samples, CALR gene mutations were also not detected using Sanger sequencing. All positive samples were confirmed by fragment analysis, as well as with Sanger- sequencing and pyro- sequencing. The described combined approach to detect mutations of the CALR gene in peripheral blood samples can be used in clinical diagnostic laboratories that have a standard set of equipment for electrophoresis of nucleic acids and a PCR-RT. We also propose a confirmatory test based on the pyrosequencing of DNA using the system of genetic analysis "PyroMark Q24"., Competing Interests: The authors declare no conflict of interest.

The results of development of a method for detection and genotyping of the bacteria Pasteurella multocida capsular five groups and Mannheimia haemolytica Al based on the multiplex polymerase chain reaction (PCR) with electrophoretic detection are submitted. Diagnostic sensitivity of the developed method was 103 CFU/ml in the study of the pure cultures and 105 CFU/g in the study of biological material. A study of 260 samples of biological material from infected animals revealed Pasteurella multocida in 50.0%, and Mannheimia haemolytica in 11.2% of the investigated samples. Circulation among the tested livestock of capsular groups B and E of Pasteurella multocida was not revealed. The majority of the tested samples contained group A, in some cases, group D, and, in one case, group F. On the basis of the phylogenetic analysis circulation of two different genetic types of Pasteurella multocida of the capsular group A was revealed.

Development of the genotyping methods of glanders agent is urgent due to its high pathogenicity, lack of effective preventive measures and threat of the use of Burkholderia mallei as a biological weapon. In this work we proposed a scheme for the typing of the B. mallei strains based on different region analysis (DFR). The choice of variable loci differentially presented in various strains of glanders agents was performed by analyzing annotated whole-genome sequences of the B. mallei strains. Primers and fluorescence probes were designed for 9 selected loci. The amplification conditions for different regions were optimized in two variants: with electrophoretic detection and hybridization-fluorescence detection in the strip format. The possibility of applying the DFR analysis to genetic characterization of strains was assessed in 14 B. mallei strains. The genetic profiles of the studied B. mallei strains revealed that the developed DFR-typing scheme was characterized by high discrimination power (Hunter-Gaston index value was 0.92), reproducibility, rapidity, easy interpretation, and applicability for epidemiological surveillance of glanders.

The article presents the results of development and practical implementation of system of polymerase chain reaction testing in real-time operation mode to detect agent of plague infield material. In laboratory conditions the system demonstrated good results and hence it was applied in conditions of field laboratory of epidemiologic team during planned epizootologic examination of Gorno-Altaisk hot spot of plague. The sampling consisted of more than 1400 objects. It was demonstrated that high sensitivity and specificity is immanent to proposed system. The adaptation of the system to the real time amplifier "Smart Cycler" (Cephid, USA) having some specific technical characteristics makes it possible to consider the proposed test-system as an effective sensitive and precise instrument for screening studies in the process of regular epizootologic examinations of hot spots of plague.

The article deals with the results of selection and testing of oligonucleotide primers to gene 16S pRN4 of a number of agents of community'-acquired pneumonia (Streptococcus pneumoniae. Haemorhilus influenzae. Moraxella catarrhalis, Klebsiella pneumonia. Pseudomonas aeruginosa. StaphyIococcus aureus) for their highly specified detection in clinical material on application of polymerase chain reaction. The comparison with bacteriological method (golden standard) is used to demonstrate the high sensibility, specificity and diagnostic effectiveness of product enabling to apply it in practice to diagnose communitly-acquired pneumonia regardless of duration, severity and phase of disease and to develop corresponding diagnostic systems.

A real-time fluorescent polymerase chain reaction was used to develop a RealBest HIV RNA kit that was clinically suitable for the detection of HIV-1 RNA and for the estimation of virus load in plasma and serum samples. Due to the selection of a highly conserved target region and to the experimental study of the impact of different primer-template and probe-template mismatches on RT-PCR with subsequent selection of the optimum oligonucleotide set, the developed assay can detect and measure the concentration of all subtypes of HIV-1, group M. The assay provides a high reproducibility and sensitivity and a wide dynamic range of virus loads (20 to 10 million IU/ml of plasma or serum).

There has been recently a rise in referrals for Ixodes tick bites in the spring and summer periods in the Kamchatka Territory. Among the dominant tick species, there has been the taiga tick Ixodes persulcatus habiting the extensive areas of the southern and central parts of the peninsula. Examination of 84 I. persulcatus females collected from human beings and domestic animals in 2003 to 2007 detected DNA of the pathogens of tick-borne borreliosis (B. burgdorferi sensu lato), rickettsiasis (R. tarasevichiae and R. helvetica), and Ehrlichiosis/anaplasmosis (A. phagocytophilum). Tick-borne encephalitis RNA and antigens and babesiasis DNA were not found in the study samples. Despite the small number of taiga ticks in Kamchatka, the detection of the pathogens of various infectious diseases in the ticks suggests that there may be a risk for contamination of the peninsula's population with these pathogens.

An electron microscopy study of large-sized DNA microparticles produced in PCR with different gene-specific primers and plasmid DNAs is described. DNA microspheres of two distinct types were revealed in the all studied samples, namely smooth moderately electron-dense microspheres, and highly electron-dense particles with large thorns and offshoots. Singular microspheres have the average diameter of 1 mum, and their aggregates were up to 3 mum in dimensions. In addition, rare so-called three-dimensional net-like structures with various size (up to several micrometers) were observed. They consisted of different amounts of DNA nanoparticles, having the special compact topology. In some studied samples the discs (nanodiscs) of several dozens nm in thickness and up to 3 mum in diameter were revealed. It was shown that the quantity of net-like structures and nanodiscs sharply increases in asymmetric PCR. We also observed DNA nanowires of different length and thickness, nanodots, nanoparticles in the form of shits of paper as well as electron-dense spherical nanoparticles of big size. Aqueous suspensions of DNA microparticles were heated at 94 degrees C for 5 min and analyzed by electron microscopy. It was shown that microspheres in heated suspensions underwent partial melting; they lost a part of DNA, therefore details of their structure (ultrastructure) can be recognized. At the some time numerous tangles of nanowires appeared. Molecular mechanisms of the DNA micro- and nanoparticles formation are discussed.

Phylogenetic and taxonomic relationships in the genus Marmota were examined using inter-SINE PCR. The primers used were complementary to the consensus sequences of two short retroposons, MIR and B1-dID. The results suggest long-term genetic isolation of Nearctic and Palearctic marmots, but do not support subgeneric subdivision because of relatively low genetic differences between the marmot groups. Confirmation was received for the isolation of bobak and camtschatica, but not the caudata intrageneric species groups. Based on comparison of the mitochondrial and nuclear genome differences, the possibility of ancient hybridization between M. menzbieri and M. caudata was recognized. Species independence of M kastschenkoi within the suggested superspecies of M. baibacina was supported.

The authors have studied the species-specific composition of major periodontopathogenic bacteria (P. gingivalis, P. intermedia, B. forsythus, A. actinomycetemcomitans, and T. denticola), by applying the Russian reagent kit developed and made by HPF "GENETECH", and some fungi, microorganisms, and viruses among the Moscow Region inhabitants with chronic generalized periodontitis in 2006-2008. The studies have shown it possible to use molecular genetic methods for diagnosis, drug therapy monitoring, and epidemiological studies in periodontology and implantology.

DNA barcoding was proposed as a method for recognition and identification of eukaryotic species through comparison of sequences of a standard short DNA fragment--DNA barcode--from an unknown specimen to a library of reference sequences from known species. This allows identifying an organism at any stage of development from a very small tissue sample, fresh or conserved many years ago. Molecular identification of plant samples can be used in various scientific and applied fields. It would also help to find new species, which is particularly important for cryptogamic plants. An optimal DNA barcode region is a small fragment present in all species of a major taxonomic group, having invariable nucleotide sequence in all members of the same species, but with sufficient variation to discriminate among the species. This fragment should be flanked by low-variable regions for use of universal primers in PCR for amplification and sequencing. The DNA barcode that is well established in animals is a sequence of a fragment of the mitochondrial cytochrome c oxidase gene CO1. However, searching for DNA barcode in plants proved to be a more challenging task. No DNA region universally suitable for all plants and meeting all of the necessary criteria has been found. Apparently, a multilocus or two-stage approach should be applied for this purpose. Several fragments of the chloroplast genome (trnH-psbA, matK, rpoC, rpoB, rbcL) in combinations of two or three regions were suggested as candidate regions with highest potential, but more representative samples should be examined to choose the best candidate. The possibility is discussed to use as DNA barcode internal transcribed spacers (ITS) of nuclear rRNA genes, which are highly variable, widely employed in molecular phylogenetic studies at the species level, but also have some limitations.

DNA microparticle formation in the course of a polymerase chain reaction (PCR) is reported. PCR with gene-specific and partially complementary primers and yeast genomic DNA as a template was shown to yield spherical DNA-composed microparticles as well as their aggregates and conglomerates, along with routine linear DNA. Microparticles were formed at late PCR stages and could be easily identified by the reaction with fluorescently labeled oligonucleotide primers or by staining of the PCR mixture with fluorescent dyes (acridine orange, propidium iodide or DAPI). According to the data of epifluorescent and electron microscopy, the microparticle size varied from 500 nm to 3-4 microm and the particles were multimeric star-shaped spheres or aggregates formed by several fused microspheres. Some properties of the microspheres were studied. It was found that the Mg2+ cations comprising the PCR buffer played a key role in the formation of microparticles and the stabilization of their structures.

The authors present the results of a comparative appraisal of a set of species-specific primers in the polymerase chain reaction (PCR) identification of typical and atypical plague pathogen strains. A hundred and seventy-three strains with a species specificity of Y. pestis and 67 heterologous strains were used to appraise the well-known Y. pestis chromosomal DNA-based primers: "3a", "yp2769ms06", "vlml2for/ISrev216", "vlm33/ISfor1754", as well as "JS" specific to Y. pseudotuberculosis". In some plague bacterial strains, the DNA sequences complementary to the primers "3a" and "yp2769ms06" were not found. All conceivable plague pathogen strains were detectable only with the primers "vlml2for/ISrev216" and "vlm33/ISfor1754". The primers "JS" recognized only the strains of pseudotuberculosis causative agent. The DNA of other microorganisms failed to react with none species of primers. For the effective detection and differentiation of typical and atypical Y. pestis strains, the authors propose PCR using a few pairs of primers with the merits of the primers of the group "vlm" and 'JS".

A test system for the diagnostics and identification of seven toxigenic fungi causing fusarioses of cereals (Fusarium graminearum, F. culmorum, F. poae, F. sporotrichioides, F. langsethiae, F. avenaceum, and F. tricinctum) was developed using PCR. The identification of pathogen is based on the specific amplification of a DNA fragment of the gene of translation elongation factor 1 alpha (tef-1alpha) and subsequent detection of the results by the fluorescent amplification-based specific hybridization method. The system was tested on 38 isolates of different fungi of the genus Fusarium.

Two tests for measurements of hepatitis B virus DNA in human serum and plasma, VERSANT HBV 3.0 (based on the use of branched DNA chains) and Biotitre-B (a real-time PCR variant), were compared. Serum specimens from patients (n=56) with documented viral hepatitis were tested. For specimens with DNA values in the linear range in both tests the correlations were evaluated by Pearson's method. The sensitivity of the two tests, and reproducibility of the results of HBV DNA measurements were evaluated using a panel of recombinant virus DNA dilutions with a step of 1 lg HBV DNA copies per ml (from 7 to 3 lg). Three measurements for each concentration of the reference sample were carried out in each test. Clinical specificity of the two tests was evaluated by the analysis of HBV-negative serum samples, collected from donors not reactive by HBsAg and anti-HBc (n=60). Of 56 samples with documented infection, the results of testing by VERSANT HBV 3.0 and Biotitre-B did not agree in 4 (7.1%) samples. Pearson's correlation coefficient of for results obtained in VERSANT HBV 3.0 and Biotitre-B in linear range for both tests was 0.712. Evaluation of reproducibility of the tests using a panel of recombinant HBV DNA showed higher reproducibility of VERSANT HBV 3.0 test with coefficient of variations from 0.79 to 2.79% vs. 2.39-10.69% for Biotitre-B reference test. All 60 serum samples from donors areactive by HBsAg and anti-HBc were negative by HBV DNA when tested by VERSANT HBV 3.0 and Biotitre-B. Hence, clinical specificity of both tests was 100%. The results indicate high specificity of both tests and good agreement of their results, the reproducibility of VERSANT HBV 3.0 test being higher.

A natural focus of Dobrava hantavirus was first revealed in an arid zone of the Astrakhan Region, by using molecular genetic methods. A polymerase chain reaction was employed to examine 389 lung tissue suspension samples taken from 9 species. Hantavirus RNA was found in 35 samples from 22 field mice (Apodemus agrarius), 8 tamarisk gerbils (Meriones temariscinus), 4 common voles (Microtus arvalis), and 1 house mouse (Mus musculus). Sequencing determined the taxonomic affiliation of 18 new isolates to the Dobrava species and 1 isolate to the Puumala species although the new Dobrava isolated greatly differed from all known strains of this virus in the nucleotide sequence of the genomic M and S segments. Hantavirus-infected rodents were found in 3 of 4 districts of the Astrakhan Region, located in the steppe and semidesert zones and at the intrazonal stations of the Volga-Akhtuba flood-lands. The high (up to 12%) hantavirus infection rates were ascertained in a tamarisk gerbil, the typical inhabitant of desert and semidesert ecosystems.

Results of studies of biological samples from patients with community-acquired pneumonia and samples from environment during Legionnaires' disease outbreak in town Verkhnyaya Pyshma (Sverdlovsk region) in July-August 2007 performed by PCR analysis are presented.

The aim of the study was a comparative analysis of diagnostic value of different laboratoty methods conducted on the basis of results of examination of patients during Legionnaires' disease outbreak in town Verkhnyaya Pyshma. Retrospective analysis of laboratory data from 74 patients with diagnosis of Legionnaires' disease was performed. Complex of laboratory methods was used (polymerase chain reaction (PCR), enzyme immunoassay (EIA), immunochromatography). In group of patients with Legionnaires' disease, the highest proportion of positive results (73%) was obtained by the EIA determining total specific antibodies in urine. Determination of antigen in urine by immunochromatographic express-test yielded 52% of positive results. PCR testing of blood specimens yielded positive results in 65% of samples but was low specific, due to that in 19% of patients from control group false-positive results were obtained. Testing of 3 autopsy samples showed that all specimens contained DNA of the causative agent. Performed analysis allowed to recommend complex use of immunochromatographic express-test of antigen detection and identification of total specific antibodies by EIA during mass people examination.

A PCR system in the fluorescent amplification-based specific hybridization (FLASH) format was developed for the detection and identification of two important wheat pathogenic fungi Septoria tritici (teleomorph of Mycosphaerella graminicola and Stagonospora nodorum (teleomorph of Phaeosphaeria nodorum), which cause spots on leaves and glumes, respectively. The pathogen detection system is based on the amplification of a genome fragment in the internal transcribed spacer 1 (ITS 1) region and a site encoding the 5.8S ribosomal RNA. The forward primers to ITS1 and a universal reverse primer and a Beacon type probe to the 5.8S ribosomal RNA region were chosen to provide the detection of the products in the FLASH format. This system was tested on different isolates of the pathogens, and on infected soil, leaf, and seed samples.

Cryptosporidium is an important protozoan that cause diarrheal illness in humans and animals. Different species of Cryptosporidium have been reported and it is believed that species characteristics are an important factor to be considered in strategic planning for control. We therefore analyzed oocysts from human and animal isolates of Cryptosporidium by PCR-RFLP to determine strain variation in Isfahan. In total, 642 human fecal samples from children under five years of age, immunocompromised patients, and high risk persons and 480 randomly selected rectal specimens of cows and calves in Isfahan were examined. Microscopic examination showed that 4.7% (30/642) of human samples and 6.2% (30/480) of animal samples were infected with Cryptosporidium. After identification of the samples infected with the parasite, oocysts were purified and their DNA was extracted. We used PCR-RFLP analysis of a 1750-bp region of 18S rRNA gene to identify Cryptosporidium species. The human samples were infected with Cryptosporidium parvum II, C. muris, C. wrairi, and a new genotype of Cryptosporidium (GenBank accession numbers: DQ520951). The cattle samples were identified as C. parvum II, C. muris, C. wrairi, C. serpentis, C. baileyi, and a new genotype of Cryptosporidium (GenBank accession numbers: DQ520952). Also we found a new genotype infecting both human and cattle samples (GenBank accession numbers: DQ520950). In addition to demonstrating the widespread occurrence of most species of Cryptosporidium, C. parvum, we also observed extensive polymorphism within species. Furthermore, the occurrence of the same species of parasite in both animal and human samples shows the importance of the animal-human cycle.

Method of site-directed mutagenesis that gave an opportunity to the site-independent manipulation with enough extended exposed mutagenesis fragments of gene cloning in plasmid vector was developed. With the help of this method hybrid constructions representing peas Pisum sativum (PSL) lectine with carbohydrate-binding fragment changing on that lectines from another legume plants were created. This method is suitable for obtaining the expression constructions in plasmid vectors, when is necessary to put a coding part under the control of promoter in a certain reading frame.

Influenza viruses are spread worldwide and cause the disease in birds and mammals, including human beings. Moreover, birds are the natural reservoir of influenza A viruses. Early detection of newly emerging influenza viruses requires permanent monitoring. Traditional viral shedding methods using cell cultures and chicken embryos are time-consuming and laborious analysis when a large number of samples are examined. The paper describes the use of polymerase chain reaction-based test systems to detect influenza A virus and to differentiate its two subtypes: H5 and H7. The developed test systems were evaluated on 19 reference influenza A virus strains. Thereafter, more than 1500 samples from wild and domestic birds, collected in the Russian Federation in 2004-2006, were studied. The data of these test systems were compared with the techniques of viral shedding in the cell cultures and chicken embryos.

A simple and sensitive method has been developed to determine the genome composition of the reassortant on the basis of B/USSR/60/69 by the restrictase analysis of DNA copies of RNA sites containing the nucleotide replacements typical of B/USSR/60/69.

Glanders and melioidosis are severe infectious diseases of people and animals. The causative agents of these infections refer to the potential agents of bioterrorism of group B. In this work the possibility of use of flagellin-based primers for the identification of B. mallei and B. pseudomallei and for diagnosis of experimental glanders and melioidosis was studied. The obtained results permit to make a conclusion that PCR using the developed primers may be recommended for the incorporation in the scheme of laboratory diagnosis of glanders and melioidosis both for the identification of clean cultures and in experimental clinical material.

Irkutsk PCR O-genotyping of 117 Yersinia pseudotuberculosis serotype O:1-O:4 strains isolated in Siberia and Far East was performed. These methods allowed both O-genotype and its variants (a, b, c) to be detected. It was demonstrated that three Y. pseudotuberculosis O-genotypes were circulated in Siberia (O:1a, O:1b and 0:3) and six genotypes (O:1a, O:1b, O:1c, O:2a, O:3 and O:4b) were circulated at Far East. Genotype O:1b dominates at both regions (87.8%). PCR-algorithm for identification of Y. pseudotuberculosis O-genotypes having epidemic significance was developed. The identification of the etiological agent O-genotype without bacteriological isolation of the stimulus is possible by PCR analysis of the clinical material.

This paper describes a technique for the development of a positive control for use in a nested PCR to show that the PCR has worked correctly with both outer and inner primers designed for diagnostic amplification of 618 bp and 317 bp products respectively. This positive control produces a larger product than the diagnostic sample that can be discriminated on an agarose gel. This technique is advantageous over traditional cloning of the diagnostic PCR product itself by: 1) making it visually easy to detect plasmid contamination and thus, prevent false positives from the plasmid; 2) develop a positive control when the target organism is at a very low prevalence so initial detection is not relied on for cloning positive controls. This will ensure the PCR is working correctly prior to diagnostic sampling, reducing false negatives; or 3) for developing a PCR and determining the sensitivity prior to the use of diagnostic samples. The methods used to produce this nested positive control demonstrates how to use large oligonucleotide primers in PCR without non-specific binding occurring.

Linear DNA molecules amplified by the polymerase chain reaction were visualized by atomic force microscopy. The measured contour length of the PCR product of 1414 bp sequence was 435 +/- 15 nm. Considering that the calculated value of the distance between the nucleotides along the duplex axis is 0.31 nm, it was assumed that linear DNA molecules on the surface of mica, which serve as a support in the atomic force microscopy method, are in the A form. The influence of surface properties of the mica and the sample drying procedure on the conformation of adsorbed DNA molecules is discussed. Possible reasons for the Gaussian distribution of the contour length of the synthesized amplicon are considered.

The study was carried out in hospital patients as well as in outpatients at the National Centre of Tuberculosis and Lung Diseases of Georgia (2002-2004). The group consisting of 32 patients with tuberculosis of urogenital system has been studied (newly detected forms). Except clinical laboratory, culture and X-ray contrast methods, two additional methods were used in testing of this group of patients. The examination of their urine, at the same time, was carried out by the Polymerase Chain Reaction method in order to detect Kochi bacillus and by three-time bacterioscopy of urine for acid resistant bacteria. Mycobacterium tuberculosis in urine has been detected in 26 (81,25%) patients by PCR method, and by urine bacterioscopy--acid fast bacilli (AFB+) in 18 (56,25%) patients. The histo-morphological investigation of specimens obtained by surgery confirmed the TB diagnosis in all patients. This study on patients suspected of Tuberculosis of genital-urinary system gives us an opportunity to update the diagnostic algorithm by including the modern molecular methods. This algorithm will help in timely detection of Tuberculosis, in selection of adequate therapy and in prevention of the further progression of the disease.

Both hospitalized patients and outpatient clinic patients at the National Centre of Tuberculosis and Lung Diseases of Georgia have been investigated. The group of patients with the tuberculosis of urogenital system, has been studied (newly detected cases), 70 cases in total. The examination of the urine was carried out by the Polymerase Chain Reaction (PCR) method in order to detect Kochi bacillus and by three-time bacterioscopy of urine on acid resistant bacterium. Mycobacterium tuberculosis in urine has been detected in 57 (81,43%) patients by PCR method, and by urine bacterioscopy acid fast bacilli (AFB)(+) in 36 (51,43%) patients. 50 hospitalized patients were examined as a separate group. They had the tuberculosis of lungs and insignificant pathological changes in urogenital system. Among them there was active bacillus secretion in 45 cases by phlegm bacterioscopy AFB(+). Out of 50 patients the mycobacterium tuberculosis in urine was detected in 30 (60%) cases by PCR method. It should be mentioned that according to the urine two-time bacterioscopy, carried out on 50 patients, has not been detected AFB(-) bacillus secretion. It may be concluded that the PCR method is advantageous in detecting of Kochi bacillus in urine. Introduction of this method in medical practice will give us the possibility to determine the risk-group for development of tubercular changes in urinary system associated with lung tuberculosis, in order to control such patients and to carry out the adequate urological examinations.

Data on the investigation of pseudotuberculosis epidemic outbreaks with the use of the polymerase chain reaction (PCR), are presented. Specific fragments of Yersinia pseudotuberculosis DNA were detected in 81.25% of patients, in 46.83% of cases confirmed by the isolation of Y. pseudotuberculosis cultures. The study of washings from vegetables and equipment in vegetable stores and kitchens yielded positive results in PCR in 8.52% and the survey of rodents--in 3.85% of cases. In the course of the bacteriological study of these specimens 6 Y. pseudotuberculosis cultures were isolated: 3--from vegetable products, 1--from a Norway rat and 2--from house mice. The coincidence of the data obtained by bacteriological study and PCR showed that the latter method gave objective results, while being capable of ensuring rather rapid analysis. PCR should be regarded as a signal test for the bacteriological search of the definite infective agent in the material under study.

A total of 188 children and adolescents were examined. In all the children, blood Mycobacterium tuberculosis (MBT) DNA was determined by polymerase chain reaction (PCR) and MBT antigens (AG) and antibodies (AB) were by enzyme immunoassay. The studies have shown that it is expedient to concurrently determine MBT DNA and MBT AT in order to identify local forms of tuberculosis in children from risk groups. If the tests are positive, a comprehensive examination for tuberculosis is required; the presence of the syndrome of common disturbances is generally associated with tuberculous infection. When a local form of tuberculosis is excluded, preventive chemotherapy should be performed. Further negative tests for MBT DNA and lower MBT AT may be a criterion for the efficiency of preventive treatment. In children with tuberculosis, the results of repeated blood and urine tests for MBT DNA provide a way of evaluating the course of a tuberculous process and the efficiency of chemotherapy. PCR used to determine blood and urine MBT DNA is a highly specific test as positive results were in 79% of the children with tuberculosis.

Two hundred and ten newborns from among a high risk group were examined for intrauterine infections by polymerase chain reaction (PCR) with the aim of assessing the clinical information density of the method in the diagnostic sphere. The patients were examined for the presence of DNA/RNA viruses and bacteria. Genetic samples of various causative agents were detected in 49% of the newborns. No correlation was established between the clinical pattern and the presence of DNA of certain microorganisms. The most severe condition of newborns was noted in cases when a DNA causative agent was found in several different biological samples. The highest mortality (in per cent) was registered in the group of newborns with detected DNA of Herpesviridae viruses. The PCR clinical value was affirmed for establishing a disease etiology in newborns.

Real-time RT-PCR using fluorescence dyes (e.g. SYBR Green I) is currently the most sensitive and precise method for investigation of RNA level and has long been widely used for absolute and relative quantification of mRNA in the cell. This highly sensitive method allows measurement of different type RNA level in the cell based on the kinetics of the corresponding double-stranded cDNA amplification. Upon its binding to the minor groove of double-stranded DNA, SYBR Green I dye increases its fluorescence about 100-fold, and this increase can be recorded even at early cycles of amplification. During the real-time RT-PCR procedure the level of amplified DNA is measured after every cycle of amplification, which permits to perform quantification at the cycles when amplification curve has not yet reached the "plateau" range and corresponds to the range of exponential increase in DNA amount. This approach makes it possible to avoid misinterpretation of data typical of conventional PCR methods "in the end point" and caused by a deficiency of one or more reaction components at the late PCR cycles. We applied for the first time real-time RT-PCR using SYBR Green I for the measurement of the class III genes RNA-product level, that is, small stable non-translated RNAs--ribosomal 5S rRNA, initiator transfer RNAiMet1, and Alu-RNA, synthesized by DNA-dependent RNA polymerase III. We investigated the level of 5S rRNA-, tRNA- and Alu-gene expression in the cell being in different states: with prolonged generation period, activated to proliferation, and apoptotic. The expression level was judged from the content of corresponding RNA-products in the total cellular RNA. The used approach enabled us to find out the specific RNA share in the total cell RNA. Human epidermoid carcinoma cells A431 were used as a model for investigating class III gene expression level in vivo. These cells expose on their surface an abnormally large amount of receptors to epidermoid growth factor (EGF), and the result of EGF action on A431 cells depends on the growth factor concentration. Low concentrations of EGF (0.1 ng/ml) cause active proliferation of A431 cells, but its high concentrations (10-100 ng/ml) cause apoptosis in these cells. Besides, upon growing in serum-free media, A431 cells continue to proliferate, but by this extending the generation period to 48 h, against 30 h on growing in serum-containing media. Hence, A431 cells can serve as a useful model for investigation of specific gene expression level in cells being in different physiological states, in both slowly and actively proliferating cells, and in apoptotic cells. For successful use of real-time RT-PCR in 5S rRNA, tRNAi(Met)1 and Alu-RNA level quantification, we optimized the amplification reaction conditions. We took into account that the share of each particular RNA in the cell may vary--the share of ribosomal RNA is high, tRNAi(Met)1--low, and Alu-RNA--very low. Moreover, the level of some small RNAs (e.g. Alu-RNA) can vary significantly in cells of different lines. This explains why the amount of cDNA, gained by reverse transcription of total cellular RNA, and the concentration of specific primers used for PCR were different in each case. We showed that the expression of different class III genes--5S rRNA-, tRNA- and Alu-genes, was not similarly regulated in response to external stimuli, causing prolongation of generation period, activation of proliferation and apoptosis. 5S rRNA level was practically the same in A431 cells both having prolonged generation period and being activated by EGF in low concentration, but in apoptotic cells this level dramatically fell about 8-fold. Alu-RNA level was equal in cells with prolonged generation period and in apoptotic cells, and increased about 2-fold in cells activated by EGF in low concentration. The initiator tRNAi(Met)1 level in cells activated by EGF in low concentration and in apoptotic cells was by almost two times higher than in cells with prolonged generation period. The data obtained testify that the real-time RT-PCR method using SYBR Green I yields highly reliable and reproducible quantification for the level of class III gene RNA-products--small stable RNAs (5S rRNA, tRNA and Alu-RNA). Examination of each specific RNA level requires individual selection for the amplification reaction conditions: the amount of cDNA and primer concentration in the sample. This is primarily caused by different expression levels in some particular class III genes within the frames of the cells, and by different levels of some small stable RNAs (e. g. Alu-RNA) in different cell lines. Special attention must be paid to the internal control for discriminating between specific RNA levels in proliferating and apoptotic cells, as in the late apoptosis RNAs of most types are degraded (for example, mRNA of "house-keeping" gene for RPLP0 protein, used as a possible internal control in our experiments). As far as the applied approach allows estimation of a specific RNA share in the total cellular RNA, we propose to chose as internal control mRNA, whose share doesn't change during the total RNA degradation in apoptosis and thus, mRNA degradation is not selective (in relation to other type RNAs). In that way, the real-time RT-PCR method, which is currently the most sensitive and precise method for quantification of RNA in the cell, holds much promise for the investigation of not only different mRNAs, but also small stable RNAs, synthesized by RNA polymerase III.

Variation in the ISSR patterns was studied in queens of five species of bumble bee habitating on the territory of Ukraine and Belarus. Species-specific characteristics were obtained for B. sylvarum, B. lapidarius, B. pascuorum, B. terrestris, and B. hortorum with using (GTG)7A and (AGC)6G as primers. Some ISSR markers were common for the groups of species. From 73 to 100% ISSR markers were invariant within the species. Individual characteristics of B. terrestris from geographically distant populations from Kiev and Minsk Oblasts were similar. The level of intraspecific variation was different among the species. Taking into account intraspecific constancy of the major ISSR-PCR patterns and in the same time their susceptibility to individual differences we proposed these markers could be used to elucidate some complicated questions of bumble bee taxonomy.

Congenital deafness affects 0.05-0.1% children. 90% of them were born from parents with normal hearing. There were no hearing defects in their families. In 70% of deafness cases deafness is the only symptom of the disease and is thought to be non-syndromal. Hypoacusis of unclear etiology may be congenital in 50% cases. Half of cases of severe autosomic-recessive non-syndromal hypoacusis (deafness) appear because of changes in only one gene--gene of connexine-26. Two thirds of the defects in this gene arise because of one mutation--35delG. Mean rate of this mutation carriage in Russia is over 2%. Identification of mutation 35delG in gene Cx26 is performed with the use of polymerase chain reaction. Genetic examination of 75 children with isolated hypoacusis (deafness) has detected deletion 35delG in both gene copies in 23 children (30%). 10 patients (13.4%) had mutation 35delG only in one copy of gene Cx26. Hearing defects in the latter may be related with the presence of another mutation in the same gene. A total of 33 patients (42%) carried deletion 35delG. According to our findings, the changed genotype is characterized primarily by bilateral neurosensory hypoacusis of the third-fourth degree. Weaker loss of hearing is rare. Thus, mutations in connexine gene26 present a problem for parents with normal hearing. Therefore, families with a deaf child should be referred for medicogenetic consultations.

Polymerase chain reaction (PCR) was used to verify the tuberculous origin of L-forms isolated from clinical non-respiratory samples from patients with extrapulmonary tuberculosis. PCR was made by using cultured L-forms obtained from negative and positive cultures. PCR used a total of 60 cultured L-forms different in the morphology of colonies and the rate of growth. The total count of L-forms yielding positive amplification with M. tuberculosis complex-specific primers was 51 (85%). L-form passages were subjected to PCR analysis. A total of 14 third-generation L-forms were examined. They turned out to be positive. Thus, the fact that L-forms isolated from nonrespiratory clinical samples from patients with tuberculosis are most commonly L-forms of M. tuberculosis was genetically substantiated.

Examination of 37 children and adolescents with long-term subfebrility and other manifestations of intoxication without local pathological changes included blood test (polymerase chain reaction) for M. tuberculosis. Tuberculous bacteriemia was discovered in 16(43.2%) cases. Non-acute onset and long-lasting intoxication, rejection of other reasons, tuberculin sensitivity, reduction or disappearance of the intoxication in antituberculous treatment, negative results of repeated PCR for M-tuberculosis gave grounds to suspect active tuberculosis in 13 children of 16. Clinical and x-ray picture corresponded to tuberculous intoxication in children and adolescents.

Comparative assessment of the sensitivity of detection of Mycobacteria tuberculosis by using bacterioscopy after Ziehl-Nilsen, the cultural study on the Finn II yolk medium and Middlebrook 7H11 agar medium, and polymerase chain reaction indicated that a complex microbiological study with all available laboratory methods should be used.

The study was undertaken to assess the role of polymerase chain reaction (PCR) analysis in complex bacteriological studies in diagnosing pulmonary tuberculosis in 197 patients by bacterioscopy, inoculation, and PCR. It was shown that in addition to conventional bacteriological methods, PCR might be used as an additional laboratory study in making a diagnosis in patients with restrictive pulmonary tuberculosis. A combination of cultural inoculation and PCR analysis enhances the sensitivity of bacteriological diagnosis and reduces its duration in oligo- and abacillary patients with tuberculosis. PCR analysis enhances the efficiency of laboratory control over antituberculous chemotherapy.