Canine parvovirology - A brief updated review on structural biology, occurrence, pathogenesis, clinical diagnosis, treatment and prevention.

Canine parvovirus (CPV) is a major cause of hemorrhagic diarrhea and mortality in puppies worldwide. There are 2 types of Parvovirus which affects canines: Canine parvovirus 2 (CPV-2) and Canine parvovirus 1 (CPV-1) or the Minute Virus of Canine (MVC). CPV-2 originated from Feline panleukopenia virus and has undergone genetic variation to give rise to its three variants (CPV-2a, CPV-2b and CPV-2c). Amino acid substitutions in VP2 capsid protein have led virus to adapt new host range. The original CPV-2 was known to be dominant in Japan, Belgium, Australia as well as USA and later circulated throughout the world. Clinically, CPV-2 infection is characterized by anorexia, lethargy, depression, vomiting, leukopenia and severe hemorrhagic diarrhea. Several diagnostic tests have been developed to detect parvoviral infections which are categorized into immunological tests (latex agglutination test, SIT-SAT and ELISA etc.) and molecular based tests (PCR, mPCR and RT-PCR etc.). To control and manage the disease several treatments like fluid therapies, antibiotics, and adjunctive treatments are available and some are in various stages of development. Apart from this, many vaccines are also commercially available and some are in developmental stages. The present review contains detailed information regarding structural biology, occurrence, pathogenesis, clinical diagnosis, treatments and prevention in order to understand the need and the growing importance of CPV-2.

A fluorescent aptasensor for the detection of Aflatoxin B1 by graphene oxide mediated quenching and release of fluorescence.

Aflatoxin B1 contamination in food and agro commodities has been major concern of global food safety and trade industry. There is an urgent need to develop sensitive and on-site detection methods for aflatoxins mainly, AFB1 monitoring. In the present study, a fluorophore (Alexa Fluor 488) based aptamer biosensor was devised in combination with graphene oxide (GO) for the detection of Aflatoxin B1 (AFB1). The optimized diagnostic procedure consisted of a fluorescent modified aptamer (Ax-AFLA5) as detection probe and GO mediated quenching of the same; to the quenched system AFB1 was added resulting in subsequent release of fluorescence. The principle of GO based adsorption of ssDNA and successive desorption in the presence of target mycotoxin was utilised in development of the bioassay. In presence of target mycotoxin, the GO adsorbed ssDNA attained a structural conformation resulting in desorption and subsequent release of fluorescence. Assay parameters such as concentration of fluorescent probe, GO and incubation time were evaluated and optimized. The optical signal thus generated could determine presence of AFB1 in the given sample. Selectivity of the method with other mycotoxins was evaluated. The linear range of AFB1 from 0.2-200 ppb was assessed. Visible green fluorescence release was observed at 20 ppb under UV transilluminator and the detection limit of the developed assay was interpreted as 20 ppb of AFB1. The suitability of the assay for AFB1 quantification in groundnut and natural samples was also evaluated. Thus, the developed assay can be a field deployable, reliable and rapid alternative tool for onsite screening method of aflatoxins and other mycotoxins at field level.

Selection and Characterization of Cell Surface Specific Aptamer and Development of Fluorescence Assay for Detection of Shigella flexneri from Water Samples.

The present study demonstrates, development of ssDNA aptamers against whole cell of S. flexneri employing a whole bacterium-based Systemic Evolution of Ligands by Exponential Enrichment (SELEX). After ten rounds of SELEX, cell surface specific aptamer pool was cloned, sequenced and divided based on sequence similarities and secondary structure. Binding affinity of FITC labelled aptamer from different group were carried out by flow cytometry analysis. The dissociation constant (Kd) values for specific and higher binder were evaluated to range from 144 to 329 nM. Six high binding aptamers with lower dissociation constant was chosen for selectivity study. Aptamer SHI 23, SHI 37 and SHI 42 showed higher selectivity towards S. flexneri in comparison with other related bacteria. Further applicability of selected aptamer was proven by fluorescence assay for convenience detection of target cell from spiked water sample and natural contaminated water samples. Altogether, aptamer generated in this study can be alternative DNA ligands for detection of S. flexneri compared to available ligands.

Dual Aptamer-DNAzyme based colorimetric assay for the detection of AFB1 from food and environmental samples.

In the present study, a colorimetric biosensor strategy is devised in combination with apta-magnetic separation assisted with DNAzyme based colorimetric detection of Aflatoxin B1 (AFB1). The optimized analytical procedures consisted of the capture of AFB1 by biotinylated aptamer conjugated to streptavidin magnetic beads and detection by a colorimetric signal from a DNAzyme modified aptamer in presence hemin and H2O2/TMB (3', 3', 5, 5'- tetramethylbenzidine). The DNA concentration, incubation time, hemin, and NaCl concentrations were evaluated and optimized. The visual optical signal thus generated could determine the presence of AFB1 in the given sample. The selectivity of the method with other mycotoxins was evaluated. The linear range of AFB1 from 0 to 200 ppb was assessed and detected as low as 40 ppb visually. The absorbance of blue color generated by the catalytic reaction was in a linear correlation with AFB1 concentrations and was able to detect as low as 22.6 ppb (LOD). The suitability of the assay for AFB1 quantification in sorghum and natural samples was also evaluated. Thus, the developed assay could be a reliable, inexpensive, alternative tool for possible use as a screening method for aflatoxins and other mycotoxins.

Highly Sensitive Colorimetric Biosensor for Staphylococcal Enterotoxin B by a Label-Free Aptamer and Gold Nanoparticles.

A simple, sensitive and selective colorimetric biosensor for the detection of Staphylococcal enterotoxin B (SEB) was developed using SEB-binding aptamer (SEB2) as recognition element and unmodified gold nanoparticles (AuNPs) as colorimetric probes. The assay is based on color change from red to purple due to conformational change of aptamer in the presence of SEB, and the phenomenon of salt-induced AuNPs aggregation which could be monitored by naked eye or UV-vis spectrometer. Results showed that the AuNPs can effectively differentiate the SEB induced conformational change of the aptamer in the presence of a given high salt concentration. A linear response in the range of 50 µg/mL to 0.5 ng/mL of SEB concentration was obtained. The assay was highly specific to SEB as compared to other related toxins. The limit of detection (LOD) of SEB achieved within few minutes was 50 ng/mL visually and spectrometric method improved it to 0.5 ng/mL. Robustness of the assay was tested in artificially spiked milk samples and cross-checked using in house developed sandwich ELISA (IgY as capturing and SEB specific monoclonal as revealing antibody) and PCR. This colorimetric assay could be a suitable alternative over existing methods during biological emergencies due to its simplicity, sensitive and cost effectiveness.

Colorimetric DNAzyme Biosensor for Convenience Detection of Enterotoxin B Harboring Staphylococcus aureus from Food Samples.

In the present study, a colorimetric DNAzymes biosensor strategy was devised in combination with immunomagnetic separation for rapid and easy detection of enterotoxin B harboring Staphylococcus aureus from food and clinical samples. The method employs immunocapture of S. aureus and amplification of seb gene by DNAzyme complementary sequence integrated forward primer and with specific reverse primer. The DNAzyme sequence integrated dsDNA PCR products when treated with hemin and TMB (3,3',5,5'-tetramethylbenzidine) in the presence of H2O2 produce colorimetric signal. A linear relationship of optical signal with the initial template of seb was obtained which could be monitored by visually or spectrophotrometrically for qualitative and quantitative detection. The limit of detection for the assay was approximately 102 CFU/mL of seb gene harboring target. This method is convenient compared to gel based and ELISA systems. Further, spiking studies and analysis on natural samples emphasized the robustness and applicability of developed method. Altogether, the established assay could be a reliable alternative, low-cost, viable detection tool for the routine investigation of seb from food and clinical sources.

Capture and detection of Staphylococcus aureus with dual labeled aptamers to cell surface components.

In the present study, a high throughput whole cell SELEX method has been applied successfully in selecting specific aptamers against whole cells of Staphylococcus aureus, a potent food poisoning bacterium. A total ten rounds of SELEX and three rounds of intermittent counter SELEX, was performed to obtain specific aptamers. Obtained oligonucleotide pool were cloned, sequenced and then grouped into different families based on their primary sequence homology and secondary structure similarity. FITC labeled sequences from different families were selected for further characterization via flow cytometry analysis. The dissociation constant (Kd) values of specific and higher binders ranged from 34 to 128nM. Binding assays to assess the selectivity of aptamer RAB10, RAB 20, RAB 28 and RAB 35 demonstrated high affinity against S. aureus and low binding affinity for other bacteria. To demonstrate the potential use of the aptamer a sensitive dual labeled sandwich detection system was developed using aptamer RAB10 and RAB 35 with a detection limit of 102CFU/mL. Furthermore detection from mixed cell population and spiked sample emphasized the robustness as well as applicability of the developed method. Altogether, the established assay could be a reliable detection tool for the routine investigation of Staphylococcus aureus in samples from food and clinical sources.

Immuno Affinity SELEX for Simple, Rapid, and Cost-Effective Aptamer Enrichment and Identification against Aflatoxin B1.

Aflatoxins are naturally occurring mycotoxins that contaminate food and agro commodities, leading to acute and chronic health conditions in human and animals. In the present work, an attempt was made to generate high-affinity single stranded DNA aptamers that specifically bind to Aflatoxin B1 (AFB1) by a modified Systemic Evolution of Ligands by Exponential Enrichment (SELEX) procedure with the aid of Immunoaffinity columns. Ten rounds of SELEX and alternating three counter SELEX rounds with a cocktail of related and other mycotoxins were performed to enhance the specificity. Resultant 105 aptamers were clustered into 12 groups according to their primary sequence homology. Candidates with lowest Gibbs free energy (dG value) and unique stem loop structures were selected for further characterization. Aptamers, AFLA5, AFLA53, and AFLA71 exhibiting lower Kd values (50.45 ± 11.06, 48.29 ± 9.45, and 85.02 ± 25.74 nM) were chosen for development of ELONA and determination of purification ability of toxin. The detection limit (LOD) of AFLA5 and AFLA71 was 20 and 40 ng/ml, respectively. HPLC analysis implied that selected aptamers were able to recover and quantify 82.2 to 96.21% (LOQ - 53.74 ng) and 78.3 to 94.22% (LOQ - 66.75 ng) of AFB1 from spiked corn samples, respectively. These findings indicate, immunoaffinity based SELEX can pave an alternative approach to screen aptamers against mycotoxin detection and purification.

Selection and Characterization of Aptamers Using a Modified Whole Cell Bacterium SELEX for the Detection of Salmonella enterica Serovar Typhimurium.

This study describes the selection of single-stranded DNA (ssDNA) aptamers against Salmonella enterica serovar Typhimurium using a modified whole cell systematic evolution of ligands by exponential enrichment (whole cell SELEX). For evolving specific aptamers, ten rounds of selection to live Salmonella cells, alternating with negative selection against a cocktail of related pathogens, were performed. The resulting highly enriched oligonucleotide pools were sequenced and clustered into eight groups based on primary sequence homology and predicted secondary structure similarity. Fifteen sequences from different groups were selected for further characterization. The binding affinity and specificity of aptamers were determined by fluorescence binding assays. Aptamers (SAL 28, SAL 11, and SAL 26) with dissociation constants of 195 ± 46, 184 ± 43, and 123 ± 23 nM were used to develop a nanogold-based colorimetric detection method and a sedimentation assay. The former showed a better sensitivity limit of 10(2) CFU/mL using aptamer SAL 26. This approach should enable further refinement of diagnostic methods for the detection of Salmonella enterica serovar Typhimurium and of other microbial pathogens.

A combinatorial systematic evolution of ligands by exponential enrichment method for selection of aptamer against protein targets.

Aptamers are synthetic DNA recognition elements which form unique conformations that enable them to bind specifically to their targets. In the present study, an attempt was made to standardize a new modified combinatorial method comprising of Ni-NTA affinity Systematic Evolution of Ligands by Exponential Enrichment (SELEX; based on affinity between His tag protein and Ni-NTA), membrane SELEX (based on immobilization of protein on nitrocellulose membrane), and microtiter plate based SELEX (to monitor affinity and to enrich the selected aptamers) for protein targets. For experimental evaluation, staphylococcal interotoxin B was the molecule chosen. The new combinatorial method enhanced selection ability up to 51.20 % in comparison with individual conventional procedures. Employing this method following six rounds of selection, high-affinity aptamers with very different properties could be obtained with a dissociation constant (K d) value as low as 34.72 ± 25.09 nM. The optimal aptamers could be employed in fluorescence binding assay, enzyme-linked oligonucleotide assays, and aptamer-based Western blot assay for characterization and detection. These results pave a potential path without using of any robotics for high-throughput generation of aptamers with advantages in terms of rapidity, simplicity, and ease in handling.