[Analysis of genetic determinants of multidrug and extensively drug-resistant Mycobacterium tuberculosis using oligonucleotide microchip].

Steadily growing resistance of the tuberculosis causative agent towards a broad spectrum of anti-tuberculosis drugs calls for rapid and reliable methods for identifying the genetic determinants responsible for this resistance. In this study, we present a biochip-based method for simultaneous identification of mutations within rpoB gene associated with rifampin resistance, mutations in katG, inhA, ahpC genes responsible for isoniazid resistance, mutations within the regions of gyrA and gyrB genes leading to fluoroquinolones resistance, and mutations in the rrs gene and the eis promoter region associated with the resistance to kanamycin, capreomycin and amikacin. The oligonucleotide microchip, as the core element of this assay, provides simultaneous identification of 99 mutations in the format "one sample--one PCR--one microchip", and it makes it possible to complete analysis of multi-drug-resistant and extensively drug-resistant tuberculosis within a single day.

[Identification of genetically modified vegetable sources in food and feed using hydrogel oligonucleotide microchip].

A method of multiplex polymerase chain reaction (PCR) followed by the hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in feed and food products. The biochip contained 22 immobilized probes intended for (i) detection of plant DNA; (ii) plant species determination (soybean, maize, potato, rice); (iii) identification of transgenic elements, including 35S CaMV, 35S FMV, rice actine gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, nptII marker genes. The limit of detection was 0.

[Method for automated extraction and purification of nucleic acids and its implementation in microfluidic system].

A method and a microfluidic device for automated extraction and purification of nucleic acids from biological samples have been developed. The method involves disruption of bacterial cells and/or viral particles by combining enzymatic and chemical lysis procedures followed by solid-phase sorbent extraction and purification of nucleic acids. The procedure is carried out in an automated mode in a microfluidic module isolated from the outside environment, which minimizes contact of the researcher with potentially infectious samples and, consequently, decreases the risk of laboratory-acquired infections.

[Molecular genetic and bacteriological methods for the diagnosis of multidrug resistant M. tuberculosis].

For the early diagnosis of multidrug resistant tuberculosis, 67 sputum samples obtained from primary patients with different clinical forms of pulmonary tuberculosis were examined by the molecular genetic test using the TB-Biochip test system. Having a high sensitivity and specificity, the molecular genetic test for determining the drug sensitivity of Mycobacterium tuberculosis substantially accelerates its diagnosis (2-3 days) before the real-time mode of a patient's admission to the clinic. The method allows identification of mutations in the rpoB (resistance to R), katG, inhG, and ahpC (resistance to H) genes, which permits timely correction of performed specific treatment.

[Investigation of Mycobacterium tuberculosis sensitivity to fluoroquinolone, by revealing gyrA gene mutations].

The resistance of Mycobacterium tuberculosis (MBT) to fluoroquinolones is associated with the mutations concentrated in the gyrA gene that is a structural gene of a gyrase A subunit. Detection of mutations in this portion of the gene allows the sensitivity of MBT to this group of drugs to be rapidly determined.

[Biological microchip for identification of orthopoxviruses and agents causing the same clinical picture as in smallpox].

A method based on hybridization of simultaneously amplified gene fragments of orthopoxviruses and herpesviruses with oligonucleotide probes immobilized on a microarray has been developed. The method permits identification of 6 orthopoxvirus species and three types of herpesviruses, including Varicella zoster, within 6 hours.

[Detection of single base polymorphism in p53 gene by ligase detection reaction and rolling circle amplification on microarrays].

We combined three modern technologies of single base polymorphism detection in human genome: ligase detection reaction, rolling circle amplification and IMAGE hydro-gel microarrays. Polymorphism in target DNA was tested by selective ligation on microarray. Product of the ligase reaction was determined in microarray gel pads by rolling circle amplification.

[The genetic variability of the protease-encoding region in HIV-1 A isolates and in CRF03_AB recombinants as observed in the CIS territory].

Protease-encoding nucleotide sequences of 27 HIV-1 variants isolated in Russia and other CIS countries from seropositive intravenous drug-users were analyzed. None of the above persons did ever take antiretroviral drugs. The nucleotide sequences were shown to belong to subtypes A and to be have a high degree of genetic homogeneity (0.

[New technologies in the determination of drug susceptibility in Mycobacterium tuberculosis].

A variety of mutations in the genes rpoB, katG, inhA, ahpC, kasA was studied by using different molecular biological methods (conformational polymorphism of single-chain fragments, heteroduplex analysis, biochips) in rifampicin- and isoniazid-resistant Mycobacterium tuberculosis (MBT) strains isolated from patients with pulmonary tuberculosis. Twenty-nine mutation combinations were identified in the MBT strains. The use of biochips is the most promising method for identifying the type of mutations responsible for the simultaneous resistance to rifampicin and isoniazid.

[Microchips based on three dimensional gel cells: history and perspective].

The review describes the history of creation and development of the microchip technology and its role in the human genome project in Russia. The emphasis is placed on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D.

[Identification of Mycobacterium tuberculosis strains and a simultaneous identification of their drug resistance by the hybridization method on oligonucleotide microchips].

A method of multiplex polymerase chain reaction (PCR) with subsequent hyoridization on oligonucleotide microchips was worked out to identify the Mycobacterium tuberculosis complex and to determine simultaneously the bacterial sensitivity to 2 first-line drugs, i.e. rifampin and isoniazid.

[Identification of orthopoxvirus species using oligonucleotide micro-chips].

A method for describing the Orthopoxviruses that are pathogenic both to man and animals is described in the article. The method is based on hybridization of a fluorescently labelled amplified DNA sample with oligonucleotides, which were immobilized in a microchip. Species-specific regions within the crmB gene encoding a viral analogue of the tumor necrosis factor receptor, i.

[Molecular genetic methods for the detection of rifampicin-resistant Mycobacterium tuberculosis strains].

RCR-heteroduplex (GDA) and chip methods were used to detect rifampricin-resistant (RR) and rifampicin-sensitive (RS) Mycobacterium tuberculosis (MTB) in the samples from patients (sputum) and in the clinical isolates of MTB from these patients (MB/BacT liquid medium and Lowenstein Jensen's (LJ) solid medium. The efficiency of detecting RR and RS of MTB (from the sputum) is 100 and 92.3% in the chip and GDA tests, respectively.