Francisella tularensis is a bacterial pathogen that can contaminate drinking water and food products, causing tularemia - a severe zoonotic disease that affects both humans and animals. This infection is dangerous to human health and may be fatal without diagnosis and treatment. Therefore, rapid, sensitive, and selective detection of this microorganism is in high demand for clinical diagnostics, environmental monitoring, and ensuring food safety. This study presents the development of immunochromatographic (lateral flow) tests for revealing F. tularensis cells in natural and drinking water samples. A special feature of the test system is catalytically active Au@Pt nanoparticles (a peroxidase-like nanozyme) used as a label for specific antibodies. Au@Pt nanozyme can catalyze the oxidation of the peroxidase substrate, followed by the formation of a colored product, which amplifies the colorimetric signal on the test strip and significantly improves detection sensitivity. The tests allow for the detection of F. tularensis cells in concentrations down to 102-103 cells per mL, depending on the strain, with visual result assessment. The application of Au@Pt nanozyme decreased the detection limits by 321-9600 times compared to gold nanoparticles commonly used in immunochromatography. The assay duration is 14 min, including the catalytic enhancement step. Monoclonal antibodies against bacterial lipopolysaccharide provide selective detection of virulent strains, excluding cross-reactions with non-pathogenic strains of F. tularensis and other microorganisms that may also contaminate water sources. The effective testing of natural and tap water samples conducted with no preliminary sample preparation has proven the relevance of the developed approach.

CRISPR/Cas12a systems coupled with lateral flow tests (LFTs) are a promising route to rapid, instrument-free nucleic acid diagnostics due to conversion target recognition into a simple visual readout via cleavage of dual-labeled single-stranded DNA reporters. However, the conventional CRISPR/Cas12a-LFT system is constructed in a format where the intact reporter should block nanoparticle conjugate migration and can produce false-positive signals and shows strong dependence on component stoichiometry and kinetics. Here, we present the first combined experimental and theoretical analysis quantifying these limitations and defining practical solutions. The experimental evaluation included 480 variants of LFT configuration with reporters differing in the concentration of interacting components and the kinetic conditions of the interactions. The most influential factor leading to 100% false-positive results was insufficient interaction time between the components; pre-incubation of the conjugate with the reporter for 5 min eliminated these artifacts. Theoretical analysis of the LFT kinetics based on a mathematical model confirmed kinetic constraints at interaction times below a few minutes, which affect the detectable signal. Reporter concentration and conjugate architecture represented the second major factors: lowering reporter concentration to 20 nM and using smaller gold nanoparticles with multivalent fluorescent reporters markedly improved sensitivity. The difference in sensitivity between various LFT configurations exceeded 50-fold. The combination of identified strategies eliminated false-positive reactions and enabled the detection of up to 20 pM of DNA target (the hisZ gene of Erwinia amylovora, a bacterial phytopathogen). The strategies reported here are general and readily transferable to other DNA targets and CRISPR/Cas12a amplification-free diagnostics.

To ensure the safety of foodstuffs, widespread non-laboratory monitoring for pathogenic contaminants is in demand. A suitable technique for this purpose is lateral flow immunoassay (LFIA) which combines simplicity, rapidity, and productivity with specific immune detection. This study considered three developed formats of LFIA for Salmonella Typhimurium, a priority pathogenic contaminant of milk. Common sandwich LFIA with all immunoreagents pre-applied to the test strip (format A) was compared with incubation of the sample and (gold nanoparticle—antibody) conjugate, preceding the lateral flow processes (format B), and sequential passages of the sample and the conjugate along the test strip (format C). Under the chosen conditions, the detection limits and the assay times were 3 × 104, 1 × 105, and 3 × 105 cells/mL, 10, 15, and 20 min for formats A, B, and C, respectively. The selected format A of LFIA was successfully applied to test milk samples. The sample’s dilution to a fat content of 1.0% causes pathogen detection, with 70–110% revealing and 1.5–8.5% accuracy. The obtained results demonstrate that the developed LFIA allows the detection of lower concentrations of Salmonella cells and, in this way, accelerates decision-making in food safety control.

Sequence-specific endonuclease Cas12-based biosensors have rapidly evolved as a strong tool to detect nucleic acids. Magnetic particles (MPs) with attached DNA structures could be used as a universal platform to manipulate the DNA-cleavage activity of Cas12. Here, we propose nanostructures of trans- and cis-DNA targets immobilized on the MPs. The main advantage of the nanostructures is a rigid double-stranded DNA adaptor that distances the cleavage site from the MP surface to ensure maximum Cas12 activity. Adaptors with different lengths were compared by detecting the cleavage by fluorescence and gel electrophoresis of the released DNA fragments. The length-dependent effects for cleavage on the MPs' surface were found both for cis- and trans-targets. For trans-DNA targets with a cleavable 15-dT tail, the results showed that the optimal range of the adaptor length was 120-300 bp. For cis-targets, we varied the length and location of the adaptor (at the PAM or spacer ends) to estimate the effect of the MP's surface on the PAM-recognition process or R-loop formation. The sequential arrangement of an adaptor, PAM, and a spacer was preferred and required the minimum adaptor length of 3 bp. Thus, with cis-cleavage, the cleavage site can be located closer to the surface of the MPs than with trans-cleavage. The findings provide solutions for efficient Cas12-based biosensors using surface-attached DNA structures.

Introduction. Epizootological monitoring of the area contamination with the causative agent of tularemia implies the collection and analysis of a variety of field specimens. The analysis of such objects is time- and labour-consuming. In this context, simple and fast diagnostic techniques are needed to analyze specimens under resource-limited conditions. Aim. To study the possibility of using immunomagnetic separation for accelerated detection of Francisella tularensis cells in soil samples using immunochromatography. Materials and methods. Immunomagnetic particles (IMPs) were produced by using monoclonal antibodies to lipopolysaccharide (LPS) of the tularemia causative agent. Soil specimens weighing 1 g with preliminary introduced inactivated F. tularensis 15/10 cells were used in the study. The samples were suspended in an extraction buffer (EB) and filtered. Tularemia cells were separated by IMP suspension. The particles were washed, resuspended in EB and heated at 100C for 5 minutes. The supernatant was analyzed with test strips based on F. tularensis IC-test kit. Results. A combination of the immunomagnetic separation method and the IC test to detect F. tularensis cells identified up to 1 106 cells of the tularemia pathogen in analyzed soil samples, while 1 107 cells were detected in soil washouts in the absence of immunomagnetic separation. Conclusion. The developed technique combining immunomagnetic separation and IC tests opens up prospects for express diagnostics of soil sample contamination in tularemia foci. The analysis takes about 3 hours, and its sensitivity is 1 106 cells/g of soil. The technique is simple, not requiring sophisticated expensive equipment. It can be easily adapted for testing other specimen types (water, grain, etc.). In addition, separated bacterial cells can be used for F. tularensis detection by other methods.

The paper presents data on the identification of a new staphylolytic enzyme from the cultural liquid of Staphylococcus hyicus B-8870. The primary sequence of the enzyme has the maximum similarity to the CHAP domain of N-acetylmuramoyl-L-alanine amidase from Staphylococcus sciuri DD 4747. The enzyme is active against a wide range of microorganisms of the Staphylococcus genus, including MRSA strains. The molecular weight of the enzyme is 13993 Da, the absorption coefficient at 280 nm is \(\varepsilon \frac{{{\text{mg}}}}{{{\text{ml}}}}\) 3.94, the value of the isoelectric point pI 10.35. The specific activity of the enzyme in relation to the cell suspension of S.aureus FDA 209P is 1518 U/mg with an optimum pH of 7.7 and a temperature of 40°C.

The paper presents development and characterization of a new bioanalytical test system for rapid detection of lipopolysaccharide (LPS) and whole cells of Francisella tularensis, a causative agent of tularemia, in water samples. Gold nanoparticles (AuNPs) coated by the obtained anti-LPS monoclonal antibodies were used for the assay. Their contact with antigen in tested samples leads to aggregation with a shift of absorption spectra from red to blue. Photometric measurements at 530 nm indicated the analyte presence. Three preparations of AuNPs with different diameters were compared, and the AuNPs having average diameter of 34 nm were found to be optimal. The assay is implemented in 20 min and is characterized by detection limits equal to 40 ng/mL for LPS and 3 × 104 CFU/mL for whole cells of F. tularensis. Thus, the proposed simple one-step assay integrates sensitivity comparable with other immunoassay of microorganisms and rapidity. Selectivity of the assay for different strains of F. tularensis was tested and the possibility to choose its variants with the use of different antibodies to distinguish virulent and non-virulent strains or to detect both kinds of F. tularensis was found. The test system has been successfully implemented to reveal the analyte in natural and tap water samples without the loss of sensitivity.

Tularemia is a natural focal zoonotic infection that can cause epidemic manifestations of an emergency nature. The purpose of the study is to develop a method for detecting DNA strains of the tularemia pathogen Francisella tularensis by loop mediated isothermal amplification (LAMP). Primers for the selected targets were calculated using the on-line Primer Explorer 5 program and tested for the specificity using the BLAST program. The primers were synthesized by Synthol, Moscow. Isolation of DNA from vaccine and virulent strains of epidemically significant subspecies of the tularemia microbe, as well as pathogens of other infectious diseases, was performed using the commercial DNA-sorb-B kit (InterLabService, Russia). The amplification reaction was carried out at a temperature of 63°C for 60 min (without loop primers) or 30 min (with loop primers) with preliminary heating at a temperature of 92°C for 2 min on a Tertsik amplifier (DNA-Technology, Russia) in the presence of the thermostable SD polymerase. Sequences of the genes encoding acid phosphatase A ( acpA ), outer membrane protein ( fopA ), and a region of the iglC gene of the pathogenicity island were chosen as DNA targets for the detection of the tularemia pathogen. Of the two sets of outer, inner, and loop original primers synthesized for each selected marker gene, the sets acpFt101, fopFt132, and iglCFt1 reproducibly and specifically detected DNA of 100–1000 F. tularensis microbial cells. The opportunity to reduce the analysis time by half appeared due to the introduction of loop primers and visual detection of amplification products stained with the SYTO 82 intercalating dye without subsequent electrophoresis and visualization of the gel after staining with ethidium bromide. An easy-to-use test that does not require sophisticated equipment is proposed for clinical and field diagnostics of the tularemia pathogen.

Tularemia is an especially dangerous infection caused by the gram-negative bacterium Francisella tularensis. It belongs to natural focal infections, and therefore is under continuous control by quarantine services. When carrying out their activities they use a whole range of diagnostic tools. The objective of this research is to develop an enzyme immunoassay based on highly specific monoclonal antibodies and immunomagnetic particles for monitoring the tularemia pathogen. To produce hybridomas mice were immunized with cells of the vaccine strain F. tularensis subsp. holarctica 15 NIIEG. After cell fusion hybridomas were selected by a solid-phase enzyme immunoassay (ELISA) using lipopolysaccharide (LPS) of the tularemia microbe. As a result, two hybridomas, 1C2 and 3F5, were produced. MABs of the hybridomas were obtained by using BALB / c mice. The MABs were purified by sepharose A affinity chromatography and used for conjugation with magnetic particles, and for biotinylation followed by matching a pair for ELISA. The pair of IMPs and MABs 3F5 as well as biotinylated FB11-x MABs was the best in detecting tularemia cells. The use of this MAB pair in ELISA allowed the identification of 105 microbial cells/ml in a 4 ml sample and 5×103 microbial cells/ml in a 45ml sample. Interaction with F. tularensis subsp. novicida Utah112 cells was absent.

The current COVID-19 pandemic has increased the demand for pathogen detection methods that combine low detection limits with rapid results. Despite the significant progress in methods and devices for nucleic acid amplification, immunochemical methods are still preferred for mass testing without specialized laboratories and highly qualified personnel. The most widely used immunoassays are microplate enzyme-linked immunosorbent assay (ELISA) with photometric detection and lateral flow immunoassay (LFIA) with visual results assessment. However, the disadvantage of ELISA is its considerable duration, and that of LFIA is its low sensitivity. In this study, the modified LFIA of a specific antigen of the causative agent of COVID-19, spike receptor-binding domain, was developed and characterized. This modified LFIA includes the use of gold nanoparticles with immobilized antibodies and 4-mercaptobenzoic acid as surface-enhanced Raman scattering (SERS) nanotag and registration of the nanotag binding by SERS spectrometry. To enhance the sensitivity of LFIA-SERS analysis, we determined the optimal compositions of SERS nanotags and membranes used in LFIA. For benchmark comparison, ELISA and conventional colorimetric LFIA were used with the same immune reagents. The proposed method combines a low detection limit of 0.1 ng/mL (at 0.4 ng/mL for ELISA and 1 ng/mL for qualitative LFIA) with a short assay time equal to 20 min (at 3.5 h for ELISA and 15 min for LFIA). The results obtained demonstrate the promise of using the SERS effects in membrane immuno-analytical systems.

The purpose of this study was the identification of genetic lineages and antimicrobial resistance (AMR) and virulence genes in Klebsiella pneumoniae isolates associated with severe infections in the neuro-ICU. Susceptibility to antimicrobials was determined using the Vitek-2 instrument. AMR and virulence genes, sequence types (STs), and capsular types were identified by PCR. Whole-genome sequencing was conducted on the Illumina MiSeq platform. It was shown that K. pneumoniae isolates of ST14(K2), ST23(K57), ST39(K23), ST76(K23), ST86(K2), ST218(K57), ST219(KL125/114), ST268(K20), and ST2674(K47) caused severe systemic infections, including ST14(K2), ST39(K23), and ST268(K20) that were associated with fatal incomes. Moreover, eight isolates of ST395(K2) and ST307(KL102/149/155) were associated with manifestations of vasculitis and microcirculation disorders. Another 12 K. pneumoniae isolates of ST395(K2,KL39), ST307(KL102/149/155), and ST147(K14/64) were collected from patients without severe systemic infections. Major isolates (n = 38) were XDR and MDR. Beta-lactamase genes were identified: bla(SHV) (n = 41), bla(CTX-M) (n = 28), bla(TEM) (n = 21), bla(OXA-48) (n = 21), bla(NDM) (n = 1), and bla(KPC) (n = 1). The prevalent virulence genes were wabG (n = 41), fimH (n = 41), allS (n = 41), and uge (n = 34), and rarer, detected only in the genomes of the isolates causing severe systemic infections-rmpA (n = 8), kfu (n = 6), iroN (n = 5), and iroD (n = 5) indicating high potential of the isolates for hypervirulence.

Three techniques were compared for lowering the limit of detection (LOD) of the lateral flow immunoassay (LFIA) of the receptor-binding domain of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) based on the post-assay in situ enlargement of Au nanoparticles (Au NPs) on a test strip. Silver enhancement (growth of a silver layer over Au NPs-Au@Ag NPs) and gold enhancement (growth of a gold layer over Au NPs) techniques and the novel technique of galvanic replacement of Ag by Au in Au@Ag NPs causing the formation of Au@Ag-Au NPs were performed. All the enhancements were performed on-site after completion of the conventional LFIA and maintained equipment-free assay. The assays demonstrated lowering of LODs in the following rows: 488 pg/mL (conventional LFIA with Au NPs), 61 pg/mL (silver enhancement), 8 pg/mL (galvanic replacement), and 1 pg/mL (gold enhancement). Using gold enhancement as the optimal technique, the maximal dilution of inactivated SARS-CoV-2-containing samples increased 500 times. The developed LFIA provided highly sensitive and rapid (8 min) point-of-need testing.

Despite the fact that the incidence of leprosy in Russia is sporadic, the number of newly identified patients has increased in recent years. In 2017–2018, 4 new cases of leprosy were registered in Russia. The standard methods of research for diagnosis, in addition to the clinical picture, are bacterioscopic study of the skin scarificates and histological study of the skin biopsy specimen. Currently, additional methods are being developed and used to confirm the diagnosis of leprosy, namely, modern serological and genetic diagnostic methods. To use methods such as enzyme-linked immunosorbent assay (ELISA) and membrane immunochromatographic analysis (leprosy LF serotest), it is appropriate to use domestic synthetic mycobacterial antigens (SMA). Key words: Mycobacterium leprae, leprosy, synthetic mycobacterial antigens, PGL-1(phenolic glycolipid-1), LAM (lipoarabinomannan), serodiagnostics, enzyme-linked immunosorbent assay (ELISA), lateral flow (LF) test, BSA (bovine serum albumin)

Here, «host-vector» expression system of Brevibacillus choshinensis was developed and used for producing a recombinant lysostaphin with high-output. The recombinant plasmid pNCMO2/lsf12 was constructed, and its expression in Brevibacillus choshinensis (strain Brevibacillus choshinensis/pNCMO2/lsf12) provided a synthesis of the 27-kDa protein, which was secreted into the culture medium. Its specific staphylolitic activity being 557 U/mg at optimal pH (7.5-8.0) and temperature (50-55 °C) values was comparable with the natural and recombinant analogs. We hope that developed methods of a deep cultivation of the recombinant Brevibacillus choshinensis/pNCMO2/lsf12 strain for a high-yield production (up to 90 mg/L) and a single-stage purification of lysostaphin (up to 90% homogeneity) become the basis for the production of the enzyme on an industrial scale. Brevibacillus choshinensis, ion-exchange chromatography, lysostaphin The work was financially supported by the Grant No. 050 of Rospotrebnadzor «Monitoring of borreliosis pathogens circulation in regions of the Russian Federation and improvement of diagnostic tools for borreliosis»

The glycoconjugates with BSA (bovine serum albumin) were synthesized using a next saccharide: disaccharide derivative M.leprae PGL-1 (phenolic glycolipid-1); a complex of the disaccharide fragment and the branched hexasaccharide fragment LAM (lipoarabinomannan); diarabinofuranose fragment LAM. These glycoconjugates were used as antigenic components for leprosy rapid serotest construction in immunochromatographic format (leprosy LF serotest). The data obtained with sera of leprosy patients, patients who have been in contact with leprosy, and healthy donors indicate that the most promising antigenic component is a BSA conjugate with two synthetic epitopes - a disaccharide derivative of PGL-1 and a branched hexasaccharide fragment of LAM. The leprosy LF serotest with such glycoconjugate demonstrated the greatest diagnostic sensitivity for main forms of leprosy - paucibacillary (PB) and multibacillary (MB).

Causative agents of melioidosis and glanders are among the most dangerous bacterial pathogens for human. Moreover, Burkholderia pseudomallei and Burkholderia mallei are considered to be potential bioterrorism agents. In connection with this, timely diagnostics of such bacteria is of high importance. In our study, we made an attempt to develop an approach for detecting pathogenic Burkholderia spp. by combining species-specific amplification and strain-specific dot blotting assay with monoclonal antibodies. The following pathogenic Burkholderia strains were used in experiments: B. mallei (C-4, C-5, t-12, B-120, P-1, Мuksuwar-11, Z-12,Zagreb, Ivanovich, 5534), and B. pseudomallei (100, 102, 115, 116, 132, 135, 301, 51274, 60913, 61503). Real-Time PCR (RT-PCR) and dot blotting with monoclonal antibodies against surface Burkholderia epitopes were used to detect such pathogens. RT-PCR was carried out by using primers designed to recognize DNA fragments in B. mallei IS407A-fliP and the gene Orf12 from B. pseudomallei . For this, DNA was isolated from bacterial cells suspended at 1 × 104 microbial cells/ml. accumulation of the end reaction products was visualized by staining with dye SYBR Green I. Specificity of amplification reaction was determined by measuring melting temperature (Tm) for end products followed by running gel electrophoresis. It was demonstrated that all ten strains of either B. mallei or B. pseudomallei examined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. It was demonstrated that all ten strains of either B. mallei or B. pseudomallei examined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. Importantly, no signals specific to heterologous microbial DNA (isolated from bacterial cell suspension at concentration of 1 × 107 microbial cells/ml) were detected by using RT-PCR. Thus, RT-PCR provides an opportunity for assessing an inter-species diversity among pathogenic Burkholderia species. A genus-specificity was observed by using monoclonal antibodies 3D3 which bind to both Burkholderia strains, whereas antibodies 2D11 exhibited no selective binding to strain Р1 B. mallei and strain 100 B. pseudomallei, thereby displaying a strain-specific interaction. Thus, it allowed to conclude that combining a species-specific DNA amplification particularly RT-PCR together with immune-based assay such as dot blotting by using a panel of monoclonal antibodies seems to be a promising approach for assessing intra-species diversity among pathogenic Burkholderia.