Detection of Rotavirus from stool samples using a standardized immuno-PCR ("Imperacer") method with end-point and real-time detection

Immuno-PCR (IPCR) has been studied to increase the detection sensitivity of current enzyme-linked immuno-sorbent assays (ELISA) as a novel approach for the early detection of Rotavirus infection, a major source for serious diarrhoea for susceptible risk groups. IPCR utilizes specific antibody-DNA conjugates with subsequent amplification of the marker-DNA. An antibody-DNA conjugate specific for Rotavirus antigen VP6 was synthesized and used in combination with a commercially available Rotavirus-ELISA kit. IPCR was carried out using reagents and protocols of the standardized Imperacer system. Real-time PCR monitoring of the marker-DNA amplification was compared to endpoint quantification of amplified haptene-labeled PCR products, using a microtiterplate-based PCR-ELISA. In spiked calibration samples, as few as 100 virus particles/ml could be clearly detected using the IPCR method and either real-time or end-point quantification compared to about 100,000 virus particles/ml in ELISA. Rotavirus positive and negative stool samples were correctly identified by IPCR with a clear separation even of a 10,000-fold dilution of the positive stool samples from the negative control. (c) 2005 Elsevier Inc. All rights reserved.