[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.

[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.

[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.

[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. Two different methods were compared. In first, selective ligation of short oligonucleotides immobilized on microarray was used with subsequent amplification on preformed circle probe ("common circle"). The circle probe was designed especially for human genome research. In second variant, allele-specific padlock probes that may be circularized by selective ligation were immobilized on microarray. Polymorphism of codon 72 in human p53 gene was used as a biological model. It was shown that LDR/RCA on microarray is a quantitative reaction and gives high discrimination of alleles. Principles and perspectives of selective ligation and rolling circle amplification are being discussed.

[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.

[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.

[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).

[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.