Real-time or quantitative loop-mediated isothermal amplification (qLAMP) is a promising technique for the accurate detection of pathogens in organisms and the environment. Here we present a comparative study of the performance of six fluorescent intercalating dyes-SYTO-9, SYTO-13, SYTO-82, SYBR Green I, SYBR Gold, EvaGreen-in three different qLAMP model systems. SYTO-9 and SYTO-82, which had the best results, were used for additional enzyme and template titration studies. SYTO-82 demonstrated the best combination of time-to-threshold (Tt) and signal-to-noise ratio (SNR).

At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNA-binding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants—urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance.

Immobilization of biocatalysts on several inorganic monolithic supports has been studied. Transition metals added to TiO:, cz-A1203 and ah~minosilicate change the specific protein binding and support capacity. It is shown that immobilized biocatalysts are stable and can be used to run complex biotechnological processes.

The DNA-binding domain of the DNA ligase from Pyrococcus abyssi (PabDBD) was mapped and cloned into two expression vectors. The resulting 6X His-tagged proteins, with a predicted molecular mass of approximately 30 kDa, were overexpressed, purified using Ni-NTA resin, and biochemically characterized. Both PabDBD derivatives bound to double-stranded DNA fragments at the temperature range of 40-70 °C, and both were inactivated via heating at 95 °C for 15 min. Complexes of the PabDBD variants with either double- and single-stranded DNA fragments were less stable than the native DNA ligase of P. abyssi. Inclusion of the C-terminally 6X His-tagged PabDBD in the reaction mixture during long-range polymerase chain reaction (PCR) increased the efficacy of amplification and eliminated the inhibitory effect of heparin.

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We investigated whether levels and repertoires of anti-interleukin-18 (IL-18) autoantibodies (auto-Abs) differ in multiple sclerosis (MS) patients and healthy donors (HDs). IL-18 concentration in MS patients' sera was higher than in HD, but the level of anti-IL-18 auto-Abs was lower in MS patients. Correlation patterns of IL-18/anti-IL-18 auto-Abs system differed in HD and MS patients, so we have compared segment composition of the anti-IL-18 single-chain variable fragments (scFvs) selected from MS and naïve phage display libraries. Considerable differences between anti-IL-18 auto-Abs of these libraries were found. MS panel contained auto-Abs displaying both signs of "fetal" and somatically hypermutated repertoires. Naïve panel mainly contained the naïve antibodies. These variations from the norm are possible results of abnormal regulation of the repertoire perhaps determined by remodeling of the molecular mechanisms involved in the V(D)J recombination and/or abnormal selection by antigen in MS pathogenesis.