Detecting viral antigens at low concentrations in field samples can be crucial for early veterinary diagnostics. Proximity ligation assays (PLAs) in both solution and solid-phase formats are widely used for high-performance protein detection in medical research. However, the affinity reagents used, which are mainly poly-and monoclonal antibodies, play an important role in the performance of PLAs. Here, we have established the first homogeneous and solid-phase proximity-dependent DNA aptamer ligation assays for rapid and accurate detection of Newcastle disease virus (NDV). NDV is detected by a pair of extended DNA aptamers that, upon binding in proximity to proteins on the envelope of the virus, are joined by enzymatic ligation to form a unique amplicon that can be sensitively detected using real-time PCR. The sensitivity, specificity, and reproducibility of the assays were validated using 40 farm samples. The results demonstrated that the developed homogeneous and solid-phase PLAs, which use NDV-selective DNA aptamers, are more sensitive than the sandwich enzymatic-linked aptamer assay (ELAA), and have a comparable sensitivity to real-time reverse transcription PCR (rRT-PCR) as the gold standard detection method. In addition, the solid-phase PLA was shown to have a greater dynamic range with improved lower limit of detection, upper-and lower limit of quantification, and minimal detectable dose as compared with those of ELAA and rRT-PCR. The specificity of PLA is shown to be concordant with rRT-PCR.
Sickle cell disease(SCD)is a monogenic disease, in which the severity and symptoms vary widely.The sickle cell mutation is the origin of all functional and structural alterations of red blood cell(RBC)which induces the acceleration of eryptosis and formation of Circulating blood cells microparticules(MPs)which has the consequence of acute anemia and increasing the thrombotic risks in sickle cell patients. Eryptosis is the suicidal erythrocyte death characterized by erythrocyte membrane scrambling by phosphatidylserine(PS)externalization. MPs are an extracellular vesicules released following cellular activation, stress or apoptosis. These parameters are able to modulate many biological functions, and considered as biomarkers of preventive diagnostic of SCD. This present study aims to determine the cellular biomarkers to implementation new and innovative methods of the preventive diagnosis of SCDacute complications. We propose to study the mechanisms involved in the triggering of eryptosis and to quantify microparticles derived from platelets and erythrocytes of homozygous sickle cell patients. Following clinical diagnosis, homozygous SCDpatients and healthy donors were sampled for hematological and cellular assays. The exploration of eryptosis and MPs was performed by a flow cytometry by determining the viability parameters of red blood cells. The outcome of our study indicated that Eryptosis in sickle cell patients is triggered essentially by high Ca2 + entry and narrowing of red blood cells. However, the pathway of ceramides and ERO can also considered a stimulating factor of eryptosis. Eryptosis ensures healthy erythrocyte quantity in circulation whereas excessive eryptosis is the cause of acute anemia and may contribute to vaso-occlusive crisis in SCD patients.For MPs study,microparticles derived from platelets and erythrocytes clearly increased in SCD patients. This suggests that erythrocytes MPs may contribute to thrombotic risk andchronic hemolytic anemia. Key messages Potentiel biomarkers in the preventive diagnosis of sickle cell disease. Eryptosis and plasmatic microparticules as potentiel biomarkers.
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