RESUMEN
Typhoid fever is an acute illness caused by Salmonella Typhi and the current diagnostic gap leads to inaccurate, over-diagnosis of typhoid leading to excessive use of antibiotics. Herein, to address the challenges we describe a new rapid color-shift assay based on a novel bifunctional nanobioprobe (Vi-AgNP probe) that is functionalized with specific biomarker Vi polysaccharide and also has the co-presence of Ag as urease inhibitor. The immunoreactions between the Vi with specific antibodies (Abs) present in typhoid patient sample forms a shielding barrier over Vi-AgNP probe rendering the urease to be active, generating colored output. Vi polysaccharide coating on the AgNP was visualized using HRTEM. TEM was performed to get insight into shielding barrier formation by the Abs. MST (microscale thermophoresis) data showed less binding Kd of 7.43 µM in presence of Abs whereas probe with urease showed efficient binding with Kd 437 nM. The assay was validated using 53 human sera samples and proven effective with 100% sensitivity. The assay showed relative standard deviation (RSD) of 4.3% estimated using rabbit anti-Vi Abs. The entire procedure could be completed within 15 min. Unlike lateral flow based assays, our assay does not require multiple combination of Abs for detection. The assay format was also found compatible in paper strip test that provides promising opportunities to develop low-cost on-spot assay for clinical diagnostics.
Asunto(s)
Técnicas Biosensibles , Fiebre Tifoidea , Animales , Humanos , Conejos , Anticuerpos Antibacterianos , Polisacáridos Bacterianos , Salmonella typhi , Fiebre Tifoidea/diagnóstico , UreasaRESUMEN
The developmental asymmetry of fission yeast daughter cells derives from inheriting 'older Watson' versus 'older Crick' DNA strand from the parental cell, strands that are complementary but not identical with each other. A novel DNA strand-specific 'imprint', installed during DNA replication at the mating-type locus (mat1), imparts competence for cell type inter-conversion to one of the two chromosome replicas. The catalytic subunit of DNA Polymerase α (Polα) has been implicated in the imprinting process. Based on its known biochemical function, Polα might install the mat1 imprint during lagging strand synthesis. The nature of the imprint is not clear: it is either a nick or a ribonucleotide insertion. Our investigations do not support a direct role of Polα in nicking through putative endonuclease domains but confirm its indirect role in installing an alkali-labile moiety as the imprint. While ruling out the role of the primase subunit of Polα holoenzyme, we find that mutations in the Polα-recruitment and putative primase homology domain in Mcm10/Cdc23 abrogate the ribonucleotide imprint formation. These results, while confirming the ribonucleotide nature of the imprint suggest the possibility of a direct role of Mcm10/Cdc23 in installing it in cooperation with Polα and Swi1.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , ADN Polimerasa I/metabolismo , Replicación del ADN/genética , Genes del Tipo Sexual de los Hongos/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Ribonucleótidos/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Dominio Catalítico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , ADN Polimerasa I/química , ADN Polimerasa I/genética , ADN Primasa/química , ADN Primasa/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Mantenimiento de Minicromosoma/química , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
We report a novel aptamer functionalized MoS2-rGO based electrochemical method for Vi polysaccharide antigen mediated detection of enteric fever. Herein, highly selective anti-Vi aptamers were screened from a pool of oligonucleotides using a microtitre based SELEX approach and characterized for its specificity and stability. The MoS2-rGO nanocomposite was synthesized using a liquid assisted exfoliation by taking optimum ratio of MoS2 and rGO. The nanocomposite presented synergistic effect owing to easy biomolecular functionalization and enhanced conductivity. The screened anti-Vi aptamers were embedded on the MoS2-rGO nanocomposite via thiol linkage to give a stable biointerface. The developed aptasensor was characterized and further evaluated for its performance with different concentrations of Vi antigen using ferrocene labeled boronic acid as an electroactive probe. The aptasensor responded linearly in the range between 0.1â¯ngâ¯mL-1 to 1000â¯ngâ¯mL-1with a detection limit of 100â¯pgâ¯mL-1, and did not show any cross-reactivity with other bacterial polysaccharides indicating high specificity. The applicability of the developed aptasensor was further validated in urine and sera specimens spiked with Vi antigen.