Optimization of Total DNA Extraction from Dried Blood Samples.

While dried blood spots are a convenient source of genetic material, they are usually associated with a lower DNA yield than from a native sample. The study evaluated the DNA yield from dried blood samples prepared on glass fibre and cellulose membranes and investigated the reasons for the yield reduction. The extraction of total DNA from membrane-dried blood samples was optimized by spin-column extraction method. It was shown that preliminary short-term (20 min) solubilization of a dried matrix in an aqueous medium, followed by standard extraction protocols for the mixture of the eluate with membranes, provides the highest DNA yield. The yield of DNA from a glass fibre membrane was 40-50% lower compared to a native sample, but on average, two times higher than from a conventional cellulose membrane (filter paper). The reduction of DNA yield when using a dried sample was found to be due to partial retention of nucleic acids by the membrane material during the lysis stage.

Multiplex gradient immunochip for detection of post-vaccinal antibodies in poultry.

Multiplex analysis as an immunochip-in-a well format for simultaneous detection of post-vaccinal antibodies to three poultry infections (Newcastle disease, infectious bronchitis and bursal disease) in one chicken sera was developed. The immunochip had a microarray format printed on the bottom of a standard microtiter plate well and consisted of 36 microspots (d = 400 µm each) with three lines of viral antigens absorbed in a gradient of five decreasing concentrations. Optimization of assay conditions revealed the necessity of careful choice of the reaction buffer due to the high tendency of chicken IgY to exhibit unspecific binding. The best results were obtained for PBS buffer (pH 6.0) supplied with 0.1% Tween 20. Assay results were visualized by a number of coloured microspots that were correlated with the specific antibody titre in the analysed serum. High (> 8000), medium (3000-8000) or low (1000-3000) antibody titre level for each of three infections could be quickly assessed in one probe visually or with the help of smartphone. ELISA results (antibody titres) and visual gradient immunochip results interpretation (high, medium, low antibody level/titre) for 63 chicken sera with multiple levels of post-vaccinal antibodies against Newcastle disease, infectious bronchitis and bursal disease were in good correlation.

Dried Blood Spots technology for veterinary applications and biological investigations: technical aspects, retrospective analysis, ongoing status and future perspectives.

Dried Blood Spots (DBS) technology has become a valuable tool in medical studies, however, in veterinary and biological research DBS technology applications are still limited. Up-to-date no review has comprehensively integrated all the evidence existing across the fields, technologies and animal species. In this paper we summarize the current applications of DBS technology in the mentioned areas, and provide a scope of different types of dried sample carriers (cellulose and non-cellulose), sampling devices, applicable methods for analyte extraction and detection. Mammals, birds, insects and other species are represented as the study objects. Besides the blood, the review considers a variety of specimens, such as milk, saliva, tissue samples and others. The main applications of dried samples highlighted in the review include epidemiological surveys and monitoring for infections agents or specific antibodies for disease/vaccination control in households and wildlife. Besides the genetic investigations, the paper describes detection of environmental contaminants, pregnancy diagnosis and many other useful applications of animal dried samples. The paper also analyses dried sample stability and storage conditions for antibodies, viruses and other substances. Finally, recent developments and future research for DBS technology in veterinary medicine and biological sciences are discussed.

Antiviral adsorption activity of porous silicon nanoparticles against different pathogenic human viruses.

New viral infections, due to their rapid spread, lack of effective antiviral drugs and vaccines, kill millions of people every year. The global pandemic SARS-CoV-2 in 2019-2021 has shown that new strains of viruses can widespread very quickly, causing disease and death, with significant socio-economic consequences. Therefore, the search for new methods of combating different pathogenic viruses is an urgent task, and strategies based on nanoparticles are of significant interest. This work demonstrates the antiviral adsorption (virucidal) efficacy of nanoparticles of porous silicon (PSi NPs) against various enveloped and non-enveloped pathogenic human viruses, such as Influenza A virus, Poliovirus, Human immunodeficiency virus, West Nile virus, and Hepatitis virus. PSi NPs sized 60 nm with the average pore diameter of 2 nm and specific surface area of 200 m2/g were obtained by ball-milling of electrochemically-etched microporous silicon films. After interaction with PSi NPs, a strong suppression of the infectious activity of the virus-contaminated fluid was observed, which was manifested in a decrease in the infectious titer of all studied types of viruses by approximately 104 times, and corresponded to an inactivation of 99.99% viruses in vitro. This sorption capacity of PSi NPs is possible due to their microporous structure and huge specific surface area, which ensures efficient capture of virions, as confirmed by ELISA analysis, dynamic light scattering measurements and transmission electron microscopy images. The results obtained indicate the great potential of using PSi NPs as universal viral sorbents and disinfectants for the detection and treatment of viral diseases.

Strip-dried blood sampling: applicability for bovine leukemia virus detection with ELISA and real-time PCR.

We recently proposed a new so-called strip-dried format aimed for convenient use of dried biomaterial in diagnostic purposes. In this work, 334 blood samples obtained in strip-dried form were used for bovine leucosis analysis with ELISA and real-time PCR methods. High percentage of seropositive animals (18.3%) let us estimate both indirect (serological) and direct methods applicability for the analysis of strip-dried blood samples and also to compare them (PCR results concurred with ELISA in 93.4% cases). Parallel analysis of native and corresponding strip-dried samples approved the proposed format as a reliable analytical way of sampling being in 100% concordance with conventional serum/whole blood ELISA and PCR analysis. Even distribution of antibodies against bovine leukemia virus along the membrane carrier was demonstrated by square-to-square analyzing of the sample strip (CV not exceeded 7%). Also, strip-dried blood samples showed enhanced stability at elevated temperatures comparing to liquid serum. The proposed strip-blood format is a promising way of sampling, storage and transportation and can find application in veterinary practice for infectious disease monitoring.

Detection of Antibodies Against Foot-and-Mouth Disease Virus Serotypes A, O and Asia-1 by ELISA in Strip-Dried Samples from Vaccinated Bovines.

Antibodies against foot-and-mouth disease virus serotypes A, O and Asia-1 were detected by ELISA in liquid and strip-dried samples from vaccinated bovines. The results showed high concordance of the results in both types of serum samples (coefficient of correlation varied from 0.89 to 0.96). Stability studies showed that anti-foot-and-mouth disease virus antibodies can be detected in strip-dried serum samples stored at different temperatures on a level of that in native samples. Preliminary study in strip-dried whole blood samples demonstrated good potential of this sample pretreatment procedure for the following anti-foot-and-mouth virus antibodies testing.