[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. The tests on 63 Mycobacterium tuberculosis clinical isolates with different resistance profiles using the developed approach allows us to reveal the spectrum of drug-resistance associated mutations, and to estimate the significance of the inclusion of extra genetic loci in the determination of M. tuberculosis drug resistance.

[Acinetobacter: microbiological, pathogenetic and resistant properties].

Species of the genus Acinetobacter represent opportunistic bacteria with a growing clinical significance. In this literature review, we focus on the current role of Acinetobacter in infectious pathology and describe physiology, taxonomy, ecology, pathogenicity, and antibiotic resistance of these bacteria. Molecular pathogenesis and regulation of virulence factors in Acinetobacter spp. are described in detail. The majority of acinetobacterial infections are associated with A. baumannii and occur predominantly in an immunocompromised host. Usually, acinetobacterial infections are characterized by local purulent inflammation; in severe cases, meningitis and sepsis may develop. Antibiotic resistance ofAcinetobacter is a major clinical problem; therefore we give special attention to laboratory testing of resistance as well as identification of Acinetobacter. In addition, treatment and prophylaxis of acinetobacterial infections are discussed.

[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 0.5% of genetically modified (GM) soybean and maize in analyzed samples. Identification of transgenic DNA in food and feed products using either the developed approach or real-time PCR led to virtually identical results. The assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of the GM component based on the identified transgene.

[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. The module includes reservoirs with lyophilized components for lysis and washing buffers; a microcolumn with a solid-phase sorbent; reservoirs containing water, ethanol, and water-ethanol buffer solutions for dissolving freeze-dried buffer components, rinsing the microcolumn, and eluting of nucleic acids; and microchannels and valves needed for directing fluids inside the module. The microfluidic module is placed into the control unit that delivers pressure, heats, mixes reagents, and flows solutions within the microfluidic module. The microfluidic system performs extraction and purification of nucleic acids with high efficiency in 40 min, and nucleic acids extracted can be directly used in PCR reaction and microarray assays.

[The first break-through of the genotype 2.3.2 of high-virulence influenza A virus A/HSN1, which is new for Russia, in the Far East].

The epizootic etiologically associated with highly pathogenic avian influenza H5N1 genotype 2.3.2 that is new for Russia among wild and domestic birds in the south of the Primorye Territory during spring migration in April 2008 has been decoded. About 25% of the wild birds of a water complex, which include European teals (Anas crecca), mallard ducks (Anas platyrhynchos), great-crested grebes (Podiceps cristatus), are involved in viral circulation in the area of the Suifun-Khankai plain. Chicken embryos and the cell lines MDCK, SPEV, BHK-21, SW-13 were used to isolate 3 strains from recently deceased hens (A/chicken/Primorje/1/08, A/chicken/Primorje/11/08, and A/chicken/Primorje/12/08) and one strain from a European teal (A/Anas crecca/Primorje/8/08). The strains were deposited in the State Collection of Viruses of the Russian Federation, D. I. Ivanovsky Research Institute of Virology, Russian Academy of Medical Sciences. The nucleotide sequences of the full-sized genomes of A/chicken/Primorje/1/08 and A/Anas crecca/Primorje/8/08 were sent to the International databank GenBank. The strains from domestic and wild birds were shown to be identical. The isolated strains are most close to the strains Alchicken/Viet Nam/10/05, A/chicken/Guangdong/178/04, and A/duck/Viet Nam/12/05. Molecular genetic analysis has indicated that the strains isolated are susceptible to rimantadine and ozeltamivir and less adapted to mammalian cells (particularly, they contain E627 in RV2, which agrees with the biological properties of these strains in vitro). Penetration of the newly isolated virus into the Far East ecosystem provides in the foreseeable future a way for infecting the birds wintering in America and Australia in the nesting places, with further carriage of viral populations there in the period of autumn migrations.

Detection of mutations in Mycobacterium tuberculosis genome determining resistance to fluoroquinolones by hybridization on biological microchips.

We developed a method of identification of Mycobacterium tuberculosis with simultaneous evaluation of the sensitivity to fluoroquinolones on a biological microchip array. The method of multiplex two-staged PCR followed by hybridization of a biochip makes it possible to detect 8 mutant variants of gyrA gene occurring in fluoroquinolone-resistant strains (approximately 85% all resistant forms) within 1 day. Using this method we analyzed 107 cultures isolated from patients with tuberculosis and 78 sputum samples. Mutations in gyrA gene were detected in 48 (92%) resistant strains. Natural S95T polymorphism in gyrA gene was detected in all resistant and in 76% sensitive strains. The sensitivity and specificity of the proposed method calculated on the basis of the analysis of sputum samples (n=78) were 94 and 100%, respectively.

[New indodicarbocyanine dyes for the biological microchip technology].

New indodicarbocyanine dyes with the carboxybutyl group in position 3 of the indolenine fragment bearing methyl and sulfonic groups in positions 5 and 7 of the cycle were synthesized in order to find the most effective fluorescent labels for the biological microchip technology. The position of absorption and fluorescence maxima, the total charge of the dye molecule, and water solubility depend on the location and the total amount of methyl and sulfonic groups. The spectral characteristics of the dyes synthesized were determined. The relative fluorescence efficiencies of the dyes at equal concentrations were measured at excitation wavelengths of 635 and 655 nm and emission wavelengths of 670 and 690 nm, respectively.

[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.

[Development of microchips for the detection of mutations of HIV-1 variability to protease inhibitors and the usage results]. / Razrabotka biologicheskikh mikrochipov dlia vyiavleniia mutatsii ustoichivosti VICH-1 k ingibitoram proteazy i rezul'taty ikh primeneniia.

An original biochip was constructed for the detection of 34 mutations of HIV-1 resistance to protease. A technology was worked out, which is based on the hybridization of a fluorescence-labeled amplified fragment of the pol gene of the HIV-1 provirus DNA with a set of specific oligonucleotides immobilized in 3-D hydrogel pads of the biological microchip. The biochip was used to analyze 115 samples of the subtype-1 provirus HIV-1 DNA isolated from untreated IDUs and their sexual partners in 15 regions of former USSR countries. Substitution of Val/IIe in position 77 of protease (V771) is known as secondary mutation of resistance to Nelfinavir detected in 55 (47.8%) of 115 HIV-1 variations. Its first appearance was registered in a patient with HIV in April 1997 in Tver, where its carrying variant caused an HIV outbreak. It is demonstrated that the V771-substitution variant, that dominates in Moscow, caused outbreaks in Irkutsk and Yekaterinburg and spread into separate districts of Perm and Perm Region. At the same time, no V771 HIV-1 was detected in any of the HIV studied cases diagnosed before 1998 in Moldova, Ukraine and Rostov Region.

[The genetic variability of the protease-encoding region in HIV-1 A isolates and in CRF03_AB recombinants as observed in the CIS territory]. / Geneticheskaia izmenchivost' oblasti, kodiruiushchei proteazu, u izoliatov VICH-1 podtipa A i rekombinantov CRF03_AB na territorii SNG.

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 (0.00-3.23; mean--1.38 +/- 0.79). No isolates contained any primary mutations of resistance to protease inhibitors. At the same time, above one half of the isolates bore the V771 substitution, which, according to published data, is the secondary mutation of resistance that conditions a higher resistance to Nelfinavir. Moreover, the substitution was associated with 2 synonymous mutations in triplets 31 and 78, which denotes a single origin for all V771 variants.

[New technologies in the determination of drug susceptibility in Mycobacterium tuberculosis]. / Novye tekhnologii opredeleniia lekarstvennoi chuvstvitel'nosti 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. Detection of several MBT strains in one patient requires the use a combination of molecular biological and microbiological studies.

[Microchips based on three dimensional gel cells: history and perspective]. / Mikrochipy na osnove trekhmernykh iacheek gelia: istoriia i perspektivy.

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. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous versions. The microchips are manufactured by photo-initiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical strength and stability, which allowed one to work with the DNA fragments of up to 500 nt in length, as well as with rather large protein molecules. At present, the gel-based microchips are widely applied to address different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA-protein interactions. The oligonucleotide microchips are a cheap and reliable tool of diagnostics designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; for diagnostics of orthopoxviruses, including the smallpox virus; for diagnostics of the anthrax pathogen; and for identification of chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteomics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which open a wide potential for creation biosensors on the basis of microchips.

Hydrogel drop microchips with immobilized DNA: properties and methods for large-scale production.

Although gel-based microchips offer significant advantages over two-dimensional arrays, their use has been impeded by the lack of an efficient manufacturing procedure. Here we describe two simple, fast, and reproducible methods of fabrication of DNA gel drop microchips. In the first, copolymerization method, unsaturated groups are chemically attached to immobilized molecules, which are then mixed with gel-forming monomers. In the second, simpler polymerization-mediated immobilization method, aminated DNA without prior modification is added to a polymerization mixture. Droplets of polymerization mixtures are spotted by a robot onto glass slides and the slides are illuminated with UV light to induce copolymerization of DNA with gel-forming monomers. This results in immobilization of DNA within the whole volume of semispherical gel drops. The first method can be better controlled while the second one is less expensive, faster, and better suited to large-scale production. The microchips manufactured by both methods are similar in properties. Gel elements of the chip are porous enough to allow penetration of DNA up to 500 nucleotides long and its hybridization with immobilized oligonucleotides. As shown with confocal microscope studies, DNA is hybridized uniformly in the whole volume of gel drops. The gels are mechanically and thermally stable and withstand 20 subsequent hybridizations or 30-40 PCR cycles without decrease in hybridization signal. A method for quality control of the chips by staining with fluorescence dye is proposed. Applications of hydrogel microchips in research and clinical diagnostics are summarized.

[Identification of Mycobacterium tuberculosis strains and a simultaneous identification of their drug resistance by the hybridization method on oligonucleotide microchips]. / Opredelenie shtammov Mycobacterium tuberculosis s odnovremennym opredeleniem ikh lekarstvennoi ustoichivosti metodom gibridizatsii na oligonukleotidnykh mikrochipakh.

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. The method provides for detecting above 95% of rifampin-resistant and around 80% of isoniazid-resistant strains within 1 day.

Typing of Mycobacterium tuberculosis strains resistant to rifampicin and isoniazid by molecular biological methods.

Mutations in the rpoB, katG, inhA, oxyR/ahpC genes in rifampicin- and isoniazid-resistant M. tuberculosis strains isolated from residents of Moscow, Astrakhan', and Moldova Republic were studied by molecular biological methods (heteroduplex analysis, single strand conformational polymorphism, biochips). Twenty-five combinations of mutations were detected. Some differences in the type distribution of detected mutations were found. The use of biochips is the most perspective method for determining the type of mutation.

[Identification of orthopoxvirus species using oligonucleotide micro-chips]. / Vidovaia identifikatsiia ortopoksvirusov na oligonukleotidnykh mikrochipakh.

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.e. an important gene determining the pathogenicity of the mentioned Orthopoxviruses type, were used as a target for identification. The identification procedure takes around 6 hours and does not demand any costly equipment (a portable fluorescent microscope can be used).

[Clinical value of microchip technology in determination of drug resistance of Mycobacterium tuberculosis]. / Klinicheskoe znachenie mikrochipovoi tekhnologii opredeleniia lekarstvennoi ustoichivosti mikobakterii tuberkuleza.

The patients with multiresistant tuberculosis were divided into 2 groups: the sensitivity of Mycobacteria tuberculosis to antituberculous drugs was evaluated in Group 1 by the methods of absolute concentrations and in Group 2 by biological microchips determining mutations in the rpo3 gene responsible for rifampicin resistance. The results of the drug sensitivity test were obtained after 3 months of treatment in Group 1 and several days prior treatment in Group 2. By taking into account the test results, reserve drugs was used in Group 2 patients. Subsequently, the results of the drug sensitivity tests carried out by the bacteriological method in Group 2 patients showed that isoniazid resistance was simultaneously noted if there were mutations in the rpo-B gene. Timely treatment with reserve drugs exhibited higher efficiency of treatment with its shorter duration in Group 2 than in Group 1.

[Molecular genetic methods for the detection of rifampicin-resistant Mycobacterium tuberculosis strains]. / Molekuliarno-geneticheskie metody vyiavleniia rifampitsinrezistentnykh shtammov Mycobacterium tuberculosis.

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. Correlations between GDA (sputum) and drug test (LJ) were 91.7%, that of chip (sputum) and drug test LJ, 88.5%, chip (sputum) and chip clinical isolates (LJ), 100%. The efficacy of GDA and chip in the detection of RR of MTB strains is under discussion.