[Recombinase Polymerase Amplification for Rapid Detection of Human Bacterial Pneumonia Pathogens].

A diagnostic system based on recombinase polymerase amplification (RPA) has been developed to identify six bacterial pathogens of human pneumonia. Species-specific primers have been designed and optimized to conduct a multiplex reaction in one common volume. Labeled primers were used for reliable discrimination of amplification products that are similar in size. Identification of the pathogen was carried out by visual analysis of an electrophoregram. The analytical sensitivity of the developed multiplex RPA was 10^(2)-10^(3) copies of DNA. The specificity of the system was determined by the absence of cross-amplification of the studied DNA samples of pneumonia pathogens for each pair of primers, as well as for the DNA of Mycobacterium tuberculosis H37rv, and amounted to 100%. The execution time of the analysis is less than an 1 h, including the electrophoretic reaction control. The test system can be used in specialized clinical laboratories for rapid analysis of samples from patients with suspected pneumonia.

[New Method for Evaluating the Substrate Efficiency of Modified Deoxynucleoside Triphosphates for Selex].

A new method for evaluating the substrate efficiency of deoxynucleoside triphosphates containing functional groups for the selection of modified aptamers (mod-SELEX) is proposed. The method involves conducting three consecutive rounds of PCR with a combinatorial library and a modified dNTP candidate for mod-SELEX. The conclusion about the applicability of a specific dNTP derivative is made by the nature of the change in the amplification curve during the three rounds of PCR in real time and does not require SELEX rounds. If the library degenerates during amplification (becomes less representative), it means that the specific modification of dNTP cannot be used with the selected polymerase and the other selected library amplification conditions, since it leads to competitive amplification. When the nature of the signal accumulation curve does not change, it is concluded that the modified triphosphate does not affect the distribution of oligonucleotides with different sequences in the library, that is, it does not lead to a change in its composition from the point of view of the applied detection method. It is these derivatives that can be applied with the selected conditions for the selection of aptamers. The method is applicable for quick assessment of the substrate suitability of modifications introduced into deoxynucleoside triphosphates for mod-SELEX and will be useful in the selection of aptamers for clinical diagnostics, medicine and scientific research.

[Study of Multiple Enzymatic Incorporation of Modified Nucleotides of Purine and Pyrimidine Nature in the Growing DNA Chain].

The substrate properties of nitrogen-base modified derivatives of purine and pyrimidine deoxynucleoside triphosphates during their simultaneous pairwise insertion into the growing DNA strand have been studied. Modified nucleotides were introduced using real-time PCR and the primer extension reaction; in one reaction, derivatives with both different and similar functional substituents were used. Genomic bacterial DNA, specially constructed synthetic DNA fragments, and SELEX libraries were used as templates. The reactions were performed using DNA polymerases with no 3'-5' correcting exonuclease activity: Taq, Vent (exo-), DeepVent (exo-), and KOD XL. It was shown that the substrate efficiency is affected by both the size of the substituent group and the chemical nature of deoxynucleoside triphosphate. The effectiveness varies significantly depending on the polymerase used. The most effective of the studied substrates are pyrimidine deoxynucleoside triphosphates in combination with Vent (exo-) DNA polymerase. DNAs modified by pairs of dissimilar nucleotides (dU + dC, dU + dA, dC + dA) with similar and different functional substituents were obtained.

[Development of Multiplex RT-PCR with Immobilized Primers for Identification of Infectious Human Pneumonia Pathogens].

A prototype of a system for the detection of infectious human pneumonia pathogens based on multiplex solid-phase reverse transcription PCR (RT-PCR) was developed. Primers were designed to identify the DNA of six bacterial pneumonia pathogen strains, and the RNA of two viral pathogens of pneumonia: influenza A and SARS-CoV-2. The signal accumulation of elongated immobilized primers occurs due to the incorporation of fluorescently labeled nucleotides in the chain. The signal is detected after all the components of the mixture are removed, which significantly reduces the background signal and increases the sensitivity of the analysis. The use of a specialized detector makes it possible to read the signals of elongated primers directly through the transparent cover film of the reaction chamber. This solution is designed to prevent cross-contamination and is suitable for simultaneous testing of a large number of test samples. The proposed platform is able to detect the presence of several pathogens of pneumonia in a sample and has an open architecture that allows expansion of the range of pathogenic bacteria and viruses that can be detected.

Multiplex PCR for Identification of Bacterial Pathogens of Infectious Pneumonia.

A multiplex PCR system has been developed and optimized for rapid detection of the five main pathogens of bacterial pneumonia. The system can be expanded to analyze viral pathogens of pneumonia (DNA- and RNA-containing viruses), as well as those of a fungal nature.

[Enzymatic Preparation of Modified DNA: Study of the Kinetics by Real-Time PCR].

The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP-template-polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature-time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).

[Substrate Properties of New Fluorescently Labeled Deoxycytidine Triphosphates in Enzymatic Synthesis of DNA with Polymerases of Families A and B].

The efficiency of the incorporation of fluorescently labeled derivatives of 2'-deoxycytidine in DNA synthesized de novo has been studied using PCR with Taq and Tth polymerases of family A and Vent (exo-) and Deep Vent (exo-) polymerases of family B. Four derivatives of 5'-triphosphate-2'-deoxycytidine (dCTP) have different chemical structures of the indodicarbocyanine dye and Cy5 analogue attached to position 5 of cytosine. The kinetics of the accumulation of the PCR products and the intensity of the fluorescent signals in the hybridization analysis with immobilized DNA probes depend on the modification of the fluorescently labeled dCTP counterpart, its concentration, and the type of DNA polymerase. All labeled triphosphates showed some inhibitory effects on PCR. The best balance between the efficiency of incorporating labeled cytidine derivatives and the negative effect on the PCR kinetics has been shown in the case of Hot Taq polymerase in combination with the Cy5-dCTP analogue, which contains containing electrically neutral chro-mophore, the axis of which is a continuation of the linker between the chromophore and the pyrimidine base.

[Preparation of Modified Combinatorial DNA Libraries via Emulsion PCR with Subsequent Strand Separation].

A modification of the enzymatic method for the preparation of combinatorial random DNA libraries, which combines amplification in isolated microvolumes with the simultaneous incorporation of modified nucleotides and subsequent separation of DNA strands, was developed. Deoxyuridine triphosphate with hydrophobic substituents such as structural analogues of amino acid side chains in the C5 position of the pyrimidine ring was used to introduce modifications into DNA. To prevent competitive amplification, which reduces the representativeness of combinatorial libraries, PCR in inverse emulsion was used. The separation of the strands of PCR products was carried out. There were six single-stranded DNA libraries with complete substitution of deoxythymidine via modified analogues with various functional groups. These DNA libraries are suitable for generating aptamers to protein targets through additional hydrophobic interactions from the introductions of appropriate modifications, and are completely compatible with the SELEX aptamer selection methodology.

Comparative Study of Novel Fluorescent Cyanine Nucleotides: Hybridization Analysis of Labeled PCR Products Using a Biochip.

This study investigated the synthesis and substrate properties of Cy5-labeled dUTP derivatives with different substituents, linkers between the dye unit and pyrimidine heterocycle and fluorophore charges. Fluorescently labeled nucleoside triphosphates were studied as substrates using multiplex PCR with Taq and Vent (exo-) DNA polymerases, the typical representatives of the A and B polymerase families. The efficiency of nucleotide incorporation during PCR was assessed with a multi-parameter hybridization analysis using a diagnostic DNA microarray. The hybridization analysis indirectly estimates the incorporation efficiency of dye-labeled nucleotides in multiplex PCR. Our results demonstrated higher efficiencies of substrates with electrically neutral dyes than electropositive and electronegative Cy5 residues.

[Enzymatic synthesis of high-modified DNA].

Here, we describe the synthesis and purification of six deoxyuridine triphosphate derivatives that contain protein-like functional groups and alkene linkers of various lengths. Using KOD XL and Deep Vent polymerases, these derivatives have been incorporated into single-stranded DNA, achieving a high degree of DNA modification. These polymerases are able to utilize highly modified DNA strands as templates for synthesizing unmodified DNA. The synthesized deoxyuridine triphosphate derivatives are promising as substrates for producing modified aptamers to various target proteins using, e.g., the systematic evolution of ligands by exponential enrichment (SELEX) methodology.

[Infrared fluorescent markers for microarray DNA analysis on biological microchip].

To expand the informational capabilities of molecular genetic research, on the biological microchips, new indotricarbocyanine dyes that fluoresce in the near infrared (IR) spectral region have been synthesized. The developed IR dyes were studied using a biochip-based test system for detection of mutations in the BRCA1/BRCA2 and CHECK2 genes associated with breast cancer. The fluorescent label was introduced to the analyzed DNA during PCR using primers labeled with the synthesized IR dyes. An analyzer that allows recording and processing of images of fluorescent microarrays in the IR spectral region was designed and manufactured. It has been shown that the use of the synthesized dyes enables to conduct analysis in the IR region and improve the reliability of medical diagnostic tests due to low fluorescence intensity of sample components as well as of a biochip substrate and the reagents used for analysis.

[FEES for neurogenic dysphagia: training curriculum of the German Society of Neurology and the German Stroke Society]. / FEES für neurogene Dysphagien : Ausbildungscurriculum der Deutschen Gesellschaft für Neurologie und Deutschen Schlaganfall-Gesellschaft.

Neurogenic dysphagia is one of the most frequent and prognostically relevant neurological deficits in a variety of disorders, such as stroke, parkinsonism and advanced neuromuscular diseases. Flexible endoscopic evaluation of swallowing (FEES) is now probably the most frequently used tool for objective dysphagia assessment in Germany. It allows evaluation of the efficacy and safety of swallowing, determination of appropriate feeding strategies and assessment of the efficacy of different swallowing manoeuvres. The literature furthermore indicates that FEES is a safe and well-tolerated procedure. In spite of the huge demand for qualified dysphagia diagnostics in neurology, a systematic FEES education has yet not been established. The structured training curriculum presented in this article aims to close this gap and intends to enforce a robust and qualified FEES service. As management of neurogenic dysphagia is not confined to neurologists, this educational program is applicable to other clinicians and speech language therapists with expertise in dysphagia as well.

[Antagonistic acttvity of Bacillus amyloliquefaciens strains--potential biocontrol agents for cereal crops].

The antagonistic activity of strains Bacillus amyloliquefaciens YKM B-7243 and YKM B-7404 against bacterial and fungal agents of winter wheat diseases was demonstrated. Low antagonistic level against bacterial agents of plant diseases was shown. Antifungal activity of strains against agents of winter wheat was on the low level. High biological efficiency was shown under binary compositions of the studied strains. It was concluded that the enhancing of fungicide and growth-stimulation effects in binary compositions were found.

[Synthesis of extracellular phythohormones by Bacillus strains isolated from different ecological sources].

Synthesis of extracellular phytohormones by bacteria strains of the genus Bacillus isolated from different ecological sources (soil and plant) has been researched. It has been shown that phytohormone's synthesis ability of the endophytic and free-living Bacillus strains is significantly different as regards the level and spectrum of synthesized compounds. It was concluded that such differences in synthesis of extracellular phytohormones by bacilli are associated with different type of interactions between the studied microorganisms and host plant.

Development of Multiplex RT-PCR with Immobilized Primers for Identification of Infectious Human Pneumonia Pathogens.

A prototype of a system for the detection of infectious human pneumonia pathogens based on multiplex solid-phase reverse transcription PCR (RT-PCR) was developed. Primers were designed to identify the DNA of six bacterial pneumonia pathogen strains, and the RNA of two viral pathogens of pneumonia: influenza A and SARS-CoV-2. The signal accumulation of elongated immobilized primers occurs due to the incorporation of fluorescently labeled nucleotides in the chain. The signal is detected after all the components of the mixture are removed, which significantly reduces the background signal and increases the sensitivity of the analysis. The use of a specialized detector makes it possible to read the signals of elongated primers directly through the transparent cover film of the reaction chamber. This solution is designed to prevent cross-contamination and is suitable for simultaneous testing of a large number of test samples. The proposed platform is able to detect the presence of several pathogens of pneumonia in a sample and has an open architecture that allows expansion of the range of pathogenic bacteria and viruses that can be detected.

[Evaluation of the diversity of random DNA-libraries by the shape of amplification curves for estimation of the efficiency of aptamer selection]. / Otsenka raznoobraziia kombinatornykh DNK-bibliotek na osnove analiza formy amplifikatsionnykh krivykh dlia monitoringa éffektivnosti selektsii aptamerov.

Using random (combinatorial) DNA-libraries with various degrees of diversity, it was shown that their amplification by polymerase chain reaction in real time resulted in appearance of a maximum on amplification curves. The relative decrease of fluorescence after passing the maximum was directly proportional to the logarithm of the number of oligonucleotide sequence variants in the random DNA-library provided that this number was within in the interval from 1 to 104 and remained practically unaltered when the number of variants was in the interval from 105 to 108. The obtained dependence was used in the course of SELEX to evaluate changes in the diversity of random DNA-libraries from round to round in selection of DNA-aptamers to the recombinant SMAD4 protein. As a result, oligonucleotides containing sequences able to form a site of SMAD4-DNA interactions known as SBE (SMAD-binding element) have been selected thus indicating that the SMAD4-SBE interaction dominates the aptamer selection.

[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.

Evaluation of hybridisation on oligonucleotide microarrays for analysis of drug-resistant Mycobacterium tuberculosis.

A molecular approach was developed to identify drug-resistant strains of Mycobacterium tuberculosis by means of biochips with oligonucleotides immobilised in polyacrylamide gel pads. The technique was based on multiplex PCR, followed by hybridisation on an oligonucleotide microarray, and detected > 95% of rifampicin-resistant and c. 80% of isoniazid-resistant M. tuberculosis isolates within 12 h. In total, 220 drug-resistant isolates and 131 clinical samples were tested using biochips. The sensitivity and specificity of the developed method were comparable with those of standard bacteriological testing of M. tuberculosis drug resistance.

Discrimination between perfect and mismatched duplexes with oligonucleotide gel microchips: role of thermodynamic and kinetic effects during hybridization.

The efficiency of discrimination between perfect and mismatched duplexes during hybridization on microchips depends on the concentrations of target DNA in solution and immobilized probes, buffer composition, and temperature of hybridization and is determined by both thermodynamic relationships and hybridization kinetics. In this work, optimal conditions of discrimination were studied using hybridization of fluorescently labeled target DNA with custom-made gel-based oligonucleotide microchips. The higher the concentration of immobilized probes and the higher the association constant, the higher the concentration of the formed duplexes and the stronger the corresponding fluorescence signal, but, simultaneously, the longer the time needed to reach equilibrium. Since mismatched duplexes hybridize faster than their perfect counterparts, perfect-to-mismatch signal ratio is lower in transient regime, and short hybridization times may hamper the detection of mutations. The saturation time can be shortened by decreasing the probe concentration or augmenting the gel porosity. This improves the detection of mutations in transient regime. It is shown that the decrease in the initial concentration of oligonucleotide probes by an order of magnitude causes only 1.5-2.5-fold decrease of fluorescence signals after hybridization of perfect duplexes for 3-12 h. At the same time, these conditions improve the discrimination between perfect and mismatched duplexes more than two-fold. A similar improvement may be obtained using an optimized dissociation procedure.

[Some properties of Bacillus subtilis strains active against rotting agents on strawberries and fruit].

This work was aimed at the study of biological and genetic peculiarities of the strains Bacillus subtilis 2/10 and 63z which were selected for protection of strawberries from Botrytis fructigena and for apples under storage. The obtained strains were shown to possess a broad spectrum of antagonistic activity against phytopathogenic fungi and bacteria and this activity was manifested at low temperatures specific for the agriculture production storage. These cultures were not toxigenic or pathogenic in respect of plants and hot-blooded. By RAPD-typing and sequencing of the cloned parts of DNA the genetic specificity of the selected cultures was shown compared with other Bacillus subtilis strains which had not been so active. A set of genetic primes was selected to allow one to reliably identify the given strains during production of biopreparations and to control their use in the field conditions.