Development of a PCR assay for the detection of human herpes virus type 7.

A PCR assay has been developed to identify the DNA of the human herpes virus type 7. The search and selection of conserved regions was carried out by comparing the whole genome nucleotide sequences of HHV-7. A fragment duplicated in the HHV-7 genomes was chosen as a target for amplification. The performance of the assay was tested on a synthetic matrix and clinical samples. The developed assay has high sensitivity and specificity and showed good efficiency in detecting HHV-7 DNA in clinical samples.

Detection of Y-chromosome marker in plasma of pregnant women using real time PCR: diagnostic accuracy depending on gestation age.

The evaluation of the clinical significance of the test for the detection of the Y-chromosome marker in the plasma of a pregnant woman at different stages of pregnancy by real-time PCR was carried out. The blood samples of 4616 women at 4 to 32 gestation weeks were studied. Identification of the Y-chromosome marker was carried out based on the amplification of a region of the TSPY gene. The Y-chromosome marker was unambiguously identified in 2131 samples, which accounted for 46.2% of the total number of analyzed samples. In 233 samples (5%), the Y-chromosome marker was detected with reduced reliability, and in 15 samples (0.3%), an unambiguous conclusion about the presence or absence of Y-specific DNA in plasma could not be made during the initial study. The diagnostic accuracy of the Y-chromosome marker determination in the plasma of a pregnant woman at the 4-6th gestation week was 95.5%, and from the 7th week and at later stages of pregnancy it reached 97.3-98.2%. Testing from the 7th gestation week may be recommended for reliable prenatal sex determination of the fetus by real-time PCR analysis of extracellular circulating fetal DNA.

[COMPARATIVE EVALUATION OF THE TRANSGENE EXPRESSION EFFICIENCY PROVIDED BY THE MODEL GENETIC CONSTRUCTS OF DIFFERENT STRUCTURE.]

Comparative evaluation of the transgene expression efficiency provided by the model genetic constructs of different structure is an important stage in the development of new expression methods and optimization of the existing expression vectors. However, presently there is no versatile approach to this problem. The goal of this work was to suggest an experimental system for comparative evaluation of the expression efficiency provided by nonviral genetic vectors of various size and topology in human cell cultures. Such system is based on the gene of the green fluorescence protein used as a reporter as well as flow cytofluorometry for evaluation of the expression level and quantitative PCR for adequate selection of the transfection conditions. This system was tested in two model constructs: linear molecule of DNA and plasmid.

[PCR study of the human herpes virus type 6 and other viruses of the herpes group in eye diseases].

To study the role of the HHV-6 type in the development of eye diseases PCR tests of blood (152), cornea biopsies (61), and intraocular fluids (11) for HHV-6 and other viruses of the herpes group (HSV type 1 and 2, CMV, EBV) were conducted. It was found that the HHV-6, along with other representatives of the Herpesviridae, can be detected in patients with different clinical forms of ophthalmopathology (174 patients were surveyed). Viral DNA was detected in blood, cornea, and in the anterior chamber fluid. The obtained data allow that the HHV-6 to be suggested as a possible cause of the ophthalmic herpes along with the other viruses of this group. It makes finding the virus DNA an essential step towards setting the etiologic diagnosis of the ophthalmological patients.

[Specific motifs in the genomes of the family Chlamydiaceae].

Specific motifs in the genomes of the family Chlamydiaceae were discussed. The search for genetic markers ofbacteria identification and typing is an urgent problem. The progress in sequencing technology resulted in compilation of the database of genomic nucleotide sequences of bacteria. This raised the problem of the search and selection of genetic targets for identification and typing in bacterial genes based on comparative analysis of complete genomic sequences. The goal of this work was to implement comparative genetic analysis of different species of the family Chlamydiaceae. This analysis was focused to detection of specific motifs capable of serving as genetic marker of this family. The consensus domains were detected using the Visual Basic for Application software for MS Excel. Complete coincidence of segments 25 nucleotide long was used as the test for consensus domain selection. One complete genomic sequence for each of 8 bacterial species was taken for the experiment. The experimental sample did not contain complete sequence of C. suis, because at the moment of this research this species was absence in the database GenBank. Comparative assay of the sequences of the C. trachomatis and other representatives of the family Chlamydiaceae revealed 41 common motifs for 8 Chlamydiaceae species tested in this work. The maximal number of consensus motifs was observed in genes of ribosomal RNA and t-RNA. In addition to genes of r-RNA and t-RNA consensus motifs were observed in 5 genes and 6 intergene segments. The gene CTL0299, CTLO800, dagA, and hctA consensus motifs detected in this work can be regarded as identification domains of the family Chlamydiaceae.

[Determination of antibodies against cyclic citrullinated peptide in rheumatoid arthritis].

The ability of the synthetic peptide IMG-5, that reproduces one of the antigenic determinants of the protein filaggrin, to show antigenic activity was studied when anti-cyclic citrullinated peptide antibodies (ACCPAb) to filaggrin were found in the serum samples of patients with rheumatoid arthritis (RA). The binding of IMG-5 to ACCPAb has been shown to be specific (dose-dependent and reversible). The serum samples from patients with RA, controls, and donors show a significant difference in the interaction of the synthetic peptide with ACCPAb (p < 0.005 and p < 0.0001, respectively). The level of IgM rheumatoid factor (RF) detected in patients with RA differs greatly from that in the controls. In the patients with RA versus the controls, the specificity of ACCPAb determination was as high as 87; and the sensitivity was 40.5%. When ACCPAbs were determined using the commercial kit CCP, the specificity and sensitivity were 94 and 47.3%, respectively. The specificity of RF detection was equal to 50% and the sensitivity was 70%. The sensitivity of the test using IMG-5 is a maximum in X-ray stage IRA (69.2%) and falls in its stage III (26.7%). On the contrary, the sensitivity of the commercial kit and RF determination increases from X-ray stage I (46.2 and 53.8%, respectively) to II (66.7 and 80%). The sensitivity of the used tests in varying RA activities has demonstrated that they are most effective in patients with moderate RA activity. The concurrent detection of ACCPAb and RF increases the probability of differentiating RA from other rheumatic diseases.

[The population structure of Mycobacterium tuberculosis in Mongolia as assessed using large sequence polymorphism analysis].

112 strains of M. tuberculosis isolated from lung tuberculosis patients in Mongolia were genotyped using RD9, RD7, TbD1, RD105, and RD750 loci. The genotypes of all the strains studied were characterized using the conservation of RD7, RD9, and RD750 loci and the presence of the deletion in the locus TbD1. RD105 was detected in 65 isolates (58%). The isolate was classified into two groups--East-Asian and Euro-American.

Detection of new epitopes of antibodies to filaggrin in filaggrin protein molecule.

Immunogenic characteristics of filaggrin protein molecule as an antigen for antibodies to filaggrin, markers of early rheumatoid arthritis, were studied. Two new peptide motives, possible epitopes for antibodies to filaggrin, were shown in the filaggrin molecule by predictive analysis using programmed algorithms. Only IMG-3 and its cyclic form IMG-4 exhibited antigenic reactivity with sera from rheumatoid arthritis patients, differing significantly from the reactivity with donor sera. The immunogenic characteristics of IMG-3 differed from the characteristics of a previously described epitope.

[Tabular excel editor for analysis of aligned nucleotide sequences].

Excel platform was used for transition of results of multiple aligned nucleotide sequences obtained using the BLAST network service to the form appropriate for visual analysis and editing. Two macros operators for MS Excel 2007 were constructed. The array of aligned sequences transformed into Excel table and processed using macros operators is more appropriate for analysis than initial html data.

[Comprehensive examination of the female urogenital tract infection by polymerase chain reaction using multiplex test systems].

The results of the polymerase chain reaction studies performed in 2006-2008 were used to make a retrospective analysis of the detection of urogenital herpesvirus infections in reproductive-aged women constituting the urban population of the central region of Russia. The study used both monotarget and mutiplex test systems to detect herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus types 6 and 7. A total of about 7.500 referrals; the detection rate for HSV was about 1%; that for CMV was from 0.3 in 2006 to 1% in 2008; that for EBV was from 0.1% in 2006 to 0.3% in 2008. More than a half of HSV-, CMV-, or EBY-positive samples also contained DNA of other causative agents and some samples did two pathogens or more. Multiplex test systems for herpesviruses considerably enhance the efficiency of diagnostic studies and reduce the material and time costs of diagnosis.

[Genotyping of varicella zoster virus strains isolated in Mongolia].

This study analyzed 50 varicella zoster virus (VZV) samples collected during 2004 to 2007 from patients with VZV infection, who were treated at the National Center of Communicable Diseases, Ulan-Bator, Mongolia. The method based on amplification of specific DNA fragments of the ORF21, ORF22, and ORF50 genes was used, followed by the sequencing and detection of the status of characteristic point mutations in these fragments. The results indicated that the collected samples belonged to genotypes J (62%), M1 (18%), E1 (12%), E2 (4%), and M2 (4%). Moreover, restriction endonuclease polymorphism in ORF 62 for the cleavage site Smal and Mspl, in ORF 38 and ORF 54 for the cleavage site Pstl and Bgll were analyzed. All the samples were Sma- Msp-. All samples with genotype E were Pst+ Bgl-; all samples with genotype M1 and M2 were Pst+ Bgl+. Out of 31 samples with genotype J, 29 and 2 were Pst+ Bgl+ and Pst+ Bgl+, respectively. The study could identify the genotypes of VZV circulating in Mongolia and confirmed the absence of mutations characteristic for the vaccine strain.

[Bioinformatic analysis of nucleotide sequences records retrieved from GenBank].

Methods and macros used for processing of samples of NP bacteria retrieved from GenBank are described. The goal of the processing is to transform lists of NP bacteria retrieved from GenBank into Excel table with classification of data concerning bacterial genes, species, and genomes of bacteria, as well as accompanying information about NP bacteria. The list is processed using several macros and the result of processing is stored in table. Each line of the table contains information about one record of initial list of NP bacteria. Information about genes, species, and genomes of NP bacteria is contained in columns of table. The capacity of the macros is demonstrated using the list of NP bacteria of the genus Ureaplasma. The developed macros can be applied to lists of NP bacteria and viruses available from GenBank. This information can be used in studies of interspecies and intraspecies genetic polymorphism and genetic targets for various problems of molecular biology (genotyping of viruses and bacteria).

[Rd7 genotyping of M. tuberculosis strains isolated from patients with lung tuberculosis in different areas of Kazakhstan].

A three-primer PCR assay was designed for detection of possible deletions in the RD7 region of the Mycobacterium tuberculosis complex chromosome. The assay produced amplicons of different size depending on the presence or absence of the deletions. The PCR assay was applied to 176 isolates from patients with lung tuberculosis collected in different areas of Kazakhstan in summer 2004. The isolates were initially characterized by culture and biochemical tests. The RD7 genotyping results demonstrated no polymorphism and the absence of deletions in the RD7 genome region. Some strains were additionally characterized using PCR-RFLP analysis of gyrB and hsp64 genes. The RFLP-patterns obtained corresponded to the M. tuberculosis genotypes. The results of this work are consistent with certain previous studies, indicating population stability of the RD7 region in M. tuberculosis strains. Species characterization of the isolates shows that M. tuberculosis sensu stricto is the principal causative agent of human lung tuberculosis in Kazakhstan.

[Polymorphism of herpes simplex virus type 1 and 2 DNA from laboratory strains and clinical material from patient with genital herpes]. / Polimorfizm DNK virusa prostogo gerpesa tipov 1 i 2 iz laboratornykh shtammov i klinicheskogo materiala ot patsientov s genital'nym gerpesom.

Polymerase chain reaction (PCR), that can amplify a fragment of the DNA-polymerase gene of 4 herpes viruses, i.e. herpes simplex viruses, type 1 (HSV-1), herpes simplex viruses, type 2 (HSV-2), Epstein-Barr virus and cytomegalovirus, was made use of to study the genetic polymorphism of HSV-1 and HSV-2 strains. The obtained amplicons were analyzed by the method of restriction-size fragments' polymorphism (RSFP) with restrictases Rsal, Taql and Hinfl. Four HSV-1 strains had an identical restriction profile. Strain G (HSV-2) also displayed the expected restriction profiles, however, contradictory results were obtained for strain BH (HSV-2): the restriction profiles with restrictases Hinfl and Rsal corresponded to HSV-2, and the restriction profile with Taql corresponded to HSV-1. The sequencing of appropriate fragments of strains G and BH revealed a dot-type mutation localized in Taql restriction site. The thus worked out PCR was used jointly with RSFP in the genotyping of 75 urogenital samples obtained from women with genital herpes who were treated at Moscow patient-care facilities. HSV-1 and HSV-2 were detected in 18 (24%) and 57 (76%) of samples, respectively. No changes were registered in the restriction profile for HSV-2 among the investigated samples and all of them had the restriction profile similar to that of strain G. The conclusion is that genital herpes associated with HSV-2 is genetically stable within its Moscow population.

Detection of sequence variation in PCR-amplified fragments of omp2 gene from three species of the family Chlamydiaceae using agarose gel electrophoresis containing bisbenzimide-PEG.

A simple technique providing a means for rapid genetic differentiation of chlamydial strains is described. The technique is based on a single-step sequence-specific separation of PCR-amplified DNA fragments by electrophoresis in an agarose gel containing a DNA ligand - bisbenzimide-PEG. A hypervariable region at the 5' end of the omp2 gene of Chlamydiaceae species encoding the 60-kDa cysteine-rich outer membrane protein was selected as a target for PCR. The appropriate fragments were amplified from strains of Chlamydia trachomatis, Chlamydophila pneumoniae, and Chlamydophila psittaci, and the PCR products originating from different species were electrophoretically separated in the presence of the DNA ligand. We therefore demonstrated that PCR with a single pair of primers followed by simple agarose gel electrophoresis with bisbenzimide-PEG can be applied to the differentiation of three members of the family Chlamydiaceae which are commonly recognized as human pathogens.

Application of nested-PCR technique for the diagnosis of tuberculosis.

A test-system based on amplification of IS 986 fragment (nested-PCR) was developed for the detection of Mycobacterium tuberculosis and M. bovis in different biological samples. We constructed external primers and selected appropriate amplifications parameters (annealing temperatures for states I and II, the number of cycles for each amplification stage, components of the amplification mixture, and pretreatment conditions for different biological samples). The developed parameters make the detection of mycobacteria more efficient and less expensive compared to commercial Cobas Amplicor system.

[The use of the polymerase chain reaction in the diagnosis of pulmonary tuberculosis]. / Ispol'zovanie polimeraznoi tsepnoi reaktsii v diagnostike tuberkuleza legkikh.

The data on the use of the polymerase chain reaction (PCR) with primers INS1 and INS2 for the diagnosis of pulmonary tuberculosis are presented. All stages of PCR are described: from the treatment of biological material to the conditions necessary for carrying PCR and the registration of the results. Simultaneously with this reaction, PCR in a Cobas Amplicor apparatus was carried out and Mycobacterium tuberculosis culture was grown in a liquid medium in an MB/BacT apparatus. The study revealed that the method of PCR in pure M. tuberculosis culture made it possible to detect even the DNA of those cells which formed no colonies on Löwenstein--Jensen medium. The detection of M. tuberculosis in clinical samples (sputum, pleural exudate) taken from 31 patients with different pulmonary pathology showed that in 87.1% of cases diagnostics with the use of PCR carried out in a Cobas Amplicor apparatus and with primers INS1 and INS2 yielded similar results. In patients with pulmonary tuberculosis the results of PCR were positive, while the results of the analysis made with use of an MB/BacT apparatus were negative. The proposed primers INS1 and INS2, the conditions of amplification and detection make up the test system for the detection of mycobacteria of the tuberculosis complex.